Impaired Endothelium-dependent Relaxation Research Articles

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis

AimsVascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. Methods and resultsThe case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17–13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated β-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE−/− mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. ConclusionsSAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

Epigallocatechin-3-gallate exerts antihypertensive effects and improves endothelial function in spontaneously hypertensive rats

Objective: To investigate the effect of epigallocatechin-3-gallate (EGCG) on endothelial dysfunction in spontaneously hypertensive rats (SHR). Methods: Wistar-Kyoto (WKY) rats and SHR were divided into four groups; WKY control, SHR control and SHR treated with EGCG (50 mg/kg/day) or losartan (10 mg/kg/day). The treatment was given daily for 4 weeks by oral gavage and the blood pressure was monitored by tail-cuff method every 3 days. Acetylcholine-induced endothelium-dependent relaxations were assessed in isolated phenylephrine-precontracted aortic rings at the end of treatment. The vascular levels of reactive oxygen species, nitric oxide, tetrahydrobiopterin, and cyclic guanosine monophosphate were also measured. Moreover, the expression of angiotensin II type 1 (AT1) receptor protein was determined. Results: The systolic blood pressure was significantly decreased in SHR treated with EGCG. The impaired endothelium-dependent relaxation was significantly improved in aortic ring isolated from the EGCG-treated SHR group. EGCG also significantly increased the levels of nitric oxide, tetrahydrobiopterin, and cyclic guanosine monophosphate, while decreasing the level of reactive oxygen species and the protein expression of AT1 receptor in SHR. Conclusions: EGCG attenuates endothelial dysfunction in SHR by decreasing oxidative stress and increasing vascular nitric oxide bioavailability, which may be modulated partly by inhibition of vascular AT1 receptors. An increase in endothelium-dependent relaxation may contribute to a decrease in blood pressure in hypertensive animals.

Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.

Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR). We used Dkk3-/- mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR. Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the central nervous system (CNS). Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke. These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt (protein kinase B)/eNOS hypotensive axis.

Tcell derived HIV proteins promote oxidative stress, endothelial dysfunction and hypertension via Interleukin 1alpha-mediated mechanisms in mice

While antiretroviral therapy (ART) has extended the life of people living with HIV (PLWH), they experience accelerated development of hypertension and cardiovascular disease (CVD) compared to seronegative individuals. However, the etiology and individual contributions of ART and repressed viral infection remain ill-defined. While ART prevents viral replication, viral reservoirs persist, and viral proteins remain in the circulation leading us to hypothesize that Tcell-derived HIV proteins contribute to endothelial function and hypertension. We utilized a Tg26 transgenic mouse model ubiquitously expressing 7/9 viral proteins. These mice display increased blood pressure (BP) and enhanced neurogenic control of BP (BP response to ganglionic blockade) as well as impaired endothelial vasorelaxation in aorta and mesenteric arteries in both sexes. Bone marrow transplant (BMT) from Tg26 mice into WT (Tg26->WT) increased systolic BP and impaired endothelium dependent relaxation in conduit and resistance vessels (P<0.05), a precursor to hypertension. To identify the immune cell subtype responsible for the CVD, thoracic aortas from WT male and female mice were co-cultured with either WT or Tg26 primary CD3+ Tcells. Vessels exposed to Tg26 Tcells displayed impaired endothelial relaxation versus WT (P<0.05). Vessels incubated in Tg26 CD4+ Tcells, the main target of HIV, also yielded endothelial dysfunction and inhibition of Tcell activation via Abatacept treatment in intact Tg26 mice lowered BP, restored vasorelaxation and reduced sympatho-activation to WT levels in Tg26 mice (P<0.05). A cytokine panel revealed increased production of interleukin 1a (IL-1a) in Tg26 CD3+ T cells (P<0.05). Similarly, Tg26->WT BMT increased plasma IL-1a levels (P<0.1), while Abatacept decreased plasma IL-1a levels in intact Tg26 mice (P<0.05). Treatment with anti-IL-1a antibody abolished Tcell-mediated endothelial dysfunction and primary human microvascular endothelial cells (hMVEC) treated with IL-1a for 24 hours increased ROS mediated DNA damage (8-OHdG), leading us to investigate the source of vascular ROS. Quantitative RT-PCR of the IL-1a exposed hMVEC displayed increases in NADPH oxidase NOX1, NOXO1, and NOX4 while BMT aortas had increased in NOX1, NOXO1, and NOXA1 and inhibition of NOX1 via GKT771 ex vivo restored vasorelaxation. Additionally, anti-IL-1a restored vasorelaxion in intact Tg26 mice. Ultimately, we found that Tcells expressing viral proteins promote the production of IL-1a, increasing endothelial NOX1 and ROS production, leading to endothelial dysfunction, which in addition to sympatho-activation promote hypertension. 1R01HL147639, 21PRE830396, 19EIA34760167 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ovarian-derived sex hormones do not mediate the sex-specific mechanism of hypertension in obese mice

