Endothelial Sirtuin Research Articles

Liraglutide prevents cellular senescence in human retinal endothelial cells (HRECs) mediated by SIRT1: an implication in diabetes retinopathy.

Diabetes mellitus (DM) is a chronic metabolic disorder affecting millions of people worldwide, characterized by dysregulated glucose homeostasis and hyperglycemia. Diabetic retinopathy (DR) is one of the serious multisystemic complications. Aging is an important risk factor for DR. Endothelial sirtuin 1 (SIRT1) plays an important role in regulating the pathophysiology of glucose metabolism, cellular senescence, and aging. Liraglutide, an analog of Glucagon-like peptide 1 (GLP-1), has been widely used in the treatment of DM. However, the effects of Liraglutide on DR are less reported. Here, we investigated whether treatment with Liraglutide has beneficial effects on high glucose (HG)-induced injury in human retinal microvascular endothelial cells (HRECs). First, we found that exposure to HG reduced the expression of glucagon-like peptide 1 receptor 1 (GLP-1R). Additionally, Liraglutide ameliorated HG-induced increase in the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin 6 (IL-6). Importantly, Liraglutide ameliorated cellular senescence and increased telomerase activity in HG-challenged HRECs. Liraglutide also reduced the levels of p53 and p21. Mechanistically, Liraglutide restored the expression of SIRT1 against HG. In contrast, the knockdown of SIRT1 abolished the protective effects of Liraglutide in cellular senescence of HRECs. Our findings suggest that Liraglutide might possess a benefit on DR mediated by SIRT1.

SIRT1 attenuates blood-spinal cord barrier disruption after spinal cord injury by deacetylating p66Shc

Disruption of the blood-spinal cord barrier (BSCB) leads to inflammatory cell infiltration and neural cell death, thus, contributing to poor functional recovery after spinal cord injury (SCI). Previous studies have suggested that Sirtuin 1 (SIRT1), an NAD+-dependent class III histone deacetylase, is abundantly expressed in endothelial cells and promotes endothelial homeostasis. However, the role of SIRT1 in BSCB function after SCI remains poorly defined. Here, we report that SIRT1 is highly expressed in spinal cord endothelial cells, and its expression significantly decreases after SCI. Using endothelial cell-specific SIRT1 knockout mice, we observed that endothelial cell-specific knockout of SIRT1 aggravated BSCB disruption, thus, resulting in widespread inflammation, neural cell death and poor functional recovery after SCI. In contrast, activation of SIRT1 by the agonist SRT1720 had beneficial effects. In vitro, knockdown of SIRT1 exacerbated IL-1β-induced endothelial barrier disruption in bEnd.3 cells, whereas overexpression of SIRT1 was protective. Using RNA-seq and IP/MS analysis, we identified p66Shc, a redox protein, as the potential target of SIRT1. Further studies demonstrated that SIRT1 interacts with and deacetylates p66Shc, thereby attenuating oxidative stress and protecting endothelial barrier function. Overall, our results indicate that SIRT1 decreases endothelial ROS production and attenuates BSCB disruption by deacetylating p66Shc after SCI, and suggest that SIRT1 activation has potential as a therapeutic approach to promote functional recovery against BSCB disruption following SCI.

Endothelial SIRT-1 has a critical role in the maintenance of capillarization in brown adipose tissue

Brown adipose tissue (BAT) has critical roles in thermogenesis and systemic metabolism. Capillary rarefaction was reported to develop in BAT with dietary obesity, and previous studies showed that suppression of vascular endothelial growth factor A (VEGF-A) reduced capillary density in BAT, promoting the functional decline of this organ. Capillarization is regulated through the balance between angiogenesis and vasculogenesis on the one hand and apoptosis of endothelial cells (ECs) on the other; however, the role of EC apoptosis in BAT remained to be explored. In studies testing the role of boysenberry polyphenols (BoyP) in BAT, we found that BoyP decreased EC apoptosis, enhanced capillarization in BAT, and ameliorated dietary BAT dysfunction, which was associated with the upregulation of nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin 1 (SIRT-1) in ECs. Our studies suggest that EC SIRT-1 would be one of the potential targets of BoyP that contributes to BAT capillarization and function.

