Early Vascular Aging (EVA): New Directions in Cardiovascular Protection

Greece

Preface

Peter M. Nilsson, Malmö, Sweden

Michael H. Olsen, Odense, Denmark

Stéphane Laurent, Paris, France

We welcome the reader to this book on different aspects of Early Vascular Aging (EVA), a concept that has attracted considerable attention since it was first described in 2008. A number of skilled authors have contributed to provide a multifaceted description of the pathophysiological and clinical aspects that are associated with EVA. Previous research has for decades described and investigated atherosclerosis, a process that starts in the intima layer of the arterial wall and becomes proximal to many cardiovascular events caused by athero-thrombotic disease. As the core component of EVA is arterial stiffness, arteriosclerosis, which is mainly influenced by morphological changes in the arterial media layer, but also in other layers, we have focused on different characteristics and mechanisms associated with stiffness of the large elastic arteries. We also consider EVA based on an integrated view linking the macro- with the microcirculation. This is because hemodynamic forces influenced by stiffness may also cause harm to the peripheral smaller vessels due to the increased pulsatile energy that is transmitted, for example, in the brain. Another aspect of a more integrated approach identifies important contributing factors for EVA also from the intima (endothelial dysfunction) and the adventitia (impaired function of vasa vasorum and innervation, accompanied by increased secretion of cytokines from the perivascular adipose tissue causing local inflammation) when impaired glucose metabolism could further contribute to stiffening by glycosylation. Therefore, we consider EVA to be a fruitful scientific concept to promote research on early changes of the arterial wall, programmed already in utero and early life and influenced by genetic and environmental factors. As meta-analyses have documented that arterial stiffness (increased aortic pulse wave velocity, aPWV) is an independent risk marker for future cardiovascular risk and total mortality, adjusted for conventional risk factors, we consider it of importance to find new ways to find, diagnose, and treat subjects with signs of EVA. Still however, neither an exact definition nor a targeted treatment exists for EVA, but several attempts are being made to find such alternatives. We therefore invite the reader to contribute to the lively discussion on EVA with data from different populations and ethnic groups, as well as with data from basic and clinical science. This could contribute to early detection of at-risk individuals, for example, from at-risk families with early onset cardiometabolic disease, for prevention based on improved life style as well as drug therapy when needed. This is not to deny the importance of atherosclerosis and the evidence-based methods that exist to prevent cardiovascular events by control of hypertension and hyperlipidemia as well as smoking cessation, but we consider that EVA is a feature starting early in life and that later in life components of arteriosclerosis and atherosclerosis will be intertwined in further promoting cardiovascular disease risk.

In an historical perspective, the interest in arterial function and stiffness contributing to hemodynamic changes predates the clinical measurement of blood pressure and diagnosis of hypertension as we know it. In London, the physician Fredrik Akbar Mahomed carried out studies on pulse wave properties in arteria radialis with his own sphygmograph and published in 1877:

It is very common to meet with people apparently in good health who have no albumen in the urine, who constantly present a condition of high arterial tension when examined by the aid of the sphygmograph. [1]

We therefore date the interest in large arteries and stiffness to an era before clinical hypertension was recognized [2], and thus the EVA concept [3–5] attempts to bridge more than a century to revive the importance of large arteries and their properties in cardiovascular medicine.

Frederick H. H. Akbar Mahomed

(c. 1849–1884), arterial studies

Scipione Riva-Rocci

(1863–1937), measurement of systolic blood pressure

References

1. Mahomed FA. Remarks on arterio-capillary fibrosis and its clinical recognition. Lancet. 1877;110(2816):232–234.

2. Riva-Rocci S. Un nuovo sfigmomanometro. Gazz Med Torino. 1896;50–51:1001–1007.

3. Nilsson PM, Lurbe E, Laurent S. The early life origin of vascular ageing and cardiovascular risk. J Hypertens. 2008;26:1049–1057.

4. Nilsson PM, Boutouyrie P, Laurent S. Vascular aging: a tale of EVA and ADAM in cardiovascular risk assessment and prevention. Hypertension. 2009;54:3–10.

5. Nilsson PM, Boutouyrie P, Cunha P, et al. Early vascular ageing in translation: from laboratory investigations to clinical applications in cardiovascular prevention. J Hypertens. 2013;31:1517–1526.

Chapter 1

Historical Aspects and Biology of Aging

Peter M. Nilsson,    Department of Clinical Sciences, Lund University, Skåne University Hospital, Malmö, Sweden

Aging is a universal finding in all mammals, shaped by evolutionary selection and environmental influences. Without a deeper understanding of the biology of aging it is not possible to disentangle the complicated web of causation behind age-related chronic disease such as cardiovascular and metabolic disorders. The genetic program for longevity and life span is influenced by nutrition, that is, calorie intake and nutrients, as well as reproduction when the number of progeny may impact on the biology of their mothers. Epigenetic imprinting plays an important role. New research on the importance of early life factors for the programming of adult health and disease has contributed to the paradigm of a life course perspective on cardiometabolic disease. In this chapter these factors are further discussed and linked to the process of vascular aging, one reflection of the biology of aging in general for humans, leading to arteriosclerosis (arterial stiffness) and later on to atherosclerosis. New understanding can also bring new treatment, for example based on studies in either long-lived subjects or patients with premature aging, that is, included in the progeroid syndromes.