The longstanding dogma that female sex hormones protect premenopausal women against cardiovascular disease perpetuated the belief that sex hormones are the main determinant for cardiovascular risk. However, increasing global trends for obesity and recent clinical evidence indicate that obesity abrogates the protective effects of female sex and predisposes women to vascular dysfunction and hypertension. Previously, our laboratory demonstrated that obesity-associated hypertension manifests via sex-specific mechanisms: leptin-mediated sympatho-activation in males and leptin-mediated aldosterone production in females. Whether sex hormones are the source for these sex differences remains unknown. We hypothesized that loss of ovarian-derived sex hormones with ovariectomy (OVX) will alter the mechanism by which obese female mice develop hypertension and impair vascular function. Obese agouti yellow mice and lean controls underwent OVX or sham surgery at 12 weeks. At 15 weeks of age, mice were implanted with radiotelemeters to record blood pressure (BP) at baseline and in response to leptin receptor blockade (Allo-Aca, 0.05mg/kg/day s.c. osmotic minipump). To determine the effect of OVX on autonomic control of BP, we recorded BP and heart rate (HR) responses to ganglionic blockade, muscarinic and β-adrenergic receptor antagonists. At 18 weeks, mice were euthanized and mesenteric arteries isolated to measure vascular function via wire myography. Obesity significantly increased (2-way ANOVA, P<0.05) mean arterial pressure (MAP), systolic BP, diastolic BP, and HR but surprisingly OVX did not further elevate BP. However, leptin receptor blockade significantly decreased (2-way ANOVA, P<0.05) MAP in obese mice, indicating that BP elevation remains leptin-dependent in OVX obese female mice. OVX did not alter MAP or HR responses to ganglionic blockade, muscarinic and β-adrenergic receptor antagonists, suggesting no further contribution of autonomic control of BP. Mesenteric wire myography revealed obesity significantly impaired (2-way ANOVA, P<0.05) endothelial-dependent relaxation to acetylcholine but interestingly, loss of ovarian-derived sex hormones did not cause further impairment. Western blot analysis showed no alteration in adrenal aldosterone synthase expression in obese OVX mice and LC-MS revealed no difference in plasma aldosterone levels. These results indicate that in the absence of ovarian-derived female sex hormones, hypertension remains leptin-dependent and aldosterone-mediated in obese female mice and female sex steroids likely play a limited role. This work challenges the notion that sex hormones mediate the sex-specific mechanism of hypertension in the context of obesity suggesting sex chromosomes as the underlying origin for the difference in mechanism. R01HL130301, R01HL155265 (E.J.B.) T32 HL155011-01A1 (C.T.B.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Selective Mineralocorticoid Receptor Blocker, Esaxerenone, Attenuates Vascular Dysfunction in Diabetic C57BL/6 Mice.

Pharmacological blockade of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications since MRs play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vessel function. We investigated the effect of a novel nonsteroidal selective MR blocker, esaxerenone, on diabetes-induced vascular dysfunction. Diabetes was induced by a single dose of streptozotocin in 8-week-old male C57BL/6 mice. Esaxerenone (3 mg/kg/day) or a vehicle was administered by gavage to diabetic mice for 3 weeks. Metabolic parameters, plasma aldosterone levels, and parameters related to renal function were measured. Endothelium-dependent or -independent vascular responses of the aortic segments were analyzed with acetylcholine or sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study. Induction of diabetes elevated plasma aldosterone level (P＜0.05) and impaired endothelium-dependent vascular relaxation (P＜0.05). The administration of esaxerenone ameliorated the endothelial dysfunction (P＜0.01) without the alteration of metabolic parameters, blood pressure, and renal function. Esaxerenone improved the eNOSSer1177 phosphorylation in the aorta obtained from diabetic mice (P＜0.05) compared with that in the vehicle-treated group. Furthermore, a major MR agonist, aldosterone, decreased eNOSSer1177 phosphorylation and increased eNOSThr495 phosphorylation in HUVECs, which recovered with esaxerenone. Esaxerenone ameliorated the endothelium-dependent vascular relaxation caused by aldosterone in the aortic segments obtained from C57BL/6 mice (P＜0.001). Esaxerenone attenuates the development of diabetes-induced endothelial dysfunction in mice. These results suggest that esaxerenone has potential vascular protective effects in individuals with diabetes.

