Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Physiological determinants of mechanical efficiency during advanced ageing and disuse.

This study aimed to determine which physiological factors impact net efficiency (ηnet) in oldest-old individuals at different stages of skeletal muscle disuse. To this aim, we examined ηnet, central haemodynamics, peripheral circulation, and peripheral factors (skeletal muscle fibre type, capillarization and concentration of mitochondrial DNA [mtDNA]). Twelve young (YG; 25 ± 2 years), 12 oldest-old mobile (OM; 87 ± 3 years), and 12 oldest-old immobile (OI; 88 ± 4 years) subjects performed dynamic knee extensor (KE) and elbow flexors (EF) exercise. Pulmonary oxygen uptake, photoplethysmography, Doppler ultrasound and muscle biopsies of the vastus lateralis and biceps brachii were used to assess central and peripheral adaptations to advanced ageing and disuse. Compared to the YG (12.1 ± 2.4%), the ηnet of lower-limb muscle was higher in the OM (17.6 ± 3.5%, P < 0.001), and lower in the OI (8.9 ± 1.9%, P < 0.001). These changes in ηnet during KE were coupled with significant peripheral adaptations, revealing strong correlations between ηnet and the proportion of type I muscle fibres (r = 0.82), as well as [mtDNA] (r = 0.77). No differences in ηnet were evident in the upper-limb muscles between YG, OM and OI. In view of the differences in limb-specific activity across the lifespan, these findings suggest that ηnet is reduced by skeletal muscle inactivity and not by chronological age, per se. Likewise, this study revealed that the age-related changes in ηnet are not a consequence of central or peripheral haemodynamic adaptations, but are likely a product of peripheral changes related to skeletal muscle fibre type and mitochondrial density. KEY POINTS: Although the effects of ageing and muscle disuse deeply impact the cardiovascular and skeletal muscle function, the combination of these factors on the mechanical efficiency are still a matter of debate. By measuring both upper- and lower-limb muscle function, which experience differing levels of disuse, we examined the influence of central and peripheral haemodynamics, and skeletal muscle factors linked to mechanical efficiency. Across the ages and degree of disuse, upper-limb muscles exhibited a preserved work economy. In the legs the oldest-old without mobility limitations exhibited an augmented mechanical efficiency, which was reduced in those with an impairment in ambulation. These changes in mechanical efficiency were associated with the proportion of type I muscle fibres. Recognition that the mechanical efficiency is not simply age-dependent, but the consequence of inactivity and subsequent skeletal muscle changes, highlights the importance of maintaining physical activity across the lifespan.

Is It Good to Have a Stiff Aorta with Aging? Causes and Consequences.

Aortic stiffness increases with advancing age, more than doubling during the human life span, and is a robust predictor of cardiovascular disease (CVD) clinical events independent of traditional risk factors. The aorta increases in diameter and length to accommodate growing body size and cardiac output in youth, but in middle and older age the aorta continues to remodel to a larger diameter, thinning the pool of permanent elastin fibers, increasing intramural wall stress and resulting in the transfer of load bearing onto stiffer collagen fibers. Whereas aortic stiffening in early middle age may be a compensatory mechanism to normalize intramural wall stress and therefore theoretically "good" early in the life span, the negative clinical consequences of accelerated aortic stiffening beyond middle age far outweigh any earlier physiological benefit. Indeed, aortic stiffness and the loss of the "windkessel effect" with advancing age result in elevated pulsatile pressure and flow in downstream microvasculature that is associated with subclinical damage to high-flow, low-resistance organs such as brain, kidney, retina, and heart. The mechanisms of aortic stiffness include alterations in extracellular matrix proteins (collagen deposition, elastin fragmentation), increased arterial tone (oxidative stress and inflammation-related reduced vasodilators and augmented vasoconstrictors; enhanced sympathetic activity), arterial calcification, vascular smooth muscle cell stiffness, and extracellular matrix glycosaminoglycans. Given the rapidly aging population of the United States, aortic stiffening will likely contribute to substantial CVD burden over the next 2-3 decades unless new therapeutic targets and interventions are identified to prevent the potential avalanche of clinical sequelae related to age-related aortic stiffness.

