Advancing age leads to an accumulation of senescent endothelial cells (ECs) within arteries. Senescent cells have undergone permanent cell cycle arrest, are pro‐oxidative and pro‐inflammatory, and therefore represent a likely cause of age‐related EC dysfunction. Yet, the molecular mechanisms and physiological consequences of EC senescence remain incompletely understood. Telomeres are repeat DNA sequences that cap chromosomes. Telomeres shorten with each cell division and are highly susceptible to oxidative damage. When telomeres become critically short or damaged, they become uncapped, which activates the DNA damage response and leads to cellular senescence. Here, we tested the hypothesis that aging results in EC telomere uncapping that induces senescence, leading to physiological hallmarks of aging. To assess whether aging results in EC telomere uncapping, we compared ECs from young (~3 mo) and old (~27 mo) mice. Aging resulted in ~4‐fold greater EC telomere uncapping (p<0.001, Figure 1A). To determine if EC telomere uncapping induces senescence, we deleted the telomere capping protein, TRF2, in ECs of young (~3.5mo) mice (TRF2‐ecKO). Compared to wildtype (WT) littermate controls, TRF2‐ecKO mice had a ~78% reduction in TRF2 gene expression (p<0.0001). TRF2 deletion reduced EC division by ~47% (p<0.0001, Figure 1B), indicative of senescence. To examine the physiological consequences of EC senescence, we examined hallmarks of vascular aging including perfused microvascular density and endothelium‐dependent dilation (EDD). TRF2‐ecKO mice had an ~18% reduction in perfused mesenteric microvessels between 5‐25 µM (p<0.001, Figure 1C). Likewise, TRF2‐ecKO displayed ~26% reduction in mesenteric artery EDD compared to WT mice (p<0.05, Figure 1D). Furthermore, TRF2‐ecKO arterial EDD was ameliorated by the superoxide scavenger, TEMPOL (p>0.05, Figure 1D). Endothelium‐independent dilation to the exogenous nitric oxide donor sodium nitroprusside was not different between WT and TRF2‑ecKO mice (p>0.05). These data suggest EC telomere uncapping leads to senescence that reduces perfused microvascular density, and to elevated oxidative stress that suppresses EDD, similar to advanced age. To assess metabolic and muscle function, we performed an intraperitoneal glucose tolerance test (GTT, 2g/kg body mass) and a forelimb grip strength test. TRF2‐ecKO mice had ~21% greater area under the curve during GTT compared to WT mice (p<0.05, Figure 2A) as well as a ~14% reduction in grip strength (p<0.05, Figure2B). Taken together, these data provide evidence that aging results in EC telomere uncapping that induces senescence and age‐related physiological dysfunction.
Background: Systemic inhibition of mammalian target of rapamycin (mTOR) improves age-related arterial and metabolic dysfunction. Although the mechanisms and tissues involved in this amelioration remain unknown, endothelial cells (ECs), key regulators of arterial and metabolic function, may be responsible for these effects. Hypothesis: The beneficial effects of systemic mTOR inhibition will be recapitulated after EC specific mTOR deletion in old mice. Methods: We studied 22-24 mo old wildtype (WT) and EC specific tamoxifen-inducible mTOR knockout (KO) mice. The arterial function was determined by assessing aortic stiffness by measuring pulse wave velocity (PWV) using Doppler ultrasound as well as endothelium-dependent dilation (EDD) and nitric oxide (NO) bioavailability using pressure myography. Carotid artery superoxide production was measured using electric paramagnetic resonance. Glucose (2g/kg, ip) and lipid (3mL/kg, oral) tolerance tests were performed to examine metabolic function. Results: Two weeks after tamoxifen administration (4mg/day, 4 days, oral), we found a ~ 60% reduction in EC mTOR protein (p=0.04). Aortic PWV was lower in KO compared to WT mice (Fig A), indicating lower aortic stiffness. EDD to acetylcholine (ACh) was higher in mesenteric arteries from KO compared to WT mice (Fig B) due to an increase in NO bioavailability (31.6 ± 8.6 vs 8.4 ± 2.3; p=0.01). Superoxide scavenger, TEMPOL, increased EDD in both WT and KO mice (Fig C), eliminating differences between groups. Superoxide was lower in carotid arteries of KO compared to WT (Fig D), suggesting that oxidative stress impairs EDD in WT mice and ablation of EC selective mTOR ameliorates this effect. Aged KO mice demonstrated greater glucose and lipid tolerance compared to WT (Fig E, F). Conclusions: Our findings demonstrate that EC specific deletion of mTOR provides beneficial effects on arterial function via reductions in oxidative stress and prevents metabolic dysfunction in old mice.
