EJINME-03354; No of Pages 7

European Journal of Internal Medicine xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

European Journal of Internal Medicine

journal homepage: www.elsevier.com/locate/ejim

Original Article

Impaired flow-mediated dilation in hospitalized patients with

community-acquired pneumonia
Lorenzo Loffredo a, Roberto Cangemi a, Ludovica Perri a, Elisa Catasca a, Camilla Calvieri b, Roberto Carnevale a,
Cristina Nocella a, Francesco Equitani c, Domenico Ferro a, Francesco Violi a,⁎, in collaboration with
the SIXTUS study group:
SIXTUS (thromboSIs-related eXTra-pulmonary oUtcomeS in pneumonia) study group: Simona Battaglia a,
Giuliano Bertazzoni a, Elisa Biliotti a, Tommaso Bucci a, Cinzia Myriam Calabrese a, Marco Casciaro a,
Andrea Celestini a, Maurizio De Angelis d, Paolo De Marzio a, Rozenn Esvan d, Marco Falcone e, Lucia Fazi a,
Lucia Fontanelli Sulekova d, Cristiana Franchi d, Laura Giordo a, Stefania Grieco d, Elisa Manzini a,
Paolo Marinelli e, Michela Mordenti e, Sergio Morelli a, Paolo Palange e, Daniele Pastori a, Pasquale Pignatelli a,
Marco Rivano Capparuccia d, Giulio Francesco Romiti a, Elisabetta Rossi a, Eleonora Ruscio a, Alessandro Russo e,
Maria Gabriella Scarpellini a, Luisa Solimando a, Gloria Taliani d, Stefano Trapè a, Filippo Toriello a
a
I Clinica Medica, Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Rome, Italy
b
Department of Cardiovascular, Respiratory, Nephrology, Anesthesiology and Geriatric Sciences, Sapienza University of Rome, Rome, Italy
c
Transfusion Medicine and Immuno-Hematology Unit, Santa Maria Goretti Hospital, Latina, Italy
d
Infectious and Tropical Diseases Unit, Department of Clinical Medicine, Sapienza University of Rome, Rome, Italy
e
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Background: Community-acquired pneumonia (CAP) is complicated by cardiovascular events as myocardial in-
Received 22 March 2016 farction and stroke but the underlying mechanism is still unclear. We hypothesized that endothelial dysfunction
Received in revised form 21 June 2016 may be implicated and that endotoxemia may have a role.
Accepted 9 September 2016 Methods: Fifty patients with CAP and 50 controls were enrolled. At admission and at discharge, flow-mediated
Available online xxxx
dilation (FMD), serum levels of endotoxins and oxidative stress, as assessed by serum levels of nitrite/nitrate
(NOx) and isoprostanes, were studied.
Keywords:
Pneumonia
Results: At admission, a significant difference between patients with CAP and controls was observed for FMD
Flow-mediated dilation (2.1 ± 0.3 vs 4.0 ± 0.3%, p b 0.001), serum endotoxins (157.8 ± 7.6 vs 33.1 ± 4.8 pg/ml), serum isoprostanes
Oxidative stress (341 ± 14 vs 286 ± 10 pM, p = 0.009) and NOx (24.3 ± 1.1 vs 29.7 ± 2.2 μM). Simple linear correlation analysis
Infection showed that serum endotoxins significantly correlated with Pneumonia Severity Index score (Rs = 0.386, p =
0.006). Compared to baseline, at discharge CAP patients showed a significant increase of FMD and NOx (from
2.1 ± 0.3 to 4.6 ± 0.4%, p b 0.001 and from 24.3 ± 1.1 to 31.1 ± 1.5 μM, p b 0.001, respectively) and a significant
decrease of serum endotoxins and isoprostanes (from 157.8 ± 7.6 to 55.5 ± 2.3 pg/ml, p b 0.001, and from 341 ±
14 to 312 ± 14 pM, p b 0.001, respectively). Conversely, no changes for FMD, NOx, serum endotoxins and
isoprostanes were observed in controls between baseline and discharge. Changes of FMD significantly correlated
with changes of serum endotoxins (Rs = −0.315; p = 0.001).
Conclusions: The study provides the first evidence that CAP is characterized by impaired FMD with a mechanism
potentially involving endotoxin production and oxidative stress.
© 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Abbreviation: CAP, community-acquired pneumonia; FMD, flow-mediated dilation; 1. Introduction