Therapeutic strategies targeting the endothelial glycocalyx.

PURPOSE OF REVIEW: This review will highlight recent studies that have examined the endothelial glycocalyx in a variety of health conditions, as well as potential glycocalyx-targeted therapies. RECENT FINDINGS: A degraded glycocalyx is present in individuals that consume high sodium diet or have kidney disease, diabetes, preeclampsia, coronavirus disease 2019 (COVID-19), or sepsis. Specifically, these conditions are accompanied by elevated glycocalyx components in the blood, such as syndecan-1, syndecans-4, heparin sulfate, and enhanced heparinase activity. Impaired glycocalyx barrier function is accompanied by decreased nitric oxide bioavailability, increased leukocyte adhesion to endothelial cells, and vascular permeability. Glycocalyx degradation appears to play a key role in the progression of cardiovascular complications. However, studies that have used glycocalyx-targeted therapies to treat these conditions are scarce. Various therapeutics can restore the glycocalyx in kidney disease, diabetes, COVID-19, and sepsis. Exposing endothelial cells to glycocalyx components, such as heparin sulfate and hyaluronan protects the glycocalyx. SUMMARY: We conclude that the glycocalyx is degraded in a variety of health conditions, although it remains to be determined whether glycocalyx degradation plays a causal role in disease progression and severity, and whether glycocalyx-targeted therapies improve patient health outcomes. Future studies are warranted to investigate therapeutic strategies that target the endothelial glycocalyx.

T cells mediate cell non-autonomous arterial ageing in mice.

KEY POINTS: Increased large artery stiffness and impaired endothelium-dependent dilatation occur with advanced age. We sought to determine whether T cells mechanistically contribute to age-related arterial dysfunction. We found that old mice exhibited greater proinflammatory T cell accumulation around both the aorta and mesenteric arteries. Pharmacologic depletion or genetic deletion of T cells in old mice resulted in ameliorated large artery stiffness and greater endothelium-dependent dilatation compared with mice with T cells intact. ABSTRACT: Ageing of the arteries is characterized by increased large artery stiffness and impaired endothelium-dependent dilatation. T cells contribute to hypertension in acute rodent models but whether they contribute to chronic age-related arterial dysfunction is unknown. To determine whether T cells directly mediate age-related arterial dysfunction, we examined large elastic artery and resistance artery function in young (4-6 months) and old (22-24 months) wild-type mice treated with anti-CD3 F(ab'2) fragments to deplete T cells (150 µg, i.p. every 7 days for 28 days) or isotype control fragments. Old mice exhibited greater numbers of T cells in both aorta and mesenteric vasculature when compared with young mice. Old mice treated with anti-CD3 fragments exhibited depletion of T cells in blood, spleen, aorta and mesenteric vasculature. Old mice also exhibited greater numbers of aortic and mesenteric IFN-γ and TNF-α-producing T cells when compared with young mice. Old control mice exhibited greater large artery stiffness and impaired resistance artery endothelium-dependent dilatation in comparison with young mice. In old mice, large artery stiffness was ameliorated with anti-CD3 treatment. Anti-CD3-treated old mice also exhibited greater endothelium-dependent dilatation than age-matched controls. We also examined arterial function in young and old Rag-1-/- mice, which lack lymphocytes. Rag-1-/- mice exhibited blunted increases in large artery stiffness with age compared with wild-type mice. Old Rag-1-/- mice also exhibited greater endothelium-dependent dilatation compared with old wild-type mice. Collectively, these results demonstrate that T cells play an important role in age-related arterial dysfunction.

Mechanisms of Dysfunction in the Aging Vasculature and Role in Age-Related Disease.

