Retinopatia Diabetica

Progress in Retinal and Eye Research 51 (2016) 156e186
Contents lists available at ScienceDirect
Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/prer
Review
The progress in understanding and treatment of diabetic retinopathy

Alan W. Stitt a, *, 1, Timothy M. Curtis a, 1, Mei Chen a, 1, Reinhold J. Medina a,
Gareth J. McKay b, 1, Alicia Jenkins a, c, 1, Thomas A. Gardiner a, 1, Timothy J. Lyons a, 1,
e, 1, Noemi Lois a, 1
Hans-Peter Hammes d, 1, Rafael Simo
a
Centre for Experimental Medicine, Queen's University Belfast, Northern Ireland, UK

Centre for Public Health, Queen's University Belfast, Northern Ireland, UK
c
NHMRC Clinical Trials Centre, Sydney Medical School, The University of Sydney, Australia
d
Department of Medicine and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany
e
Institut de Recerca Hospital Universitari Vall d'Hebron (VHIR), Barcelona, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2015
Received in revised form
12 August 2015
Accepted 13 August 2015
Available online 18 August 2015
Diabetic retinopathy is the most frequently occurring complication of diabetes mellitus and remains a
leading cause of vision loss globally. Its aetiology and pathology have been extensively studied for half a
century, yet there are disappointingly few therapeutic options. Although some new treatments have
been introduced for diabetic macular oedema (DMO) (e.g. intravitreal vascular endothelial growth factor
inhibitors (anti-VEGFs) and new steroids), up to 50% of patients fail to respond. Furthermore, for people
with proliferative diabetic retinopathy (PDR), laser photocoagulation remains a mainstay therapy, even
though it is an inherently destructive procedure.
This review summarises the clinical features of diabetic retinopathy and its risk factors. It describes
details of retinal pathology and how advances in our understanding of pathogenesis have led to identication of new therapeutic targets. We emphasise that although there have been signicant advances,
there is still a pressing need for a better understanding basic mechanisms enable development of reliable
and robust means to identify patients at highest risk, and to intervene effectively before vision loss
occurs.
2015 Elsevier Ltd. All rights reserved.
Keywords:
Diabetic retinopathy
Diabetic macular oedema
Diabetes
Retina
Pathogenesis
Contents
1.
2.
3.
The global significance of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Classification of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Identification of risk factors for diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3.1.
The diabetic milieu and retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3.1.1.
The importance of glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3.1.2.
The importance of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
3.1.3.
The importance of blood pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.1.4.
Is systemic control of diabetes enough to prevent diabetic retinopathy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.2.
Screening for diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.3.
Retinal vessel profiling during diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.3.1.
Vessel analysis as a prognostic indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.3.2.
Oxygen saturation in the retinal vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.3.3.
Wide angle imaging in the diabetic retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3.4.
Systemic and tissue specific biomarkers for retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
* Corresponding author. Centre for Experimental Medicine, Queen's University Belfast, Belfast, BT9 7BL Northern Ireland, UK.
E-mail address: a.stitt@qub.ac.uk (A.W. Stitt).
1
Percentage of work contributed by each author in the production of the manuscript is as follows: Stitt 20%; Curtis 10%; Chen 5%; Medina 5%; McKay 5%; Jenkins 5%,
Gardiner 5%; Lyons 10%; Hammes 10%; Simo 10%; Lois 15%.
http://dx.doi.org/10.1016/j.preteyeres.2015.08.001
1350-9462/ 2015 Elsevier Ltd. All rights reserved.
A.W. Stitt et al. / Progress in Retinal and Eye Research 51 (2016) 156e186
4.
5.
6.
7.
8.
157
3.5.
Genetic, epigenetics and miRNA profiling and diabetic retinopathy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Modelling diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
4.1.
Culture-based models of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
4.2.
Rodent models of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
4.3.
Large animals as models of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
4.4.
Models of end-stage diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Molecular & cellular pathology of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.1.
Features of retinal microvascular dysfunction during diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.1.1.
Blood flow changes in diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.1.2.
Blood retinal barrier dysfunction in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
5.1.3.
BM thickening in retinal blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
5.1.4.
Pericyte death in diabetic retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
5.1.5.
Capillary endothelial cell death in diabetic retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
5.2.
Diabetes-related changes to the RPE-choroid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
5.2.1.
RPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
5.2.2.
Choroid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
5.3.
Retinal neuron and glial dysfunction during diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
5.4.
Inflammation and immune cell activation in diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
5.5.
Retinal ischaemia during diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Therapeutic options in diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
6.1.
Treatments for DMO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
6.2.
Treatments for PDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Developing new therapies for diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
7.1.
Understanding pathogenesis as a basis for new drug development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
7.2.
Difficulties of translating discoveries into benefits for patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
7.3.
New therapeutic angles for diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
7.4.
Time to consider a precision medicine approach for diabetic retinopathy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
1. The global signicance of diabetic retinopathy

The global incidence of diabetes mellitus is set to rise dramatically from an estimated 382 million people in 2013 to 592 million
by 2030 (Derakhshan et al., 2012; Guariguata et al., 2014;
Rathmann and Giani, 2004). Type 1 diabetes (T1D) arises from an
autoimmune destruction of beta cells in the endocrine pancreas
causing severe insulin deciency. Type 2 diabetes (T2D) is characterized by impaired insulin action (insulin resistance) which is
multifactorial in origin, and in its later stages, leads to impaired
insulin production. More than 90% of patients contributing to the
steep rise in disease incidence have T2D and this relates to
increased consumption of calorie-dense foods of low nutritional
value, lack of exercise, and an increased prevalence of obesity
(Kanguru et al., 2014). T2D has reached epidemic proportions, most
recently in developing nations as they adopt western dietary and
lifestyle habits. T1D also shows some increase in incidence
although the reasons behind this are unclear (Derakhshan et al.,
2012; Guariguata et al., 2014; Rathmann and Giani, 2004).
The morbidity caused by diabetes threatens to overwhelm
already stretched healthcare systems in advanced nations but also
in the developing world. Much of this burden is caused by the
vascular complications of diabetes. Patients with long-duration
diabetes (whether T1D or T2D) develop macrovascular complications (heart disease, stroke and peripheral artery disease) from
which 70% eventually die, and microvascular complications which
affect the kidneys (nephropathy), eyes (retinopathy) and peripheral
nerves (neuropathy).
Diabetic retinopathy is the most common microvascular
complication of diabetes (Antonetti et al., 2012), and after two
decades of disease, almost all T1D patients will have some degree
of retinopathy, as will more than 80% of insulin-treated T2D
patients and 50% of those not requiring insulin (Klein et al., 1989;
Romero-Aroca et al., 2010). Among people with diabetes, the
overall prevalence of diabetic retinopathy worldwide is about one
third, with increasing risk associated with longer disease duration, higher haemoglobin A1C (HbA1c) and presence of hypertension (Yau et al., 2012). Recent estimates suggest that the
number of people with diabetic retinopathy will increase from
127 million in 2010 to 191 million by 2030 (Zheng et al., 2012).
The World Health Organisation (WHO) has estimated that diabetic retinopathy accounts for 15e17% of total blindness in
Europe and the USA (Resnikoff et al., 2004) although there are
apparent ethnic variations in the occurrence of vision loss
(Sivaprasad et al., 2012). In other parts of the world, the burden of
blindness arising from diabetes is considerably greater (Resnikoff
et al., 2004).
It is important to note that not every patient who develops
diabetic retinopathy will experience severe vision loss which occurs only in advanced stages typied by diabetic macular oedema
(DMO) and/or proliferative diabetic retinopathy (PDR). In addition
to the personal and nancial implications for the patient, the societal burden of diabetic retinopathy is substantial. Healthcare costs
for patients with diabetic retinopathy are almost doubled
compared to those without the condition (Heintz et al., 2010). It
follows that any treatment that could block or inhibit the progression of diabetic retinopathy would be of immense value, both
to individuals with diabetes and to society as a whole.
DMO is the more common form of advanced diabetic retinopathy, and thus constitutes a major healthcare challenge. Using
England as an example, Minassian et al. (2012) estimated that
there were over 167,000 patients with DMO in one or both eyes in
2010 (representing ~7% of the diabetic population). Almost 65,000
of these had clinically signicant reduction in visual acuity in at
158
least one eye. In monetary terms, they estimated the annual cost of
DMO in England to be 117M. This estimate was made before
expensive intra-vitreal drugs targeting vascular endothelial
growth factor (VEGF) were approved for use in the UK National
Health Service.
2. Classication of diabetic retinopathy
Diabetes is thought to affect almost all of the 30 or more cell
types in the retina. However, because the inner retinal vasculature
is so readily visualized the classication and grading of retinopathy
has been based on the severity of vascular lesions. The commonly
used and now standard Early Treatment of Diabetic Retinopathy
Study (ETDRS) scale (ETDRS, 1991) is based on the anatomical
features of the retina and on the number of photographically
detectable microvascular lesions. It must be noted that being
anatomically-based, the ETDRS severity scale is not a true quantitative measure, and may not reect important functional decits.
The fact that retinopathy is geographically disperse across an individual retina, and between the two eyes of an individual person,
makes it difcult to obtain quantitative data for analysis in clinical
research. As technologies improve, there is an opportunity for
improved assessments that more accurately dene retinopathy and
its effects, including those on subtle but signicant changes in visual function in the early stages of disease. Thus, functional as well
as structural and biochemical biomarkers for diabetic retinopathy
are now being developed.
Diabetic retinopathy may be very broadly classied into two
stages based on the level of microvascular degeneration and
related ischemic damage: non-proliferative diabetic retinopathy
(NPDR) (Fig. 1) and advanced, proliferative diabetic retinopathy
(PDR) (Fig. 2). NPDR can be sub-classied into i) mild NPDR
(presence of microaneurysms in the retina); ii) moderate NPDR
(more than mild but less than severe NPDR; and iii) severe NPDR
(>20 intraretinal hemorrhages in each of the four quadrants,
venous beading in at least two quadrants, and intraretinal
microvascular abnormalities (IRMA) in at least one quadrant in
the absence of PDR). The progression of diabetic retinopathy is
related to abnormalities of the vasculature including permeability
of the blood retina barrier (BRB), progressive microvascular
damage with vascular endothelial cell and pericyte loss, subsequent occlusion of capillaries, thickening of vascular basement
membrane (BM) (Fig. 3), and retinal neuronal and glial
abnormalities.
When diabetic retinopathy affects the macula it is termed diabetic maculopathy. This vascular leakage and accompanying
swelling of the macula is called DMO and this endpoint constitutes
Fig. 1. NPDR lesions in the diabetic retina. Colour fundus photographs (left) and fundus
uorescein angiogram (right) obtained from the left eye of a patient with nonproliferative diabetic retinopathy. Retinal haemorrhages, microaneurysms and hard
exudation are seen (left). Microaneurysms are more apparent on uorescein angiography where areas of ischaemia are also detected (white arrow).
Fig. 2. Clinical prole of PDR. Colour fundus photographs (left) and fundus uorescein
angiogram (right) obtained from the left eye of a patient with PDR. A large pre-retinal
haemorrhage (left, black arrow) and areas of retinal neovessels (left, detailed magnied) were detected on fundus examination. Areas of retinal ischaemia (white arrows,
left) and leak from retinal neovessels (white arrow heads) were seen on uorescein
angiography (right).
the most common cause of blindness in diabetic patients (Klein

et al., 1989). Although DMO can occur at any stage during the
development of retinopathy, it is most prevalent during the later
phases, following progressive vascular and neural damage
(Antonetti et al., 2012). Diabetic maculopathy may be classied as
central or non-central, depending on whether the pathology affects the fovea. It can also be focal or diffuse, based on the extent
of the oedema, and ischemic or non-ischemic, based on the
preservation or involvement of the perifoveal capillary network
(Fig. 4). Diabetic maculopathy may also be classed as tractional or
non-tractional depending on whether or not traction from an
incompletely detached posterior hyaloid or pre-retinal proliferation becomes a feature.
3. Identication of risk factors for diabetic retinopathy
3.1. The diabetic milieu and retinopathy
3.1.1. The importance of glucose
Normalisation of blood glucose levels is the ideal in the management of people with diabetes, but actual targets for glycaemia
must be tailored to the individual, taking into account risks of
hypoglycaemia, especially in T1DM patients. In older patients,
short-term risks of hypoglycaemia, risks associated with medications, and disruption of life must be carefully balanced against any
longer-term benets of tight control. The benets of achieving
tight control early in the course of diabetes, and sustaining it over
time are now well established, based most clearly on the Diabetes
Control and Complications Trial (DCCT) (DCCT, 1993) for T1D, and
on the UK Prospective Diabetes Study (UKPDS) for T2D (UKPDS,
1998a).
In the DCCT, 1441 T1D patients were randomised to intensive
(INT) or conventional (CON) therapy between 1984 and 1989, and
studied until 1993. INT therapy aimed for glycaemia as close to
normal as safely possible, while CON continued the standard care of
the time. The two groups maintained a separation of HbA1c levels
of two percentage points throughout the randomisation phase
(INT: 7.5%; CON: 9.5%), and retinopathy served as the primary study
outcome. The DCCT had a dramatic and clear-cut outcome, and the
study had to be terminated one year early because of the salutatory
effect of INT on development (primary prevention group) and
progression (secondary intervention group) of retinopathy: they
were reduced by 76% and 54% respectively.
Immediately after the DCCT, an observational follow-up study,
called the Epidemiology of Diabetes, Interventions and Complications (EDIC) was initiated and almost all DCCT subjects were
159
Fig. 4. Clinical prole of DMO. Colour fundus photograph (top left), optical coherence
tomography (top right), and early (bottom left) and late (bottom right) phase uorescein angiogram of a patient with DMO. Hard exudation (top left, white arrow
heads), retinal thickening from intraretinal uid (top right, white arrows) and leakage
(bottom left) predominantly from areas with microaneurysmatic changes (bottom
right) are seen.
Fig. 3. Vascular histology of human NPDR. Trypsin digest specimen of post-mortem

non-proliferative diabetic retinopathy from a type-2 diabetic patient. PAShaematoxylin staining of the retinal vasculature shows a retinal artery anked above
and below by capillary beds. Numerous microaneurysms occur a peri-arterial distribution pattern (arrows) (A). B: Higher magnication trypsin digests show a precapillary arteriole (arrow) in which smooth muscle covering is lost and downstream of this
vessel there is a number of microaneurysms and conuent areas of (non-perfused)
acellular capillaries (AC). C: Trypsin digest showing precapillary arterioles with arteriolar pathology (smooth muscle loss) and many microaneurysms (arrowheads). D:
The central retina from a T2D patient showing IRMA (*) between pre-capillary arterioles and adjacent post-capillary venules. The IRMA occur in an area of acellular
capillaries and are drained by venules (arrows).
enrolled. In the continuing EDIC, study subjects are assessed