NO, nitric oxide; NOx, nitrite/nitrate; MI, myocardial infarction; NSTEMI, non-ST elevation
myocardial infarction; STEMI, ST-elevation MI; T2DM, type 2 diabetes mellitus; LPS, lipo-
polysaccharides; CHF, congestive heart failure; PSI, Pneumonia Severity Index.
⁎ Corresponding author at: I Clinica Medica, Department of Internal Medicine and
Medical Specialties, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome,
Italy.
E-mail address: francesco.violi@uniroma1.it (F. Violi).
Community-acquired pneumonia (CAP) is the most common
infection leading to hospitalization in intensive care units and the
most common cause of death associated with infectious disease [1].
Epidemiological studies have shown that respiratory tract infections
are associated with an increased risk for cardiovascular events such as

http://dx.doi.org/10.1016/j.ejim.2016.09.008
0953-6205/© 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
2 L. Loffredo et al. / European Journal of Internal Medicine xxx (2016) xxx–xxx
acute myocardial infarction (MI), stroke and cardiac arrhythmia such as
atrial fibrillation [2,3]. The relationship between CAP and cardiovascular
events is corroborated by studies indicating that influenza vaccination
lowers the risk for CAP hospitalization, heart disease, cerebrovascular
disease and death from any cause during flu seasons in the elderly [4].
The mechanism accounting for the development of MI in the early
phase of CAP is still unclear [2,5]. Platelets have been suggested to
play a role as markers of in vivo platelet activation; soluble CD40 Ligand
and P-selectin have been associated with an enhanced risk of MI [6].
Furthermore, an observational study demonstrated that CAP patients
treated with aspirin had a lower risk of experiencing MI during a
30-day follow-up compared to non aspirin users [7]. Changes in artery
vasodilation could be another mechanism accounting for MI occurrence.
Thus, we have recently showed that CAP is essentially associated with
non-ST elevation myocardial infarction (NSTEMI), suggesting that
incomplete coronary occlusion or enhanced oxygen demand could
account for the increased risk of MI in CAP patients [6]. Accordingly,
we speculated that impaired artery dilation could complicate the
clinical course of CAP and potentially precipitate in acute coronary syn-
drome. Flow-mediated dilation (FMD) is an established marker of artery
dilation, which is associated with cardiovascular outcomes; [8,9] thus,
impaired FMD increases the risk for cardiovascular disease [8,9]. To ad-
dress if acute phase of CAP is associated with impaired artery vasodila-
tion, FMD was measured at admission and at discharge in patients
with CAP and in hospitalized patients with clinical disease unrelated
to any infection. Furthermore, to investigate the underlying mechanism,
we explored the interplay among endotoxemia, oxidative stress and
FMD.
2. Methods
2.1. Patients
In this cross-sectional study, we included 50 consecutive patients
with CAP recruited between October 2014 and March 2015 at the
Internal Medicine ward of “Sapienza” University of Rome. All patients
admitted to the medical ward with diagnosis of CAP through the emer-
gency department were consecutively recruited. Patients who fulfilled
the following criteria were enrolled in the study after giving written
informed consent: (1) age 18 years or over; (2) clinical presentation
of an acute disease with 1 or more of the following signs or symptoms
suggesting pneumonia: presence of rales, bronchial breath sounds,
rhonchi, tachycardia, fever (N38.0 °C), dyspnea, chills, coughing, or
chest pain; and (3) presence of new consolidation(s) on chest X-ray.
Pneumonia was considered as CAP if it was diagnosed upon hospitaliza-
tion and the patient had not been discharged from an acute care facility
within 14 days preceding the clinical presentation. Patients were
excluded from the study if any of the following criteria applied: criteria
for health care-associated pneumonia [10], radiographic evidence of
a preexisting infiltrates, presence of malignancy, pregnancy or
breastfeeding, documented severe allergy to antibiotics, or refusal to
sign informed consent.
In the same period of CAP enrollment, 50 hospitalized patients with-
