Impaired Endothelium-dependent Vasodilation Research Articles

Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies.

The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1β monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by increasing tetrahydrobiopterin via regulation of GTP-cyclohydrolase 1 and reducing uncoupled endothelial nitric oxide synthase activity

Background and aimsThe apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. MethodsLow-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. ResultsUnexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. ConclusionsHypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Coronary Microvascular Dysfunction.

[Figure: see text].

The Diabetic Coronary Microcirculation is Regulated by MicroRNA‐21

Introduction Coronary microvascular dysfunction is particularly common among diabetes and overlaps with impaired endothelial-dependent vasodilation. These impairments are seen in early stages of the disease, but detailed mechanisms have yet to be elucidated. Nitric oxide (NO) is the major endothelial-dependent mediator of vasodilation in the healthy coronary circulation, but this mediator changes to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a risk factor for CAD, we hypothesized that a similar switch would occur. Methods Coronary arteries were isolated, and endothelial-dependent vasodilation was assessed using myography (DMT). Reactive oxygen species (ROS) in coronary endothelial cells were measured using electron paramagnetic resonance (EPR). Quantitative polymerase chain reaction (qPCR) was performed for gene expression analysis. In vivo myocardial blood flow (MBF) was measured by contrast echocardiography during increased cardiac work achieved through the administration of norepinephrine. Results Our ex vivo and in vivo data show nitric oxide synthase inhibitor (L-NAME) inhibited vasodilation in wild type (WT) mice, but the H2O2 scavenger (PEG-catalase) had no effect. In contrast, vasodilation in diabetic mice was blunted by PEG-catalase, but not L-NAME. This suggested that the mediator of coronary vasodilation switched from NO to H2O2 in diabetes. Importantly, we found that microRNA-21 (miR-21) was upregulated in diabetes and the deficiency modulated the mediator switch from NO to H2O2 in diabetic mice. Conclusions The switch in the mediator of coronary vasodilation from NO to H2O2 contributed to microvascular dysfunction in diabetes and miR-21 regulated this switch. Further genetic profiling will elucidate the pathways and mechanisms converging with miR-21 to regulate microvascular function in diabetes. This is the first mouse model that recapitulates the switch in mediator of coronary vasodilation from NO to H2O2 seen in CAD patients.

Role of Nicotine and Exposure Duration in the Cardiovascular Toxicity of Electronic Cigarettes and Tobacco Cigarettes in a Long‐term Mouse Exposure Model

Background Electronic cigarette (e-cig) vaping (ECV) has been proposed as a safer alternative to tobacco cigarette smoking (TCS); however, this remains controversial due to a lack of long-term comparative studies. Therefore, we developed and applied a chronic mouse exposure model, which mimics human vaping and allows comparison to TCS. This model was applied to determine the role of nicotine levels and exposure duration in the onset of cardiovascular changes and toxicity. Methods Longitudinal studies were performed to evaluate alterations in cardiovascular function with TCS and ECV exposure durations of up to 60 weeks. For ECV, e-cig liquid formulations were used with box-mod and for TCS, 3R4F-cigarettes. C57/BL6 male mice were exposed 2 hours/day, 5 days/week to TCS, ECV, or air-control. The role of vape nicotine levels was evaluated using e-cig-liquids with 0, 6 or 24 mg/ml nicotine. Results Following 16 weeks of exposure, increased constriction to phenylephrine and impaired endothelial-dependent and endothelial-independent vasodilation were observed in aortic segments of the TCS and ECV exposure groups. This paralleled the onset of systemic hypertension, with elevations in systemic vascular resistance. These changes were seen in all ECV and TCS exposed groups but were most severe with TCS and ECV with 24 mg/ml nicotine. Following 32 weeks, TCS and ECV induced cardiac hypertrophy with concentric left ventricular wall thickening. All of these abnormalities further increased out to 60 weeks of exposure, with elevated heart weight and aortic wall thickness along with increased superoxide production measured in vessels and cardiac tissues of both ECV and TCS mice. Conclusion While ECV-induced abnormalities were seen in the absence of nicotine, these occurred earlier and were more severe with higher nicotine exposure. Thus, long-term vaping of e-cig, can induce cardiovascular disease similar to TCS, and the severity of this toxicity increases with exposure duration and vape nicotine content.