Reactive Oxygen Species in the Aorta and Perivascular Adipose Tissue Precedes Endothelial Dysfunction in the Aorta of Mice with a High-Fat High-Sucrose Diet and Additional Factors.

Metabolic syndrome (Mets) is the major contributor to the onset of metabolic complications, such as hypertension, type 2 diabetes mellitus (DM), dyslipidemia, and non-alcoholic fatty liver disease, resulting in cardiovascular diseases. C57BL/6 mice on a high-fat and high-sucrose diet (HFHSD) are a well-established model of Mets but have minor endothelial dysfunction in isolated aortas without perivascular adipose tissue (PVAT). The purpose of this study was to evaluate the effects of additional factors such as DM, dyslipidemia, and steatohepatitis on endothelial dysfunction in aortas without PVAT. Here, we employed eight-week-old male C57BL/6 mice fed with a normal diet (ND), HFHSD, steatohepatitis choline-deficient HFHSD (HFHSD-SH), and HFHSD containing 1% cholesterol and 0.1% deoxycholic acid (HFHSD-Chol) for 16 weeks. At week 20, some HFHSD-fed mice were treated with streptozocin to develop diabetes (HFHSD-DM). In PVAT-free aortas, the endothelial-dependent relaxation (EDR) did not differ between ND and HFHSD (p = 0.25), but in aortas with PVAT, the EDR of HFHSD-fed mice was impaired compared with ND-fed mice (p = 0.005). HFHSD-DM, HFHSD-SH, and HFHSD-Chol impaired the EDR in aortas without PVAT (p < 0.001, p = 0.019, and p = 0.009 vs. ND, respectively). Furthermore, tempol rescued the EDR in those models. In the Mets model, the EDR is compromised by PVAT, but with the addition of DM, dyslipidemia, and SH, the vessels themselves may result in impaired EDR.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

Sitagliptin Ameliorates Creb5/lncRNA ENSMUST00000213271-Mediated Vascular Endothelial Dysfunction in Obese Mice.

Obesity is mediated by the changes in dyslipidemia, oxidative stress, and inflammation, leading to vascular endothelial dysfunction. Glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 inhibitors prevent the development of endothelial dysfunction. However, the underlying mechanism still remains largely unclear. Long non-coding RNAs (lncRNAs), one class of non-coding small RNAs, have been shown to exert a regulatory impact on the endothelial function in obesity. This study aimed to investigate whether the elevation of GLP-1 by a DPP-4 inhibitor sitagliptin improved vascular endothelial function by modulating lncRNAs in obese mice and to clarify the underlying molecular mechanism. Male C57BL/6J mice were fed a high-fat diet for 4 months to induce obesity and some obese mice were treated with sitagliptin for the last 1 month. Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and glucagon-like peptide-1 (GLP-1) in plasma were detected by ELISA. LncRNA expression profile was analyzed via microarray. Aortic relaxations were examined by myograph. Protein expressions and phosphorylations were determined using western blot. The differentially expressed lncRNAs were validated using qRT-PCR. Obese mice exhibited increased levels of TC and LDL, decreased concentrations of HDL and GLP-1 in plasma, and impaired aortic endothelium-dependent relaxations; such effects could be reversed by sitagliptin. Moreover, the altered expression profile of lncRNAs in the obese mouse aortae could be modulated by sitagliptin. Consistent with microarray analysis, qRT-PCR also revealed that lncRNA ENSMUST00000213271 was up-regulated in obese mouse aortae and aortic endothelial cells (ECs), which could be down-regulated by sitagliptin. Creb5 silencing reduced lncRNA ENSMUST00000213271 in obese mouse ECs. Knockdown of either Creb5 or lncRNA ENSMUST00000213271 restored the activation of AMPK/eNOS in obese mouse ECs. Furthermore, sitagliptin also suppressed Creb5 and lncRNA ENSMUST00000213271 and increased the phosphorylations of AMPK and eNOS in obese mice. Creb5/lncRNA ENSMUST00000213271 mediated vascular endothelial dysfunction through inhibiting AMPK/eNOS cascade in obesity. Elevation of GLP-1 by sitagliptin possibly improved endothelial function by suppressing Creb5/lncRNA ENSMUST00000213271 and subsequently restoring AMPK/eNOS activation in obese mice. This study will provide new evidence for the benefits of GLP-1 against vasculopathy in obesity.