The inhibitory effects of Dulaglutide on cellular senescence against high glucose in human retinal endothelial cells

Diabetic nephropathy is one of the most important chronic microvascular complications of diabetes, and its main feature is diabetic glomerulosclerosis. Endothelial sirtuin 1 (SIRT1) expression is related to aging, and reducing SIRT1 expression promotes endothelial cell aging. Plasminogen activator inhibitor-1 (PAI-1) can be synthesized in a variety of cells, such as endothelial cells. Dulaglutide is a glucagon-like peptide-1 (GLP-1) drug, and it can activate the GLP-1 receptor and promote the conversion of intracellular adenosine triphosphate to adenylate cyclase, thereby activating phosphokinase A, and regulating blood glucose levels effectively in the body. We analyzed the effects of Dulaglutide on inhibiting cell senescence by studying the effects of its different concentrations on telomerase activity and senescence-related gene expression. Our results suggest that Dulaglutide can alleviate high-glucose-induced oxidative stress in human retinal endothelial cells by restoring the expressions of SIRT1 and endothelial nitric oxide synthase (eNOS), thereby inhibiting the expression of PAI-1, and restoring telomerase activity. This suggests that the activity of retinal endothelial cells can be controlled by regulating the expression of SIRT1, so as to achieve the effect of treating diabetic retinopathy.

Childhood psychosocial stress is linked with impaired vascular endothelial function, lower SIRT1, and oxidative stress in young adulthood.

Adverse childhood experiences (ACEs) are psychosocial stressors that occur during sensitive developmental windows and are associated with increased lifetime cardiovascular disease (CVD) risk in a dose-dependent manner. Vascular endothelial dysfunction is a pathophysiological mechanism that promotes hypertension and CVD and may be a mechanism by which ACEs contribute to lifetime CVD risk. We examined whether exposure to ACEs is associated with reduced vascular endothelial function (VEF) in otherwise healthy, young adult women (20.7 ± 3 yr) with (ACE+) versus without (ACE-) ACEs, explored whether differences in circulating sirtuin 1 (SIRT1) or systemic oxidative stress could explain ACEs-related differences in VEF, and examined the ability of a pilot, 8-wk exercise intervention to augment VEF and SIRT1 or reduce oxidized LDL cholesterol (oxLDL) in ACE+ young adult women. Forty-two otherwise healthy young adults completed this study. Prior to the intervention, VEF (P = 0.002) and SIRT1 (P = 0.004) were lower in the ACE+ than ACE- group, but oxLDL concentrations were not different (P = 0.77). There were also significant relationships (P ≤ 0.04) among flow-mediated dilation (FMD), SIRT1, and oxLDL in the ACE+, but not ACE- group. Adjusting for circulating SIRT1 and oxLDL reduced the differences in FMD observed between groups (P = 0.10), but only SIRT1 was a significant adjuster of the means (P < 0.05). Finally, the exercise intervention employed was unable to enhance VEF or SIRT1 in the ACE+ exercise group. Our data suggest that ACEs likely increase susceptibility to hypertension and CVD by causing endothelial dysfunction, perhaps through a SIRT1 pathway-related mechanism.NEW & NOTEWORTHY Our study provides novel evidence that young adult women with moderate-to-severe adverse childhood experience (ACE) exposure present impaired endothelial function and lower circulating sirtuin 1 (SIRT1) concentrations than age-matched controls. However, an 8-wk exercise intervention was unable to augment endothelial function or SIRT1 concentrations in a subset of those with ACEs. Our data suggest that ACEs-related impairments in endothelial function may be secondary to decreased NO bioavailability via SIRT1 and/or oxidative stress-related mechanisms.

Abstract MP55: Endothelial Sirtuin-1 Deficiency Promotes the Rupture of Intracranial Aneurysm