out acute infections and matched for sex, age, and comorbidities including
diabetes, dyslipidemia, hypertension, chronic obstructive pulmonary
disease (COPD), congestive heart failure (CHF) and renal failure were
used as controls. Frequency matching procedures were applied to select
controls. Matched controls were selected such that the distribution of
the relevant characteristics in this group was similar to the distribution
in the cases. Fifty out of 532 patients hospitalized between October
2014 and March 2015 were selected. They were hospitalized for CHF
(n = 10), syncope (n = 4), hypertension crisis (n = 4), COPD exacerba-
tion (n = 16), decompensated diabetes mellitus (n = 9), new onset
arrhythmias (n = 5), and renal failure (n = 2).
Baseline treatments were defined according to patients' pharmaco-
logical histories.
Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
Severity of disease at presentation was assessed by the Pneumonia
Severity Index (PSI), a validated prediction score for 30-day mortality
in patients with CAP [11,12].
Systemic inflammatory response syndrome (SIRS), was defined as
previously described [13], i.e. the occurrence of at least two of the
following criteria: fever N38.0 °C or hypothermia b 36.0 °C, tachycardia
N90 beats/min, tachypnea N20 breaths/min, leukocytosis N12*109/l or
leucopoenia b4*109/L. Sepsis was defined as SIRS in addition to a
documented or presumed infection [13].
Type 2 diabetes mellitus (T2DM), hypertension, history of coronary
heart disease, dyslipidemia, CHF and COPD were defined as previously
described [14–16]. ST-elevation MI (STEMI) and NSTEMI were defined
as previously reported [17] and were confirmed by cardiologists.
Daily diet was based on the hospital guidelines and was tailored
according to age, nutritional status and severity of the comorbidities.
In all subjects we performed FMD and collected blood samples to
analyze markers of oxidative stress as assessed by serum isoprostanes,
nitrite/nitrate (NOx), and serum endotoxins (Lipopolysaccharides,
LPS) at admission and at discharge (10 ± 3 days in average). Given
that dietary changes could potentially influence serum NOx [18], we
paid attention that the daily diet of each patient was not modified
during the intra-hospital stay.
This study was conducted according to the principles stated in the
Declaration of Helsinki. The institutional review board approved this
prospective, observational study, which was registered at ClinicalTrials.
gov (Identifier: NCT01773863).
2.2. FMD
Ultrasound assessment of basal brachial diameter and endothelial de-
pendent FMD of brachial artery were investigated according to current
guidelines [19] and as previously described [20]. FMD was performed
in all patients by the same operator. To evaluate the reproducibility of
FMD ten hospitalized patients underwent FMD measurement on 2
separate occasions (baseline AND after 1 week). Variability of different
measurements was assessed by using intra-class correlation coefficient
(ICC). ICC for brachial diameter at rest and FMD was 0.98 and 0.89,
respectively.
2.3. Blood sampling
Blood samples were collected between 8.00 and 9.00 am for routine
biochemical evaluation, including fasting total cholesterol and glucose,
and for oxidative stress analysis. Blood samples were collected in
Vacutainers (Vacutainer Systems, Belliver Industrial Estate, Plymouth,
UK) after an overnight fast (12 h). Samples were centrifuged at 300 g
for 10 min and the supernatant was collected and stored at − 80 °C
until dosage.
2.4. Serum isoprostanes (8-iso-PGF2α-III) assays
Analysis of isoprostanes was performed measuring serum 8-iso-
PGF2α-III by a validated enzyme immunoassay method (Cusabio).
Values were expressed as pM; intra-assay and inter-assay coefficients
of variation were 5.8% and 5.0%, respectively.
2.5. Serum nitrite and nitrate (NOx)
NOx were assessed in serum by the measurements of metabolic
end-products i.e. (Tema Ricerca). Intra- and inter-assay coefficients of
variation were 2.9% and 1.7%, respectively.
2.6. Endotoxemia
Serum levels of endotoxins (Lipopolysaccharides, LPS) were mea-
sured by a validated enzyme immunoassay method (Cusabio). Briefly,
 L. Loffredo et al. / European Journal of Internal Medicine xxx (2016) xxx–xxx 3