Endothelial Cell (EC) dysfunction has classically been viewed as a consequence of systemic metabolic dysfunction. However, recent evidence suggests that EC dysfunction may precede and contribute to systemic metabolic dysfunction, although the underlying mechanisms remain unclear. Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6), a small GTPase of the Ras superfamily that regulates cellular proliferation and motility, has been found to be dysregulated in a host of human vascular pathologies. We sought to test the hypothesis that endothelial Arf6 plays a critical role in vascular and systemic metabolic function. To do so, we used a model of constitutive EC specific Arf6 deletion, tie2‐cre (tie2Arf6 KO). To achieve efficient EC deletion of Arf6 in this model, the EC KO was created on a whole body Arf6 heterozygous (HET) background. Using ex vivo pressure myography, we found insulin‐mediated vasodilation to be blunted (p<0.05) in adipose tissue arteries, via an NO dependent mechanism (Fig 1A). Importantly, this was not observed in response to either acetylcholine or sodium nitroprusside, suggesting that Arf6 influences EC insulin signaling specifically. To explore this, we assessed insulin‐stimulated phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs with and without siRNA‐mediated Arf6 knockdown or NAV‐2729‐induced inhibition of Arf6. We found insulin‐mediated phosphorylation of both Akt (p<0.05) and eNOS (p<0.05) to be lower compared to scramble or vehicle control treated cells (Fig 1B). Concomitantly, insulin sensitivity (1U/kg, ip, p<0.01), but not glucose tolerance (2g/kg, ip), was impaired in tie2Arf6 KO compared to whole body HET mice. Because heterozygosity for Arf6 in the whole body may impact systemic metabolism and activation of tie2‐cre may induce changes in other cell types including macrophages, we developed a superior EC specific, tamoxifen inducible Arf6 knockout (ECArf6KO) mouse using a VECAD‐cre. In agreement with our previous model, insulin sensitivity, but not glucose tolerance, was impaired in ECArf6KO compared to controls (CON) (Fig 2A). Furthermore, both HOMA‐IR and ‐B% were ~40% higher (both p<0.01) in ECArf6KO mice compared to CON mice. These results support a role for EC Arf6 in the development of systemic insulin resistance as well as suggest a role in beta cell function. Thus, we assessed glucose stimulated insulin secretion and found that both fasting (4–5 hr) and glucose‐stimulated (2g/kg, ip, 5 min) insulin secretion were higher in ECArf6KO mice compared to CON mice (Fig 2B). Taken together, we demonstrate a novel role of EC Arf6 signaling as both a modulator of vascular function and as a critical regulator of systemic metabolic function. The findings provide insight into a therapeutic target to improve insulin sensitivity. Support or Funding Information NIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492. Endothelial cell (EC) Arf6 deletion leads to blunt...
Objective Early diagnosis of systemic sclerosis (SSc) is imperative and Raynaud’s phenomenon (RP) is an important component of progressive vasculopathy. Nailfold videocapillaroscopy (NVC) is a wellestablished tool that can quantify structural vascular abnormalities. Digital thermal monitoring (DTM) assesses microvascular functional dysfunction related to thermoregulation. In this study, we investigated the correlation of NVC patterns and DTM variables in SSc patients. Methods Patients with SSc by 2013 ACR/EULAR criteria that were consented into the clinical care registry had NVC and DTM performed. For NVC, the number of capillaries (density), measurement of apical diameter (dimension), presence or absence of hemorrhages and number of abnormal shapes were assessed to categorize three different qualitative patterns: ‘early’, ‘active’ and ‘late’. For DTM, Doppler ultrasound hyperemic, low frequency, blood velocity of radial artery and fingertip vascular function was assessed and a vascular reactive index (VRI) measurement was automated. Statistical evaluation was performed by non-parametric tests to assess the correlation of NVC and VRI. Results Thirty-one SSc subjects with interpretable NVC and DTM performed on the same day were included in the study. VRI was progressively higher in SSc patients with the ‘early’, ‘active’ and ‘late’ NVC patterns of microangiopathy (p< 0.0001). There was a significant negative correlation between VRI and microhemorrhages score (r=-0.363, p=0.044). Conclusion Our study suggests that more advanced vasculopathy correlates to reduced microvascular function as detected by DTM and more advanced structural abnormalities detected by NVC. NVC and DTM may provide different aspects of vasculopathy quantification and complement each other as investigative tools.
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