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

PS-B01-2: ASSOCIATION BETWEEN IMPAIRED METABOLIC FUNCTION AND VASCULAR DYSFUNCTION IN METABOLIC SYNDROME

Objectives: Metabolic syndrome is a leading cause for vascular dysfunction and cardiovascular diseases. We have previously shown that vascular functional responses in metabolic syndrome exhibit exaggerated vasocontractility and impaired endothelium-dependent relaxation. However, it remains unclear if a correlation between vascular abnormalities and metabolic disturbances exists. Accordingly, we aimed to explore the association between vascular dysfunction and changes in metabolic parameters in a rodent model of metabolic syndrome, the high fat diet (HFD)-streptozotocin (STZ)-induced diabetes mellitus rat (HFD-D). Design and method: Five weeks old male Wistar albino rats (n = 24) were fed with either HFD (45 kcal% fat) or control diet (10 kcal% fat) for 10 weeks. On week 6, 40 mg/kg STZ and saline were injected intraperitoneally into the HFD and control groups, respectively. At the end of the treatment, metabolic data were collected then rats were euthanized for blood and kidney collection and to measure maximal vasoconstrictor and vasodilator responses of the abdominal aortic rings. Recorded measures also included fasting blood glucose (FBG), plasma total cholesterol (TC), plasma triglyceride (TG), plasma high density lipoprotein (HDL), plasma creatinine (Cr), urinary protein: urinary creatinine (UPC) and kidney index (KI). Results: HFD-D rats had higher FBG, TG, Cr, UPC, and KI compared with controls. HDL was lower in HFD-D while TC remained unchanged. Maximal vasoconstriction to norepinephrine (NE Rmax) was greater in the HFD group compared with controls. Maximal endothelium-dependent vasorelaxation to acetylcholine (ACh Rmax) was blunted in the HFD group relative to controls. A positive correlation between NE Rmax and FBG was found, whereas ACh Rmax was negatively correlated with FBG, KI, UPC and TG. Conclusions: Impaired glucose metabolism, lipid profile and renal function are likely key contributors to vascular dysfunction in metabolic syndrome; however, further mechanistic investigations are required to verify these assumptions.

Sex hormones and vascular reactivity: a temporal evaluation in resistance arteries of male rats

The role of androgens in vascular reactivity is controversial, particularly regarding their age-related actions. The objective of this study was to conduct a temporal evaluation of the vascular reactivity of resistance arteries of young male rats, as well as to understand how male sex hormones can influence the vascular function of these animals. Endothelium-mediated relaxation was characterized in third-order mesenteric arteries of 10-, 12-, 16-, and 18w (week-old) male rats. Concentration-response curves to acetylcholine (ACh, 0.1 nmol/L-10 µmol/L) were constructed in arteries previously contracted with phenylephrine (PE, 3 µmol/L), before and after the use of nitric oxide synthase or cyclooxygenase inhibitors. PE concentration-response curves (1 nmol/L-100 μmol/L) were also built. The levels of vascular nitric oxide, superoxide anion, and hydrogen peroxide were assessed and histomorphometry analysis was performed. The 18w group had impaired endothelium-dependent relaxation. All groups showed prostanoid-independent and nitric oxide-dependent vasodilatory response, although this dependence seems to be smaller in the 18w group. The 18w group had the lowest nitric oxide and hydrogen peroxide production, in addition to the highest superoxide anion levels. Besides functional impairment, 18w animals showed morphological differences in third-order mesenteric arteries compared with the other groups. Our data show that time-dependent exposure to male sex hormones appears to play an important role in the development of vascular changes that can lead to impaired vascular reactivity in mesenteric arteries, which could be related to the onset of age-related cardiovascular changes in males.