annually, with the primary goal of dening the effects of prior DCCT
randomisation on the development of cardiovascular and microvascular complications. Importantly, there was no therapeutic
intervention in EDIC, and HbA1c values in the two former DCCT
randomisation groups rapidly converged (in 1994) and they have
remained identical for the past 20 years at ~8.5%. Remarkably, over
90% of the surviving DCCT cohort are still followed in EDIC in 2015.
Over the rst 10 years of EDIC (1994e2004), despite the equalization of HbA1c between the former randomisation groups, the cumulative incidence of retinopathy continued to diverge, with an
overall hazard reduction of 56% in the former INT vs CON group
(White et al., 2010). More recent data up to EDIC year 18 (2012)
show that the cumulative annual incidence of retinopathy progression has now become similar between the former randomisation groups (i.e. lines are now parallel instead of diverging, and
therefore the severity of retinopathy remains lower in former INT
therapy subjects) (Diabetes et al., 2015). Another recent publication
shows that subjects in the former INT vs CON group have signicantly lower requirement for ocular surgery (e.g. 42% fewer vitrectomies). This inter-group difference has emerged only in recent
years, is still divergent (Group et al., 2015).
The persistence and even increasing benet of intensive management after the end of the DCCT randomisation phase was
observed during EDIC not only for retinopathy, but also for other
vascular complications of diabetes (nephropathy, neuropathy, and
cardiovascular disease). It has led to a concept of glycaemic or
metabolic memory (Silva et al., 2014; Team, 2003) suggesting that
an early period of good glycaemic control has durable benecial
effects. The mechanism(s) underlying such effects are not clearly
understood and are the subject of intensive research. Unfortunately, the converse may also be true, i.e. that an early period of
poor control may lead to lasting damage that is not amenable to
future good control. While a reverse DCCT study, i.e. prospective
study of the effects of deliberate poor glycaemic control, would
clearly not be ethical in humans, such a study was conducted in
dogs in a 5-year experiment by Engerman and Kern. After 2.5 years
of poor glycaemic control, and as yet with no evident retinopathy,
diabetic dogs were switched to good glycaemic control for a second
2.5 year period. At the ve year time point, these dogs exhibited
severe retinopathy: just as severe as dogs that had been in poor
control throughout (Engerman and Kern, 1987). This study clearly
shows that the stage may be set for diabetes-related damage in a
retina that, by standard anatomical criteria, appears entirely
normal.
This latter observation is pertinent to studies of T2D patients, in
whom the onset of disease is gradual and asymptomatic, making
disease duration and cumulative exposure to prior hyperglycaemia/hyperlipidaemia difcult to quantify. Many such studies
involve older patients who often have disease of long and poorlydened duration, co-morbidities, and who take numerous
160
medications. Perhaps for these reasons, prospective studies to

assess the efcacy of improved glycaemic control in preventing
cardiovascular disease in T2D have failed to show benet for their
primary outcomes e.g. ACCORD, ADVANCE, VADT which all
enrolled relatively elderly cohorts and followed them for a few
(~5) years. ACCORD showed marginal benet for retinopathy in its
intensive glycaemic control arm (but was stopped early because of
higher mortality in that group) (Ismail-Beigi et al., 2010).
ADVANCE showed no benet for retinopathy (Group et al., 2008).
Interestingly, and consistent with the considerations above, VADT
demonstrated a benet of intensive management for retinopathy
in younger participants, but a deleterious effects in older participants (Azad et al., 2014).
In contrast to these studies, the UKPDS, enrolled newlydiagnosed T2D patients at a somewhat younger age (25e65
years, mean: 53 years), and, recognising that complications develop
over decades, followed them for much longer (1977e1997) (UKPDS,
1998a). Differing in design from DCCT, the prospective UKPDS was
not a straightforward randomised control trial of conventional vs
intensive treatment; rather, within its intensive arm, it also
compared different therapies (sulphonylurea, insulin, metformin).
Its primary questions were whether improved glycaemic control
would prevent clinical complications, and whether it mattered
which treatment modality was employed. In the study, 4209
newly-diagnosed T2D patients were randomised to conventional
(CON) (n 1138) or intensive (INT) (n 2729; 1153 to sulphonylurea and 1156 to insulin) therapy. Metformin was used as a rstline agent in 342 study subjects who were overweight at randomisation. Overall, the study maintained a difference in HbA1c of
0.9% between its CON and INT groups following randomisation,
although in contrast to DCCT, both drifted upwards over time. At
study-end, intensive therapy reduced cumulative microvascular
end-points by 25% (p < 0.01). For retinopathy, INT conferred
benecial effects on progression that became evident within 6
years of randomisation and were sustained thereafter, reducing risk
by 21% after 12 years (p 0.015). Importantly, INT was also associated with lower overall mortality, reduced CVD by some measures, and an even greater reduction in progression of nephropathy
than for retinopathy. For an analysis of the UKPDS cohort 10 years
after study close-out (and without subsequent study intervention),
about one third of the original study subjects were available, and
among these, prior randomisation to INT vs CON conferred a 24%
reduction in risk for a composite microvascular end-point, but
specic effects for retinopathy were not provided (Holman et al.,
2008).
Overall, the clinical evidence shows clearly that good blood
glucose control implemented early in the course of T1D or T2D
reduces the development and progression of retinopathy and other
vascular complications of diabetes. As duration of diabetes increases, longer-term exposure to environmental stresses (as
experienced by the macro and micro-vasculature) associated with
the presence of diabetes, continued injury mediated by glycaemic
memory, and the establishment of cumulative structural vascular
damage, may mean that the benets of achieving better glycaemia
decline as patients age.
3.1.2. The importance of lipids
Hyperglycaemia is, by denition, essential for making a diagnosis of diabetic retinopathy, and perhaps for this reason, it tends
to be viewed as the sole or primary cause of retinal injury. However,
this conclusion is likely to be an over-simplication, because (a)
altered insulin action perturbs not only carbohydrate metabolism,
but also fat and protein metabolism and this may contribute
directly to retinopathy, and (b) many effects of hyperglycaemia are
indirect, e.g. through increasing oxidative stress and/or through the
formation of advanced glycation/lipoxidation products. Therefore

other factors related to these processes may play critical roles (e.g.
variations in antioxidant defense status, or the nature of substrates
exposed to oxidative stress) (Kowluru et al., 2015; Narayanan et al.,
2013; Zhang et al., 2015).
Plasma lipid and lipoprotein levels are established markers of
risk for atherosclerosis. To a large extent, this is because they provide a surrogate index of lipoprotein penetration of the arterial
intima, where atherosclerosis actually begins. Lipoproteins in the
vessel wall, not in the plasma, are the primary drivers of atherogenesis; thus oxidation of intimal (extravasated) LDL promotes
foam cell and plaque formation, and HDL promotes reverse
cholesterol transport from tissues to liver. In contrast to the arteries, the retinal circulation has inner and outer BRBs which normally prevent lipoprotein extravasation so that in the absence of
diabetes, hyperlipidaemia is irrelevant. Once diabetes compromises
BRB integrity, plasma lipoproteins leak into the retinal tissue,
elevated glucose levels and lipoxidative stress promote proteineprotein cross-linking and lipoprotein entrapment, and this
entrapment inevitably results in modication (oxidation, glycation), rendering the lipoprotein cytotoxic. It follows that in the
retina, the existence and extent of lipoprotein extravasation into
tissue is an important concern. Variations in plasma lipoprotein
levels are likely of secondary importance, and, given the difference
in proportionality, cannot be as closely associated with retinopathy
as with atherosclerosis. Importantly, however, this should not
disguise the danger: once extravasated, lipoproteins may
contribute to propagation of retinopathy just as much as they do in
atherogenesis (Yu and Lyons, 2013).
Assocations between plasma lipid/lipoprotein levels are generally weak and not sufcient to dene risk for retinopathy in individual people. Many studies, both cross-sectional and longitudinal,
have addressed this question. Prominent among these, the Pittsburgh Epidemiology of Diabetes Complications study (Lloyd et al.,
1995), showed that in T1D, serum triglycerides, and to some
extent LDL, were associated cross-sectionally with retinopathy, and
longitudinally with progression to PDR. In our own cross-sectional
study of T1D patients in the DCCT/EDIC cohort, retinopathy was
positively associated with serum triglycerides and negatively with
HDL, and, using nuclear magnetic resonance to analyse size-based
lipoprotein subclasses, with small, atherogenic, sub-classes of LDL
and HDL (more so in men than in women) (Lyons et al., 2004). In
T2D, the Early Treatment Diabetic Retinopathy Study (ETDRS),
studying 2709 patients (Chew et al., 1996) found that elevated LDL
cholesterol levels at baseline doubled the likelihood of retinal hard
exudates. The Hoorn study (van Leiden et al., 2002) found that
diabetic retinopathy was positively associated with cholesterol and
triglyceride levels, and that retinal hard exudates were associated
with LDL cholesterol. In the Atherosclerosis Risk In Communities
(ARIC) study (Klein et al., 2002), the presence of retinal hard exudates was associated with LDL and lipoprotein(a), but other features of retinopathy were not associated with plasma lipid levels. A
recent cross-sectional study by Sacks et al. (2014), assessing longduration T2D patients from 13 different countries, also found associations of plasma lipid levels with retinopathy, but these became
non-signicant when fully adjusted. Broadly therefore, these
studies show relatively weak, mainly cross-sectional associations
between plasma lipids and severity of retinopathy in both T1D and
T2D patients. Differences may relate to the populations studied,
including type of diabetes, age, genetics, and medications taken, as
well as variation in denitions and stages of diabetic retinopathy,
and differences in laboratory and statistical techniques used. The
most challenging elements of these studies are the slow evolution
of retinopathy over years and decades with the need to assess covariates over these prolonged time-frames.
Lipoprotein extravasation and modication occur in retinopathy, and this may play a role in disease progression. These studies
have demonstrated that in the diabetic retina there is signicant
extravasation of LDL, but that this does not occur in healthy, nondiabetic retina (Wu et al., 2012). The extent of LDL extravasation,
and the amount of intra-retinal oxidised LDL, is proportionate to
the severity of retinopathy, but the appearance of extravasated
and modied lipoproteins in the retina in the pre-clincial phase
suggests relevance to the earliest stages of retinal injury. Consistent with intra-retinal effects, human cell culture studies have
demonstrated cytotoxicity of modied LDL towards retinal capillary endothelial cells (Lyons et al., 1994), pericytes (Difey et al.,
2009), RPE (Du et al., 2013), and Mller glial cells (Wu et al.,
2012), and revealed alterations in signalling pathways, gene
expression, autophagy, and apoptosis (Difey et al., 2009; Wu
et al., 2012). Furthermore, modied LDL may become immunogenic. It has been suggested that oxidised-LDL-immune complexes
are present in human diabetic retinas (Fu et al., 2012) and that, in
cultured retinal pericytes, immune-complexed oxidised LDL is
even more toxic than oxidised LDL alone (Fu et al., 2014). Further
supporting an immunologic mechanism, in decades-long followup of the DCCTeEDIC cohort, circulating levels of LDL immune
complexes predicted the progression of retinopathy (Lopes-Virella
et al., 2012). These studies on the intra-retinal effects of plasma
lipoproteins have recently been reviewed in greater detail (Yu and
Lyons, 2013).
The question arises whether lipid-lowering drugs are effective
in slowing the progression of retinopathy. From the data summarised above, it follows that improving circulating lipoprotein
levels might confer only limited benet. Complicating the question,
it is well established that the two major classes of lipid-lowering
drugs, statins and brates, have other, pleiomorphic effects in
tissues: this brings the opportunity to conate markers (an effect
on plasma levels) with mechanisims (tissue effects), especially
since they are often correlated. Statins, which predominantly lower
circulating LDL levels, have not been shown to have major independent benecial effects in retinopathy, but are nevertheless
important for reducing cardiovascular events in people with diabetes (Cholesterol Treatment Trialists et al., 2008). In contrast,
brates (classically used as triglyceride-lowering agents) have
recently attracted intense interest for a potential direct effect on the
retina. In two major prospective randomised controlled trials (the
ACCORD-Eye and the Fenobrate Intervention and Event Lowering
in Diabetes (FIELD) studies) the progression of retinopathy was
signicantly reduced by fenobrate (Chew et al., 2014; Keech et al.,
2007), and the effect was independent of the reduction of plasma
triglyceride levels. Fenobrate is a peroxisome proliferator activated-receptor alpha (PPARa) agonist, which seem key to its retinal
benets (Ding et al., 2014; Noonan et al., 2013). In addition, other
pleiotropic effects of fenobrate have been recognised and include
anti-inammatory, anti-oxidant, anti-apoptotic and antiangiogenic actions which may also explain retinal protective effects (Simo, 2013).
In conclusion, lipoproteins may play an important role in the
propagation of retinopathy, one that is under-recognised because of
relatively weak associations of plasma levels with retinopathy
severity. Understanding their role, and their capacity to establish
vicious cycles of retinal injury once extravasated, holds promise for
the development of new and effective treatments to stop the progression of diabetic retinopathy.
3.1.3. The importance of blood pressure
It has been suggested that hypertension is an important risk
factor for diabetic retinopathy, as demonstrated by the UKPDS with
patients who achieved tight control of blood pressure experiencing
161
a signicant protection against retinopathy progression (UKPDS,

1998a,b). The META-EYE study showed patients with normal
blood pressure were protected against diabetic retinopathy progression compared to those who had hypertension (BP > 140/
90 mmHg) or were already receiving anti-hypertensive drugs (Yau
et al., 2012). Hypertension is an important risk factor for macrovascular disease but this is also true for the microvascular disease in
the diabetic retina and bouts of systemic hypertension can exacerbate initiation and progression of retinopathy (Silva et al., 2010).
Patients may benet from early use of current anti-hypertensive
agents such as angiotensin-converting enzyme inhibitors and
angiotensin-2 receptor blockers (Simo and Hernandez, 2012).
However a recent Cochrane review examining the impact of blood
pressure control in T2D over 4.5 years showed no effect on diabetic
retinopathy progression (Do et al., 2015).
3.1.4. Is systemic control of diabetes enough to prevent diabetic
retinopathy?
Hyperglycaemia remains the most widely-recognised systemic
factor impacting the onset or progression of early diabetic retinopathy and it has been suggested that HbA1c underpins risk of
complications between the intensive and conventional groups in
the DCCT (White et al., 2008). However, as shown by separate
analysis of the DCCT data (Hirsch and Brownlee, 2010), even HbA1c,
may only account for around 10% of the risk of diabetic retinopathy
and together with blood pressure and total serum cholesterol, may
account for no more than 10% of the risk of diabetic retinopathy as
suggested by the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) (Klein et al., 2008). This suggests that many
unknown factors (e.g. genetic or environmental inuences) must
play an important, if unidentied, role. These outcomes present an
apparent paradox, since intensive management of the DCCT patients (ended 1993) has conferred signicant benet regarding
onset and progression of diabetic retinopathy and this persists.
Although the annual rate of new cases is now equal in the prior
randomisation groups, there is still much less retinopathy in the
former intensively controlled group of patients (Diabetes et al.,
2015). As this debate continues and as research progresses, systemic risk factors beyond the established set may become associated with diabetic retinopathy. As an example, there is growing
evidence that the occurrence of sleep apnoea in diabetic patients
could be associated with DMO (Mason et al., 2012). In addition,
epigenetic mechanisms such as DNA methylation, histone posttranslational modications in chromatin, and non-coding RNAs
can play a role in the pathogenesis of diabetic retinopathy (Perrone
et al., 2014).
3.2. Screening for diabetic retinopathy
Diabetic retinopathy may develop and progress to advanced
stages without producing any immediate symptoms to the patient. Screening for this disease is essential in order to establish
early treatment of sight-threatening retinopathy and has been
demonstrated to be successful at achieving vision loss. Different
methods of screening for diabetic retinopathy have been used,
including direct fundus examination and review of fundus photographs, obtained with or without mydriasis (Stefansson et al.,
2000). Most national screening programmes for diabetic retinopathy rely on digital fundus photographs, obtained following
mydriasis, which are then graded by trained readers, with or
without a measure of visual acuity (Looker et al., 2013; Stefansson
et al., 2000; Stratton et al., 2013; Thomas et al., 2015). Annual
screening for diabetic retinopathy is recommended for anyone
with T1D who is aged 12 or more and has had diabetes for ve
years or more, and for those with T2D, from the time of diagnosis
162
(Stefansson et al., 2000; Scanlon et al., 2008). Although screening