Keywords

Aging; arteriosclerosis; biology; cardiometabolic; evolution; longevity; vascular

Aging is a universal finding in humans, afflicting biological processes as well as maturation and deterioration of organ function. There exist a number of theories on how aging is programmed and develops as presented in gerontology, the science of normal aging. Not only the wear and tear hypothesis exists but also aging models dependent on the influence of oxidative stress, metabolic processes, and the accumulation of genetic damage on the DNA and impaired genetic repair functions [1]. Modern discoveries point to the role of longevity-regulating genes, so-called gerontogenes [2]. These gerontogenes are classified as lifespan regulators, mediators, effectors, housekeeping genes, genes involved in mitochondrial function linked to metabolism, and genes regulating cellular senescence and programmed cell death (apoptosis) [2]. Intensive research is directed to understand what regulates aging and how to control this, not at least apoptosis, of vital importance to understand organ development and changes in health and disease. The maximum lifespan recorded was 122 years for a French woman (Jean Calment, France, 1875–1977).

Even if it is very hard to disentangle the different influences on the aging process and to judge upon the accuracy of the different hypotheses to explain human aging in general, it comes natural to view aging in its evolutionary context as all aspects of human biology, and even cognitive function, are supposed to be influenced by evolutionary selection mechanisms during millennia perspectives.

Evolutionary Traits, Genes, and the Environment Influencing Aging

From an evolutionary perspective the lifespan of mammals has been formed by selective processes based on genetic regulation of survival and reproduction in relation to available nutrition, environmental hazards, and competition for resources. According to the "disposable soma hypothesis" by Kirkwood [3] there exists a trade-off between maintenance of bodily functions, depending on energy investments, and the costs of reproduction, especially for women. This is why, according to this hypothesis, women with a higher number of offspring will be at increased risk for a shorter lifespan as compared to women with fewer offspring, if basal health and social conditions tend to be equal, as studied in British noble families over many centuries [4]. This is also influenced by nutritional resources, as reproductive capacity in women tends to cease during periods of famine and starvation.

Behind such traits there must be genetic regulators, as evolution works via genetic adaption and fitness in relation to a changing environment. A further support for the genetic influence on longevity is the family resemblance of longevity as well as risk of some chronic disease conditions that tend to run in families, that is, clusters of cardiovascular disease [5] and metabolic abnormalities. According to a number of studies the genetic explanation of longevity is approximately 25% [6]. This leaves a substantial proportion of longevity to the influence of environmental factors or to epigenetic mechanisms (gene–environmental interactions). It is still unclear if true life-prolonging genes exist in humans as in other less-developed organisms (Caenorhabditis elegans), or if a long lifespan is a marker of the less strong impact or lack of disease-related genes in some individuals. According to environmental factors, there are many such detrimental factors well known to decrease lifespan, for example, smoking, infectious disease, and malnutrition, but the only environmental factor known to prolong life in mammals, at least in rodents and monkeys, is continuous calorie restriction [7]. This is believed to exert similar effects in humans but still not proven. Nevertheless some individuals have adopted a lifestyle based on calorie restriction and balanced physical activity, hoping for a prolonged life.

Changes During the Twentieth Century in Life Expectancy

There is no doubt that the rapid increase in longevity during the past twentieth century is an indication of the strong influence of environmental factors on human lifespan, reflecting better nutrition and housing, improved hygiene and conditions in early life, as well as the progress of healthcare and improved medical treatment, even if temporary setbacks have also been noticed, for example, in Russia during the 1990s [8]. The negative socioeconomic changes for many citizens in Russia during this period could be one component of the increased cardiovascular risk based on gene–environmental interactions in high-risk populations [9]. On the other hand, it is still necessary to understand the biology (and genetics) behind the aging process, as there are still many examples of differential aging also in developed countries. A proof of the role of genetic influences on aging and shortened lifespan are the rare conditions of Hutchinson–Gilford progeria in children and Werner’s syndrome in middle-aged subjects [10]. Even if these rare conditions are not possible to causally treat today, they represent an opportunity to learn more about biological changes taking place during the aging process, especially when it is upregulated in the progeria syndromes with shortened lifespan.

Early Life Programming Effects

Human life starts at the conception followed by a growth during 9 months in fetal life in utero when organs are formed and developed based on numerous cell divisions under genetic regulation. Nutritional factors are of great importance for this process, as mediated by the feto-placental unit and influenced by maternal dietary intakes. For more than 30 years now, researchers have documented the importance of fetal growth and birth weight for bodily development and health also in adult life. Starting with early observations from northern Norway by Forsdahl [11] and by Gennser [12] in Sweden, David Barker and many other colleagues developed a concept based on the detrimental health consequences of fetal growth retardation leading to the small-for-gestational age (SGA) phenotype in newborn babies. This condition in early life was associated with increased levels of cardiovascular risk factors (hypertension, dyslipidemia, and hyperglycemia) and even overt type 2 diabetes in adult life, but also with impaired neurocognitive developments and a number of other adverse health conditions, summarized in the so-called Barker hypothesis [13]. In more recent years a new paradigm has evolved with a focus not only on fetal growth and birth weight as outcomes but also on postnatal growth patterns. Of special importance for adult health is the combination of impaired fetal growth, causing SGA at birth, combined with a rapid catch-up growth pattern in the first few years of life. This has been named the mismatch growth pattern when different organs are programmed in utero for a life with scarce resources and calorie depletion but later on the newborn child will experience the opposite, an environment with a surplus of calories and nutritional abundance. This may negatively impact on organ development and increase the risk of cardiometabolic disturbances in adult life. The most well-known protagonists of the mismatch hypothesis today are Peter Gluckman and Mark Hanson, with important reviews on the topic [14]. They are both active in the Developmental Origins of Health and Disease (DOHaD) society, to further explore the mismatch hypothesis.