PS-B12-12: NOVEL PATHOPHYSIOLOGICAL ROLES OF A-SYNUCLEIN IN AGE-RELATED VASCULAR ENDOTHELIAL DYSFUNCTION AND E NHANCED PERMEABILITY.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide (NO) production in ECs. Treatment with rSNCA also suppressed TNF-α and palmitic acid induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β; gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Mechanistically, loss-of-function of SNCA upregulated the expression of β;-site amyloid precursor protein cleaving enzyme 1 (BACE1), which was reported to exacerbate eNOS inactivation and permeability in ECs. Addition of SNAP, NO donor, rescued the increment of BACE1, which indicated eNOS downregulation by loss-of-function of SNCA caused BECE1 increment in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high fat diet, displayed elevated blood pressure compared with wild-type (WT) mice; this could be eNOS dysfunction dependent because of the lower difference caused by L NAME administration. In addition, increased depositions of immunoglobulin were found in the aortas of SNCA KO compared with those of WT mice, which indicated enhanced endothelial permeability by in vivo loss-of-function of SNCA. Thus, these results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction and enhanced permeability might be partially ascribed to loss-of-function of SNCA in ECs.

Citri Reticulatae Pericarpium (Chenpi) Protects against Endothelial Dysfunction and Vascular Inflammation in Diabetic Rats.

Dried tangerine peel (Citri reticulatae Pericarpium, CRP; Chenpi in Chinese) possesses medicine and food homology with hypolipidemic, anti-inflammatory and antioxidant activities. This study aimed to explore the protective effect of CRP extract on endothelial function and inflammation in type 2 diabetic rats and the related mechanisms. Type 2 diabetes mellitus was induced by high-fat diet (HFD)/streptozotocin (STZ) in male Sprague Dawley rats, and CRP extract was orally administered at 400 mg/kg/day for 4 weeks. Rat and mouse aortas were treated with high glucose and CRP extract ex vivo. The data showed that the ethanolic extract of CRP normalized blood pressure and the plasma lipid profile as well as the plasma levels of liver enzymes in diabetic rats. Impaired endothelium-dependent relaxations in aortas, carotid arteries and renal arteries were improved. CRP extract suppressed vascular inflammatory markers and induced AMPK activation in aortas of diabetic rats. Exposure to high glucose impaired vasodilation in aortas of rats and mice, and this impairment was prevented by co-incubation with CRP extract. In conclusion, our findings suggest that CRP extract protects endothelial function by inhibiting the vascular inflammatory state on activation of AMPK in diabetic rats.

Jatrorrhizine Improves Endothelial Function in Diabetes and Obesity through Suppression of Endoplasmic Reticulum Stress.

Jatrorrhizine (JAT) is one of the major bioactive protoberberine alkaloids found in rhizoma coptidis, which has hypoglycemic and hypolipidemic potential. This study aimed to evaluate the vasoprotective effects of JAT in diabetes and obesity and the underlying mechanism involved. Mouse aortas, carotid arteries and human umbilical cord vein endothelial cells (HUVECs) were treated with risk factors (high glucose or tunicamycin) with and without JAT ex vivo and in vitro. Furthermore, aortas were obtained from mice with chronic treatment: (1) control; (2) diet-induced obese (DIO) mice fed a high-fat diet (45% kcal% fat) for 15 weeks; and (3) DIO mice orally administered JAT at 50 mg/kg/day for the last 5 weeks. High glucose or endoplasmic reticulum (ER) stress inducer tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations (EDRs) in mouse aortas, induced oxidative stress in carotid arteries and HUVECs, downregulated phosphorylations of Akt at Ser473 and eNOS at Ser1177 and enhanced ER stress in mouse aortas and HUVECs, and these impairments were reversed by cotreatment with JAT. JAT increased NO release in high-glucose-treated mouse aortas and HUVECs. In addition, chronic JAT treatment restored endothelial function with EDRs comparable to the control, increased Akt/eNOS phosphorylation, and attenuated ER stress and oxidative stress in aortas from DIO mice. Blood pressure, glucose sensitivity, fatty liver and its morphological change, as well as plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and plasma lipid profile, were also normalized by JAT treatment. Collectively, our data may be the first to reveal the vasoprotective effect of JAT that ameliorates endothelial dysfunction in diabetes and obesity through enhancement of the Akt/eNOS pathway and NO bioavailability, as well as suppression of ER stress and oxidative stress.

Aldosterone-stimulated endothelial epithelial sodium channel (EnNaC) plays a role in cold exposure-induced hypertension in rats.