for diabetic retinopathy has been shown to be cost-effective
(Javitt et al., 1994), given the increasing prevalence of diabetes
mellitus in the population, some experts contend that annual
screening may not be needed for everyone. A recent systematic
review suggests that published literature broadly supports
extending the screening intervals to every two years for people
with well controlled T2D with no diabetic retinopathy without
increasing the risk of visual loss (Taylor-Phillips et al., 2015). A
more rened approach proposed entails determining the risk of
each individual by the results of two sequential annual photographic screening visits; on this basis extending the screening
interval to 2 years for those with no evidence of retinopathy
appears to be safe and has been recommended (Stratton et al.,
2013). Personalised screening intervals, estimated using a mathematical algorithm, created based on epidemiological data, and
which takes into account information on gender, type and duration of diabetes, HbA1c or mean blood glucose level, blood
pressure and the presence and grade of retinopathy could be also
used (Aspelund et al., 2011).
Even with improved and more cost-effective strategies for
screening of diabetic retinopathy, the challenge remains of assuring
that all eligible individuals are reached and when so, that they
remain engaged in the screening programmes to reduce the risk of
non-attendance, which is closely associated with poorer long-term
outcomes and increased prevalence of diabetic complications
(Deckert et al., 1978a,b; Rhatigan et al., 1999). To this end, smart
phone ophthalmoscope technology has been suggested to provide
some value since these devices provide portability, affordability,
and connectivity (Russo et al., 2015b). A recent study suggests that
the smart phone ophthalmoscope provides agreement with dilated
retinal biomicroscopy for the grading of diabetic retinopathy and
this makes it a promising tool for community screening programs
(Russo et al., 2015a).
Providing adequate information about diabetic retinopathy to
those affected by diabetes as well as making screening more
convenient (for example, by offering appointments after working
hours) may increase the uptake of screening programmes and
reduce visual loss (Lewis et al., 2007). Furthermore, it is essential
that patients identied as having sight-threatening retinopathy at
screening are referred to Hospital Eye Services. Adequate patient
pathways that allow urgent referral and treatment to be delivered,
if required, need to be in place if the nal goal of screening programmes, namely prevention of visual loss, is to be accomplished.
3.3. Retinal vessel proling during diabetes
3.3.1. Vessel analysis as a prognostic indicator
Determination of new prognostic indicators for early identication and stratication of individuals at increased risk of diabetic
microvascular complications may offer improved opportunity for
timely implementation of effective therapeutic intervention.
Indeed changes in the retinal microvasculature have previously
been reported to offer prognostic value for the prediction of diabetic retinopathy prior to the development of pathological features
such as microaneurysms or haemorrhage (Ikram et al., 2013; Wong,
2011). As such, the retinal microvasculature provides a unique, noninvasive and opportunistic window through which to view general
vascular health. With advances in digital analysis of retinal images,
characteristics relating to retinal vessel calibre, tortuosity, fractal
dimension and bifurcating angles, can now be accurately and systematically measured in a reliable manner (Fig. 5).
Evaluations of retinal microvascular characteristics as indicators
of diabetic retinopathy are well established, with changes in retinal
venular calibre reported to predict vision loss (Rand et al., 1985).
Fig. 5. Vascular proling in the diabetic retina. Quantitative retinal fundus image
assessment using the Singapore I Vessel Assessment (SIVA) software. Arterioles are
coloured in red and venules in blue. The measured area of retinal vascular parameters
(calibre, fractal dimension, tortuosity, and branching angle) was standardised as the
region from 0.5 to 2.0 optic disc diameters from the disc margin.
The development of the Airlie House classication system of diabetic retinopathy acknowledged retinal venular widening as an
early prognostic indicator but recognised the difculties of its
precise measurement at that time and as such opted for more
qualitative measures as a sign of venular dilation, such as retinal
venous beading (Wu et al., 2013). Measurement of such retinal
characteristics from digital images has now been shown to be
precise with good interspecic and intraspecic reliability
(Hubbard et al., 1999).
Previous studies have investigated the association of retinal
vessel calibre with diabetic retinopathy in both T1D and T2D
(reviewed by (Wong, 2011)) with strong evidence showing venular
widening in both cross-sectional (Klein et al., 2003; Tsai et al., 2011)
and prospective studies (Broe et al., 2014; Klein et al., 2004; Roy
et al., 2011). In addition, narrower arteriolar calibre has been
associated with diabetic retinopathy (Klein et al., 2003, 2004) in a
cross-sectional analysis and wider arteriolar diameter in T1D in two
prospective studies (Alibrahim et al., 2006; Cheung et al., 2008)
although additional longitudinal studies have reported no correlation between retinal arteriolar diameter and diabetic microvascular complications (Klein et al., 2007, 2004). This apparent
paradox in arteriolar width in diabetic retinopathy is discussed
later in the review.
3.3.2. Oxygen saturation in the retinal vessels

It is possible to measure oxygen levels in retinal blood vessels
using non-invasive imaging techniques which rely on the different
light absorbance of oxyhaemoglobin and deoxyhemoglobin
(Hammer et al., 2009; Hardarson, 2013). Using this technology,
oxygen levels in the retinal blood vessels of diabetic patients have
been determined in cross-sectional studies which demonstrated
higher oxygen saturation in the retinal venules of diabetic patients
when compared with those of controls (Hammer et al., 2009;
Hardarson and Stefansson, 2012; Jorgensen et al., 2014). Furthermore, oxygen saturation in retinal venules was found to increase as
the severity of diabetic retinopathy advanced (Hammer et al., 2009;
Jorgensen et al., 2014). These ndings are possibly explained by a
reduced oxygen extraction in the retina, result of a loss of the
capillary bed and arterio-venous shunt formation (as evidenced by
IRMA) which would limit the distribution of oxygen in the retina, as
well as other factors such as a reduced need of oxygen in the
context of cell loss in a degenerated retina. Prospective, longitudinal studies determining changes in oxygen saturation in retinal
blood vessels and their implication with regards to development
and progression of diabetic retinopathy would be required to
determine their role on the pathogenesis of the disease. Defects in
retinal oxygen saturation during diabetes is also reected in the
observed lesions in human post-mortem retinal vascular casts

which show loops and so-called reduplications in the larger calibre
venules which show evidence of endothelial cell proliferation (Bek,
2002). It has been suggested that these loop venules are shunt
vessels which serve to bypass an occluded larger retinal vein (Bek,
1999).
3.3.3. Wide angle imaging in the diabetic retina
New wide angle imaging systems have been recently developed,
which achieve a more adequate visualisation of the mid-peripheral
and peripheral retina than previously available technologies. This
so-called ultrawide eld retinal imaging uses scanning laser
ophthalmoscope technology combined with ellipsoidal mirrors to
enabling visualisation of up to 200 (82%) of the retina and may
offer signicant prognostic value in diabetic retinopathy (Silva
et al., 2013). Thus, wide angle imaging enables identication of
peripheral lesions not detected with conventional 7 eld ETDRS
photography and suggested a more severe retinopathy level than
that determined based on standard photography in 10% of patients
(Silva et al., 2013; Wessel et al., 2012). A recently conducted systematic review and meta-analysis of published literature suggests
that digital imaging can be used widely for diabetic retinopathy
screening and that techniques that incorporate mydriasis and a
wide (100e200 ) imaging are best suited for this purpose (Shi et al.,
2015). Furthermore, using wide-angle uorescein angiography, a
relationship between the presence of peripheral retinal ischaemia
and diabetic macular oedema has been identied (Wessel et al.,
2012) which may have potential therapeutic implications. Given
the larger eld of view associated with this technology and as it
becomes more widespread, it is likely that its usefulness in the
assessment of patients with diabetic retinopathy will become even
more apparent and provide earlier treatment options.
3.4. Systemic and tissue specic biomarkers for retinopathy
A biomarker is dened as a characteristic that is objectively
measured and evaluated as an indicator of normal biological processes, or pharmacologic responses to a therapeutic intervention.
Biomarkers can be used to diagnose or predict who will get overt
disease, such as diabetic retinopathy, or who will respond to a
specic treatment. They can also be used to monitor existing disease and treatments and to explain residual risk. Importantly,
biomarkers may also be useful to explain how drugs work and to
suggest new therapeutics.
Challenges to the use of biomarkers include the slow development of end-points such as diabetic retinopathy, that their utility
must be conrmed in independent populations, and that predictors
at population level may not apply well to individuals (Cunha-Vaz
et al., 2014). In addition, there are many micro-environments,
including at tissue and cellular level, and readily accessible tissues, such as blood and urine, may not reect what is happening at
the site of disease (the retina). For example levels of the VEGF
counter-balancing peptide, pigment epithelium derived factor
(PEDF), are low in the ocular tissues of people with diabetic retinopathy (Funatsu et al., 2006), but are increased in their blood
(Jenkins et al., 2007). A site-specic uid that is readily obtainable
in a non-invasive manner is tears and it has been demonstrated
that the levels of TNF-alpha increase with the severity of diabetic
retinopathy (especially PDR) and nephropathy (Costagliola et al.,
2013).
Types of biomarkers include clinical, biochemical factors and
molecular markers. Examples relevant to diabetic retinopathy
include clinical factors (e.g. diabetes duration, obesity, smoking,
ETDRS score, electroretinography, biochemical factors (e.g. HbA1c,
lipoprotein related factors)); and molecular factors (such as the
163
results of GWAS analyses and miRNA proles (discussed below)).

Cytokines, growth factors and/or hormones have been widely used,
such as the case with adiponectin as an adipocyte-derived hormone
that regulates glucose and lipid metabolism. Adiponectin has been
shown to be signicantly higher in T1D patients with severe diabetic retinopathy than in those without, even after adjustment for
occurrence of microalbuminuria (Hadjadj et al., 2005). As retinopathy has multiple risk factors it is likely, as is increasingly used for
cardiovascular disease and suggested for diabetic nephropathy
(Elley et al., 2010; van Dieren et al., 2011; Vergouwe et al., 2010),
and more recently for retinopathy (Harris Nwanyanwu et al., 2013)
that a panel rather than an individual biomarker will prove useful
in clinical practice and in clinical research. With existence of an
increasing array of biomarkers, but with availability and cost limitations, it is likely a range of retinopathy risk scores should arise
and be tested in various settings. The value of biomarkers in understanding the pathogenesis of diabetic retinopathy and developing therapeutics is well illustrated by identication of VEGF in
ocular uids during DMO and PDR. This led to an understanding the
role of VEGF in retinal leakage, inammation and angiogenesis with
subsequent development of anti-VEGF agents for clinical practice.
3.5. Genetic, epigenetics and miRNA proling and diabetic
retinopathy?
Several diabetic microvascular complications have shown
strong genetic associations which have advanced understanding of
disease aetiology and informed new therapeutic approaches to
patients according to a dened genetic prole. This is particularly
evident in the diabetic nephropathy eld where genome-wide association studies (GWAS) in T1D patients have been conducted
alongside candidate gene meta-analyses. These have highlighted
some underlying genetic risks, including ACE, Wnt, TNF-a, COL4A1,
HIF-1alpha, SOD2, APOE, SORBS1 (reviewed by (McKnight et al.,
2014)). Consortia such as the Genetics of Nephropathy and International Effort (GENIE) study has made signicant advances, with
the future possibility of explaining diabetic nephropathy risk from
genetic data (Sandholm et al., 2012; Williams et al., 2012). In terms
of genetic association the diabetic retinopathy eld is less advanced
than that for nephropathy, although there have been a number of
worthwhile studies (reviewed by (Kuo et al., 2014)). A genomewide association study for diabetic retinopathy identied an association with a long intergenic non-coding RNA (LincRNA) sequence.
LincRNAs are non-protein coding transcripts (>200 nucleotides in
length) and the sequence called RP1-90L14 (adjacent to the CEP162
gene) has shown susceptibility to diabetic retinopathy (Awata et al.,
2014). Interestingly, other LincRNAs are also being studied for their
association with diabetic retinopathy such as MALAT1 (Yan et al.,
2014) and MIAT (Yan et al., 2015). While some interesting leads
are emerging, as yet there is no robust indication that diabetic
retinopathy has a signicant genetic component. Candidate gene
and genome-wide studies may yet nd genetic linkage to particular
retinopathy phenotypes in T1D and T2D although both diabetestypes will need to be assessed separately in view of their distinct
genetic architecture.
Interactions between environmental factors and genetic predisposition leading to epigenetic changes could provide a powerful
risk association to diabetic complications, especially in relation to
the metabolic memory phenomenon (Reddy et al., 2015). Various
epigenetic modications can occur in histones such as acetylation,
phosphorylation or ubiquitination but methylation via histone
methyltransferase (HMT) enzymes at lysine residues is likely to be
the most important in the context of diabetic complications
(Cooper and El-Osta, 2010). Histone methylation at various promoter regions can signicantly alter transcriptional regulation at
164
dened promoters and lead to suppression or inappropriately

sustained gene expression thus impacting on normal cell function.
With particular emphasis on the metabolic memory phenomenon in diabetic retinopathy and the role of epigenetics, a recent
study showed that high glucose-exposed retinal endothelial cells
undergo histone methylation which changes the transcriptional
activity of Sp1 at the Keap1 promoter (Mishra et al., 2014). In turn,
this impacts the normal transcriptional activity of Nrf2 and associated protection from anti-oxidant gene expression (Mishra et al.,
2014). Vascular endothelial cells exposed to bouts of high glucose
seem to be particularly susceptible to epigenetic changes and
exposure for only 16 hours leaves an epigenetic mark which is
still evident following a return to normal glucose levels for 6 days.
Signicantly, this results in sustained activation of NFkB at the p65
promoter and resultant oxidative stress responses (El-Osta et al.,
2008). This has far-reaching implications and demonstrates that
relatively short-term high glucose exposure induces a harmful
chromatin re-modelling and epigenetic changes that could be
linked to diabetic complications in general and retinopathy in
particular (Giacco and Brownlee, 2010; Kadiyala et al., 2012;
Perrone et al., 2014; Villeneuve and Natarajan, 2010). As investigation continues, it seems likely that the role of epigenetics in
diabetic retinopathy will become more clearly understood. This
could provide scope for novel therapeutic approaches such as use of
drugs that regulate histone deacetylases and methylases in
appropriate retinal cell-types.
In relation to the regulation of gene expression, the role of
microRNAs (miRNAs) in diabetic retinopathy has been gaining
more emphasis. miRNAs are non-coding small RNAs which
modulate post-transcriptional control of gene expression through
degradation or translational repression of key messenger RNAs.
miRNAs can be detected in serum (free, associated with proteins or
within membrane-bound particles) (Weiland et al., 2012), vitreous
(Ragusa et al., 2013) and aqueous (Dunmire et al., 2013). As
reviewed by Mastropasqua et al., miRNAs hold considerable interest for diabetic retinopathy since they can regulate important
pathogenic responses such as angiogenesis, blood ow, neural cell
dysfunction, tissue-specic inammation and glucose metabolism
(Mastropasqua et al., 2014). Although based on a small patient
sample, it has been reported that three separate miRNAs (miR-21,
miR-181c, and miR-1179) in serum of patients with diabetic retinopathy have potential to be used as biomarkers for early detection
of disease (Li et al., 2014; Qing et al., 2014). While this is still a
growing research area, miRNAs hold considerable clinical potential
in the diabetic retinopathy eld, both as possible drug-targets for
regulation of dysfunctional cell responses and as diagnostic
biomarkers.
4. Modelling diabetic retinopathy
As this review develops it will become clear that there is a
need for further understanding of the molecular and cellular
pathways involved in diabetic retinopathy and to utilise this
knowledge for drug development. While patient based studies
remain vital, there is also a requirement for models that facilitate
experimental and hypothesis-driven research in this eld. However, the inherent complexity and decades-long progression of
diabetic retinopathy presents a serious challenge for developing
models of clinical disease. For example, the various animal species used have differing retinal anatomy, metabolism, diets, lifespans etc, and while they may be able to reproduce some aspects of human diabetic retinopathy, no single model currently
exists that faithfully reproduces all aspects of the clinical scenario.
In particular, it is important to note that many important hallmark lesions which are typical of background diabetic
retinopathy (including microaneurysms, exudates, hemorrhages