An even more recent hypothesis of early life programming of adult disease risk is linked to the impact on child gut microbiota from the mother during delivery [15], as a detrimental gut microbiota pattern could be one factor increasing the risk of obesity in adult life and adverse health conditions such as cardiovascular disease [16] and type 2 diabetes [17]. It is believed that the mother’s gut bacteria will normally colonize the gastrointestinal system of the newborn child and that this will protect from overgrowth of more deleterious skin bacteria that could be associated with later disease risk [15].

It is likely that such influences in early life from nutrition, growth patterns, and microbiota patterning could also impact on aging in general and/or age-related medical conditions. These include not only defined chronic disease but also the increasing frailty, that is, related to sarcopenia and osteoporosis in old age, as well as cognitive decline [18]. Newer studies on the life of centenarians have also highlighted the role of early life influences, for example, the longevity associated with being born to younger mothers (first-born) when siblings within the same family are compared [19]. There also seem to exist large gender differences found in longevity determinants for males and females, suggesting a higher importance of occupation history for male centenarians as well as a higher importance of home environment history for female centenarians [19].

Vascular Aging in Perspective

What implications do these observations have for the concept of early vascular aging (EVA) with increased arterial stiffness as a central characteristic [20]? First of all, EVA is likely to be an expression of biological aging in general and some of the mechanisms regulating aging in other organs must also be applicable to the vascular tissue, especially in the arterial wall. This is believed to be possible to estimate by measuring leukocyte telomere length (LTL), a proposed marker of biological aging as LTL tends to shorten with every cell division. However, in a large population-based study, the Asklepios study in Belgium, no association between pulse wave velocity (PWV), a marker of arterial stiffness as the core characteristic of EVA, and LTL was seen in a cross-sectional analysis [21]. On the other hand, some associations were seen with cardiac function, which is why the authors concluded that in a generally healthy, young to middle-aged population, LTL is not related to left ventricular (LV) mass or systolic function, but might be associated with an altered LV filling pattern, especially in women. The Asklepios study purposefully selected healthy individuals for screening.

The findings of this large and more recent Belgian study contradicts earlier observations from a smaller French study [22], when it was concluded that LTL provides an additional account to chronological age with regard to variations in both pulse pressure and PWV among men, such that men with shorter telomere length are more likely to exhibit high pulse pressure and PWV, which both are indices of large artery stiffness (arteriosclerosis). The longer telomere length in women of that study suggests that for a given chronological age, biological aging of men is more advanced than that of women [22].

How to resolve these contradictory findings? It is believed that cross-sectional analyses of LTL in relation to organ function is probably not enough. Of even greater importance could be to evaluate relationship with telomere attrition rate based on repeated measurements of LTL over a time period. Few studies have applied this more laborious and costly method, and this is why more studies are needed with precise methods for measuring LTL and also attrition rate over time [23]. Before such data are available it is hard to judge on the true relationship between LTL and telomere biology, as a marker of aging, and arterial stiffness representing vascular aging. On the other hand, there are numerous studies to show associations between shorter telomeres and vascular disease based on atherosclerosis, as recently summarized [24].

New Models and Interventions to Influence Aging

If a deeper understanding can be achieved of the aging process in general, with its vascular implications, this could also lead to the establishment of new experimental models to test the reversibility (if any) of these processes. Molecules that suppress these age-related changes would provide an excellent medical intervention target for vascular disorders. Mammalian Sir2 (SIRT1, a NAD+-dependent deacetylase), previously shown to extend the lifespan of lower organisms, is a promising target molecule to influence some aspects of vascular aging. The influence of SIRT1 in various pathophysiological processes of vascular aging has been summarized and Wang et al. proposed that SIRT1 and its activators can become novel therapeutic targets for age-related vascular disease [25]. Time will tell if this intervention model will be able to shed new light on the aging process in general and vascular aging in particular (Table 1.1).

Table 1.1

Some Factors of Importance to the Shaping of Human Aging and Longevity

Genetic programming, based on evolutionary selection

Epigenetic influences (gene – environmental interaction and imprinting)

Early life programming (nutrition, growth rates, neurocognitive function)

Family patterns (sibling rank, age of parents, shared microbiota)

Adult lifestyle (smoking, nutrition, physical activity)

Telomere biology

Health problems and disease

Medical treatment and interventions

Societal factors and social support

Secular trends

Acknowledgment

This review was supported by a grant from the Research Council of Sweden for studies on early vascular aging in the population.

References

1. Kolovou GD, Kolovou V, Mavrogeni S. We are ageing. Biomed Res Int. 2014;2014:808307.

2. Moskalev AA, Aliper AM, Smit-McBride Z, Buzdin A, Zhavoronkov A. Genetics and epigenetics of aging and longevity. Cell Cycle. 2014;13:1063–1077.

3. Kirkwood TB, Rose MR. Evolution of senescence: late survival sacrificed for reproduction. Philos Trans R Soc Lond B Biol Sci. 1991;332:15–24.

4. Westendorp RG, Kirkwood TB. Human longevity at the cost of reproductive success. Nature. 1998;396:743–746.

5. Nilsson PM, Nilsson JA, Berglund G. Family burden of cardiovascular mortality: risk implications for offspring in a national register linkage study based upon the Malmö Preventive Project. J Intern Med. 2004;255:229–235.

6. Brooks-Wilson AR. Genetics of healthy aging and longevity. Hum Genet. 2013;132:1323–1338.

7. Smith Jr DL, Nagy TR, Allison DB. Calorie restriction: what recent results suggest for the future of ageing research. Eur J Clin Invest. 2010;40:440–450.