Background: Sirtuin-1 is highly expressed in vascular tissues and plays an important role in numerous metabolic and inflammatory processes. Age-related reduction in Sirtuin-1 expression has been linked to chronic vascular inflammation and senescence, and may explain the increased risk of aneurysm rupture seen in the elderly population. Our previous study showed that pharmacological inhibition of Sirtuin-1 increases the rate of aneurysm rupture. This suggests that Sirtuin-1 protects against the rupture of intracranial aneurysm. However, the cell type responsible for this effect has not been determined. Hypothesis: We hypothesize that reduced expression of Sirtuin-1 in endothelial cells promotes the rupture of intracranial aneurysm. Methods: We induced intracranial aneurysms in mice by combining systemic hypertension and a single injection of elastase into the cerebrospinal fluid. We induced aneurysms in tamoxifen-inducible vascular smooth muscle cell-specific Sirtuin-1 knockout mice (SIRT1 f/f MYH11 ER-Cre+ ) and in endothelial cell-specific Sirtuin-1 knockout mice and their littermates (SIRT1 f/f Tie2 Cre+ , SIRT1 f/f Tie2 Cre- ). We used the rate of aneurysm rupture as the primary endpoint. Results: Smooth muscle cell-specific Sirtuin-1 knockout had no effect on rate of aneurysm rupture. In contrast, SIRT1 f/f Tie2 Cre+ mice experienced a significantly increased rate of aneurysm rupture (SIRT1 f/f Tie2 Cre+ vs. SIRT1 f/f Tie2 Cre- : 86% vs. 42%, P &lt; 0.05). Conclusions: Our findings suggest that Sirtuin-1 expressed in the endothelium, but not in smooth muscle cells, protects against the rupture of intracranial aneurysm. As Tie2 is also expressed in hematopoietic cells, we are conducting experiments using SIRT1 f/f LysM Cre+ mice to exclude potential contributions of hematopoietic Sirtuin-1 to the observed effects. Endothelial Sirtuin-1 may be used as a therapeutic target for preventing the rupture of intracranial aneurysm.

A Sex-Specific Role of Endothelial Sirtuin 3 on Blood Pressure and Diastolic Dysfunction in Female Mice.

Background: Heart failure with preserved ejection fraction (HFpEF) is characterized by a diastolic dysfunction and is highly prevalent in aged women. Our study showed that ablation of endothelial Sirtuin 3 (SIRT3) led to diastolic dysfunction in male mice. However, the sex-specific role of endothelial SIRT3 deficiency on blood pressure and diastolic function in female mice remains to be investigated. Methods and Results: In this study, we demonstrate that the ablation of endothelial SIRT3 in females elevated blood pressure as compared with control female mice. Diastolic function measurement also showed that the isovolumic relaxation time (IVRT) and myocardial performance index (MPI) were significantly increased, whereas the E’ velocity/A’ velocity (E’/A’) ratio was reduced in the endothelial-specific SIRT3 knockout (SIRT3 ECKO) female mice. To further investigate the regulatory role of endothelial SIRT3 on blood pressure and diastolic dysfunction in metabolic stress, SIRT3 ECKO female mice were fed a normal diet and high-fat diet (HFD) for 20 weeks. The knockout of endothelial SIRT3 resulted in an increased blood pressure in female mice fed with an HFD. Intriguingly, SIRT3 ECKO female mice + HFD exhibited impaired coronary flow reserve (CFR) and more severe diastolic dysfunction as evidenced by an elevated IVRT as compared with control female mice + HFD. In addition, female SIRT3 ECKO mice had higher blood pressure and diastolic dysfunction as compared to male SIRT3 ECKO mice. Moreover, female SIRT3 ECKO mice + HFD had an impaired CFR and diastolic dysfunction as compared to male SIRT3 ECKO mice + HFD. Conclusions: These results implicate a sex-specific role of endothelial SIRT3 in regulating blood pressure and diastolic function in mice. Deficiency of endothelial SIRT3 may be responsible for a diastolic dysfunction in aged female.

Endothelial Sirtuin 3 Dictates Glucose Transport to Cardiomyocyte and Sensitizes Pressure Overload-Induced Heart Failure.