and cotton wool spots) seldom, if ever, occur in short-term diabetic rodent models and only appear in dog, pig or monkey
models with longer durations of diabetes (Lai and Lo, 2013).
Therefore the pathology occurring in current animal models
needs to be carefully evaluated to avoid injudicious extrapolation
to the clinical scenario. While a detailed review of the models
used for diabetic retinopathy is beyond the scope of this review,
some important aspects will be discussed.
4.1. Culture-based models of diabetic retinopathy
Cell culture-based approaches have proved valuable for examining retinal cell-types and how they respond to factors associated
with diabetes, such as high glucose, lipids, shear stress, metabolite
imbalance, cytokines and growth factors. In some cases, clinical
linkage has been provided by exposure of retinal cells in vitro to
diabetic patient vitreous (Murugeswari et al., 2014) or serum (Stitt
et al., 2005). Cell culture-based approaches continue to provide
scope for understanding key molecular pathways that are linked to
diabetic retinopathy. Importantly, they also identify novel therapeutic targets with potential for drug development. Examples of
retinal cells used for in vitro investigation of diabetic retinopathy
are pericytes (Hughes et al., 2004), retinal capillary endothelium
(Bhatwadekar et al., 2009), retinal pigment epithelium (RPE)
(Garcia-Ramirez et al., 2011; Rosales et al., 2014b), Mller glia (Jiang
et al., 2014) and microglia (Wang et al., 2007).
Of course, the disadvantage to the study of isolated cells is they
carry an inherent articiality since the retina is a complex, multicellular tissue with inter-dependability of the various constituent
cells. While co-culture systems have been deployed, such as retinal
capillary endothelium and blood monocytes (Rangasamy et al.,
2014), retinal explant approaches have been developed to overcome this problem. These ex vivo studies have provided valuable
information relating to retinal vessel autoregulatory function (Kur
et al., 2012; McGahon et al., 2007) and intricate cellecell signals
in whole retinal explants (D'Cruz et al., 2012; Knott et al., 1999).
While experimental use of retinal explants has merits the tissue
remains non-perfused and atrophic necessitating the studies to be
conducted within very limited time-spans. Any cell/organ culture
model lacks the aspect of chronicity which is so relevant when
studying something that develops over years.
4.2. Rodent models of diabetic retinopathy
In both mice and rats, diabetes leads to dysfunction or failure (or
partial failure) of various organs including the retina, kidneys,
nerves and heart. Use of these rodent models is advantageous from
the perspective of cost, genetic uniformity and opportunities for
transgenic technology, although they have obvious disadvantages
such as metabolism, diet and life-span that is very different from
humans. In addition, a systematic comparative transcriptomics
study has recently reported that gene expression changes found in
three mouse models for acute inammation correlated poorly with
their corresponding human conditions (Seok et al., 2013).
Depending on the strain used and the duration of diabetes, these
models reproduce some early lesions of retinopathy, including
capillary BM thickening, vasopermeability, loss of retinal pericytes
and capillary closure (reviewed by (Robinson et al., 2012)). Most
notably, vasoregression is the retinal lesion that simulates the most
important damage in the human diabetic retina, as all other
changes result from progressive capillary dropout (Calcutt et al.,
2009; Hammes et al., 2011). The use of rodent models of diabetic
retinopathy has added greatly to our knowledge of molecular pathology in this disease and, importantly, enabled the pre-clinical
assessment of experimental drugs (Berkowitz et al., 1999; Hammes

et al., 2003; Kowluru et al., 2011; Stitt et al., 2002b). In this regard,
the db/db mouse has recently been proposed as a particularly
useful model for investigating the underlying mechanisms involved
in diabetes-induced retinal neurodegeneration and for testing
neuroprotective agents (Bogdanov et al., 2014).
4.3. Large animals as models of diabetic retinopathy
Next to rodents, cats and dogs have been used most extensively
as models of diabetic retinopathy and they provide excellent
models of early-stage clinical disease (Budzynski et al., 2005;
Gardiner et al., 2003a, 1994; Hatchell et al., 1995; Linsenmeier
et al., 1998; Mansour et al., 1990). Dogs have been most extensively used for retinopathy studies with diabetes commonly
induced using chemical induction of diabetes (Anderson et al.,
1993) (Fig. 6). Hypergalactosaemia producing diabetes like lesions has also been extensively used through feeding with high
galactose diets (Engerman et al., 1990; Kador et al., 1994). The main
advantage of dogs over mice and rats is the duration of diabetes
that can be achieved due to the longer life-span of this species.
Typically, 5 years hyperglycaemia or galactosaemia produces lesions that are much closer to clinical diabetic retinopathy than
what is possible with rodent models (Howell et al., 2013). With
daily insulin delivery, the degree of hyperglycaemia in diabetic dogs
can be maintained to much tighter levels which provides a more
clinically-relevant disease state than can be achieved with insulintreated diabetic rodents (Anderson et al., 1993; Gardiner et al.,
1994). Notably, dogs which have been diabetic for 5 years show
microaneurysms and loss of arteriolar smooth muscle (Gardiner
165
et al., 1994), lesions that are rarely observed in diabetic rodents.

Since diabetic retinopathy in dogs is much closer to the clinical
scenario, this species has been also used to assess drug efcacy
(Gardiner et al., 2003a; Kador et al., 1990). The main disadvantages
of the model are general objections against research in large
mammals and the costs of the model. Cats have also been used as
models of diabetic retinopathy and they show lesions such as
capillary BM thickening, vessel tortuosity, BRB dysfunction, capillary degeneration and microaneurysms (Hatchell et al., 1995;
Mansour et al., 1990).
The pig retina has a cone-rich region known as the area centralis
which is somewhat analogous to the primate macula. The pig eye is
human-size and enables imaging and surgery which confers signicant advantage as a model of human diabetic retinopathy. While
this model has not been extensively reported in comparison to
rodents (and even dogs), chemical diabetes-induction in pigs is
achievable (King et al., 2011) and has often been combined with
high-fat diet (Hainsworth et al., 2002). Following 4 months diabetes, the pig retina shows pericyte degeneration, vascular BM
thickening and a compromise of the BRB (Lee et al., 2010). The pig
model (including transgenics) has also been used for studying
autoregulatory function in the retinal vasculature using electrophysiological approaches (Hansen et al., 2015; Misfeldt et al.,
2010a,b). In the context of diabetic retinopathy, Bek et al. examined retinal vessels from diabetic pigs over-expressing the gain-offunction mutant (D374Y) of the human gene PCSK9 that blocks LDL
transport and demonstrated that in the presence of both hyperglycaemia and dyslipidemia there was a marked reduction in
vasogenic tone regulation and structure of retinal arterioles (Bek
et al., 2013a).
Non-human primates provide the ultimate model of clinical
diabetic retinopathy since these animals are anatomically closest to
humans and their retina has a true macula. There have been several
research teams examining retinopathy following T1D and T2D in
rhesus monkeys. The Lutty and Tso research groups have made a
signicant contribution to this area and they have reported retinal
lesions in spontaneously T2D monkeys (Johnson et al., 2005; Kim
et al., 2004) and in monkeys with streptozotocin (STZ) induced
T1D (Buchi et al., 1996; Tso et al., 1988). These studies have
shown, perhaps unsurprisingly, that in primates diabetic retinopathy follows a human-like prole, including marked variation between subjects. Severe retinopathy has been shown in T2D models
with up to 12 years of disease, with neural abnormalities manifesting as defects in the electroretinogram (ERG) (Kim et al., 2004).
Monkeys with long-term T2D show retinal lesions which include
extensive ischaemia with presence of cotton-wool spots, numerous
microaneurysms, capillary degeneration, large vessel occlusions,
BRB damage, IRMA and atrophy of the inner retina in the macula
region (Johnson et al., 2005). Interestingly, despite extensive
ischaemia, no advanced PDR was apparent in these monkeys
(Johnson et al., 2005). The use of nonhuman primates for diabetic
retinopathy research carries signicant cost limitations and this,
combined with obvious ethical challenges, limits the use of these
models.
4.4. Models of end-stage diabetic retinopathy
Fig. 6. Diabetes-related vascular BM thickening in retinal capillaries. Transmission

electron micrographs (TEMs) of retinal capillaries from diabetic and control dogs. The
retinal capillary from a control dog shows BM enveloping the pericyte (P) and endothelial cells (E) (arrows). The retinal capillary from a dog with diabetes for 4 years
shows that the BM is considerably thickened (arrows).
There are no T1D or T2D models that reproduce either DMO or

PDR. In particular, rodent models lack a macula which impedes
translation from the model to the human condition. Recently, animal models were analysed which have a visual streak in their retina
(as an area of high cone density and enhanced visual acuity) which
shows anatomical mimicry of the human macula (Huber et al.,
2010). These models may be benecial for studying diabetic macular disease provided that diabetes can be induced reproducibly
166
with comprehensive retinal pathology. As noted, there is no animal

model that develops retinal neovascularisation, possibly due to the
particular anatomical structure, and the short lifespan of the animals. Thus scientists have relied on non-diabetic animal models,
such as oxygen induced retinopathy (OIR) which was originally
developed by the Smith laboratory to study retinopathy of prematurity (ROP) in newborns (Smith et al., 1994). Retinal hypoxia is a
feature of end-stage diabetic retinopathy and the OIR model produces this in a highly delineated manner in the central retina. In
OIR, oxygen deprivation of the retinal glia and neurons drives
expression of pro-angiogenic growth factors (such as VEGF, erythropoietin, TNF-alpha, and angiopoietin-2) and the neovascular
membranes that ensue are similar to those occurring in PDR
(Connor et al., 2009). The model has been extensively used for
assessment of anti-angiogenic agents (Gardiner et al., 2005;
Gebarowska et al., 2002; Hammes et al., 1996; McVicar et al.,
2011) and cell therapies (Medina et al., 2010, 2011). In addition, a
degree of BRB breakdown, glial and neuronal damage have been
observed in the OIR model (Vessey et al., 2011). Results from the
mouse OIR model also indicated that vascular tortuosity measured
in retinal arterioles correlated well with levels of hypoxia in the
retina (Scott et al., 2014a). No in vitro, ex vivo or in vivo models of
diabetic retinopathy can reproduce all aspects of the clinical scenario and the data acquired through their use needs to be carefully
interpreted in the context of human disease. For a detailed discussion see www.DIAMAP.eu.
5. Molecular & cellular pathology of diabetic retinopathy
5.1. Features of retinal microvascular dysfunction during diabetes
As mentioned previously, the ETDRS clinical classication of
diabetic retinopathy is based on vascular lesions since they are the
most obvious in the fundus. As a result most research into the
pathogenesis of diabetic retinopathy has concentrated on the
microvasculopathy in the retina with a focus on how this leads to
non-perfusion and hypoxia with resultant breakdown of the BRB,
oedema and/or PDR. It is well established that retinal capillary
pericytes and arteriolar smooth muscle cells are among the rst
vascular components to die by apoptosis during diabetic retinopathy (Gardiner et al., 1994; Mizutani et al., 1996) but there is now a
stronger appreciation of the complexity of the neurovascular unit in
the retina. Like the brain, retinal blood vessels are multicellular
units comprising endothelial cells, pericytes (at the capillary level),
vascular smooth muscle cells (arteriolar/arterial level) and closely
associated macro- and microglia and neurons. This cellular unit
responds dynamically to complex circulatory and neural cues to
control blood ow and regulate the blood-retinal barrier which are
signicantly altered by diabetes (Feng et al., 2012; Fletcher et al.,
2010; Klaassen et al., 2013).
5.1.1. Blood ow changes in diabetic retinopathy
A commonly described early functional defect in the diabetic
retina is a change in the rate of blood ow and loss of normal
autoregulatory capacity in response to metabolic demands of the
neuropile (Kohner et al., 1995). The intra-retinal vasculature lacks
autonomic innervation and close-regulation of blood ow through
the inner retina is dependent on cellecell signalling mechanisms
(Delaey et al., 2000). Abnormalities of retinal blood ow in diabetic
patients were reported by Wagener in the 1930's (Wagener et al.,
1934) and as technology for assessing vascular function improved
there were further indications that vessel calibre changed during
diabetic retinopathy (Ballantyne and Loewenstein, 1943; Skovborg
et al., 1969) with some predictive value (Crosby-Nwaobi et al.,
2012). In the 70's, seminal work by Kohner described
haemodynamic changes in the diabetic retinal vasculature with a

strong assertion that these abnormalities were connected to pathology and could form the basis of an early-stage indicator for
diabetic retinopathy progression (Kohner, 1993; Kohner et al.,
1975). More recently, diabetic patient-based studies have shown
that haemodynamic abnormalities occur before the onset of overt
retinopathy. For example, quantiable differences have been shown
in Doppler ow velocity waveform morphology, as recorded in the
retrobulbar circulation, in patients with impaired glucose tolerance
and diabetes prior to the development of overt diabetic retinopathy
(Lockhart et al., 2014) and signicant arteriolar vasoconstriction has
been demonstrated which is associated with decreases in total
retinal blood ow (Bursell et al., 1996; Ciulla et al., 2002; Klein et al.,
2003). Such reductions in blood perfusion of the neuropile, even in
relatively early stages of diabetes, would have implications for
meeting the O2 and nutrient requirements of the retina which has
extraordinarily high metabolic demands (Anderson and Saltzman,
1964; Buttery et al., 1991). Retinal blood vessels change as diabetes progresses and it has been shown that arterioles dilate in later
stages of retinopathy (Schmetterer and Wolzt, 1999) which results
in a shift to enhanced blood ow (Ciulla et al., 2002) which may
hasten the progression to DMO and PDR (Grunwald et al., 1992;
Klein et al., 2012) (Fig. 7).
Precisely what modulates blood ow dysregulation in arterioles
and capillaries during diabetic retinopathy remain uncertain
although the early reductions in blood ow observed in diabetic
patients and experimental animal models of diabetes have been
linked to hyperglycaemia-induced PKC activation and K channel
dysfunction on retinal arteriolar smooth muscle cells and capillary
pericytes (Aiello et al., 2006; McGahon et al., 2007). It seems likely
that diabetes-induced impairment of endothelium-dependent
vasorelaxation also contributes to this early vasoconstrictor phase
possibly through a mechanism involving downregulation of retinal
endothelial TRPV4 channels (De Vriese et al., 2000; Fitzgerald et al.,
2005; Ito et al., 2006; Kawagishi et al., 1999; Monaghan et al., 2015;
Yu et al., 2003). As diabetic retinopathy develops, the progressive
loss of pressure and metabolic autoregulatory mechanisms
(Grunwald et al., 1984; Patel et al., 1994; Rassam et al., 1995; Sinclair
et al., 1982; Trick et al., 2006) disturbances in retinal vasomotion
(Bek, 2013; Bek et al., 2013b) and the release of dilatory factors from
the hypoxic retina such as lactate, adenosine and VEGF (Clermont
et al., 1997; Gidday and Park, 1993; Hein et al., 2006; Yamanishi
et al., 2006) most likely underlies the switch to retinal hyperperfusion (Curtis and Gardiner, 2012). Further research elucidating
the basic mechanisms controlling blood ow autoregulation in the
retina will be essential if we are fully understand why this process
becomes disrupted during diabetes.
When considering abnormal blood ow in the diabetic retina,
there is also good reason to look closely at the complex neural, glial
and vascular cell interactions in both large and small retinal vessels.
As reviewed by Kur et al., the intricate mechanisms controlling
neurovascular coupling in the retina are being gradually identied
although considerably more investigation is needed (Kur et al.,
2012). Neurovascular coupling refers to the process by which
blood ow in the retina is matched to the metabolic demands of the
retinal neurons. This requires at least two intercellular signalling
steps, the rst coupling neuronal activity to glial stimulation
(neuroglial coupling) and the second linking glial activity to
relaxation of vascular smooth muscle and vasodilatation (gliovascular coupling). This response is depressed in diabetic patients
before overt retinopathy develops and the degree of this depression
correlates with the severity of retinopathy when present (Nguyen
et al., 2009b, 2009c; Pemp et al., 2009). Thus, abnormal neurovascular coupling may play an important role in mediating retinal
blood ow changes during diabetes and contribute to the
167
Fig. 7. Pathways to haemodynamic changes in diabetic retina and relation to pathology. A schematic depicting how abnormalities in haemodynamics and retinal blood ow may
contribute to disease progression and lesion formation. In the early stages of diabetic retinopathy, hypoperfusion (shown in yellow) may contribute to a low-grade, chronic vascular
inammation in the retinal capillaries (leukostasis) and contribute to progressive, irreversible hypoxia. Persistent hypoxia in the diabetic retina causes vasodilatation and enhanced
retinal blood ow which results in a switch from hypoperfusion to hyperperfusion (shown in blue). It has been suggested that when retinal hypoxia reaches a certain threshold it
may override the direct vasoconstrictive effects of diabetes causing relative hyperperfusion leading to BM thickening, loss of arterial tone, formation of microaneurysms and
acellular capillary formation which exacerbates retinal ischaemia.
development of retinal hypoxia and retinopathy. In an animal