Advancing age promotes cardiovascular disease (CVD), the leading cause of death in the United States and many developed nations. Two major age-related arterial phenotypes, large elastic artery stiffening and endothelial dysfunction, are independent predictors of future CVD diagnosis and likely are responsible for the development of CVD in older adults. Not limited to traditional CVD, these age-related changes in the vasculature also contribute to other age-related diseases that influence mammalian health span and potential life span. This review explores mechanisms that influence age-related large elastic artery stiffening and endothelial dysfunction at the tissue level via inflammation and oxidative stress and at the cellular level via Klotho and energy-sensing pathways (AMPK [AMP-activated protein kinase], SIRT [sirtuins], and mTOR [mammalian target of rapamycin]). We also discuss how long-term calorie restriction-a health span- and life span-extending intervention-can prevent many of these age-related vascular phenotypes through the prevention of deleterious alterations in these mechanisms. Lastly, we discuss emerging novel mechanisms of vascular aging, including senescence and genomic instability within cells of the vasculature. As the population of older adults steadily expands, elucidating the cellular and molecular mechanisms of vascular dysfunction with age is critical to better direct appropriate and measured strategies that use pharmacological and lifestyle interventions to reduce risk of CVD within this population.

Mechanisms of Vascular Aging.

Aging of the vasculature plays a central role in morbidity and mortality of older people. To develop novel treatments for amelioration of unsuccessful vascular aging and prevention of age-related vascular pathologies, it is essential to understand the cellular and functional changes that occur in the vasculature during aging. In this review, the pathophysiological roles of fundamental cellular and molecular mechanisms of aging, including oxidative stress, mitochondrial dysfunction, impaired resistance to molecular stressors, chronic low-grade inflammation, genomic instability, cellular senescence, epigenetic alterations, loss of protein homeostasis, deregulated nutrient sensing, and stem cell dysfunction in the vascular system are considered in terms of their contribution to the pathogenesis of both microvascular and macrovascular diseases associated with old age. The importance of progeronic and antigeronic circulating factors in relation to development of vascular aging phenotypes are discussed. Finally, future directions and opportunities to develop novel interventions to prevent/delay age-related vascular pathologies by targeting fundamental cellular and molecular aging processes are presented.

Induced Trf2 deletion leads to aging vascular phenotype in mice associated with arterial telomere uncapping, senescence signaling, and oxidative stress.

Age-related vascular dysfunction in large elastic and resistance arteries is associated with reductions in microvascular perfusion and elevations in blood pressure. Recent evidence indicates that telomere uncapping-induced senescence in vascular cells may be an important source of oxidative stress and vascular dysfunction in aging, but the causal relationship between these processes has yet to be elucidated. To test this important unexplored hypothesis, we measured arterial senescence signaling and oxidative stress, carotid and mesenteric artery endothelium-dependent vasodilatory capacity, markers of mesenteric microvascular perfusion and endothelial glycocalyx deterioration, and blood pressure in a novel mouse model of Cre-inducible whole body Trf2 deletion and telomere uncapping. Trf2 deletion led to a 320% increase in arterial senescence signaling (P < .05). There was a concurrent 29% and 22% reduction in peak endothelium-dependent vasodilation in carotid and mesenteric arteries, respectively, as well as a 63% reduction in mesenteric microvascular endothelial glycocalyx thickness (all P ≤ .01). Mesenteric microvascular perfusion was reduced by 8% and systolic blood pressure was increased by 9% following Trf2 deletion (both P < .05). Trf2 deletion also led to a pro-oxidative arterial phenotype characterized by increased in NADPH oxidase gene expression; a 210% increase in superoxide levels that was partly dependent on NADPH oxidase activity; and an oxidative stress mediated reduction in carotid artery vasodilation (all P ≤ .05). Collectively, our findings demonstrate that induced Trf2 deletion leads to telomere uncapping, increased senescence signaling, and oxidative stress mediated functional impairments in the vasculature similar to those seen in human aging.

A Focused DNA-Encoded Chemical Library for the Discovery of Inhibitors of NAD+-Dependent Enzymes.

