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.

Endothelial cell-specific reduction in mTOR ameliorates age-related arterial and metabolic dysfunction.

Systemic inhibition of the mammalian target of rapamycin (mTOR) delays aging and many age-related conditions including arterial and metabolic dysfunction. However, the mechanisms and tissues involved in these beneficial effects remain largely unknown. Here, we demonstrate that activation of S6K, a downstream target of mTOR, is increased in arteries with advancing age, and that this occurs preferentially in the endothelium compared with the vascular smooth muscle. Induced endothelial cell-specific deletion of mTOR reduced protein expression by 60-70%. Although this did not significantly alter arterial and metabolic function in young mice, endothelial mTOR reduction reversed arterial stiffening and improved endothelium-dependent dilation (EDD) in old mice, indicating an improvement in age-related arterial dysfunction. Improvement in arterial function in old mice was concomitant with reductions in arterial cellular senescence, inflammation, and oxidative stress. The reduction in endothelial mTOR also improved glucose tolerance in old mice, and this was associated with attenuated hepatic gluconeogenesis and improved lipid tolerance, but was independent of alterations in peripheral insulin sensitivity, pancreatic beta cell function, or fasted plasma lipids in old mice. Lastly, we found that endothelial mTOR reduction suppressed gene expression of senescence and inflammatory markers in endothelial-rich (i.e., lung) and metabolically active organs (i.e., liver and adipose tissue), which may have contributed to the improvement in metabolic function in old mice. This is the first evidence demonstrating that reducing endothelial mTOR in old age improves arterial and metabolic function. These findings have implications for future drug development.

Reduction of double-strand DNA break repair exacerbates vascular aging.

Advanced age is the greatest risk factor for cardiovascular disease (CVD), the leading cause of death. Arterial function is impaired in advanced age which contributes to the development of CVD. One underexplored hypothesis is that DNA damage within arteries leads to this dysfunction, yet evidence demonstrating the incidence and physiological consequences of DNA damage in arteries, and in particular, in the microvasculature, in advanced age is limited. In the present study, we began by assessing the abundance of DNA damage in human and mouse lung microvascular endothelial cells and found that aging increases the percentage of cells with DNA damage. To explore the physiological consequences of increases in arterial DNA damage, we evaluated measures of endothelial function, microvascular and glycocalyx properties, and arterial stiffness in mice that were lacking or heterozygous for the double-strand DNA break repair protein ATM kinase. Surprisingly, in young mice, vascular function remained unchanged which led us to rationalize that perhaps aging is required to accumulate DNA damage. Indeed, in comparison to wild type littermate controls, mice heterozygous for ATM that were aged to ~18 mo (Old ATM +/-) displayed an accelerated vascular aging phenotype characterized by increases in arterial DNA damage, senescence signaling, and impairments in endothelium-dependent dilation due to elevated oxidative stress. Furthermore, old ATM +/- mice had reduced microvascular density and glycocalyx thickness as well as increased arterial stiffness. Collectively, these data demonstrate that DNA damage that accumulates in arteries in advanced age contributes to arterial dysfunction that is known to drive CVD.

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments.

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.

Endothelial specific reduction in Arf6 impairs insulin-stimulated vasodilation and skeletal muscle blood flow resulting in systemic insulin resistance.

Background: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as 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. ADP ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in endothelial cell (EC) 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 EC-specific Arf6 deletion (Arf6 f/- Tie2Cre) and tamoxifen inducible Arf6 knockout (Arf6 f/f Cdh5Cre). 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. A fluorescence microsphere-based technique was used to measure tissue blood flow. Intravital microscopy was used to assess skeletal muscle capillary density. Results: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue (WAT) and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide (NO) bioavailability but independent of altered acetylcholine- or sodium nitroprusside-mediated vasodilation. In vitro Arf6 inhibition resulted in suppressed insulin stimulated phosphorylation of Akt and endothelial NO synthase. 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. Conclusion: 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.

Glycocalyx-targeted therapy ameliorates age-related arterial dysfunction.