model of diabetes, the impairment of neurovascular coupling in the
retina has been attributed to alterations in the NO signalling
pathway since the response could be restored by inhibition of the
enzyme inducible nitric oxide synthase (iNOS) (Mishra and
Newman, 2010, 2011). Interestingly, it has been reported that
glial-induced vasodilating prostanoids (i.e., epoxygenase metabolites) are active at low nitric oxide (NO) concentrations, whereas
vasoconstricting prostanoids (i.e., 20-hydroxyeicosatetraeonic
acid) are predominant at higher NO concentrations (Metea and
Newman, 2007). In addition, most of these molecules can increase or decrease blood ow depending on local oxygen concentration, but how this switch occurs is unclear (Attwell et al., 2010;
Mishra et al., 2011).
5.1.2. Blood retinal barrier dysfunction in diabetes
Passage of proteins and many other macromolecules into the
retina from the bloodstream is controlled by the inner BRB (iBRB),
formed by the intra-retinal microvasculature and the outer BRB
(oBRB) formed by the RPE. The BRB is formed by tight junctions
between adjacent cells which effectively blocks paracellular
vascular permeability (i.e. the transport of substances between
cells). Several tight junction proteins, including transmembrane
proteins, such as occludin, claudins and ZO-1 contribute to the
formation of the BRB (Erickson et al., 2007) and this has been
reviewed in detail by Klaassen et al. (Klaassen et al., 2013). BRB
compromise in diabetic retinopathy was rst shown by Cunha-Vaz
in the mid 1970's using vitreous orophotometry (Cunha-Vaz et al.,
1975; Cunha-Vaz, 1976) and became an important clinical readout
of diabetic retinopathy progression (Cunha-Vaz, 1981). In diabetic
retinopathy, the iBRB is compromised and macromolecules leak
from the intraretinal vasculature into the interstitial spaces of the
surrounding retina. Clinically, this is indicated on uorescein
angiography and, in the fundus, lipid exudates are a strong indication of retinopathy progression (Cunha-Vaz et al., 2011). Magnetic resonance imaging (MRI) also demonstrates signicant retinal
vasopermeability before clinically recognisable lesions of diabetic
retinopathy occur (Trick et al., 2008, 2005). Persistent BRB
dysfunction leads to DMO with or without cystoid degenerative
changes, photoreceptor atrophy and an irreversible loss of central
vision. This vasopermeability response is driven by hypoxia-related
growth factor/cytokine expression and, in particular, VEGF which is
elevated in the ocular uid of diabetic patients (Aiello et al., 1994).
Laser photocoagulation is an effective treatment for DMO (Aiello
et al., 2010; Diabetic Retinopathy Clinical Research et al., 2009)
while randomised trials have demonstrated that anti-VEGF drugs
can signicantly reverse DMO and improve vision in a proportion of
patients (Diabetic Retinopathy Clinical Research et al., 2010;
Korobelnik et al., 2014; Mitchell et al., 2011; Nguyen et al., 2012,
2010).
Diabetic animal models show iBRB breakdown within a relatively short time-frame following disease-induction (Erickson et al.,
2007) and this has been linked to capillary leukostasis and endothelial damage (Miyamoto et al., 1999; Sone et al., 1997). It is
interesting that iBRB compromise in animals is not always reective of the clinical scenario and the pre-clinical models never show
the overt leakage observed in DMO. Nevertheless, these models do
have value since they can enable early-stage indications of
diabetes-related damage that are impossible to assess clinically. For
example, recent evidence suggests that in addition to extravasation
of macromolecules, iBRB compromise may also consist of Mller
glial swelling which in itself could contribute signicantly to
oedema in the diabetic retina (Krugel et al., 2011). This reects
dysfunction of the active transport mechanisms of the BRB, especially at the capillary:Mller glial interface (Biedermann et al.,
2004). Mechanistically, this relates to dysfunction of the inwardly
168
rectifying channels of the Kir family, mainly Kir4.1 which is mislocalised and contributes to K channel currents and swelling of
Mller glia in diabetic retina (Pannicke et al., 2006). Changes to
Kir4.1 and aquaporins on Mller glia are consistent ndings in
diabetic animal models (Berner et al., 2012; Curtis et al., 2011; Vacca
et al., 2014) with links to aldosterone upregulation (Zhao et al.,
2010) and localised inammation (Zhao et al., 2011). Dysfunction
of the ne balance controlling ion exchange during diabetes has
obvious implications for normal function of the neurovascular unit
and retinal homoeostasis.
5.1.3. BM thickening in retinal blood vessels
An early histological change in the retinal vasculature of diabetic
patients and animal models is thickening of the capillary BM (Fig. 6)
and this phenomenon has been reviewed by Roy et al. (2010a). BM
thickening is thought to be a consequence of increased synthesis of
vascular BM components such as collagen IV, bronectin and
laminin in combination with reduced degradation by catabolic
enzymes (Cherian et al., 2009; Roy et al., 1994, 2003; Stitt et al.,
1994). BM thickening is a hallmark lesion in diabetic retinopathy
but it still remains uncertain if it is of primary or secondary
importance in the development of microvasculopathy. While it has
been suggested that BM thickening may be an epiphenomenon, it
seems likely that such radical alteration of the extracellular matrix
(including protein and proteoglycan composition, charge selectivity
and covalent crosslinking) has a signicant impact on the neurovascular unit. For example, such BM modications may contribute
to impaired endothelial e pericyte communication, defects in
capillary autoregulation or inappropriate cell interactions with
constituent BM proteins (Beltramo et al., 2002; Bhatwadekar et al.,
2008b; Padayatti et al., 2001; Roy et al., 2010b). Advanced glycation
endproducts (AGEs) accumulate in diabetes and these adducts
causes crosslinking in vascular BMs and as a result there is loss of
protein degradability and vessel elasticity with impact on vascular
cell survivability (Kalfa et al., 1995; Mott et al., 1997; Stitt et al.,
2004). Inhibition of AGE formation during diabetes has been
shown to prevent BM thickening in diabetic rats (Gardiner et al.,
2003b; Hammes HP et al., 1995; Stitt et al., 2002a).
5.1.4. Pericyte death in diabetic retina
Pericyte loss has been described as the hallmark of incipient
diabetic retinopathy since the introduction of trypsin digestion into
retinal research during the early 60s (Cogan et al., 1961) (Fig. 8).
Retinal pericytes are characterised as intramural pericytes being
enveloped by BM and their loss results in so-called ghosts.
Quantication of pericyte ghosts is the technical equivalent of
pericyte loss both leading to a precise measure of glucose-induced
vasodegenerative pathology at the early stages of diabetic retinopathy. It is believed that pericyte dropout from retinal vessels is
an important trigger preceding the other vascular abnormalities of
diabetic retinopathy. Although precise information about the onset
and progression of pericyte loss in human diabetic retina is not
available, studies in rats have shown that pericyte loss appears after
2 months of diabetes which is well before the development of
acellular capillaries, which often occurs after 6 months disease
duration (Pster et al., 2008). The loss of pericytes may weaken the
iBRB and can result in capillary instability and vascular leakage.
Interestingly, several studies have shown that pericyte dropout and
the development of other vascular abnormalities may be separate
events and independent of each other. Treatment of diabetic animals with aminoguanidine reduced acellular capillaries formation,
but did either not prevent pericyte loss (Agardh et al., 2000) or to a
modest proportion (Hammes et al., 1991). However, treatment,
with the vitamin E analogue nicanartine prevented pericyte loss,
but had no effect on the prevention of vasoregression in diabetic
Fig. 8. Vasodegeneration during experimental diabetes. Trypsin digest of the retina

from a 4-year diabetic dog shows a pericyte ghost (arrowhead) while there is an
adjacent viable pericyte also on the capillary wall (arrow) (Ai). The ultrastructural view
in the diabetic dog shows a retinal pericyte ghost (PG) represented as a pocket of
vesicular debris enveloped by BM. In this example, the endothelial cell (EC) remains
viable (Aii). Experimental diabetes of 6 months duration in the rat shows the presence
of acellular capillaries in the retina (B). The lesions are demonstrated by confocal
microscopy of at-mounted retina and the examples show vascular BM stained for
collagen IV (green) (Bi) and viable endothelium stained with isolectin (red) (Bii). The
merged image (Biii) demonstrates empty BM tubes without endothelial cells, classied
as acellular capillaries (arrows).
rats (Hammes et al., 1997). More recently, our own group has
shown that genetic deletion of the receptor for AGEs (RAGE) protected diabetic retina from acellular capillary formation, but pericyte loss remained at the same level as the C57BL/6 diabetic
controls (McVicar et al., 2015). Furthermore, in eyes donated from
T2D patients, pericytes were detected in areas of acellular capillaries (Pster et al., 2013), suggesting that pericyte and endothelial
cell loss during diabetic retinopathy may be two independent
events.
The underlying mechanism of pericyte loss during early diabetic
retinopathy remains unknown. A number of common pathophysiological pathways including the formation of AGEs (involving
activation of RAGE), inammation, activation of protein kinase C
(PKC), production of reactive oxygen species (ROS), and abnormal
signalling of Ang2-related pathways have been shown to play an
important role in diabetes-related pericyte loss in the retina. For
example, AGEs have been found to accumulate in pericytes of
diabetic retina (Stitt et al., 1997), and the use of a glycation inhibitor
LR-90 prevented pericyte loss in diabetic rats (Bhatwadekar et al.,
2008a). In vitro, treatment of pericytes with pre-formed AGEs
induced apoptosis through the p38 and JNK MAP kinase pathway
(Alikhani et al., 2010), and blocking RAGE reduced AGE-evoked ROS
generation in pericytes (Maeda et al., 2014). Pericyte death in diabetes may result from an uncontrolled immune response as illustrated by an in vitro study which demonstrated that retinal
autoantibodies can induce pericyte death by activating the complement system (Li et al., 2012). Other factors causing pericyte
death may involve lipid abnormalities. For example, oxidised LDLimmune complex is a potent inammatory insult to induce
pericyte apoptosis (Fu et al., 2014). PPARa, is an important modulator of lipid metabolism and appears to protect diabetic mice from
retinal pericyte dropout (Ding et al., 2014).
High glucose not only leads to the accumulation of AGEs and
the concomitant activation of RAGE, but can also activate other
biochemical pathways, such as the hexosamine pathway
(Kandarakis et al., 2014; Nishikawa et al., 2000). When cells are
exposed to high glucose, intracellular AGS such as methyglyoxal
can modify transcription factors which, in case of Mller cells in
the retina, or endothelial cells in the kidney, can then recruit enzymes of the hexosamine pathway such as O-GlcNAc-transferase.
The subsequent modication of the transcription factor SP3 alters
its binding to the glucose-sensitive GC box in the promoter of
angiopoietin2 leading to a substantial upregulation. Ang-2 upregulation has been found in the diabetic retina, preceding pericyte
loss, and loss and gain of function models exposed to diabetic
conditions underlined the critical role of Ang-2 in pericyte loss
(Hammes et al., 2002; Pster et al., 2013). Exposure to Ang2 induces pericyte death under high glucose conditions through the
a3b1 integrin signalling pathway (Park et al., 2014). Excessive ROS
with activation of NF-kB are regarded as key mediators for
hyperglycaemia-induced retinal cell apoptosis. More recently, a
study suggests that hyperglycaemia also activate PKC- d and P38a
MAP Kinase and increase SHP-1 (Src homology-2-domain-containing phosphatise-1) expression in pericytes, independent to the
activation of NF-kB. This signalling cascade leads to PDGFR-b dephosphorylation and results in perictye apoptosis (Geraldes
et al., 2009). Alternatively to pericyte death, pericyte migration
mediated by Ang-2 can explain the extent of the loss of these cells
in the diabetic retina (Pster et al., 2008).
5.1.5. Capillary endothelial cell death in diabetic retina
It has been proposed that the primary defect in diabetic retinopathy lies with the vascular endothelium and that the complication could be considered to be, at least in part, an
endotheliopathy (Khan et al., 2006). When endothelial cells die
retinal capillaries become acellular and this vasodegeneration is a
central tenet of progressive ischaemia during diabetic retinopathy.
Indeed, degenerative retinal vessels are a universal nding in longterm diabetic animal models and post-mortem specimens
(Gardiner et al., 2007). On trypsin digest preparations, these acellular capillaries appear as naked BM tubes where the endothelial
cells have disappeared (Gardiner et al., 2007) (Fig. 8). There have
been many studies assessing molecular and cellular responses of
isolated retinal capillary endothelium following exposure to components of the diabetic milieu (high glucose, lipids, modied proteins, growth factors, etc) and they have made a considerable
contribution to knowledge of pathogenic mechanisms in these
cells. However, it needs to be acknowledged that isolated endothelium is articial and has limits for uncovering the complexities
of the neurovascular unit and how it responds to diabetes.
Associations between uorescein angiograms and trypsin digest
preparations from donors with diabetic retinopathy show that regions of capillary acellularity correspond to non-perfused microvasculature angiographically, often downstream from areas where
microaneurysms are numerous (Bresnick et al., 1977). In the
context of abnormal blood ow in the retina, microaneurysms may
appear as dark red or white spots in the fundus. Clinically, some
microaneurysms are sclerosed and non-perfused while others can
be observed as fully or partially perfused during uorescein angiography (Kohner and Dollery, 1970; Kohner and Henkind, 1970).
Microaneurysms do not occur in diabetic rodents and therefore
histological studies have been limited although it is apparent that
hypertension, endothelial proliferation, thrombus formation and
pericyte cell-death are causal or contributory factors to their
169
formation (Amemiya and Bhutto, 2001; Baskin et al., 1999;