DNA-encoded chemical libraries are increasingly used in pharmaceutical research because they enable the rapid discovery of synthetic protein ligands. Here we explored whether target-class focused DNA-encoded chemical libraries can be cost-effective tools to achieve robust screening productivity for a series of proteins. The study revealed that a DNA-encoded library designed for NAD+-binding pockets (NADEL) effectively sampled the chemical binder space of enzymes with ADP-ribosyltransferase activity. The extracted information directed the synthesis of inhibitors for several enzymes including PARP15 and SIRT6. The high dissimilarity of NADEL screening fingerprints for different proteins translated into inhibitors that showed selectivity for their target. The discovery of patterns of enriched structures for six out of eight tested proteins is remarkable for a library of 58 302 DNA-tagged structures and illustrates the prospect of focused DNA-encoded libraries as economic alternatives to large library platforms.

Impact of high-fat diet on vasoconstrictor reactivity of white and brown adipose tissue resistance arteries.

Blood flow regulation is a critical factor for tissue oxygenation and substrate supply. Increased reactivity of arteries to vasoconstrictors may increase vascular resistance, resulting in reduced blood flow. We aimed to investigate the effect of a high-fat (HF) diet on stiffness and vasoconstrictor reactivity of white adipose tissue (WAT) and brown adipose tissue (BAT) resistance arteries and also investigated the interconversion of both adipose depots in the setting of a HF diet. Vasoconstrictor reactivity and passive morphology and mechanical properties of arteries from B6D2F1 mice (5 mo old) fed normal chow (NC) or a HF diet (8 wk) were measured using pressure myography. Receptor gene expression in WAT and BAT arteries and markers of WAT and BAT were assessed in whole tissue lysates by real-time RT-PCR. Despite greater receptor-independent vasoconstriction (in response to KCl, P < 0.01), vasoconstriction in response to angiotensin II ( P < 0.01) was lower in NC-BAT than NC-WAT arteries and similar in response to endothelin-1 ( P = 0.07) and norepinephrine ( P = 0.11) in NC-BAT and NC-WAT arteries. With the exception of BAT artery reactivity to endothelin-1 and angiotensin II, the HF diet tended to attenuate reactivity in arteries from both adipose depots and increased expression of adipose markers in BAT. No significant differences in morphology or passive mechanical properties were found between adipose types or diet conditions. Alterations in gene expression of adipose markers after the HF diet suggest beiging of BAT. An increase in brown adipocytes in the absence of increased BAT mass may be a compensatory mechanism to dissipate excess energy from a HF diet. NEW & NOTEWORTHY Despite no differences in passive mechanical properties and greater receptor-independent vasoconstriction, receptor-mediated vasoconstriction was either lower in brown than white adipose tissue arteries or similar in brown and white adipose tissue arteries. A high-fat diet has a greater impact on vasoconstrictor responses in white adipose tissue but leads to altered adipose tissue gene expression consistent with beiging of the brown adipose tissue.

The role of senescence, telomere dysfunction and shelterin in vascular aging.

In the United States and other westernized nations, CVDs are the leading cause of death in adults over 65 years of age. Large artery stiffness and endothelial dysfunction are increased with age and age-associated arterial dysfunction is an important antecedent of CVDs. One age-associated change that may contribute to vascular dysfunction and CVD risk is an increase in the number of resident senescent cells in the vasculature. Senescent cells display a pro-oxidant, pro-inflammatory phenotype known as the SASP. However, the mechanisms that drive the SASP and the vascular aging phenotype remain elusive. A putative mechanism is the involvement of oxidative stress and inflammation in telomere function. Telomeres are the end caps of chromosomes which are maintained by a six-protein complex known as shelterin. Disruption of shelterin can uncap telomeres and induce cellular senescence. Accordingly, in this review, we propose that oxidative stress and inflammation disrupt shelterin in vascular cells, driving telomere dysfunction and that this mechanism may be responsible for the induction of SASP. The proposed mechanisms may represent some of the initial changes that lead to vascular dysfunction in advanced age.

Deletion of Robo4 prevents high-fat diet-induced adipose artery and systemic metabolic dysfunction.