8. Plavinski SL, Plavinskaya SI, Klimov AN. Social factors and increase in mortality in Russia in the 1990s: prospective cohort study. BMJ. 2003;326:1240–1242.

9. Nilsson PM. Genetic and environmental determinants of early vascular ageing (EVA). Curr Vasc Pharmacol. 2012;10:700–701.

10. Ding SL, Shen CY. Model of human aging: recent findings on Werner’s and Hutchinson–Gilford progeria syndromes. Clin Interv Aging. 2008;3:431–444.

11. Forsdahl A. Are poor living conditions in childhood and adolescence an important risk factor for arteriosclerotic heart disease? Br J Prev Soc Med. 1977;31:91–95.

12. Gennser G, Rymark P, Isberg PE. Low birth weight and risk of high blood pressure in adulthood. Br Med J (Clin Res Ed). 1988;296:1498–1500.

13. Cooper C, Phillips D, Osmond C, Fall C, Eriksson J. David James Purslove Barker: clinician, scientist and father of the fetal origins hypothesis. J Dev Orig Health Dis. 2014;5:161–163.

14. Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med. 2008;359:61–73.

15. Reinhardt C, Reigstad CS, Bäckhed F. Intestinal microbiota during infancy and its implications for obesity. J Pediatr Gastroenterol Nutr. 2009;48:249–256.

16. Ettinger R, MacDonald K, Reid G, Burton JP. The influence of the human microbiome and probiotics on cardiovascular health. Gut Microbes. 2014;5:719–728.

17. Tilg H, Moschen AR. Microbiota and diabetes: an evolving relationship. Gut. 2014;63:1513–1521.

18. Langie SA, Lara J, Mathers JC. Early determinants of the ageing trajectory. Best Pract Res Clin Endocrinol Metab. 2012;26:613–626.

19. Gavrilov LA, Gavrilova NS. New developments in the biodemography of aging and longevity. Gerontology 2014; Dec 20. [Epub ahead of print] PubMed PMID: <25531147>.

20. Nilsson PM, Boutouyrie P, Cunha P, et al. Early vascular ageing in translation: from laboratory investigations to clinical applications in cardiovascular prevention. J Hypertens. 2013;31:1517–1526.

21. Denil SL, Rietzschel ER, De Buyzere ML, et al. Asklepios investigators on cross-sectional associations of leukocyte telomere length with cardiac systolic, diastolic and vascular function: the Asklepios study. PLoS One. 2014;9(12):e115071.

22. Benetos A, Okuda K, Lajemi M, et al. Telomere length as an indicator of biological aging: the gender effect and relation with pulse pressure and pulse wave velocity. Hypertension. 2001;37(2 Pt 2):381–385.

23. Nilsson PM. Mediterranean diet and telomere length. BMJ. 2014;349:g6843 <http://dx.doi.org/10.1136/bmj.g6843>. PubMed PMID: <25467755>.

24. Butt HZ, Atturu G, London NJ, Sayers RD, Bown MJ. Telomere length dynamics in vascular disease: a review. Eur J Vasc Endovasc Surg. 2010;40:17–26.

25. Wang F, Chen HZ, Lv X, Liu DP. SIRT1 as a novel potential treatment target for vascular aging and age-related vascular diseases. Curr Mol Med. 2013;13:155–164.

Chapter 2

Cellular and Molecular Determinants of Arterial Aging

Patrick Lacolleya, Pascal Challandeb, Veronique Regnaulta, Edward G. Lakattac and Mingyi Wangd,    aInstitut National de la Santé et de la Recherche Médicale—INSERM U1116; Université de Lorraine, Nancy, France,    bUniversité Pierre et Marie Curie, CNRS—UMR 7190, Paris, France,    cLaboratory of Cardiovascular Science, Intramural Research Program, Biomedical Research Center, National Institute on Aging, NIH, Baltimore, MD, USA,    dLaboratory of Cardiovascular Sciences Biomedical Research Center, Baltimore, MD, USA

A number of key signaling pathways are highly relevant to early vascular aging. These are overviewed here to provide a context for understanding the initiation and progression of hypertension, arteriosclerosis, and atherosclerosis. The elements of the pathways include the following: cytoskeletal and contractile proteins; elastin and collagen networks; adhesion proteins and metalloproteinases; cytokines; reactive oxygen species; and NO bioavailability and the renin–angiotensin–aldosterone system. Activation of these pathways leads to a proinflammatory state and tissue oxidative stress, which in turn drives age-associated remodeling processes. A progressive decrease in plasticity and increase in reprogramming potential of vascular smooth muscle cells plays a complementary role, contributing to the increase in arterial stiffness and associated cardiovascular risk factors. These key signaling pathways have become the focus of modern aging research and their elucidation will undoubtedly provide a rich resource for the development of selective drugs that will interfere with these processes and aid in the prevention of the number one cause of death in the modern world.

Keywords

Aging; inflammation; vascular smooth muscle cell; arterial stiffness; hypertension

Introduction

The aging of the world population has progressed unabated as more adults are surviving into their senior years. The heterogeneity of aging phenotypes results from genetic and epigenetic impacts on different cell types and tissues throughout a lifetime [1]. Importantly, arterial aging is intertwined with hypertension and atherosclerosis at the molecular, cellular, vascular, and clinical levels because the aged arterial wall is fertile soil for their pathogenesis. Age-associated arterial diseases account for a large part of total mortality, approximately 29% of all deaths. Hypertension is a major factor to promote arterial aging. The prevalence of hypertension is around 50% and 60% over 60 and 70 years of age, respectively [2]. It is higher in men than in women before 50 years of age, whereas in older persons, the sex difference in prevalence of hypertension is greater in women than in men [3]. The prevalence of hypertension is similar in various regions of the world [4], whereas the prevalence of stroke is 3.5-fold higher in low-income than in middle- and high-income countries [5].