Ishikawa et al., 1983; Stitt et al., 1995; Tolentino et al., 1996)
(Fig. 9). On retinal trypsin digest preparations of post-mortem
human eyes, microaneurysms occur largely on the arteriolar side
of the circulation upstream of denuded arterioles and in association
with large areas of capillary acellularity (Gardiner et al., 2007).
Interestingly, some early-stage microaneurysms have been shown
to contain large numbers of monocyte and polymorphonuclear
cells (Stitt et al., 1995), which underscores the pro-inammatory
state of diabetes and the role of leucocyte-mediated capillary occlusion in diabetic retinopathy (Miyamoto et al., 1999).
Increasing closure of capillaries may be linked with cotton-wool
spots in the neural retina and also the occurrence of IRMA which
are represented in trypsin digests by wide-calibre multicellular
Fig. 9. Microaneurysm congurations in human diabetic retina. A) Retinal trypsin

digest from a T2D patient showing a microaneurysm (arrow) which contains darkly
staining aggregates of red blood cells. B) An electron micrograph of a similar microaneurysm which is engorged with red blood cells (RBCs). The endothelial cells and
pericytes are missing and structure is contained within the vascular BM (BM). C) Some
microaneurysms become sclerosed (arrow) and ultrastructually (D) they can be seen as
acellular structures with thickened BM and brous inltrates (arrows). E) Electron
micrograph a late-stage microaneurysm containing red blood cells (RBCs) and RBC
breakdown products (arrows). In this example, there is evidence of recanalization in
the form of a blood vessel enclosed by a thin BM inltrating the lumen of the
microaneurysm (*). An acellular capillary prole (AC) can be observed adjacent to the
microaneurysm.
170
channels within the capillary bed (Bresnick et al., 1977; Gardiner

et al., 2007; Kohner and Dollery, 1970). Although there have been
few studies conducted on the nature of IRMA in the diabetic retina,
it can be observed that these structures contain large numbers of
endothelial-like cells and occur in association with acellular capillaries close to the arterial side of the circulation. IRMA show large
calibre vessels traversing ischaemic retina with direct communication between pre-capillary arterioles and post-capillary venules
(see Fig. 3) and probably represent shunt vessels and an attempt to
re-vascularise the hypoxic neuropile (Bresnick et al., 1977; Kohner
and Dollery, 1970; Stitt et al., 2011).
5.2. Diabetes-related changes to the RPE-choroid
5.2.1. RPE
Since diabetes impacts on almost all cells of the retina, is it
perhaps surprising that the function of the RPE in diabetes has
received relatively little attention. Fluid normally moves from the
retina to the choroid largely due to the osmotic pressure exerted by
the proteins in the choroidal stroma (Bill, 1975). However, there is
now strong clinical and experimental evidence that the RPE becomes dysfunctional in diabetic retinopathy and that leakage from
the choriocapillaris in unison with impaired uid clearance,
contribute to retinal oedema (Simo et al., 2010). Therefore,
although it is generally assumed that the disruption of the iBRB is
the main cause of DMO, there is growing evidence that the
disruption of the external limiting membrane (ELM) and RPE
resulting in oBRB compromise signicantly contributes to the
pathogenesis of DMO (Kowalczuk et al., 2011; Runkle and
Antonetti, 2011; Weinberger et al., 1995; Xu and Le, 2011). In itself, the ELM composed of junctional complexes presents a significant barrier within the retina and there is evidence that this
membrane becomes disrupted in diabetic animals (Omri et al.,
2010).
Most experimental studies of the RPE have been conducted
in vitro and they show that exposure of these cells to components of
the diabetic milieu, such as high glucose, causes signicant barrier
dysfunction (Beasley et al., 2014; Trudeau et al., 2011; Villarroel
et al., 2009), early apoptotic death (Kim et al., 2014; Lim et al.,
2012) and abnormal expression and/or receptor signalling of
growth factors/cytokines (Chang et al., 2014; Sugimoto et al., 2013;
Xie et al., 2014; Yokouchi et al., 2013). The Goto-Kakizaki (GK) T2D
rat model shows evidence of diabetes-mediated damage to the
oBRB with concomitant photoreceptor damage (Omri et al., 2013).
Also in the GK rat, oBRB damage is related to the occurrence of
pore-like channels in the RPE which enables enhanced inammatory cell trafc between the retina and choroid in diabetes (Omri
et al., 2011). In other diabetic animal models, the RPE has been
shown to suffer a degree of nitrosative stress (Rosales et al., 2014a).
Components of the ERG in animal models, particularly the
negative-polarity fast oscillations in the cat retina, indicate the
recovery of [K] and a [Cl]-dependent hyperpolarization of the
basal RPE membrane (Linsenmeier and Steinberg, 1982) and this
may represent a useful, non-invasive measure of RPE and interlinked photoreceptor function (Samuels et al., 2010). Recently this
parameter has been used to study the inuence of diabetes on the
function of the RPE in murine T1D and T2D and reported that
dysfunction of these cells represents an early change in both T1D
and T2D which is linked to hyperglycaemia (Samuels et al., 2015)
and may also involve aspects of polyol metabolism (Samuels et al.,
2012).
5.2.2. Choroid
The outer retina is supplied by the high-ow blood vessels of the
choriocapillaris and there have been a number of studies linking
choroidal thinning to diabetes (as reviewed by (Lutty, 2013)). This

so-called diabetic choroidopathy has been demonstrated in patients (Cao et al., 1998; Esmaeelpour et al., 2012; Fryczkowski et al.,
1989; Hua et al., 2013) where it correlates with HbA1c levels (Unsal
et al., 2014). It also occurs in diabetic animal models (Braun et al.,
2009; Hidayat and Fine, 1985; Muir et al., 2012). In diabetic patients, there is choriocapillaris BM thickening, narrowing and dropout of the choroidal capillaries as well as macroaneurysms, intrachoroidal microvascular abnormalities and, occasionally, choroidal
neovascularisation (Cao et al., 1998; Esmaeelpour et al., 2012;
Fryczkowski et al., 1989; Hua et al., 2013). Similarly to retinopathy, vascular inammation is important in diabetic choroidopathy
and it has been suggested that polymorphonuclear leukocytes
precipitate choroidal endothelial cell damage and loss and vessel
occlusion in the choriocapillaris (Lutty et al., 1997).
The inherent difculty in viewing the choroid and its layers has
limited the description of pathology the living eye. A few studies
using indocyanine green angiography (ICGA) have described hypo
and hyperuorescent spots (Hua et al., 2013; Shiragami et al., 2002;
Weinberger et al., 1998) which likely correlate with the choroidal
hypo-perfusion and macroaneurysms, intra-choroidal microvascular abnormalities and choroidal neovascularisation described in
histopathology studies. In recent years, and with the advent of
enhanced depth imaging (EDI), several studies evaluating the
thickness of the choroid and its changes as a result of diabetes have
been conducted. Most have reported reduced central choroidal
thickness in diabetic eyes when compared with controls, which
could be explained by the choroidal degeneration described in the
diabetic eye (Esmaeelpour et al., 2012; Querques et al., 2012;
Vujosevic et al., 2012). However, a cross-sectional retrospective
study found increased choroidal thickness with increased severity
of the disease, from no diabetic retinopathy to PDR (Kim et al.,
2013). Although these ndings need to be conrmed, preferably,
in prospective, longitudinal studies, the increased thickness could
potentially relate to development of brosis and choroidal neovascularisation. In this regard, newly developed technologies, such
as and wide angle ICGA and wide angle fundus autouorescence
coupled with prospective, longitudinal studies may provide the
opportunity of furthering our understanding of the choroidal and
RPE changes and their implication on the development and progression of the disease.
Basal laminar deposits (BLDs) have been described in diabetic
eyes and they appear to be associated with areas of choriocapillaris
degeneration (Cao et al., 1998). Although the origin of BLDs in the
diabetic eye is uncertain, it is plausible that they could be the result
of an inadequate clearance by the choroid of waste material produced by the RPE. Deposition of this material could reduce further
the oxygen supply to the RPE by the already compromised choroid.
Direct effects of high glucose on the RPE as well as this reduced
oxygen supply would be expected to have a deleterious effect on
this retinal layer and, subsequently, on photoreceptor cells. Interestingly, the early literature had suggested abnormalities in dark
adaptation in diabetic patients which could be also explained by
these events (Henson and North, 1979).
The mechanisms underpinning diabetic choroidopathy are unclear and this denitely warrants more research effort since
attenuated perfusion of this capillary bed is likely to have a signicant pathophysiological consequence for the integrity and
function of the outer retina as diabetes progresses.
5.3. Retinal neuron and glial dysfunction during diabetes
Retinal neurodegeneration is a concept that was clearly
described and characterized at the end of 1990's and may be
manifest before any microvascular impairment can be identied by
ophthalmoscopic examination (Barber et al., 1998; Reiter and

Gardner, 2003). This has been conrmed in several more recent
studies performed in retinas from diabetic donors without any
vascular abnormality in the ophthalmologic examination performed the year before death (Carrasco et al., 2008, 2007; GarciaRamirez et al., 2009). In these retinas, the two hallmarks of neurodegeneration (apoptosis and reactive gliosis) were clearly shown
when compared with the retinas from non-diabetic patients
matched by age (Fig. 10). These ndings suggest that, at least in
some patients, neurodegeneration antedates the microcirculatory
abnormalities that occur in diabetic retinopathy and in some respects the early stages of diabetic retinopathy could be considered a
neuropathy rather than simply a microangiopathy. This concept has
informed our understanding of the pathogenesis of diabetic retinopathy but also in the design of new therapeutic approaches (see
below).
Neural apoptosis and reactive gliosis are now recognised as
important histological features of diabetic retinopathy. Retinal
ganglion cells (RGCs), located in the inner retina, are the retinal
neurons in which the apoptotic process related to diabetes is rst
detected (Kern and Barber, 2008). RGC loss results in a reduction in
the thickness of the retinal nerve bre layer, and has been detected
in diabetic patients without or with only minimal diabetic retinopathy, by using scanning laser polarimetry or optical coherence
tomography (OCT) (Araszkiewicz et al., 2012; van Dijk et al., 2009,
2010, 2012, 2011). It has been recently demonstrated that an
imbalance between proapoptotic and survival signalling exists in
the neuroretinas of diabetic patients in the early stages of diabetic
retinopathy (Valverde et al., 2013). Glutamate accumulation in the
extracellular space, oxidative stress, an imbalance in the retinal
production of neuroprotective factors and inammation are the
main factors involved in the development of neurodegeneration in
the setting of diabetic retinopathy (Simo et al., 2014b).
Regarding reactive gliosis it should be noted that there are
broadly two types of glial cells in the retina: astrocytes and Muller
cells, and the later is unique to the retina. One of the most signicant characteristics of reactive gliosis is glial acidic brilar protein
(GFAP) overexpression. Retinal astrocytes normally express GFAP,
171
whereas Mller cells do not. However in diabetes an aberrant

expression of GFAP is shown by Mller cells (Hammes et al., 1995;
Mizutani et al., 1998) (Fig. 10). Diabetes is also associated with
activation of microglial cells, the main resident sentinel immune
cells located in the inner part of the retina, which release cytokines
that contribute to neuronal cell death (Zeng et al., 2008). At present
it is unknown whether neural apoptosis or reactive gliosis is rst in
the neurodegenerative process that occurs in the diabetic retina.
The demonstration that reactive gliosis antedates neural apoptosis
would argue against the primary hypothesis that neuronal damage
is the rst event in diabetic retinopathy. Mller cells even have
relevance following vasodegeneration and it has been shown that
S100-positive glial processes penetrate the luminal and perivascular space in association with naked vascular BM tubes in
human post-mortem eyes with diabetic retinopathy (Bek, 1997a,b).
This diabetes-associated ingrowth into the lumen of degenerative
retinal vessels would be expected to limit the opportunity for
capillary regeneration and re-perfusion (Stitt et al., 2011).
The retinal neurodegenerative process can be assessed in vivo by
using electroretinography and in particular multifocal ERG (mfERG)
which permits us to explore the retinal function in terms of electrical response in different areas of the retina. The most consistent
and widely reported aspect of the ERG that changes early in diabetic retinopathy using animal models and patients is the oscillatory potential implicit time which reect electrophysiological
communication between the amacrine cells and the bipolar cells
with interactions from ganglion cells to amacrine cells (Tzekov and
Arden, 1999). Such early indicators reect retinal neural dysfunction and perhaps progression to neurodegenerative pathology in
the later stages of disease (Bearse et al., 2006). As such, the ERG has
important clinical value in diabetic retinopathy. While ERG is a very
sensitive but is also a quite cumbersome and time-consuming examination in which corneal electrodes are necessary. The SD-OCT is
another very useful tool which complements mfERF because it
provides anatomical images and enables measurement of thickness
of the different retinal layers. This is more rapid than mfERG and
the most useful parameter in identifying diabetes-induced retinal
neurodegeneration is the reduction of the thickness of RGC layer
Fig. 10. Neurodegeneration in diabetic retinopathy. Immunohistological sections corresponding to the retina from a representative diabetic (DM) and non-diabetic donor (non-DM)
matched by age (65 years). The fundoscopic examination performed the preceding year before death did not show any microvascular abnormality. The diabetic donor presents a
higher rate of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling [TUNEL] technique) and reactive gliosis (aberrant expression of glial brillary acidic protein
[GFAP]) than the non-diabetic donor.
172
and the bre nerve layer.

There is evidence that neurodegeneration measured by either
mfERG or OCT is present in patients with normal fundoscopic examination (see reviews by (Simo and Hernandez, 2012; Simo et al.,
2014b)). The question is whether the neurodegenerative process
can be considered a predictor of microvasular disease or even an
active mediator of the microvascular impairment. The proof of
concept in the clinical setting that neurodegeneration participates
in microangiopathy is based on observational studies in which
neurodegeneration assessed by mfERG predicts which retinal locations will develop new retinopathy in the near future (that means
in a period between 1 and 3 years) (Han et al., 2004; Harrison et al.,
2011; Lim and Cheung, 2008). Therefore neurodegeneration could
be a useful index to predict the development of microvascular
disease in the diabetic retina.
The mechanisms linking retinal neurodegeneration with
microvascular abnormalities remains to be fully elucidated but glial
dysfunction seems to play a critical role (Feng et al., 2009; Simo
et al., 2014a). In this regard it should be noted that Mller cells
produce factors capable of modulating blood ow, vascular
permeability, and cell survival, and their processes surround all the
blood vessels in the retina, thus contributing to the sealing function
of the iBRB (Bringmann and Wiedemann, 2012). As discussed
earlier, there is also increasing evidence that glial cells play a key
role in the haemodynamic response that governs neurovascular
coupling in the reina (Attwell et al., 2010; Feng et al., 2012; Kur
et al., 2012; Metea and Newman, 2006, 2007). In addition, it has
been demonstrated that accumulation of advanced glycation and
lipoxidation end-products and upregulation of RAGE has a key role
in the hyperglycaemia-induced activation of Mller glia and
downstream cytokine production in the context of diabetic retinopathy (Berner et al., 2012; Curtis et al., 2011; Yong et al., 2010;
Zong et al., 2010). Diabetes has also been reported to accelerate

death of Mller glia (Feenstra et al., 2013; Hammes et al., 1995), an
effect which has recently been linked to the disruption of retinal
vascular integrity and the induction of neural cell dysfunction and
death (Shen et al., 2012). A schematic diagram summarising how
Mller glia changes are believed to contribute to the sight threatening complications of diabetic retinopathy is presented in Fig. 11.
Apart from the Mller cells, activated microglial cells adjacent to
the vessels also appear to have a key role in vasoregression, the
vascular hallmark of the early stages of diabetic retinopathy in both
animal models (McVicar et al., 2015) and diabetic patients (Scott
et al., 2014b).
5.4. Inammation and immune cell activation in diabetic
retinopathy
The persistence of inammation in the retina from the early
stages of diabetes through to the sight-threatening end-stages is
well established as reviewed by (Tang and Kern, 2011). In the
context of diabetes-related tissue damage, inammation can be
viewed as an adaptive response to the milieu (dyslipidemia,
hyperglycaemia) of circulating immune cells and vascular endothelium (Xu et al., 2009). While para-inammation may help to
maintain homoeostasis, persistent and prolonged diabetes may
exceed the protective capacity of this response and result in
detrimental chronic inammation which may damage retinal tissue. At the early stages of diabetic retinopathy, an intravascular
immune response occurs in the form of leucocyteeendothelial interactions and this illustrates the mounting inammatory response.
So-called leukostasis is initiated by activation of vascular endothelium and circulating myeloid cells such as neutrophils and
monocytes. The leukostasis phenomenon is typied by immune
Disruption of
neurovascular
coupling
Mller Glia
in
Diabetic
Retina
Vasoproliferation
Retinal Hypoxia
Release of angiogenic
& inflammatory factors
Neuronal
Cell
Death
Loss of
neurotrophic
support
Excitotoxicity
Apoptosis
Loss of vascular
integrity
Dysregulation
of retinal K+
transport
Cell swelling and