100 μl of serum sample was plated for 2 h at room temperature. After Table 1
incubation, samples were read at 450 nm. Values were expressed as Clinical characteristics of CAP patients and controls.

pg/ml; intra-assay and inter-assay coefficients of variation were b8% Patients CAP Controls p
and b 10%, respectively. (n = 50) (n = 50)

Age 74 ± 14 73 ± 12 0.877
2.7. C-reactive protein Gender males/females 30/20 27/23 0.686
PSI score 104 ± 6 n.a. n.a.
Systolic blood pressure 139 ± 25 136 ± 26 0.560
High-sensitivity C-reactive protein (CRP) was measured by commer-
Diastolic blood pressure 79 ± 14 78 ± 13 0.627
cially available immunoassay (Temaricerca). Intraassay and interassay Sepsis/SIRS 36 (72) 0 (0) b0.001
coefficients of variation were 9.5% and 9.0%, respectively. T2DM (%) 10 (20) 12 (24) 0.809
Hypertension (%) 40 (80) 42 (84) 0.794
2.8. Statistical methods Dyslipidemia (%) 12 (24) 14 (28) 0.820
Current smokers (%) 6 (12) 5 (10) 0.749
Former smokers (%) 34 (68) 35 (70) 0.829
Categorical variables were reported as counts (percentage), contin- CHD (%) 17 (34) 18 (36) 0.834
uous variables with normal distribution were expressed as mean ± CHF (%) 9 (18) 10 (20) 0.799
standard deviations (SD) unless otherwise indicated. In case of nonho- COPD (%) 14 (28) 18 (36) 0.520
Renal failure (%) 7 (14) 6 (12) 0.766
mogeneous variances, data were reported as median and interquartile
Medications
range [IQR]. Differences between categorical variables were tested • ACE-inhibitors (%) 31 (62) 33 (66) 0.835
using the χ2 test. Comparisons between patients and controls were car- • Statins (%) 18 (36) 20 (40) 0.837
ried out using Student's t test. Nonparametric tests (Kolmogorov– • Insulin (%) 7 (14) 6 (12) 0.766
Smirnov (Z) test) was used in case of nonhomogeneous variances as • Oral antidiabetic drugs (%) 3 (6) 5 (10) 0.712
• Antiplatelet drugs (%) 23 (46) 24 (48) 0.852
verified by Levene's test. Pairwise comparisons were performed by
• Corticosteroids (%) 23 (46) 12 (24) 0.036
T-test for paired data or by Wilcoxon signed rank test in case of nonho- • Fluoroquinolones 3 (6) n.a. –
mogeneous variances. • Piperacillin/Tazobactam 14 (28) n.a. –
Simple linear regression analysis was performed by Spearman test; • Macrolides 35 (70) n.a. –
• Cefalosporin 30 (60) n.a. –
Spearman's Rank Correlation Coefficient was described as Rs. P b 0.05
• Other antibiotics 3 (6) n.a. –
was considered as statistically significant. All analyses were carried
out with SPSS V.18.0 (SPSS Statistics v. 18.0, SPSS Inc. Chicago, USA). Legends. CHD: coronary heart disease; CHF: congestive heart failure; COPD: chronic
obstructive pulmonary disease; SIRS: Systemic inflammatory response syndrome;
T2DM: type-2 diabetes mellitus; n.a.: not applicable.
2.9. Sample size determination