Background: Previous studies have demonstrated that activated endothelial epithelial sodium channel (EnNaC) impairs vasodilatation, which contributes to salt-sensitive hypertension. Here, we investigate whether mesenteric artery (MA) EnNaC is involved in cold exposure–induced hypertension (CIH) and identify the underlying mechanisms in SD rats. Methods: One group of rats was housed at room temperature and served as control. Three groups of rats were kept in a 4°C cold incubator for 10 h/day; among which two groups were administrated with either benzamil (EnNaC blocker) or eplerenone (mineralocorticoid receptor antagonist, MR). Blood pressure (BP), vasodilatation, and endothelial function were measured with tail-cuff plethysmography, isometric myograph, and Total Nitric Oxide (NO) Assay kit, respectively. A cell-attached patch-clamp technique, in split-open MA, was used to determine the role of EnNaC in CIH rats. Furthermore, the plasma aldosterone levels were detected using an ELISA kit; and Western blot analysis was used to examine the relative expression levels of Sgk1 and Nedd4-2 proteins in the MA of SD rats. Results: We demonstrated that cold exposure increased BP, impaired vasodilatation, and caused endothelial dysfunction in rats. The activity of EnNaC significantly increased, concomitant with an increased level of plasma aldosterone and activation of Sgk1/Nedd4-2 signaling. Importantly, CIH was inhibited by either eplerenone or benzamil. It appeared that cold-induced decrease in NO production and impairment of endothelium-dependent relaxation (EDR) were significantly ameliorated by either eplerenone or benzamil in MA of CIH rats. Moreover, treatment of MAs with aldosterone resulted in an activation of EnNaC, a reduction of NO, and an impairment of EDR, which were significantly inhibited by either eplerenone or GSK650394 (Sgk1 inhibitor) or benzamil. Conclusion: Activation of EnNaC contributes to CIH; we suggest that pharmacological inhibition of the MR/Sgk1/Nedd4-2/EnNaC axis may be a potential therapeutic strategy for CIH.

Novel pathophysiological roles of α-synuclein in age-related vascular endothelial dysfunction.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide production in ECs. Treatment with rSNCA also suppressed TNF-α- and palmitic acid-induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β-gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high-fat diet, displayed elevated blood pressure compared with wild-type mice; this could be eNOS dysfunction-dependent because of the lower difference caused by L-NAME administration. These results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction might be partially ascribed to loss-of-function of SNCA in ECs.

Influenza A virus elicits peri-vascular adipose tissue inflammation and vascular dysfunction of the aorta in pregnant mice.

Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1–3 days post infection (d.p.i) with the levels being ~4–8 fold higher compared with the vessel wall. IAV infection also increased Ly6Clow patrolling monocytes and Ly6Chigh pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a “vascular storm”- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4+ and CD8+ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.

Vasorin plays a critical role in vascular smooth muscle cells and arterial functions.

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

Soluble Epoxide Hydrolase Inhibitor t-AUCB Ameliorates Vascular Endothelial Dysfunction by Influencing the NF-κB/miR-155-5p/eNOS/NO/IκB Cycle in Hypertensive Rats.

Epoxyeicosatrienoic acids (EETs), angiogenic mediators degraded by soluble epoxide hydrolase (sEH), have been shown to exert beneficial effects on the cardiovascular system. The current study assessed the impact of increased EETs with an sEH inhibitor, t-AUCB, on two-kidney-one-clip (2K1C)-induced renovascular endothelial dysfunction, associated with hypertension, in rats. The hypertensive rats exhibited increased systolic blood pressure, reduced renal blood flow, impaired endothelium-dependent relaxation and eNOS phosphorylation in the renal arteries, elevated ROS production in the endothelium of the renal arteries, and decreased EET levels in plasma, the renal arteries, and endothelial cells; however, t-AUCB reversed all the deleterious effects. Moreover, we found that the stimulation of AMPK/UCP2 scavenged ROS and restored endothelial function in the renal arteries of hypertensive rats undergoing therapy with t-AUCB. In addition, we were the first to reveal the potential role of miR-155-5p in the occurrence and development of vascular endothelial dysfunction in hypertension. Importantly, t-AUCB recovered NO bioavailability by regulating the NF-κB/miR-155-5p/eNOS/NO/IκB cycle after the activation of AMPK/UCP2 and the subsequent inhibition of ROS in hypertensive rat renal artery endothelial cells. This study will provide evidence for this additional new mechanism, underlying the benefits of EETs and the related agents against hypertensive vasculopathy.