BackgroundAlterations of energetic metabolism are suggested to be an important contributor to pressure overload (PO)‐induced heart failure. Our previous study reveals that knockout of endothelial Sirtuin 3 (SIRT3) alters glycolysis and impairs diastolic function. We hypothesize that endothelial SIRT3 regulates glucose utilization of cardiomyocytes and sensitizes PO‐induced heart failure.Methods and Results SIRT3 endothelial cell knockout mice and their control SIRT3 LoxP mice were subjected to PO by transverse aortic constriction for 7 weeks. The ratio of heart weight to tibia length was increased in both strains of mice, in which SIRT3 endothelial cell knockout mice+transverse aortic constriction exhibited more severe cardiac hypertrophy. Coronary blood flow and systolic function were significantly decreased in SIRT3 endothelial cell knockout mice+transverse aortic constriction compared with SIRT3 LoxP mice+transverse aortic constriction, as evidenced by lower systolic/diastolic ratio, ejection fraction, and fractional shortening. PO‐induced upregulation of apelin and glucose transporter 4 were significantly reduced in the hearts of SIRT3 endothelial cell knockout mice. In vitro, levels of hypoxia‐inducible factor‐1α and glucose transporter 1 and glucose uptake were significantly reduced in SIRT3 knockout endothelial cells. Furthermore, hypoxia‐induced apelin expression was abolished together with reduced apelin‐mediated glucose uptake in SIRT3 knockout endothelial cells. Exposure of cardiomyocyte with apelin increased expression of glucose transporter 1 and glucose transporter 4. This was accompanied by a significant increase in glycolysis. Supplement of apelin in SIRT3 knockout hypoxic endothelial cell media increased glycolysis in the cardiomyocytes.ConclusionsKnockout of SIRT3 disrupts glucose transport from endothelial cells to cardiomyocytes, reduces cardiomyocyte glucose utilization via apelin in a paracrine manner, and sensitizes PO‐induced heart failure. Endothelial SIRT3 may regulate cardiomyocyte glucose availability and govern the function of the heart.

Implication of the deacetylase sirtuin-1 on synovial angiogenesis and persistence of experimental arthritis

ObjectivesTo decipher the phenotype of endothelial cells (ECs) derived from circulating progenitors issued from patients with rheumatoid arthritis (RA).MethodsRA and control ECs were compared according to their proliferative capacities, apoptotic...

Reduced Sirtuin1 signalling exacerbates diabetic mice hindlimb ischaemia injury and inhibits the protective effect of a liver X receptor agonist

Diabetes mellitus causes endothelial dysfunction, which further exacerbates peripheral arterial disease (PAD). Improving endothelial function via reducing endothelial oxidative stress (OS) may be a promising therapy for diabetic PAD. Activation of liver X receptor (LXR) inhibits excessive OS and provides protective effects on endothelial cells in diabetic individuals. Therefore, we investigated the effects of LXR agonist treatment on diabetic PAD with a focus on modulating endothelial OS. We used a streptozotocin‐induced diabetes mouse model combined with a hindlimb ischaemia (HLI) injury to mimic diabetic PAD, which was followed by LXR agonist treatment. In our study, the LXR agonist T0901317 protected against HLI injury in diabetic mice by attenuating endothelial OS and stimulating angiogenesis. However, a deficiency in endothelial Sirtuin1 (SIRT1) largely inhibited the therapeutic effects of T0901317. Furthermore, we found that the underlying therapeutic mechanisms of T0901317 were related to SIRT1 and non‐SIRT1 signalling, and the isoform LXRβ was involved in LXR agonist‐elicited SIRT1 regulation. In conclusion, LXR agonist treatment protected against HLI injury in diabetic mice via mitigating endothelial OS and stimulating cellular viability and angiogenesis by LXRβ, which elicited both SIRT1‐mediated and non‐SIRT1‐mediated signalling pathways. Therefore, LXR agonist treatment may be a promising therapeutic strategy for diabetic PAD.

The Role of Sirtuin1 in Regulating Endothelial Function, Arterial Remodeling and Vascular Aging.

Sirtuin1 (SIRT1), which belongs to a highly conserved family of protein deacetylase, is one of the best-studied sirtuins. SIRT1 is involved in a variety of biological processes, including energy metabolism, cell proliferation and survival, chromatin dynamics, and DNA repair. In the vasculature, SIRT1 is ubiquitously expressed in endothelial cells, smooth muscle cells, and perivascular adipose tissues (PVAT). Endothelial SIRT1 plays a unique role in vasoprotection by regulating a large variety of proteins, including endothelial nitric oxide synthase (eNOS). In endothelial cells, SIRT1 and eNOS regulate each other synergistically through positive feedback mechanisms for the maintenance of endothelial function. Recent studies have shown that SIRT1 plays a vital role in modulating PVAT function, arterial remodeling, and vascular aging. In the present article, we summarize recent findings, review the molecular mechanisms and the potential of SIRT1 as a therapeutic target for the treatment of vascular diseases, and discuss future research directions.