We computed the minimum sample size with respect to a two-

tailed, one-sample Student t test considering, on the basis of data from
a previous pilot study (data not shown): (i) a difference for FMD, at ad-
mission to the ward, to be detected between CAP and controls of 2.5%;
(ii) SD of the paired differences: 3.5%; (iii) type I error probability α:
0.05 and power 1-β: 0.90. This resulted in n = 36 patients, which was
increased to n = 50.
3. Results
Demographic, clinical characteristics and therapies at admission
were similar between the two groups as shown in Table 1.
At admission, most of the CAP patients (65%) showed a severe pneu-
monia, as assessed by the PSI scoring system, belonging to PSI class IV
(41%) and V (24%). Moreover, 72% of the CAP patients met the clinical
criteria for sepsis (defined as SIRS plus infection); the remaining 28%
had infection plus one criterion for SIRS (WBC N 12,000/mm N 3 or
b4000/mm N 3: n = 10, Temp N 38 °C (100.4 °F) or b 36 °C (96.8 °F):
n = 3, Respiratory Rate N 20 or PaCO2 b 32 mmHg: n = 1). No patients
in the control group had SIRS.
No significant difference in resting diameter of brachial artery was
observed between CAP patients and controls at baseline (3.68 ± 0.69
vs 3.68 ± 0.60 mm, respectively, p = 0.984) and at discharge (3.65 ±
0.66 vs 3.69 ± 0.55 mm, respectively, p = 0.743).
At admission, a significant difference between patients with CAP and
controls was observed for FMD (2.1 ± 0.3 vs 4.0 ± 0.3%; p b 0.001),
serum endotoxins (143 [112–175] vs. 21.5 [7–44.7]; p b 0.001 pg/ml,
p b 0.001], serum isoprostanes (341 ± 14 vs 286 ± 10 pM; p =
0.009). CRP (67.1 [23.8–122] vs 2.74 [1.8–3.7] mg/l, p b 0.001), and
NOx (24.3 ± 1.1 vs 29.7 ± 2.2 μM; p = 0.036) (Fig. 1 Panels A–D).
FMD significantly correlated with serum endotoxins (Rs = − 0.291;
p = 0.003) and PSI classes (Rs = −0.376; p b 0.001); also, serum endo-
toxins significantly correlated with PSI (Rs = 0.386; p = 0.006) (Fig. 1,
Panel E). No significant association was found between FMD and CRP
(Rs = 0.148; p = 0.370). Brachial basal diameter did not correlate
with CRP (Rs = 0.211; p = 0.196), NOx (Rs = 0.007; p = 0.955) and
LPS (Rs = 0.013; p = 0.919).
The pairwise comparisons (between admission and discharge)
showed that, in CAP patients, FMD and NOx significantly increased
(from 2.1 ± 0.3 to 4.6 ± 0.4%; p b 0.001 and from 24.3 ± 1.1 to
31.1 ± 1.5 μM; p b 0.001, respectively) (Fig. 2, Panels A and D).
Conversely, compared to baseline, serum endotoxins, isoprostanes and
CRP significantly decreased at discharge (from 143 [112–175] to 54
[43–66] pg/ml; p b 0.001, from 341 ± 14 to 312 ± 14 pM; p b 0.001,
and from 67.1 [23.8–122] to 7.5 [4.2–21] mg/dl; p b 0.001; respectively)
(Fig. 2, Panels B, C and D).
Compared to baseline, at discharge no changes for FMD, NOx,
isoprostanes and serum endotoxins (from 4.0 ± 0.3 to 4.4 ± 0.3%;
p = 0.287, from 29.7 ± 2.2 to 27.7 ± 1.5 μM; p = 0.414, from 286 ±
10 pg/ml to 279 ± 7 pg/ml; p = 0.652 and from 33.1 ± 4.8 to 34.5 ±
2.4 pg/ml; p = 0.804, respectively) were observed in controls (Fig. 2,
Panel A–D).
A linear correlation analysis showed that Δ (expressed by difference
of values between admission and discharge) of FMD inversely correlat-
ed with Δ of serum endotoxins (Rs = −0.315; p = 0.001) and Δ of CRP
(Rs = −0.396; p = 0.001). Furthermore, Δ of serum endotoxins corre-
lated with Δ of CRP (Rs = 0.460; p b 0.001) and Δ of NOx (Rs = −0.459;
p b 0.001). A multiple linear regression analysis, including the variables
linearly associated with the dependent variable, was performed to de-
fine the independent predictors of FMD; only Δ of serum endotoxins
was significantly associated to Δ of FMD (SE: 0.05; standardized coeffi-
cient β: −0.257; P = 0.01).
During the intra-hospital stay, 5 CAP patients (10%) experienced
NSTEMI, within the first 48 h from admission, while no cases of myocar-
dial infarction or stroke were observed in the control group. The median
length of hospital stay was 9 days [IQR: 7–11 days] for CAP patients and
8 days [IQR: 6–10 days] for control subjects (p = 0.214).

Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
4 L. Loffredo et al. / European Journal of Internal Medicine xxx (2016) xxx–xxx

Fig. 1. Baseline values of FMD (panel A), serum endotoxins (panel B), isoprostanes (panel C) and NOx (panel D) in CAP patients and controls. Linear correlation between PSI and serum
endotoxin in CAP patients (panel E). *p b 0.01; ** b 0.05.

Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
 L. Loffredo et al. / European Journal of Internal Medicine xxx (2016) xxx–xxx
Fig. 2. Time-related changes of FMD values (panel A), serum endotoxins (panel B), serum isoprostanes (panel C) and serum NOx (panel D) in CAP patients (continuous line) and controls
(dotted line). Data are represented as mean ± SE.
4. Discussion
The study provides the first evidence that in the early phase of CAP
patients disclose an impaired FMD and suggests that bacteria-derived
LPS may play a role as a vasoconstriction molecule.
About 10% of patients with CAP experience MI during hospitalization
and are at higher risk for early and late mortality and cardiovascular
recurrence during a 1 year follow-up [6,21]. CAP patients with severe
disease, as assessed by PSI score, and history of cardiovascular disease
are more frequently associated with MI [6,22]. A peculiarity of MI in
CAP patients is that it is prevalently silent and NSTEMI [6], suggesting
that non occlusive coronary artery disease may be implicated in such
phenomenon. We speculated that endothelial dysfunction, in particular
impaired artery vasodilation, may concur to myocardial ischemia. To ex-
plore this issue at admission and discharge of CAP patients and controls,
we measured FMD, which assesses artery vasodilation. FMD is preva-
lently related to the release of nitric oxide (NO) from endothelial wall
[23] and is a surrogate marker of systemic artery dysfunction as indicat-
ed by the fact that it is significantly associated with poor cardiovascular
outcomes [8,9]. Thus, even if FMD does not directly reflect coronary
dilation, its analysis may be useful to explore the status of artery
reactivity in CAP patients. The novelty of the present study is the
demonstration that CAP patients disclose an impaired FMD in the
early phase of disease compared to a control population, which was
hospitalized for illnesses not related to infectious disease. The role of
pneumonia in eliciting impaired artery dilation was corroborated by
the fact that, in the remission phase, FMD improved reaching values
comparable with that of control population. This is in accordance with
previous studies that reported an association between reduced FMD
and acute infections [24–26].
Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
5
As FMD is essentially dependent upon NO release from endothelial
wall [23] and several studies demonstrated a significant direct correla-
tion between reduced FMD and circulating levels of nitroso compounds,
that reflect systemic NO bioactivity [20,27,28], we measured serum
levels of nitrite/nitrate in CAP and control population. Compared to con-
trol population, serum levels of nitrite/nitrate were reduced in CAP and
significantly correlated with FMD, corroborating previous data indicat-
ing that circulating levels of NO-derived compounds are predictors of
FMD [28]. We acknowledge that analysis of serum nitrite/nitrate may
be influenced by several factors, including daily dietary habits, which
could bias our finding; however, patients and controls followed a simi-
lar diet which was not modified throughout the hospital stay.
In order to explore the mechanism accounting for reduced NO gen-
eration, we focused on oxidative stress, which is crucial for NO inactiva-
tion and generation as it rapidly interacts with NO to give formation of
peroxynitrite, an important oxidant species detected in the atheroscle-
rotic plaque, and affects NO generation by inhibiting eNOS [29,30]. Fur-
thermore, previous study from our group demonstrated that Nox2,
which has a prominent role in the cellular formation of reactive oxidant
species, is up-regulated in CAP and significantly correlates with
isoprostanes [31], which are chemically stable eicosanoids with vaso-
constriction properties [32]. Accordingly with this previous finding,
serum isoprostanes were significantly higher in CAP patients compared
to control, suggesting a role for oxidative stress in inhibiting NO gener-
ation and eventually determining artery vasoconstriction. This hypoth-
esis, however, needs to be supported by an interventional study with
antioxidants to assess if this therapeutic approach is useful to counteract
artery dysfunction in CAP patients.
Finally, we investigated the mechanism potentially accounting for
oxidative stress and eventually vasoconstriction. An interesting finding
6 L. Loffredo et al. / European Journal of Internal Medicine xxx (2016) xxx–xxx