Arteriosclerosis is defined as an age-associated stiffening and dilatation of the large arteries. Atherosclerosis represents the leading cause of mortality and is characterized by four major steps: (i) an initial endothelial activation by hemodynamic factors and dyslipidemia followed by leucocyte transmigration and activation involving cytokines and innate or adaptive immunity; (ii) a promotion step, which includes development of foam cells and lipoprotein retention; (iii) a progression step by growth of complex plaques; and (iv) plaque destabilization and thrombosis. Atherosclerosis within the arterial wall leads to inflammation, accumulation of fibronectin, collagen deposition, and fibrosis.

Aging is characterized by chronically elevated levels of low-grade circulating inflammatory molecules such as monocyte chemoattractant protein-1 (MCP-1) [6]. In particular, the interactions of environmental, systemic, and local chronic stress signals are conferred to vascular cells and the matrix, which insidiously facilitate arterial adverse remodeling through proinflammatory signaling such as the angiotensin II (Ang II) signaling cascade with aging. This process leads to endothelial disruption, thrombosis, senescence, glycoxidation, fibrosis, elastin fragmentation, calcification, and amyloidosis [1,7–9]. Importantly, this proinflammatory response accelerates the cardiovascular burden of both hypertension and atherosclerosis in the elderly [7,9]. This review focuses upon the key molecules involved in inflammatory mechanisms and pathways that are implicated in the aging of the arterial wall (Figure 2.1).

Figure 2.1 Diagram of cellular and molecular determinants of arterial aging.

Cytoskeletal and Contractile Proteins in the Aging Arterial Wall

Cytoskeletal Proteins

Desmin and vimentin are the main components of intermediate filaments implicated in mechanotransduction (Figure 2.1). Both desmin and vimentin are generally found to be decreased with advancing age in rat smooth muscle cells (SMCs) [10–13]. The mechanical properties of SMCs through cytoskeletal proteins contribute to the increased stiffness of the aorta in old versus young monkeys [14]. Desmin is required in the dilatory and contractile functions of SMCs and provides an efficacious interaction between the cytoskeletal and the contractile elements to maintain the mechanical integrity of SMCs. In old SMCs there is a shift toward small vimentin fragments, and co-localization with calpain-1 argues for calcium-dependent vimentin cleavage by calpain-1 [15].

Contractile Proteins

Smooth muscle (SM) myosin heavy-chain content/isoform expression is the most discriminant marker of fully differentiated SMCs (Figure 2.1). Alteration in SM myosin has been reported in aged rats. In SMCs cultured from 30-month-old Fischer 344XNB rats or 24-month-old Wistar rats, SM myosin is decreased compared to SMCs isolated from 6-month-old rats [12,13,16]. SMCs freshly isolated from 18-month-old Wistar rat aortae showed percentages of SM-myosin-positive cells similar to those observed in newborn and young adult rat SMCs [10]. Higher tissue content of myosin heavy chain and a higher ratio of SM1/SM2 isoforms have been reported in aortae of 36-month-old Fischer 344/NNiaHSd X Brown Norway/BiNia compared to those of 6-month-old rats [17]. Interestingly, embryonic myosin in SMCs is increased in aged thickened intima in humans [18]. In addition, the contractile regulatory light-chain MyL9 is overexpressed in endothelial layers of aging rats and is associated with an increase of endothelial cell (EC) contraction resulting in endothelial hyperpermeability [19].

Cellular Matrix Structure in the Aging Arterial Wall

It is known that the elastin/collagen ratio plays an important role in arterial mechanical properties [20]. Changes in both content and organization of elastin and collagen fibers influence the arterial wall with age. When expressed as a percentage, elastin percentage is decreased while collagen percentage is increased, which causes a net decrease in the elastin/collagen ratio with aging [20]. The bulk of the elastin is highly susceptible to age-related changes, which involve an increase in associated polar amino acids, the binding and accumulation of calcium and lipids, and fragmentation due to enzymatic degradation or fatigue processes [20,21]. Advanced glycation end-products (AGEs)-mediated cross-linking of elastin increases with age in the human aorta [22]. By contrast, collagen fibrils become organized into multibranched bundles and stiffen [23].

Proinflammatory Molecular, Cellular, and Vascular Events in the Aging Arterial Wall

The Renin–Angiotensin–Aldosterone System

The components of the renin–angiotensin–aldosterone system (RAAS) are important aspects of the proinflammatory system (Figure 2.1), including angiotensin converting enzyme (ACE), Ang II, and its receptor AT1. The transcription, translation, and activity of ACE markedly increase within both ECs and vascular SMCs (VSMCs) in the arterial wall with aging in rodents, nonhuman primates, and humans [18,24,25]. In addition, an alternative angiotensin convertase, chymase, increases within the arterial wall with aging [25]. As a result, the cleaved product, Ang II protein, becomes markedly increased, particularly in the thickened intima of rats, nonhuman primates, and humans [15,18,25–27]. Furthermore, the Ang II receptor, AT1, is up-regulated within the old arterial wall [18].