MicroRNA‐33 and SIRT1 influence the coronary thrombus burden in hyperglycemic STEMI patients

Primary percutaneous coronary intervention (PPCI) is a pivotal treatment in ST-segment elevation myocardial infarction (STEMI) patients. However, in hyperglycemic-STEMI patients, the incidence of death is still significant. Here, the involvement of sirtuin 1 (SIRT1) and miR33 on the pro-inflammatory/pro-coagulable state of the coronary thrombus was investigated. Moreover, 1-year outcomes in hyperglycemic STEMI in patients subjected to thrombus aspiration before PPCI were evaluated. Results showed that hyperglycemic thrombi displayed higher size and increased miR33, reactive oxygen species, and pro-inflammatory/pro-coagulable markers. Conversely, the hyperglycemic thrombi showed a lower endothelial SIRT1 expression. Moreover, in vitro experiments on endothelial cells showed a causal effect of SIRT1 modulation on the pro-inflammatory/pro-coagulative state via hyperglycemia-induced miR33 expression. Finally, SIRT1 expression negatively correlated with STEMI outcomes. These observations demonstrate the involvement of the miR33/SIRT1 pathway in the increased pro-inflammatory and pro-coagulable state of coronary thrombi in hyperglycemic STEMI patients.

Endothelial dysfunction is a superinducer of syndecan-4: fibrogenic role of its ectodomain

Syndecan-4 (Synd4) is a member of the membrane-spanning, glycocalyx-forming proteoglycan family. It has been suggested that Synd4 participates in renal fibrosis. We compared wild-type and fibrosis-prone endothelial sirtuin 1-deficient (Sirt1endo-/-) mice, the latter being a model of global endothelial dysfunction. We performed mass spectrometry analysis, which revealed that Synd4 was highly enriched in the secretome of renal microvascular endothelial cells obtained from Sirt1endo-/- mice upon stimulation with transforming growth factor-β1; notably, all detectable peptides were confined to the ectodomain of Synd4. Elevated Synd4 was due to enhanced NF-κB signaling in Sirt1endo-/- mice, while its shedding occurred as a result of oxidative stress in Sirt1 deficiency. Synd4 expression was significantly enhanced after unilateral ureteral obstruction compared with contralateral kidneys. Furthermore, hyperplasia of renal myofibroblasts accompanied by microvascular rarefaction and overexpression of Synd4 were detected in Sirt1endo-/- mice. The ectodomain of Synd4 acted as a chemoattractant for monocytes with higher levels of macrophages and higher expression levels of Synd4 in the extracellular matrix of Sirt1endo-/- mice. In vitro, ectodomain application resulted in generation of myofibroblasts from cultured renal fibroblasts, while in vivo, subcapsular injection of ectodomain increased interstitial fibrosis. Moreover, the endothelial glycocalyx was reduced in Sirt1endo-/- mice, highlighting the induction of Synd4 occurring in parallel with the depletion of its intact form and accumulation of its ectodomain in Sirt1endo-/- mice. On the basis of our experimental results, we propose that it is the Synd4 ectodomain per se that is partially responsible for fibrosis in unilateral ureteral obstruction, especially when it is combined with endothelial dysfunction. NEW & NOTEWORTHY Our findings suggest that endothelial dysfunction induces the expression of syndecan-4 via activation of the NF-κB pathway. Furthermore, we show that syndecan-4 is shed to a greater amount because of increased oxidative stress in dysfunctional endothelial cells and that the release of the syndecan-4 ectodomain leads to tubulointerstitial fibrosis.

OS 02-02 microRNA-204 AND SIRTUIN1 REGULATE VASCULAR ENDOPLASMIC RETICULUM STRESS AND ENDOTHELIUM-DEPENDENT VASORELAXATION.