OBJECTIVE: Accumulating evidence suggests the vascular endothelium plays a fundamental role in the pathophysiology of obesity by regulating the functional status of white adipose and systemic metabolism. Robo4 is expressed specifically in endothelial cells and increases vascular stability and inhibits angiogenesis. We sought to determine the role of Robo4 in modulating cardiometabolic function in response to high-fat feeding. METHODS: We examined exercise capacity, glucose tolerance, and white adipose tissue artery gene expression, endothelium-dependent dilation (EDD), and angiogenesis in wild type and Robo4 knockout (KO) mice fed normal chow (NC) or a high-fat diet (HFD). RESULTS: We found Robo4 deletion enhances exercise capacity in NC-fed mice and HFD markedly increased the expression of the Robo4 ligand, Slit2, in white adipose tissue. Deletion of Robo4 increased angiogenesis in white adipose tissue and protected against HFD-induced impairments in white adipose artery vasodilation and glucose intolerance. CONCLUSIONS: We demonstrate a novel functional role for Robo4 in endothelial cell function and metabolic homeostasis in white adipose tissue, with Robo4 deletion protecting against endothelial and metabolic dysfunction associated with a HFD. Our findings suggest that Robo4-dependent signaling pathways may be a novel target in anti-obesity therapy.

Cerebral and skeletal muscle feed artery vasoconstrictor responses in a mouse model with greater large elastic artery stiffness.

NEW FINDINGS: What is the central question of this study? Greater large artery stiffness is associated with dysfunctional resistance artery vasodilatory responses, impaired memory and greater risk of Alzheimer's disease. However, it is unknown whether stiffer large arteries affect cerebral and skeletal muscle feed artery responses to vasoconstrictors. What is the main finding and its importance? In a mouse model with greater large artery stiffness (Eln+/- ), we find an exacerbated vasoconstrictor response to angiotensin II in cerebral arteries, but not skeletal muscle feed arteries, thus implicating altered cerebral artery angiotensin II responsiveness in the poor brain outcomes associated with greater large artery stiffness. ABSTRACT: Greater stiffness of the large elastic arteries is associated with end-organ damage and dysfunction. At the same time, resistance artery vasoconstrictor responsiveness influences vascular tone and organ blood flow. However, it is unknown whether large elastic artery stiffness modulates the responsiveness to vasoconstrictors in resistance arteries of the cerebral or skeletal muscle circulations. We previously described the elastin haploinsufficient (Eln+/- ) mouse as a model with greater aortic stiffness, but with similar cerebral and skeletal muscle feed artery stiffness to wild-type (Eln+/+ ) mice. Here, we used this model to examine the relationship between large elastic artery stiffness and resistance artery vasoconstrictor responses. In middle cerebral arteries (MCAs), vasoconstriction in response to angiotensin II (Ang II) was ∼40% greater in Eln+/- compared with Eln+/+ mice (P = 0.02), and this group difference was ameliorated by losartan, indicating a role for Ang II type 1 receptors (AT1Rs). In gastrocnemius feed arteries, Eln+/- and Eln+/+ mice did not differ in the response to Ang II. In addition, the vasoconstrictor responses to noradrenaline, endothelin-1 and potassium chloride were not different between Eln+/- and Eln+/+ mice for either MCAs or gastrocnemius feed arteries. The MCA AT1R gene expression did not differ between groups, whereas Ang II type 2 receptor gene expression was ∼50% lower in MCAs from Eln+/- versus Eln+/+ mice (P = 0.01). In conclusion, greater large elastic artery stiffness is associated with an exacerbated vasoconstriction response to Ang II in cerebral arteries, but is not associated with the responses to other vasoconstrictors in either cerebral or skeletal muscle feed arteries.

Telomere uncapping and vascular aging.

Although most telomere biology research continues to focus on telomere shortening, there is increasing evidence that telomere deprotection, or "uncapping," is more biologically and possibly clinically important. Telomeres form t-loops to prevent the chromosome ends from appearing as a double-stranded DNA break and initiating a DNA damage response. Breakdown of the t-loop structure, referred to as uncapping, can lead to cellular senescence, increased oxidative stress, and inflammation in tissues. In this review, we describe how telomere uncapping potentially leads to age-related vascular dysfunction and increased cellular senescence, oxidative stress, and inflammation. Importantly, we present evidence to argue that telomere uncapping is more biologically relevant than telomere shortening and a better marker of vascular aging and target for antiaging interventions.