of our reports was that FMD was inversely associated with PSI score
suggesting that pneumonia severity could negatively influence artery
dilatation. The fact that PSI score was significantly associated with
endotoxemia led us to hypothesize that endotoxemia could be the link
between pneumonia severity and impaired FMD. This putative interplay
between endotoxemia and artery vasoconstriction was based on previ-
ous study showing that LPS infusion in humans suppresses the response
of the resistance vessels to Acetylcholine, suggesting that LPS elicits
artery vasoconstriction via inhibition of NO generation; [33] the role of
oxidative stress in such phenomenon was supported by the fact that
infusion of ascorbic acid, which is a scavenger of superoxide anion, re-
stored artery dilation [33]. Our study demonstrated that endotoxemia
may be implicated in impairing FMD for several reasons. Comparison
of CAP with controls demonstrated that CAP patients had significantly
higher values of serum endotoxins compared to controls at baseline;
also serum endotoxins significantly decreased in CAP while no changes
were detected in the control group at discharge. Finally, serum endo-
toxins inversely correlated with FMD and NOx reinforcing the hypothe-
sis that bacteria-derived LPS are implicated in the impaired FMD of CAP
via down-regulation of NO generation. We should, however, acknowl-
edge that analysis of serum NO by nitrite/nitrate measurement is a
loosely method to study NO generation, therefore this hypothesis
must be wisely considered [34].
The study has implications and limitations. As the purpose of the
paper was to explore the potential mechanism accounting for NSTEMI
complicating the early phase of the CAP, the study could not specifically
address as to whether impaired FMD was associated with acute coro-
nary events overall because the sample size was inadequate to explore
this issue. Therefore, our hypothesis that NSTEMI may be dependent
on impaired coronary dilation occurring after lung infection should be
considered still speculative and needs to be explored with more appro-
priate study design. However, our data indicating the existence of
impaired FMD in the acute phase of the disease may represent an inter-
esting scientific background to pursue this study hypothesis. At this
regard we must also acknowledge that we have no elements to
substantiate if impaired FMD is dependent on CAP-mediated NO
downregulation because experiments with NO donors were not per-
formed. A further limitation could be represented by the small sample
that does not permit to evaluate in more details the relationship
between FMD and patients' different clinical characteristics and clinical
outcomes.
Finally, an important point of the present study was the interpreta-
tion of the increased in levels of endotoxins in these CAP patients. One
possibility is that, even if not frequent, Gram-negative bacteria may
provoke or complicate the clinical course of CAP [35]. An alternative
and intriguing possibility is that, in the acute phase of CAP, enhanced
gut permeability could be responsible for translocation of LPS in the
peripheral circulation [36].
Previous study demonstrated that bacteria-derived LPS elicits artery
vasoconstriction via an oxidative stress-mediated mechanism. We have
only indirect evidence that this mechanism may occur in vivo, therefore
further study is necessary to establish if bacteria-derived LPS impairs ar-
tery dilation in CAP patients. In this context, it would be interesting to
evaluate if, in this specific clinical setting, infusion of an antioxidant
such as ascorbic acid may be useful to restore FMD and reduce the risk
of myocardial infarction.
In conclusion, the study shows that impaired FMD is detected in the
early phase of CAP suggesting its potential implication in the cardiovas-
cular complications occurring in the early phase of the disease.
Disclosures
None. All the authors have approved the final article. The
authors report no relationships that could be construed as a conflict of
interest.
Please cite this article as: Loffredo L, et al, Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia, Eur J
Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
Acknowledgments
Author contributions
Study concept and design: LL and FV. Data collection: EC and CC. FMD
data collection: LP. Analysis and interpretation of data: RC and LL. Labora-
tory data collection: CN, FE and RCar. Drafting of the manuscript: LL, RC
and FV. Critical revision of the manuscript for important intellectual
content: DF and FV. Statistical analysis: LL and RC. Study supervision: FV.
Funding sources
This work was supported by a grant from Sapienza University of
Rome (Progetto Universitario 2012) to Prof. Violi.
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Intern Med (2016), http://dx.doi.org/10.1016/j.ejim.2016.09.008
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