Ang II stimulates aldosterone secretion. The mineralocorticoid receptor (MR) expression is increased in the arterial wall with aging [28,29]. Furthermore, aging increases the sensitivity of MR to Aldo. Increased MR activity in aged rats promotes a proinflammatory phenotype via an extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase/epidermal growth factor receptor (ERK-1/2/MAPK/EGFR)-dependent pathway, contributing to the synthetic phenotypic shift of SMCs within the aging arterial wall [28]. In addition, aldosterone mediates an increase in the expression of EGFR in SMCs with aging, further reinforcing its proinflammatory effects [28]. Notably, cardiotrophin-1 (CT-1), a proinflammatory cytokine overexpressed in SMCs by aldosterone [30], also contributes to vascular aging because CT-1 treatment increases SMC proliferation and collagen production, whereas its invalidation increases longevity in mice [31].

Increased activation of the RAAS and an increase in oxidative stress that contributes to arterial proinflammation are both implicated in age-related arterial remodeling. Chronic infusion of a physiologically relevant dose of Ang II to adult rats (8-months-old) increases expression of molecules that comprise the proinflammatory profile, that is, matrix metalloproteinase type II (MMP-2), MCP-1, calpain-1, transforming growth factor-β1 (TGF-β1), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The infusion also elicits the age-associated increase in aortic and coronary structural manifestations, that is, intimal and media thickening of old (30-month-old), untreated arteries [27]. In addition, the α-adrenergic receptor agonist, phenylephrine, increases arterial Ang II protein, causing MMP-2 activation and intimal and medial thickening [27]. In contrast, chronic ACE inhibition and AT1 receptor blockade, beginning at an early age, markedly inhibit the expression of proinflammatory molecules and delay the progression of age-associated aortic remodeling [24,32]. Interestingly, long-term AT1 blockade improves endothelial function, decreases blood pressure, and doubles the life span of hypertensive rats similar to normotensive animals [33]. Disruption of the AT1 receptor retards arterial inflammation, promotes longevity, and improves survival after myocardial infarction in mice [34].

Proteinases

Matrix Metalloproteinases

An important component of age-associated vascular remodeling is degradation and resynthesis of extracellular matrix (ECM) (Figure 2.1). Specialized enzymes known as matrix metalloproteinases (MMPs) mediate the degradation process. Among MMPs, the MMP-2 mRNA and protein increase in the aortic walls of aged rodents, nonhuman primates, and humans and is also activated by Ang II signaling [25,27,35–38]. The increased MMP-2 activity is mainly seen within the thickened intima and the inner media in rodents and monkeys [25,39]. The enhanced MMP-2/-9 activity is also observed in the aortae from human autopsy in the grossly normal vessels with aging [18]. An increase of MMP-2/-9 activity is attributable to not only an enhanced transcription and translation, but also to an imbalance of its activators, membrane-type-1 matrix metalloproteinase (MT1-MMP), urine plasminogen activator, and tissue plasminogen activator and inhibitors, tissue inhibitor of MMP-2 (TIMP-2), and plasminogen activator inhibitor [25,39].

Notably, the micro-processing of extracellular bioactive molecules via MMP activation likely facilitates the initiation and progression of hypertension. Activated MMP-2 increases the bioavailability of vasoconstrictors such as big endothelin-1 (ET-1), while decreasing the vasodilator such as adventitial calcitonin gene-related peptide (CGRP) and endothelial NO-synthase enzyme (eNOS) [7,40,41]. MMP-2-7/-9 reduces the density of the extracellular domain of β(2)-adrenergic receptor in blood vessels and enhances the arteriolar tone [42,43].

Interestingly, age-associated arterial remodeling due to arterial wall collagen deposition and elastin fragmentation known as elastolysis is associated with an increase in arterial MMP activation. Chronic administration of a broad-spectrum MMP inhibitor markedly blunts the age-associated increases in aortic gelatinase and interstitial collagenase activity and reduces the elastin network degeneration, collagen deposition, MCP-1 expression, TGF-β1 activation, and Smad-2/-3 phosphorylation [44]. Importantly, MMP inhibition also substantially diminishes pro-ET-1 activation and down-regulates Ets-1 expression [44].

Calpain-1

Calpain-1 is a calcium-dependent intracellular proteinase and is an important activator of MMP-2 [45]. Transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic SMCs from old rats compared to young animals [15]. Co-localized calpain-1 and Ang II are within the aged arterial wall [15]. Ang II induces calpain-1 expression in the aortic walls in vivo and aortic rings ex vivo and SMCs in vitro [15]. Over-expression of calpain-1 in young SMCs leads to cleavage of intact vimentin, an increase of migratory capacity, and calcification mimicking that of old SMCs [15].

In addition, communication between MMP-2 and calpain-1 is observed in aged arterial walls or SMCs. Aging induces both MMP-2 and calpain-1 expression and activation in the arterial wall [45]. Co-localization of calpain-1 and MMP-2 are observed within old rat SMCs [45]. Over-expression of calpain-1 induces MMP-2 transcription, translation, and activity, in part, due to increasing the ratio of MT1-MMPs to TIMP-2 [45]. These effects of calpain-1 over-expression-induced MMP-2 activation are linked to increased TGFβ-1/Smad-2/-3 signaling, and collagen I, II, and III production [38,45]. Cross-talk of two proteases, calpain-1 and MMP-2, synergistically modulates ECM remodeling and facilitates calcification by enhancing collagen production in SMCs with aging [45]. A switch from a de-differentiated to a pro-calcificatory phenotype of SMCs also induces vascular calcification with advancing age [46].