Advanced age is accompanied by arterial dysfunction, as well as a diminished glycocalyx, which may be linked to reduced high molecular weight-hyaluronan (HMW-HA) synthesis. However, the impact of glycocalyx deterioration in age-related arterial dysfunction is unknown. We sought to determine if manipulations in glycocalyx properties would alter arterial function. Tamoxifen-induced hyaluronan synthase 2 (Has2) reduction was used to decrease glycocalyx properties. Three weeks post-tamoxifen treatment, glycocalyx thickness was lower in Has2 knockout compared to wild-type mice (P<0.05). Has2 reduction induced arterial dysfunction, demonstrated by impaired endothelium-dependent dilation (EDD) and elevated aortic stiffness (P<0.05). To augment glycocalyx properties, old mice received 10 weeks of a glycocalyx-targeted therapy via Endocalyx™ (old+ECX), which contains HMW-HA and other glycocalyx components. Compared to old control mice, glycocalyx properties and EDD were augmented, and aortic stiffness decreased in old+ECX mice (P<0.05). Old+ECX mice had a more youthful aortic phenotype, demonstrated by lower collagen content and higher elastin content than old control mice (P<0.05). Functional outcomes were repeated in old mice that underwent a diet supplemented solely with HMW-HA (old+HA). Compared to old controls, glycocalyx properties and EDD were augmented, and aortic stiffness was lower in old+HA mice (P<0.05). We did not observe any differences between old+HA and old+ECX mice (P>0.05). Has2 reduction phenocopies age-related arterial dysfunction, while 10 weeks of glycocalyx-targeted therapy that restores the glycocalyx also ameliorates age-related arterial dysfunction. These findings suggest that the glycocalyx may be a viable therapeutic target to ameliorate age-related arterial dysfunction.

Mechanisms and consequences of endothelial cell senescence.

Endothelial cells are located at the crucial interface between circulating blood and semi-solid tissues and have many important roles in maintaining systemic physiological function. The vascular endothelium is particularly susceptible to pathogenic stimuli that activate tumour suppressor pathways leading to cellular senescence. We now understand that senescent endothelial cells are highly active, secretory and pro-inflammatory, and have an aberrant morphological phenotype. Moreover, endothelial senescence has been identified as an important contributor to various cardiovascular and metabolic diseases. In this Review, we discuss the consequences of endothelial cell exposure to damaging stimuli (haemodynamic forces and circulating and endothelial-derived factors) and the cellular and molecular mechanisms that induce endothelial cell senescence. We also discuss how endothelial cell senescence causes arterial dysfunction and contributes to clinical cardiovascular diseases and metabolic disorders. Finally, we summarize the latest evidence on the effect of eliminating senescent endothelial cells (senolysis) and identify important remaining questions to be addressed in future studies.

Aging results in DNA damage and telomere dysfunction that is greater in endothelial versus vascular smooth muscle cells and is exacerbated in atheroprone regions.

Aging increases the risk of atherosclerotic cardiovascular disease which is associated with arterial senescence; however, the mechanisms responsible for the development of cellular senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) remain elusive. Here, we study the effect of aging on arterial DNA damage and telomere dysfunction. Aging resulted in greater DNA damage in ECs than VSMCs. Further, telomere dysfunction-associated DNA damage foci (TAF: DNA damage signaling at telomeres) were elevated with aging in ECs but not VMSCs. Telomere length was modestly reduced in ECs with aging and not sufficient to induce telomere dysfunction. DNA damage and telomere dysfunction were greatest in atheroprone regions (aortic minor arch) versus non-atheroprone regions (thoracic aorta). Collectively, these data demonstrate that aging results in DNA damage and telomere dysfunction that is greater in ECs than VSMCs and elevated in atheroprone aortic regions.

Hemostatic Responses to Multiple Bouts of Firefighting Activity: Female vs. Male Differences in a High Demand, High Performance Occupation.