impaired retinal
water absorption
Edema
Impaired
glutamate
uptake and
metabolism
Fig. 11. Mller glia dysfunction in diabetic retinopathy. Schematic showing the central role of Mller glia in the various pathophysiological events in diabetic retina. Early-stage
dysfunction of these cells impacts on disruption of normal neurovascular coupling in the retina can lead impaired blood ow which can lead to hypoxia in the neuropile. Prolonged hypoxia can evoke cell and tissue damage leading to pro-inammatory pathways concomitant with increased expression of angiogenic and vasopermeability growth factors
which, if left uncheck can result in retinal neovascularization or oedema.
cells becoming trapped in narrow-channel retinal capillaries leading to occlusion and non-perfusion (Valle et al., 2013). It is interesting to note that in the advanced stages of diabetic retinopathy,
the systemic neutrophil count is increased (Woo et al., 2011) and
these cells express higher levels of myeloperoxidase and produce
more hydrogen peroxide compared to cells from non-diabetic
controls (Gorudko et al., 2012). Circulating monocytes are also
activated in T1D patients (Josefsen et al., 1994) and they express
elevated adhesion molecules (Bouma et al., 2004) with concomitant binding to endothelium (Devaraj et al., 2011). Leukostasis has
been observed in animal models of diabetes, even after a very short
disease-duration (Joussen et al., 2004). This intravascular immune
response may also occur in diabetic patients (Chibber et al., 2007)
with the adherent leukocytes showing potential to damage the
retinal vascular endothelium (Li et al., 2012, Talahalli et al., 2012).
There is a complex milieu of dysregulated pro-inammatory
factors evident in the diabetic retina such as IL-1a, IL-1b, IL-6,
IL-8, MCP-1 and TNFa (Simo-Servat et al., 2012; Tang and Kern,
2011). It should be noted that mean levels for IL-8 within the
vitreous uid have been found in the same range as those reported
in pleural effusions of patients with pneumonia or tuberculosis
and they correlated with PDR activity (Hernandez et al., 2005).
Furthermore, the increased vitreous levels of IL-6 and IL-8 correlated with the progression of PDR and the outcome of vitreous
surgery (Murugeswari et al., 2014, 2008). These ndings underscore inammation as crucial in the pathogenic events that lead to
PDR.
Although the source of these cytokines may be Mller gliarelated, the bulk of expression is probably linked to activation of
retina-resident microglia and inltrating monocytes (Fig. 12). Such
cell activation responses are known to be central to inammation in
the brain and they are becoming more recognised in the diabetic
retina. For example, a number of in vitro studies and in vivo investigations of animal models and human post-mortem specimens
indicate that activation of retinal microglia could play an important
regulatory role in diabetes-mediated retinal inammation (Zeng
et al., 2008) by modulating cytokine expression (Budzynski et al.,
2005) and other pathologic responses (Kuiper et al., 2004).
Monocytes that inltrate the retina are distinct from microglia and
they reside in proximity to blood vessels (perivascular macrophages) or within various layers of the neuropile (Xu et al., 2009).
Both monocytes and microglia have important roles in retinal
homoeostasis but they are also central to neuroinammation
occurring in both humans and animal models (Barber et al., 2005;
Rungger-Brandle et al., 2000; Zeng et al., 2000).
Innate immune responses are undoubtedly critical to inammation in the diabetic retina and so-called Pattern Recognition
Receptors (PRRs) which are present on immunologically active cells
have an important modulatory role. Examples of PRRs include Tolllike receptors (TLRs), CD36 and RAGE which coordinate innate responses (Takeuchi et al., 2010). The precise inter-play of PRRs in the
diabetic retina remains poorly understood but there has been signicant attention given to RAGE and its role in diabetic retinopathy
(Barile et al., 2005; Li et al., 2011; Zong et al., 2010). RAGE is signalling receptor for various ligands including AGEs, S100B, highmobility group box-1 (HMGB-1), amyloid-b and Mac-1 (Zong
et al., 2011). Ligand-binding and signal transduction activates
transcription of NFkB and induction of adhesion molecules, cytokines and/or oxidative stress. In the retina, RAGE is expressed by
many cells although the highest expression levels are in Mller glia
(Barile et al., 2005). S100B is neurotrophic at low levels (Donato,
2001) although upregulation occurs in the Mller glia of diabetic
animal models where it can induce inammatory cytokine
expression (Zong et al., 2010). Blockade of RAGE may be a useful
therapeutic strategy and it has been shown that the soluble RAGE
173
Fig. 12. Immune cell activation in the diabetic retina. Shown is a retinal section from a
non-diabetic and 3 month diabetic rat stained for CD11b (green) (nuclei shown in red).
The dendritic shape of the non-activated microglia is clear in the healthy retina but in
diabetes, there is a change in the phenotype of the cells with a more activated
(amoeboid) phenotype and apparent inltration of other CD11b cells into various
layers of the retinal neuropile.
fragment (known as sRAGE) can prevent Mller glia dysfunction

(Barile et al., 2005) during diabetes and retinal capillary leukostasis
in AGE-infused (non-diabetic) mice (Moore et al., 2003). Importantly, RAGE antagonists can prevent acellular capillary formation
in diabetic mice (Li et al., 2011) while mice in which RAGE has been
genetically deleted show signicant protection against diabetic
retinopathy (McVicar et al., 2015).
5.5. Retinal ischaemia during diabetes
As outlined above, the diabetic retina suffers a progressive
dysfunction of the neurovascular unit and the resulting decit in
perfusion has serious consequences for normal retinal function. As
ischaemia increases in diabetic retina, micro-infarctions are
observed in the fundus indicated by cotton wool spots which reect
disrupted axoplasmic transport. These indictors of neural cell
dysfunction occur in unison with conuent acellular capillaries and
often adjacent to IRMA (Fig. 3) which may indicate an attempt to revascularise hypoxic retina or form shunt-like channels secondary to
capillary pressure differentials (Gardiner et al., 2007). On the basis
of their predominant location on the arterial side of the retinal
microcirculation it seems improbable that IRMA (or microaneurysms) are abortive attempts at neovascularisation since such
new vessels originate mostly from venules close to areas of
ischaemia (Garner, 1993). In the diabetic retina, there is only very
limited intra-retinal re-vascularisation and IRMA are indicative of
the impaired vascular regeneration which is typical of many organs
in diabetes. This statement may appear to run counter to the widely
174
held view that diabetic retinopathy is a pro-angiogenic disorder.

However, the PDR phase in diabetic patients often only occurs after
decades of vasodegeneration, depressed vascular repair and inadequate remodelling following prolonged ischaemia.
While hypoxia of the retinal neurons and glia is associated with
DMO and PDR, it is important to note that the retina normally experiences diurnal uctuations in oxygenation and is capable of
near-normal function during transient hypoxia (Wangsa-Wirawan
and Linsenmeier, 2003; Yu et al., 2007). In contrast to most other
CNS tissues, the retina is able to withstand signicant ischaemia
(Hayreh and Weingeist, 1980) which may be related to an ability to
rely on glycolytic metabolism in low PO2 conditions, at least in the
short-term (Zager and Ames, 1988). Irrespective of an in-built
resilience to hypoxia, the retina suffers appreciably during
ischaemia and the oxygen-deprived glia and neurons produce
reactive oxygen species (ROS) (Bonne et al., 1998) and demonstrate
increased release of elevation of extracellular transmitters such as
glutamate and gamma-aminobutyric acid (GABA) which are associated with excitotoxicity (Osborne et al., 2004) and also evident in
the diabetic retina (Catalani et al., 2007; Lieth et al., 1998; MadsenBouterse and Kowluru, 2008).
In the diabetic retina, progressive loss of capillaries can create
overt hypoxia above and beyond the normal diurnal uctuations
and the rst experimental evidence of this was shown in cats
which had been diabetic for more than 6 years (Linsenmeier et al.,
1998). Using intra-retinal microelectrodes, there was signicantly
lower P02 in the diabetic cat inner retina compared to nondiabetic controls. Lower P02 occurred, even in regions where
there was no obvious perfusion decits, although some retinal
regions with microaneurysms, leukostasis and degenerative capillaries showed marked hypoxia (Linsenmeier et al., 1998). Studies
in mice have revealed that even after relatively short diabetes
duration there is a lower P02 in the inner retina, as evidenced by
deposition of the drug pimonidazole (which deposits as immunoreactive insoluble adducts at PO2 < 10 mmHg) (de Gooyer et al.,
2006) (Fig. 13). In diabetic patients, with no clinically-evident
retinopathy, breathing oxygen can reverse losses in contrast
sensitivity and colour vision (Dean et al., 1997; Grunwald et al.,
1984; Harris et al., 1996; Patel et al., 1994) which suggests the
occurrence of a degree of vascular insufciency and subtle hypoxia
very early in retinopathy progression. This also serves to underscore the risk of sleep apnoea and progression of diabetic retinopathy (Mason et al., 2012).
One of the major consequences of retinal hypoxia is expression
of hypoxia-regulated cytokines and growth factors, many of which
have been directly implicated in DMO and PDR. VEGF is the most
widely studied growth factor in the hypoxic diabetic retina (see
reviews (Miller et al. 2013; Simo et al., 2014b)). In the ischemic
retina, there is stabilisation and nuclear translocation of hypoxiainducible factor-1 alpha (HIF-1a) in the neurons and glia
(Semenza, 2000). HIF1a is one of a family of hypoxia inducible
transcription factors that includes HIF1a, HIF2a and HIF3a and
they bind to hypoxia response elements in inducible target genes
thereby increasing gene expression of proteins such as erythropoietin (EPO) (Jiang et al., 1996), VEGF (Carmeliet et al., 1998) and
glucose transporters (Iyer et al., 1998). The ischaemia-mediated
angiogenic stimulus in retina can be exceptionally strong and the
ensuing neovascular response often leads to uncontrolled growth
of new vessels that breach the internal limiting membrane of the
retina to proliferate on the retinal surface or at remote sites such
as the iris and trabecular meshwork. Likewise, VEGF can also
stimulate overt breakdown of the iBRB/oBRB as occurs in DMO
which is effectively diminished through the use of anti-VEGF antibodies (Regnier et al., 2014), laser photocoagulation (Elman et al.,
2015) or corticosteroids (Edelman et al., 2005). If left untreated,
Fig. 13. Evidence of vascular insufciency and hypoxia in the early stages of diabetic
retinopathy. Microvascular density becomes progressively reduced in diabetic mouse
retina. The retinal microvasculature was assessed by ADPase enzyme histochemistry in
retina from non-diabetic (A) and diabetic mice which had diabetes for 5 months
duration (B). Darkeld images of retinal atmounts show vascular density in the mid
and peripheral regions of at embedded retina show reduced vessels in the diabetic.
This reduction in vascular density is linked to hypoxia which occurs in retina. Hypoxia
was visualized using the bio-reductive drug pimonidazole (hypoxyprobe; HP), which
forms irreversible adducts when pO2 < 10 mmHg. In non-diabetic mouse retina, there
is minimal HP-immunoreactivity (green) at the level of the ganglion cell layer (C). By
contrast in the diabetic retina, HP-immunoreactivity is increased (D), appearing
throughout the inner retina. (GCL: ganglion cell layer, INL: inner nuclear layer, ONL:
outer nuclear layer).
retinal oedema, pre-retinal and papillary neovascularisation with

accompanying brogliosis in the inner retina and degenerative
changes at the outer retina can lead to profound visual impairment although it is still unknown why only some patients progress
to DMO or PDR or both. More research is required to understand
the phenotypic prole of diabetic retinopathy patients so that the
nature of their disease path is predictable and more effectively
treated.
6. Therapeutic options in diabetic retinopathy
At present there are few measures available to prevent diabetic
retinopathy beyond regulating hyperglycaemia (DCCT, 1993), preventing dyslipidemia (Chowdhury et al., 2002), controlling hypertension (Kohner et al., 1995) and cessation of tobacco smoking
(Schram et al., 2003). Patients who develop DMO can be treated
with laser photocoagulation, intravitreal anti-VEGF drugs and
intravitreal corticosteroids. For PDR, the standard interventions are
laser photocoagulation and vitreoretinal surgery although trials
evaluating the potential use of anti-VEGF drugs to also treat this
group of patients are ongoing. All of these treatments are focused
on end stage disease and carry signicant sight-threatening side
effects (Simo and Hernandez, 2009). Importantly, they do not
address the early and potentially reversible failure of retinal
perfusion (Spranger et al., 2001).
6.1. Treatments for DMO
Laser photocoagulation in the form of focal or grid laser
treatment has been used to treat diabetic retinopathy patients for
many decades. The ETDRS demonstrated this treatment to be

efcacious to prevent visual loss in people with DMO (ETDRS,
1991). Although in the ETDRS laser mainly prevented or slowed
loss of visual acuity recent trials suggest that laser treatment can
actually improve vision in some patients (Aiello et al., 2010).
Sometimes in conjunction with laser, corticosteroids such as
triamcinolone, dexamethasone and uocinolone have been used
also for treating DMO. While these drugs carry signicant sideeffects, they still have value for controlling vision loss and recent
derivations such as slow release implants of steroids (for example
of dexamethasone and ucinolone) may be of value in selected
patients. Non-steroidal anti-inammatory drugs (NSAIDs) have
been used widely in ophthalmology for allergic conjunctivitis and
to reduce postoperative pain. However this class of drugs may also
have value for diabetic retinopathy and DMO since they can target
important pathways which higher dosing intervals alongside
expectation of less severe side effects (Kim et al., 2010). Recent
evidence suggests that there is scope for developing aqueous
ophthalmic solutions of NSAIDs that provide better tissue penetration a topical delivery and that they have potential for treatment of diabetic retinopathy and DMO in particular (Sahoo et al.,
2015; Semeraro et al., 2015).
Based on success in the treatment of neovascular age-related
macular degeneration (nvAMD), blockade of VEGF bioactivity was
investigated and shown to have a good efcacy for the treatment of
DMO too. The value of these intravitreal anti-VEGFs has been
demonstrated by a range of randomised control trials (Do et al.,
2011; Korobelnik et al., 2014; Massin et al., 2010; Michaelides
et al., 2010; Mitchell et al., 2011; Nguyen et al., 2009a). In a general sense, these anti-VEGFs have signicant benets over the other
therapeutic options for DMO (Virgili et al., 2014). However, these
treatments require multiple intravitreal injections (estimated to be
around 12e15 in the rst three years of treatment) (Diabetic
Retinopathy Clinical Research et al., 2012). Furthermore, even
following monthly injections of anti-VEGF (Ranibizumab) for 2
years ~20% of patients had still residual oedema, as detected by OCT
and in ~70% some degree of leakage is still detected by fundus
uorescein angiography (FFA) (Nguyen et al., 2012). A relatively
high proportion of patients (46%) may still require focal or grid laser
treatment despite of anti-VEGF injections and in ~40% limited visual acuity improvement (<10 ETDRS letters) is achieved (Diabetic
Retinopathy Clinical Research et al., 2012). There have also been
concerns that anti-VEGF therapy could compromise retinal neuroglial and resident microvascular survival (Benjamin et al., 1998;
D'Amore, 2007; Nishijima et al., 2007; Saint-Geniez and D'Amore,
2004; Saint-Geniez et al., 2008). Like in the case of exudative agerelated macular degeneration, in which anti-VEGF treatment has
been linked to the development of geographic atrophy
(Chakravarthy et al., 2013; Lois et al., 2013; McBain et al., 2011), it is
still possible that other unwanted effects of anti-VEGF therapy may
be recognize as further experience is gained with this treatment in
diabetic patients. On balance, while anti-VEGF therapy has revolutionised ophthalmic care, there is a need for caution surrounding
long-term use of these approaches (Kim et al., 2012; Kurihara et al.,
2012; Sang and D'Amore, 2008).
6.2. Treatments for PDR
Unlike DMO, treatment advancements for PDR have been relatively modest. Pan-retinal laser photocoagulation (PRP) remains
the mainstay treatment for PDR (Fig. 14) although vitreoretinal
surgery is used now in earlier stages (rather than as a treatment of
last resort). Indeed, recent data supports better visual and
anatomical outcomes and reduced number of complications of
vitrectomy in patients with PDR compared with earlier data
175
Fig. 14. Current therapy for PDR. Fundus photograph of the right eye of a patient with
PDR treated successfully with panretinal photocoagulation. Note the chorioretinal
scars result of the laser treatment in the midperipheral retina.
published by the Diabetic Retinopathy Vitrectomy Study (DRVS)