Transforming Growth Factor-β1

Arterial TGF-β1 mRNA and protein are abundantly present in the aged arterial wall (Figure 2.1) [27,38]. Co-expression of both TGF-β1 and TGF-β1 receptor II (TβIIR) proteins increases in rat aortae at 30 versus 8 months of age [38]. TGF-β1 plays an important role in arterial fibrosis [27,29,37,38]. TGF-β1 expression is tempo-spatially associated with the collagen expression and local fibrosis in the aging arterial wall [27,39]. In vitro studies show that ECs and VSMCs treated with TGF-β1 increase collagen types I and III mRNA, and this is attenuated by a TβIIR blocker [47,48]. Importantly, enhanced expression of active TGF-β1 and collagen deposition in the thickened vascular wall of aged rats is, in part, produced by exaggerated MMP-2 activation of latent transforming protein-1 [38]. Furthermore, the increased MCP-1 co-localizes with TGF-β1, which suggests an interaction may exist between MCP-1 production and TGF-β1 activity [37]. Indeed, TGF-β1 transcription, translation, and activity increase in VSMCs treated in response to MCP-1 and enhance production of ECM [37].

Monocyte Chemoattractant Protein-1

MCP-1, a downstream molecule of Ang II signaling, is a potent inflammatory cytokine (Figure 2.1). With aging, MCP-1 mRNA increases within aortic walls in FXBN rats [49]. The increased MCP-1 protein is predominantly localized to the thickened intima [49]. The increased MCP-1 also co-localizes with TGF-β1, suggesting an interaction between MCP-1 production and TGF-β1 activity [37]. Indeed, TGF-β1 transcription and translation increase in SMCs treated with MCP-1 and exposure of SMCs to MCP-1 increases TGF-β1 activity [37]. Thus, MCP-1 signaling also initiates the fibrosis of aging. Notably, MCP-1 dimerization is necessary for chemoattractant activity [50]. MCP-1 forms dimers at local high concentrations such as in the aged arterial wall, which is likely to strongly attract the invasion of SMCs [26].

Reactive Oxygen Species and NO Bioavailability

Non-phagocytic NAD(P)H oxidase, which generates arterial cell reactive oxygen species (ROS) in the vascular system, is activated by Ang II signaling (Figure 2.1). NAD(P)H oxidase membrane-bound components p22phox and gp91phox are increased in the endothelium of aortae from old versus young rats [51]. Further, cytosolic component p47phox also increases in the arterial wall with aging in rodents [52,53]. Importantly, anti-oxidant Cu/Zn superoxide dismutase (SOD1), Mg SOD (SOD2), and ECM superoxide dismutase (ECM-SOD/SOD3) decrease in the arterial wall, which accompanies aging in rats [54–57]. Indeed, with aging, a loss of balance between oxidase and dismutase has been observed in the coronary arterial wall and aortic wall of rats, consequently resulting in an increase of superoxide and hydrogen superoxides [58–61].

Nitric oxide (NO) is a diffusible gas that can act as an intracellular and intercellular messenger in the arterial wall that is avidly scavenged by superoxide anions. The main source of NO is the ECs in the arterial wall. Endothelial production of NO becomes reduced with advancing age [55,62,63]. NO is generated from the metabolic conversion of L-arginine into L-citrulline by the activity of the NOS. Two major classes of NOSs have been described in the vascular system. One isoform is constitutively expressed (eNOS) under basal conditions and is involved in the endothelium-dependent vasodilation response. Another isoform, iNOS, is inducible by inflammation [64]. While iNOS is absent in the aortic segments of young rats, a marked expression of the iNOS protein is observed in segments of aging rats [64]. The expression of both eNOS and iNOS is altered in the arterial wall with aging [55,64,65].

Augmented release of ROS subsequently inactivates NO with increasing age [61]. Reactive nitrogen species are also important modulators of NO bioavailability [66]. The interaction of NO and free radicals will result in subsequent formation of peroxynitrite (ONOO–). Strong experimental evidence has recently been presented for a close association between the formation of ONOO– and age-associated vascular endothelial dysfunction [66].

The aging arterial wall is a frequent target of modifications by reactive oxidative compounds such as NADPH oxidase and reducing sugars known as glycoxidation [8,67–70]. AGEs are easily formed by a reaction between sugar chains and biologic amines of oxidized collagen. Stabilized glycated proteins accumulate over a lifetime and contribute to age-associated multiple structural and physiologic changes in the vascular system such as increased vascular stiffness, endothelial dysfunction, and inflammation [8].

MFG-E8, Fibronectin, and Integrin Receptors

MFG-E8 and Its Fragment Medin

A high-throughput proteomic screening identified milk fat globule-EGF-8 (MFG-E8) (Figure 2.1), a cell adhesion protein, as an important Ang II signaling signature of aging arterial walls [26]. Levels of arterial MFG-E8 and its degradation fragment, medin, both increase and accumulate in the aorta with aging in rodents, nonhuman primates, and humans [26,71,72]. MFG-E8 is induced by Ang II and itself induces the expression of MCP-1 in SMCs within the aortic wall of old rats [26].

Integrins comprise a widely distributed family of cell surface α/β heterodimeric adhesion receptors that bind cells to components of the ECM such as fibronectin. They act as sensing and signaling transmembrane receptors. Integrin α5β1 and αvβ3/5 expressions are increased in the arterial wall of old hypertensive rats, contributing to arterial stiffening [73,74]. Co-expression and increased physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in SMC in vitro, promoting SMC invasion and proliferation with aging [26,75].

Increased amyloid deposition is a characteristic of the aged arterial wall. A specific amyloid protein, known as medin, is deposited in the aortic media in the majority of Caucasians over 50 years of age. In addition, both medin and MFG-E8, in an amyloid protein complex, bind to tropoelastin [76–78]. Thus, MFG-E8/medin amyloid may likely be a factor in the increased aortic stiffness that accompanies advancing age. Indeed, serum MFG-E8 levels and pulse wave velocity, an index of arterial stiffening, correlate with cardiovascular risk factors in old humans with type 2 diabetes [79].