Advanced age results in a diminished endothelial glycocalyx.

Age-related microvascular dysfunction is well characterized in rodents and humans, but little is known about the properties of the microvascular endothelial glycocalyx in advanced age. We examined the glycocalyx in microvessels of young and old male C57BL6 mice (young: 6.1 ± 0.1 mo vs. old: 24.6 ± 0.2 mo) using intravital microscopy and transmission electron microscopy and in human participants (young: 29 ± 1 yr vs. old: 60 ± 2 yr) using intravital microscopy. Glycocalyx thickness in mesenteric and skeletal muscle microvessels was 51-54% lower in old compared with young mice. We also observed 33% lower glycocalyx thickness in the sublingual microcirculation of humans in advanced age. The perfused boundary region, a marker of glycocalyx barrier function, was also obtained using an automated capture and analysis system. In advanced age, we observed a 10-22% greater perfused boundary region in mice and humans, indicating a more penetrable glycocalyx. Finally, using this automated analysis system, we examined perfused microvascular density and red blood cell (RBC) fraction. Perfused microvascular density is a marker of microvascular function that reflects the length of perfused microvessel segments in a given area; RBC fraction represents the heterogeneity in RBC presence between microvessel segments. Compared with young, the perfused microvascular density was 16-21% lower and RBC fraction was 5-14% lower in older mice and in older humans. These data provide novel evidence that, across mammalian species, a diminished glycocalyx is present in advanced age and is accompanied by markers of impaired microvascular perfusion. Age-related glycocalyx deterioration may be an important contributor to microvascular dysfunction in older adults and subsequent pathophysiology. NEW & NOTEWORTHY Advanced age is characterized by microvascular dysfunction that contributes to age-related cardiovascular diseases, but little is known about endothelial glycocalyx properties in advanced age. This study reveals, for the first time, lower glycocalyx thickness and barrier function that is accompanied by impaired microvascular perfusion in both mice and humans in advanced age.

Attenuated nitric oxide bioavailability in systemic sclerosis: Evidence from the novel assessment of passive leg movement.

NEW FINDINGS: What is the central question of this study? Do systemic sclerosis patients exhibit impaired nitric oxide-mediated vascular function of the lower limb and are these decrements correlated with plasma biomarkers for inflammation and oxidative stress? What is the main finding and its importance? Findings indicate impaired nitric oxide-mediated vascular function, linked to the incidence of digital ulcers and a milieu of inflammation and oxidative stress. However, the absence of significant correlations between individual biomarkers and blood flow responses suggests that the vasculopathy observed in systemic sclerosis may not be solely the result of derangements in the redox balance or inflammatory signalling. ABSTRACT: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, which may be the consequence of inflammation and oxidative stress that ultimately leads to a reduced nitric oxide (NO) bioavailability. Passive leg movement (PLM) is a novel methodology for assessing lower limb vascular function that is predominantly NO dependent. We combined this vascular assessment with a comprehensive panel of plasma biomarkers to assess the axis of inflammation, oxidative stress and NO in SSc patients (n = 12; 62 ± 11 years of age) compared with healthy control subjects (n = 17; 60 ± 16 years of age). The PLM-induced changes in leg blood flow (LBF; 191 ± 104 versus 327 ± 217 ml min-1 ) and LBF area under the curve (39 ± 104 versus 125 ± 131 ml) were reduced in SSc compared with control subjects. Stratification of patients according to history of digital ulcer (DU) formation revealed a further reduction in LBF area under the curve in DU (-13 ± 83 ml) versus non-DU (91 ± 102 ml) patients. Biomarkers of inflammation (C-reactive protein) and oxidative stress (malondialdehyde and protein carbonyl) were all elevated in SSc (C-reactive protein, 3299 ± 2372 versus 984 ± 565 ng ml-1 ; malondialdehyde, 3.2 ± 1.1 versus 1.1 ± 0.7 µm; and protein carbonyl, 0.15 ± 0.05 versus 0.12 ± 0.03 nmol mg-1 ), and C-reactive protein was further elevated in patients with a history of DU (4551 ± 2752 versus 2047 ± 1019 ng ml-1 ) compared with non-DU, although these were not individually correlated with changes in LBF. These findings of impaired NO-mediated vascular function, linked to DU and a milieu of inflammation and oxidative stress, suggest that redox balance plays an important, but not necessarily deterministic, role in the vascular pathophysiology of SSc.