Objective: Endoplasmic reticulum (ER) stress leads to vascular endothelial dysfunction in hypertension. The role of microRNAs in vascular ER stress is not fully understood. Here we examined the role of vascular microRNA-204 (miR-204) in inducing endothelial ER stress and dysfunction by governing the expression of the SIRTUIN1 (SIRT1) lysine deacetylase. Design and Method: Human Umbilical Vein cells (HUVECs) were treated with tunicamycin or transfected with miR-204 Mimic or siRNA SIRT1. In other set of experiments, HUVEC cells were treated with tunicamycin and either transfected with miR-204 inhibitor or transfected with adenovirus of SIRT1. Expression of ER stress markers were determined by Western blots, RTPCR and Immunostaining in all these conditions. Additionally, we treated C57/b6 mice and endothelial specific knockout mice for SIRT1 with tunicamycin and we infused them with miR-204 inhibitor and we studied the vessels reactivity and the expression of ER stress markers. Results: miR-204 mimic decreased expression of SIRT1 and promoted ER stress in HUVECs. Tunicamycin induced endothelial ER stress was also mediated by miR-204. Moreover, knockdown of SIRT1 induced ER stress in HUVECs, which were rescued by a miR-204 antagomir. Tunicamycin-induced ER stress upregulated miR-204, and downregulated SIRT1 in HUVECs. Similarly and in endothelium of aortas and mesenteric arteries of mice, antagonism of miR-204, or SIRT1 overexpression, rescued endothelial cells from tunicamycin-induced ER stress. Similarly, antagonism of miR-204 in vivo with anti-miR-204 mitigated tunicamycin-induced vascular ER stress, decline in vascular SIRT1 and impairment of endothelium-dependent vasorelaxation. Moreover, tunicamycin-induced vascular ER stress and endothelial dysfunction were exacerbated in mice with conditional deletion of endothelial SIRT1. Finally, Vascular miR-204 was upregulated with high-fat diet feeding, anti-miR-204 prevented vascular ER stress stimulated by high-fat diet feeding. Conclusions: These findings show an important role for miR-204 as a mediator of vascular ER stress, and ER stress-induced endothelial dysfunction, via downregulation of endothelial SIRT1.

Endothelial sirtuin 1 inactivation enhances capillary rarefaction and fibrosis following kidney injury through Notch activation

Peritubular capillary (PTC) rarefaction along with tissue fibrosis is a hallmark of chronic kidney disease (CKD). However, molecular mechanisms of PTC loss have been poorly understood. Previous studies have demonstrated that functional loss of endothelial sirtuin 1 (SIRT1) impairs angiogenesis during development and tissue damage. Here, we found that endothelial SIRT1 dysfunction causes activation of endothelial Notch1 signaling, which leads to PTC rarefaction and fibrosis following kidney injury. In mice lacking functional SIRT1 in the endothelium (Sirt1 mutant), kidney injury enhanced apoptosis and senescence of PTC endothelial cells with impaired endothelial proliferation and expanded myofibroblast population and collagen deposition. Compared to wild-type kidneys, Sirt1 mutant kidneys up-regulated expression of Delta-like 4 (DLL4, a potent Notch1 ligand), Hey1 and Hes1 (Notch target genes), and Notch intracellular domain-1 (NICD1, active form of Notch1) in microvascular endothelial cells (MVECs) post-injury. Sirt1 mutant primary kidney MVECs reduced motility and vascular assembly and enhanced senescence compared to wild-type kidney MVECs. This difference in the phenotype was negated with Notch inhibition. Concurrent stimulation of DLL4 and transforming growth factor (TGF)-β1 increased trans-differentiation of primary kidney pericytes into myofibroblast more than TGF-β1 treatment alone. Collectively, these results indicate that endothelial SIRT1 counteracts PTC rarefaction by repression of Notch1 signaling and antagonizes fibrosis via suppression of endothelial DLL4 expression.

MicroRNA‐204, SIRTUIN1, and Caveolin‐1 regulate vascular endoplasmic reticulum stress and endothelium‐dependent vasorelaxation