While the fire service has long been a male-dominated occupation, women's participation in this strenuous, high risk, high performance activity has increased in recent years. Firefighting induces significant cardiovascular strain, including hemostatic disruption; however, the effect of sex on hemostatic responses has not been investigated despite evidence that there are sex-related differences in hemostatic variables at rest and following exercise. Thus, we investigated hemostatic responses in age- and BMI-matched male and female firefighters who performed 3-4 evolutions of firefighting drills over a 3 h period. Venous blood samples were collected before and after the firefighting training drills and hemostatic variables were assessed. Firefighting significantly increased platelet count and factor VIII, tissue plasminogen activator (t-PA) antigen, and t-PA activity, and decreased activated partial thromboplastin time and plasminogen activator inhibitor (PAI-1) activity. Females had lower values for epinephrine-induced platelet closure time, antithrombin III, PAI-1 activity, and PAI-1 antigen. There were no interactions between sex and time for any variables assessed. In conclusion, multiple bouts of firefighting activity resulted in a procoagulatory state. Although there were sex differences for several hemostatic variables, male and female firefighters did not differ in their hemostatic response to multiple bouts of firefighting.

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.

High-fat diet induced obesity and age influence the telomere shelterin complex and telomerase gene expression in mouse adipose tissue.

Obesity and aging are linked to inflammation and increased risk of chronic disease. Telomeres are the endcaps of chromosomes that are regulated by telomerase, the enzyme that elongates telomeres, as well as a protein complex known as shelterin. Telomere dysfunction is associated with inflammation, aging, and disease. However, the effect of high-fat diet (HFD) induced obesity and advancing age on the shelterin complex and telomerase in adipose tissue is unknown. The present study investigated the effects of obesity and aging on C57BL/6J mice adipose tissue mRNA expression of shelterin complex genes. Young (YG) mice (3 mo) were randomly assigned to be fed either a high-fat diet (YG + HFD; 60% kcal from fat) or a low-fat diet (YG + LFD; 10% kcal from fat). A subset of mice were aged until 16 months. Body weight and epididymal white adipose tissue (EWAT) weight increased with age or a HFD. There was a trend for increased Terf2 expression, as expression was increased in HFD + YG by ~47% and aged mice by ~80%. Pot1b expression was increased in aged mice by ~35%-60% compared to YG, independent of diet. mTert, the gene that codes for the catalytic subunit of telomerase, was significantly elevated in aged mice. Changes in telomere associated gene expression was accompanied by changes in expression of inflammatory markers Mcp1 and Tnfα. These findings suggest obesity and age impact expression of shelterin complex and telomerase related genes in adipose, perhaps altering telomere function in adipose tissue thereby increasing inflammation and risk of chronic disease.

Firefighting Induces Acute Inflammatory Responses that are not Relieved by Aspirin in Older Firefighters.

OBJECTIVE: Sudden cardiac events account for 40% to 50% of firefighter line-of-duty deaths. Inflammatory proteins are strong biomarkers of cardiovascular inflammation. The present study investigated the effects of aspirin supplementation on inflammatory biomarkers following firefighting. METHODS: Using a randomized, placebo-controlled, double-blind crossover design, 24 male firefighters (48.2 ±â€Š5.9 years) were allocated into four conditions: acute (81 mg; single-dose) aspirin and placebo supplementation, and chronic (81 mg; 14 days) aspirin and placebo supplementation. Inflammatory proteins [interleukin (IL)-6, C-reactive protein (CRP), intracellular adhesion molecule (ICAM)-1, P-selectin, matrix metalloproteinase-9 (MMP-9)] and antioxidant potential [total antioxidant capacity (TAC)] were measured pre- and post-structural firefighting drills. RESULTS: Firefighting activities significantly increased IL-6, MMP-9, and P-Selectin; however, no changes in TAC and ICAM-1 were detected. Neither acute nor chronic aspirin supplementation attenuated this inflammatory response. CONCLUSION: Firefighting significantly increases inflammatory biomarkers and neither acute nor chronic low-dose aspirin mitigates this response.

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.

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.

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.

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.

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.