(Arrigg and Cavallerano, 1998). However, vitreoretinal surgery is
still predominantly used when sight threatening complications of
PDR (e.g. vitreous haemorrhage, tractional retinal detachment,
epimacular proliferation) have developed. Randomised controlled
trials have reported reduced retinopathy progression in patients
receiving intravitreal anti-VEGFs or steroids (Korobelnik et al.,
2014; Massin et al., 2010; Nguyen et al., 2012). While these treatments may reduce the development of PDR, there is a pressing need
for new therapeutic options in this patient-group.
7. Developing new therapies for diabetic retinopathy
The development of new therapies capable of preventing or
slowing the onset and progression of diabetic retinopathy remains
a priority. A wide range of new approaches based on retinal glial,
neuronal and vascular pathophysiology during diabetes are being
developed although, thus far, none have entered clinical use for
early-stage diabetic retinopathy. Our ability to prevent initiation
and progression of diabetic retinopathy is due to several factors: i)
lack of understanding of what pathogenic pathways are clinically
relevant and drive retinopathy at each phase of the disease; ii) the
degree of patient variability and inability to predict patients who
will progress rapidly and suffer DMO and/or PDR; iii) difculty of
designing and nancing clinical trials that are focused on earlystage retinopathy and iv) uncertainty with regards to the costeffectiveness of potential new early interventions in diabetic retinopathy, given the length of time required for people with diabetes
to develop sight threatening disease and the fact that the latter
occurs in only a proportion of people with diabetes.
7.1. Understanding pathogenesis as a basis for new drug
development
Diabetic retinopathy is complex and multi-factorial and a range
of hypotheses have been proposed as the pathogenic basis of this
complication. A detailed analysis of these pathogenic pathways and
their role in diabetic retinopathy is beyond the scope of this review
since they have been discussed in detail previously (for example see
(Antonetti et al., 2012; Curtis et al., 2009; Heng et al., 2013; Stitt
et al., 2013)). Nevertheless, it is important to note that diabetesrelated biochemical processes such as increased aldose reductase
activity, oxidative stress, nitrosylation, activation of the hexosamine
176
Fig. 15. Aspects of hyperglycaemia-related vascular cell dysfunction. Hyperglycaemia-induces a range of pathways in cells such as endothelium, and these include the polyol
pathway, ROS formation, and AGEs formation. Excess glucose in endothelial cells enters polyol pathway; the electron donors like reduced nicotinamide adenine dinucleotide
(NADH) and Flavin adenine dinucleotide (FADH2) accumulate in the mitochondria, thus affecting the electron transport chain; the excess electrons increase ROS in mitochondria;
ROS triggers accumulation of AGEs; ROS and AGEs create mitochondrial DNA damage and mitochondrial dysfunction; protein kinase C (PKC) and AGE mediated activation of NFkB
activate the expression of inammation proteins, tumour suppressor p53, and iNOS; increased NO by iNOS is highly reactive with superoxide anions; the peroxynitrite thus
generated acts as a strong oxidant and completes the vicious cycle of oxidative stress by increasing ROS production; accumulation of AGEs also increases ROS production independent of glucose levels.
pathway ux, accumulation of AGEs and over-activation of protein

kinase C (PKC) should not necessarily be regarded as independent
phenomena (Fig. 15). Indeed the overproduction of superoxide has
been proposed as common denominator of these seemingly independent pathways (Brownlee, 2001) with follow-up evidence that
the transketolase activator, benfotiamine, shifts the glycolytic ux
from the toxic to a non-toxic pathway, thus preventing several aspects of experimental diabetic retinopathy in vitro and in vivo
(Beltramo et al., 2009; Berrone et al., 2006; Hammes et al., 2003;
Tarallo et al., 2012). A clinical trial tested the ability of benfotiamine to reduce plasma and urinary AGEs but showed no benet
(Alkhalaf et al., 2012) and in T1D patients, 24 month treatment with
benfotiamine demonstrated no change in peripheral nerve function
or soluble inammatory biomarkers (Fraser et al., 2012). The
appropriateness of the clinical endpoints used in this study have
been questioned (Ziegler et al., 2012). Benfotiamine may yet offer
benet in some aspects of impaired ow-mediated dilatation in the
brachial artery of T2D patients (Stirban et al., 2013). It has yet to be
evaluated in clinical diabetic retinopathy.
7.2. Difculties of translating discoveries into benets for patients

In many other cases, drugs that show considerable promise in
animals never even reach a clinical trial. This indicates signicant
shortcomings in how we reconcile target discovery and drug
development in animal models with clinical scenarios. This is
especially the case in diabetic retinopathy which develops over
decades and for which most models only represent the very early
stages of disease. Drug trials in diabetic retinopathy almost always
concentrate on late-stage DMO and PDR since they are shorter-term
and the clinical outcome measures are well-established. There is a
need for identication of imaged-based or blood-circulating
markers combined with functional outcomes which show robust
association with disease initiation and early-stage progression in
diabetic patients with highly dened retinopathy phenotypes.
7.3. New therapeutic angles for diabetic retinopathy

As previously mentioned, neurodegeneration is an early event in
the pathogenesis of diabetic retinopathy and, therefore, it is
reasonable to propose therapeutic strategies based on neuroprotection as a new and targeted approach for treating the early
stages of diabetic retinopathy. The reduction of oxidative stress and
the administration of neuroprotective agents (i.e. PEDF, SST, NGF,
BDNF) are among the most important therapeutic strategies based
on neuroprotection (Simo et al., 2014a). From the clinical point of
view, the early identication of neurodegeneration (by SD-OCT
and/or mfERG) will be crucial for implementing an early treatment based on drugs with a neuroprotective effect. However, at
these stages patients may be asymptomatic and, therefore,
aggressive treatments such as intravitreal injections would not be
appropriate. Emerging experimental evidence has already shown
the neuroprotective effects of topical administration of PEDF, SST,
NGF, brimonidine and insulin (Fort et al., 2011; Hernandez et al.,
2013; Lambiase et al., 2009; Liu et al., 2012). In addition, the
topical administration of drugs limits their action to the eye and
minimizes the associated systemic effects (Aiello, 2008). Therefore,
topical therapies could revolutionize the care of diabetic patients.
However, apart from preveting or arresting neurodegeneration it is
crucial to examine whether the neuroprotective drugs have any
benecial effect on microvascular abnormalities. The ongoing
clinical trial EUROCONDOR (EudraCT Number: 2012-001200-38),
the rst Randomised Controlled Trial (RCC) to assess the safety and
effectiveness of neuroprotection in diabetic patients, will help in
answering this question. This study has been funded by the European Commission in the setting of the FP7-HEALTH-2011 (see more
details in http://eurocondor.eu).
For patients who develop sight-threatening complications of
diabetic retinopathy, intra-vitreal delivery of VEGF inhibitors undoubtedly represents a major advance for DMO. As a result, many
therapeutic strategies have been attempting to provide more convienent or better-tolerated ways of impacting the VEGF pathway or
to devise new delivery methods and/or slow-release systems

(Gross et al., 2013; Kontturi et al., 2014; Lovett et al., 2015; Rauck
et al., 2014). The fact that ~40% of DMO patients fail to respond to
VEGF blockade (Ford et al., 2013) (this proportion may be even
higher in clinical practice, outside randomised trials), suggests that
other pathways may be important in this disease. New therapeutics
targetting VEGF-independent mechanisms may offer scope for a
wider treatment response in DMO, as exemplied by positive results from trials using inhibitors to other targets such as TNF-alpha
(Skakis et al., 2010). At the least they could offer additional therapeutic advantage as an adjunctive treatment alongside antiVEGFs. New drugs with a non-VEGF mode of action could provide
a much-needed option for diabetic patients who are classed as
non-responders to anti-VEGF therapy. In this context, inhibitors
of the pre-kallikrein-kinin (PKK) pathway have shown promise for
DMO (Giusti et al., 2001; Liu and Feener, 2013) with an VEGFindependent mode of action (Kita et al., 2012). Importantly, better
predictors of treatment response should be sought so that stratied
approaches could be implemented.
7.4. Time to consider a precision medicine approach for diabetic
retinopathy?
As outlined in this review and elsewhere, one of the main
challenges to effectively treat diabetic retinopathy is to address the
fact that not all patients present with the same clinical phenotype
(even taking into account systemic variance and duration of disease). Individuals often progress through the stages of disease at
different and unpredictable rates, manifest distinct types of endstage disease and respond very differently to drug therapy. We
are now in an era of so-called precision medicine as recognized by
the Obama administration in the USA which recently announced a
research initiative that aims to accelerate progress for better patient outcomes across many areas of medicine (https://www.
whitehouse.gov/the-press-ofce/2015/01/30/remarks-presidentprecision-medicine). This initiative has been strongly embraced by
the cancer eld which is increasingly using patient-specic
tailoring of available chemotherapeutic drugs to achieve
improved responses (Rubin, 2015). Precision medicine represents a
step beyond evidence-based medicine and has signicant benets
for ophthalmology in general and diabetic retinopathy in particular.
The concept is simple: to identify and harness prevention and
treatment strategies that take individual variability into account
(Collins and Varmus, 2015). The scientic and clinical diabetic
retinopathy research communities possesses large, wellcharacterized clinical cohorts with potential for them to be married to biological and mechanistic data (such as proteomics,
metabolomics, genomics, molecular pathways, cellular assays) with
analysis using powerful bioinformatics approaches. This will
require true, two-way translational research conjoining laboratory
and clinical research. Even though the challenge is signicant,
achieving tailored therapies may enable the provision of the right
drug at the right dose at the right time-point to the most appropriate diabetic retinopathy patient (Collins and Varmus, 2015).
8. Summary
Diabetic retinopathy involves a highly complex interplay between biochemical and metabolic abnormalities occurring in almost
all cells of the retina. Identication of a precise pathogenesis that
links the neuroglial and microvascular damage occurring in the
diabetic retina remains a valid goal. In the years to come, continued
progress is anticipated as our understanding of the molecular and
cellular basis of diabetic retinopathy improves in unison with
patient-based research. We are also entering a new, exciting era in
177
which the rst pharmacological treatments based on an understanding of the causative mechanisms of diabetic retinopathy may
soon become available. These will include vascular and neuroprotection therapies that can halt progression of retinopathy in the
early stages. In view of the complexity of diabetic retinopathy, it
seems likely that combinatory therapy will be required with pathways targeted in different cell types at different stages of the disease
process. With genome-wide, metabolomics and proteomic assessments for patients becoming a possibility, the likelihood that a
personalised, precision approach to diabetic retinopathy will be
achievable to the benet of patients who will receive the most efcacious therapy at the most appropriate time-point in their disease.
Acknowledgements
The authors would like to acknowledge nancial support from
Fight for Sight (UK) (FFS 1316), The Sir Jules Thorn Trust (05SC/02A),
The Medical Research Council (MRC) (MRC G0801962), The European Union (EC FP7 e Health 2012-305736), The British Heart
Foundation (PG1/11/99/29027), British Heart Foundation, The
Biotechnology and Biological Sciences Research Council (BBSRC)
(BBSRC e BB/H005498/1), The Royal Society (WM100045), and the
Juvenile Diabetes Research Foundation (JDRF-5-CDA-2014-225-AN).
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Progress in Retinal and Eye Research 51 (2016) 156e186

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

The progress in understanding and treatment of diabetic retinopathy

Centre for Experimental Medicine, Queen's University Belfast, Northern Ireland, UK

The global significance of diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

1. The global signicance of diabetic retinopathy

the most common cause of blindness in diabetic patients (Klein

Fig. 3. Vascular histology of human NPDR. Trypsin digest specimen of post-mortem

enrolled. In the continuing EDIC, study subjects are assessed

medications. Perhaps for these reasons, prospective studies to

formation of advanced glycation/lipoxidation products. Therefore

a signicant protection against retinopathy progression (UKPDS,

(Stefansson et al., 2000; Scanlon et al., 2008). Although screening

3.3.2. Oxygen saturation in the retinal vessels

observed lesions in human post-mortem retinal vascular casts

results of GWAS analyses and miRNA proles (discussed below)).

dened promoters and lead to suppression or inappropriately

retinopathy (including microaneurysms, exudates, hemorrhages

assessment of experimental drugs (Berkowitz et al., 1999; Hammes

et al., 1994), lesions that are rarely observed in diabetic rodents.

Fig. 6. Diabetes-related vascular BM thickening in retinal capillaries. Transmission

There are no T1D or T2D models that reproduce either DMO or

with comprehensive retinal pathology. As noted, there is no animal

haemodynamic changes in the diabetic retinal vasculature with a

development of retinal hypoxia and retinopathy. In an animal

Fig. 8. Vasodegeneration during experimental diabetes. Trypsin digest of the retina

formation (Amemiya and Bhutto, 2001; Baskin et al., 1999;

Fig. 9. Microaneurysm congurations in human diabetic retina. A) Retinal trypsin

channels within the capillary bed (Bresnick et al., 1977; Gardiner

choroidal thinning to diabetes (as reviewed by (Lutty, 2013)). This

ophthalmoscopic examination (Barber et al., 1998; Reiter and

whereas Mller cells do not. However in diabetes an aberrant

and the bre nerve layer.

Zong et al., 2010). Diabetes has also been reported to accelerate

Cell swelling and

fragment (known as sRAGE) can prevent Mller glia dysfunction

held view that diabetic retinopathy is a pro-angiogenic disorder.

retinal oedema, pre-retinal and papillary neovascularisation with

many decades. The ETDRS demonstrated this treatment to be

published by the Diabetic Retinopathy Vitrectomy Study (DRVS)

pathway ux, accumulation of AGEs and over-activation of protein

7.2. Difculties of translating discoveries into benets for patients

7.3. New therapeutic angles for diabetic retinopathy

to devise new delivery methods and/or slow-release systems

hypoinsulinemia in diabetes induced retinal cell death: evidence for retinal

International Diabetes Federation, Diabetes Atlas. International Diabetes

Glob. Health Action 7, 23987.

deviations in diabetic retinopathy. Diabetologia 42, 387e405.

retinovascular leakage in diabetic maculopathy. Br. J. Ophthalmol. 79, 728e731.

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