BackgroundEndoplasmic reticulum (ER) stress leads to vascular endothelial dysfunction in disease states such as obesity, diabetes and hypertension. The role of microRNAs in vascular ER stress is not fully understood. Here we examined the role of vascular microRNA‐204 (miR‐204) in inducing endothelial ER stress and dysfunction by governing the expression of the SIRTUIN1 (SIRT1) lysine deacetylase and the Caveolin‐1 (Cav1).Methods &amp; resultsmiR‐204 mimic decreased expression of SIRT1 and Cav1, and promoted ER stress in endothelial cells. Tunicamycin induced endothelial ER stress was also mediated by miR‐204. Moreover, knockdown of SIRT1 induced ER stress and reduced Cav1 in endothelial cells, both of which were rescued by a miR‐204 antagomir. Tunicamycin‐induced ER stress upregulated miR‐204, and downregulated SIRT1 and Cav1, in endothelial cells. Similarly and in endothelium of aortas and mesenteric arteries of mice. Antagonism of miR‐204, or SIRT1 overexpression, rescued endothelial cells from tunicamycin‐induced ER stress. Similarly, antagonism of miR‐204 in vivo with anti‐miR‐204 mitigated tunicamycin‐induced vascular ER stress, decline in vascular SIRT1 and Cav1, and impairment of endothelium‐dependent vasorelaxation. Moreover, tunicamycin‐induced vascular ER stress and endothelial dysfunction were exacerbated in mice with conditional deletion of endothelial SIRT1. Additionally, overexpressing Cav1 ex‐vivo, with adenovirus, in tunicamycin‐induced vascular ER stress or in mice with conditional deletion of endothelial SIRT1, reduced ER stress markers and miR‐204 in endothelial cells and was associated with an improvement in vascular endothelial dependent relaxation. Finally, Vascular miR‐204 was upregulated with high‐fat diet feeding, anti‐miR‐204 prevented vascular ER stress stimulated by high‐fat diet feeding.ConclusionsThese findings show an important role for miR‐204 as a mediator of vascular ER stress, and ER stress‐induced endothelial dysfunction, via downregulation of endothelial SIRT1 and cav1.Support or Funding InformationT32‐ Institutional Training Grant (HL007121)

Abstract 14050: The Gut Flora Promotes Endothelial Dysfunction Through Vascular MicroRNA-204-mediated Down-regulation of Endothelial Sirtuin1

The gut flora contributes to development of atherosclerosis. Endothelial dysfunction, one manifestation of which is impaired endothelium-dependent vasorelaxation, accompanies and promotes atherosclerotic vascular disease. Here we show that gut flora impair endothelium-dependent vasorelaxation by remotely up-regulating microRNA-204 (miR-204) which downregulates SIRTUIN1 (SIRT1) in the vascular wall. Microarray analysis in aortas of germ-free mice revealed a set of down-regulated microRNAs, including miR-204, which target SIRT1. Suppression of gut flora in mice with antibiotics in drinking water decreased aortic miR-204, increased aortic SIRT1, and improved endothelium-dependent vasorelaxation, effects that were reversed with discontinuation of antibiotics. In addition, miR-204 mimic impaired endothelium-dependent aortic vasorelaxation ex vivo. Moreover, high-fat diet feeding stimulated aortic miR-204, suppressed SIRT1, and impaired endothelial function, all of which were mitigated by administration of antibiotics, and reversed with stoppage of antibiotics. In contrast, antibiotics did not improve high-fat diet-induced endothelial dysfunction in mice conditionally lacking endothelial SIRT1. In addition, anti-miR-204 delivered systemically prevented high-fat diet-induced endothelial dysfunction and vascular SIRT1 decrease. Finally, serum from mice on antibiotics suppressed miR-204, and increased SIRT1, in endothelial cells, effects that were not observed with serum from mice in which antibiotics were discontinued. Therefore, the gut flora remotely downregulates endothelial SIRT1 through miR-204, leading to impairment of endothelial function.

Sirtuin 1 ablation in endothelial cells is associated with impaired angiogenesis and diastolic dysfunction.

Discordant myocardial growth and angiogenesis can explain left ventricular (LV) hypertrophy progressing toward heart failure with aging. Sirtuin 1 expression declines with age; therefore we explored the role played by angiogenesis and Sirtuin 1 in the development of cardiomyopathy. We compared the cardiac function of 10- to 15-wk-old (wo), 30-40 wo, and 61-70 wo endothelial Sirtuin 1-deleted (Sirt1(endo-/-)) mice and their corresponding knockout controls (Sirt1(Flox/Flox)). After 30-40 wk, Sirt1(endo-/-) animals exhibited diastolic dysfunction (DD), decreased mRNA expression of Serca2a in the LV, and decreased capillary density compared with control animals despite a similar VEGFa mRNA expression. However, LV fibrosis and hypoxia-inducible factor (HIF)1α expression were not different. The creation of a transverse aortic constriction (TAC) provoked more severe DD and LV fibrosis in Sirt1(endo-/-) compared with control TAC animals. Although the VEGFa mRNA expression was not different and the protein expression of HIF1α was higher in the Sirt1(endo-/-) TAC animals, capillary density remained reduced. In cultured endothelial cells administration of Sirtuin 1 inhibitor decreased mRNA expression of VEGF receptors FLT 1 and FLK 1. Ex vivo capillary sprouting from aortic explants showed impaired angiogenic response to VEGF in the Sirt1(endo-/-) mice. In conclusion, the data demonstrate 1) a defect in angiogenesis preceding development of DD; 2) dispensability of endothelial Sirtuin 1 under unstressed conditions and during normal aging; and 3) impaired angiogenic adaptation and aggravated DD in Sirt1(endo-/-) mice challenged with LV overload.