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation.

RATIONALE: Telomerase is a nuclear regulator of telomere elongation with recent reports suggesting a role in regulation of mitochondrial reactive oxygen species. Flow-mediated dilation in patients with cardiovascular disease is dependent on the formation of reactive oxygen species. OBJECTIVE: We examined the hypothesis that telomerase activity modulates microvascular flow-mediated dilation, and loss of telomerase activity contributes to the change of mediator from nitric oxide to mitochondrial hydrogen peroxide in patients with coronary artery disease (CAD). METHODS AND RESULTS: Human coronary and adipose arterioles were isolated for videomicroscopy. Flow-mediated dilation was measured in vessels pretreated with the telomerase inhibitor BIBR-1532 or vehicle. Statistical differences between groups were determined using a 2-way analysis of variance repeated measure (n≥4; P<0.05). L-NAME (N(ω)-nitro-L-arginine methyl ester; nitric oxide synthase inhibitor) abolished flow-mediated dilation in arterioles from subjects without CAD, whereas polyethylene glycol-catalase (PEG-catalase; hydrogen peroxide scavenger) had no effect. After exposure to BIBR-1532, arterioles from non-CAD subjects maintained the magnitude of dilation but changed the mediator from nitric oxide to mitochondrial hydrogen peroxide (% max diameter at 100 cm H2O: vehicle 74.6±4.1, L-NAME 37.0±2.0*, PEG-catalase 82.1±2.8; BIBR-1532 69.9±4.0, L-NAME 84.7±2.2, PEG-catalase 36.5±6.9*). Conversely, treatment of microvessels from CAD patients with the telomerase activator AGS 499 converted the PEG-catalase-inhibitable dilation to one mediated by nitric oxide (% max diameter at 100 cm H2O: adipose, AGS 499 78.5±3.9; L-NAME 10.9±17.5*; PEG-catalase 79.2±4.9). Endothelial-independent dilation was not altered with either treatment. CONCLUSIONS: We have identified a novel role for telomerase in re-establishing a physiological mechanism of vasodilation in arterioles from subjects with CAD. These findings suggest a new target for reducing the oxidative milieu in the microvasculature of patients with CAD.

Implications of endothelial shear stress on systemic sclerosis vasculopathy and treatment.

There are no Federal Drug Administration approved drugs for the treatment of systemic sclerosis vascular digital ulcers (DU) in the United States, which are thought to be an end-stage result of prolonged ischaemia due to severe, prolonged Raynaud's phenomenon. Most therapeutics for vasodilation used in SSc work different pathways to target the smooth muscle to induce vessel relaxation. Longitudinal studies of vascular function allow insight into the effects of medications used for Raynaud's phenomenon in the SSc patient population. In this review, we discuss vascular tone, the function of the endothelium in SSc, and provide the rationale for longitudinal studies of vascular function and therapeutics that target the endothelial shear stress in addition to vasodilation for treatment and prevention of DU. This review provides the rationale for vasodilatory medication use for treatment of SSc-related DU and justifies access to non-FDA approved medications for this indication.

Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P < 0.05), respectively, in ECs obtained from antecubital veins of older sedentary (60 ± 1 yr, n = 12) versus young sedentary (22 ± 1 yr, n = 9) adults. These age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 (r = -0.49, P = 0.003), p21 (r = -0.38, P = 0.03), and p16 (r = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P < 0.05), respectively, in ECs sampled from brachial arteries of healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed (P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age.NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function.