Fibronectin

In large arteries, the increase in α5β1 and fibronectin participates in the adaptation to mechanical stress in aged spontaneously hypertensive rats through increased numbers of cell–matrix attachments and phenotypic changes [80]. Pressure and age induce accumulation of fibronectin and more specifically the EIIIA isoform [21]. Paralleling the increase in integrins in the aging vasculature are marked increases in fibronectin levels [73]. Inhibition of αvβ3 integrin increases senescence of SMCs [81], which suggests an up-regulation of this integrin with aging. Interestingly, the level of integrin β4 increases in the endothelium of mouse aorta with aging, which contributes to vascular EC senescence by affecting the levels of p53 and ROS [82].

Transcription Factors

Cytoskeletal Serum Response Transcription Factor

Serum response factor (SRF) (Figure 2.1) is a MADS (MCM1, Agamous, Deficiens, SRF) box transcription factor that regulates numerous cytoskeletal SMC genes, which produce SM-actin, SM-myosin heavy chain, calponin, troponin, dystrophin, and desmin through specific CArG-element-binding sites. SRF has also been implicated in EC migration during sprouting angiogenesis [83]. SRF is highly expressed in SMCs compared to most other tissues and appears to increase with aging (personal data) and in cerebral arteries of Alzheimer’s patients [84]. The development of hypertension in spontaneously hypertensive rats is also linked to an increased SRF-binding affinity to the CArG box present in the SM-myosin light-chain kinase promoter, resulting in higher phosphorylation of the myosin light chain [85]. VSMC phenotypic modifications are induced by SRF and control vascular tone as well as carotid stiffness via modulation of genes coding for components of the contractile apparatus and integrins without changes in collagen, elastin, fibronectin, and MMPs [86].

Proinflammatory Transcription Factors Ets-1 and NF-κB

Pronflammatory transcription factors Ets-1 and nuclear factor kappaB (NF-κB) associated with Ang II signaling are both increased within the arterial wall with aging (Figure 2.1). Elevated Ets-1 activity is closely associated with increased transcription of ET-1, MCP-1, TGF-β1, and MMP-2 within the old arterial wall [44]. Activated NF-κB regulates the activity of MMP-2/-9, calpain-1, MCP-1, TGF-β1, and ROS, which deliver multiple signals and potentially drive arterial aging [7,87].

Anti-Inflammatory Molecule SIRT1

Sirtuins, including SIRT1, are members of a small family of enzymes that require nicotinamide adenine dinucleotide (NAD+) for their deacetylase or ADP-ribosyltransferase activity (Figure 2.1). The mRNA expression of the seven isoforms with unique subcellular localization and distinct functions in ECs is reduced with aging. SIRT1, located predominantly in the nucleus but also found in cytoplasm, is highly expressed in vascular ECs. Expression of SIRT1 is reduced in ECs from older versus younger mice and older versus younger healthy human adults. Decreases in arterial expression and activity of SIRT1 with advancing age are associated with increased acetylated eNOS, which inhibit eNOS activity and in turn contribute to vascular endothelial dysfunction [88]. The transcription factors p53, NF-κB, and forkhead box-containing protein type O subfamily (FOXO) have also been identified as deacetylation substrates of SIRT1, thereby down-regulating stress-induced premature senescence in ECs. SIRT1 also regulates oxidative stress at the chromatin level via decrease in acetylated histone H3 binding to the ShcA adapter protein P66Shc promoter region [89].

Recent reports have brought particular emphasis to the implication of sirtuins in healthy aging. Among the sirtuins, SIRT1 has been the most extensively characterized for its protective role in aging and cardiovascular diseases, which depends upon the tissue and its degree of activation. Low to moderate over-expression of SIRT1 in mouse hearts reduces cardiac dysfunction and senescence markers, while high levels of SIRT1 expression are associated with cardiomyopathy and high levels of oxidative stress [90]. The protective role of SIRT1 is also related to its ability to decrease the age-associated impairment in endothelium-dependent dilatation without affecting endothelium-independent dilatation. Transfection of ApoE−/− mice with a truncated inactive SIRT1 increases DNA damage, inflammation, and atherothrombotic lesions [91]. Inflammation and endothelial dysfunction shift the hemostatic balance in favor of thrombosis in aging, and that in turn, can further enhance inflammation. Production and secretion of coagulation enzymes and cofactors as well as von Willebrand factor by vascular cells increase as the vascular wall function deteriorates with age [92]. In addition, the age-associated irreversible cellular senescence process, leading to a progressive decrease in plasticity and reprogramming potential of SMCs, plays a complementary signaling role and contributes to the increase in oxidation, fibrosis, calcification, and arterial stiffness [46,53,81].

Conclusion

Several new altered molecular and cellular pathways in the aging arterial remodeling have emerged and prompted the development of selective drugs such as inhibitors of Ang II signaling or downstream molecules MMP, MCP-1, and TGF-β; integrin antagonists; and SIRT1 activators. Preliminary studies of these interventions provide promising results in attenuating age-related decline in physiological functions. However, several major challenges involving simultaneous multidrug usage on several of the above-mentioned systems need to be addressed. This may require new pharmacological design of specific drugs with careful concern for key signaling system nodes or targeting more than one of the compensatory networks.

Acknowledgment

The authors would like to thank Robert E. Monticone for his editorial assistance in preparing this document. This research was supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.

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