Endothelial Sirtuin 1 Deficiency Perpetrates Nephrosclerosis through Downregulation of Matrix Metalloproteinase-14

Sirtuin 1 (SIRT1) depletion in vascular endothelial cells mediates endothelial dysfunction and premature senescence in diverse cardiovascular and renal diseases. However, the molecular mechanisms underlying these pathologic effects remain unclear. Here, we examined the phenotype of a mouse model of vascular senescence created by genetically ablating exon 4 of Sirt1 in endothelial cells (Sirt1(endo-/-)). Under basal conditions, Sirt1(endo-/-) mice showed impaired endothelium-dependent vasorelaxation and angiogenesis, and fibrosis occurred spontaneously at low levels at an early age. In contrast, induction of nephrotoxic stress (acute and chronic folic acid-induced nephropathy) in Sirt1(endo-/-) mice resulted in robust acute renal functional deterioration followed by an exaggerated fibrotic response compared with control animals. Additional studies identified matrix metalloproteinase-14 (MMP-14) as a target of SIRT1. In the kidneys of Sirt1(endo-/-) mice, impaired angiogenesis, reduced matrilytic activity, and retention of the profibrotic cleavage substrates tissue transglutaminase and endoglin accompanied MMP-14 suppression. Furthermore, restoration of MMP-14 expression in SIRT1-depeleted mice improved angiogenic and matrilytic functions of the endothelium, prevented renal dysfunction, and attenuated nephrosclerosis. Our findings establish a novel mechanistic molecular link between endothelial SIRT1 depletion, downregulation of MMP-14, and the development of nephrosclerosis.

Redox Factor-1 Activates Endothelial SIRTUIN1 through Reduction of Conserved Cysteine Sulfhydryls in Its Deacetylase Domain

Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE1/Ref-1) is a reductant which is important for vascular homeostasis. SIRTUIN1 (SIRT1) is a lysine deacetylase that also promotes endothelium-dependent vasorelaxation. We asked if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function. APE1/Ref-1 maintains sulfhydryl (thiol) groups of cysteine residues in SIRT1 in the reduced form and promotes endothelial SIRT1 activity. APE1/Ref-1 stimulates SIRT1 activity by targeting highly conserved vicinal thiols 371 and 374 which form a zinc tetra-thiolate motif in the deacetylase domain of SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for reducing SIRT1 and stimulating its activity. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation of sulfhydryls and from inactivation. APE1/Ref-1 also promotes lysine deacetylation of the SIRT1 target endothelial nitric oxide synthase (eNOS). SIRT1 mutated at cysteines 371 and 374, which renders it non-reducible by APE1/Ref-1, prevents lysine deacetylation of eNOS by APE1/Ref-1. SIRT1 free thiol (reduced sulfhydryl) content and deacetylase activity are diminished in all examined tissues of APE1/Ref-1+/− mice, including the vasculature. Overexpression of SIRT1 in aortas of APE1/Ref-1+/− mice restores endothelium-dependent vasorelaxation and bioavailable nitric oxide (NO) to levels similar to those observed in wild-type mice. Thus, APE1/Ref-1, by maintaining functionally important cysteine sulfhydryls in SIRT1 in the reduced form, promotes endothelial SIRT1 activity. This reductive activation of endothelial SIRT1 by APE1/Ref-1 mediates the effect of APE1/Ref-1 on eNOS acetylation, promoting endothelium-derived NO and endothelium-dependent vasorelaxation.