Chronic Nicotine Inhalation‐Induced Vascular Dysfunction is Mediated by the Alpha 7 Nicotinic Acetylcholine Receptor

Cigarette smoking remains a leading risk factor for cardiopulmonary pathologies. However, the effects of nicotine, the addictive component of cigarettes, on cardiopulmonary function is not fully understood. Our laboratory has shown previously that chronic nicotine inhalation in mice leads to the development of pulmonary hypertension with right ventricular (RV) remodeling. This study aims to further examine the molecular mechanisms of nicotine-induced vascular dysfunction. We hypothesize that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nicotinic acetylcholine receptor (nAChR). To investigate our hypothesis, male C57BL6/J wildtype (WT) and α7-nAChR global knockout (KO) mice were exposed to air (WT, n=10; α7-nAChR KO, n=7) or nicotine vapor (WT, n=10; α7-nAChR KO, n=10) daily, 12 hour on/12 hour off, for 3 months. After the 3-month exposure, mice were subjected to right heart catheterization to measure right ventricular systolic pressure (RVSP) as an index of pulmonary artery pressure. In contrast to WT mice, which developed increased RVSP (31.4 ± 1.5 mmHg in nicotine-exposed compared to 24.0 ± 1.3 mmHg in air controls, p = 0.002), α7-nAChR KO mice were protected from these nicotine effects (25.7 ± 0.8 mmHg in nicotine-exposed compared to 21.6 ± 2.0 mmHg in air controls, p = 0.218). In addition, thoracic aortas were isolated from WT and α7-nAChR KO mice following air or nicotine exposure and subjected to RNA-sequencing analysis. Aortas isolated from WT mice showed 123 differentially expressed genes (112 up and 11 down) in nicotine-exposed compared to controls, with 18 upregulated and 3 downregulated pathways as identified by the Ingenuity Pathway Analysis (IPA). Many of the top upregulated pathways are involved in inflammatory response, including acute phase response signaling (p = 4.91E-06), complement system (p = 5.71E-06), and interleukin (IL)-6 signaling (p = 1.88E-03). Furthermore, the Angiotensin II receptor type 1, an important effector controlling blood pressure and volume, was upregulated 4.48-fold (Adjusted p = 0.038). In contrast, only 7 none-overlapping genes were differentially regulated in α7-nAChR KO mice by nicotine compared with the air controls. Finally, vasoreactivity assays were performed on aortas isolated from air and nicotine-exposed WT mice, and aortas isolated from nicotine-exposed mice exhibited impaired endothelium-dependent vasodilation. In conclusion, we found that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nAChR.

Alpha-mangostin improves endothelial dysfunction in db/db mice through inhibition of aSMase/ceramide pathway.

Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha‐mangostin has been reported to have anti‐diabetic capacity in recent years. Here, we investigated the protective function of alpha‐mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha‐mangostin improved impaired endothelium‐dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up‐regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha‐mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha‐mangostin increased phosphorylation of eNOS and NO production in high glucose‐treated aortas. Alpha‐mangostin normalized high glucose‐induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha‐mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation.

Long-term electronic cigarette exposure induces cardiovascular dysfunction similar to tobacco cigarettes: role of nicotine and exposure duration.

Electronic cigarette (e-cig) vaping (ECV) has been proposed as a safer alternative to tobacco cigarette smoking (TCS); however, this remains controversial due to a lack of long-term comparative studies. Therefore, we developed a chronic mouse exposure model that mimics human vaping and allows comparison with TCS. Longitudinal studies were performed to evaluate alterations in cardiovascular function with TCS and ECV exposure durations of up to 60 wk. For ECV, e-cig liquid with box-mod were used and for TCS, 3R4F-cigarettes. C57/BL6 male mice were exposed 2 h/day, 5 days/wk to TCS, ECV, or air control. The role of vape nicotine levels was evaluated using e-cig-liquids with 0, 6, or 24 mg/mL nicotine. Following 16-wk exposure, increased constriction to phenylephrine and impaired endothelium-dependent and endothelium-independent vasodilation were observed in aortic segents, paralleling the onset of systemic hypertension, with elevations in systemic vascular resistance. Following 32 wk, TCS and ECV induced cardiac hypertrophy. All of these abnormalities further increased out to 60 wk of exposure, with elevated heart weight and aortic thickness along with increased superoxide production in vessels and cardiac tissues of both ECV and TCS mice. While ECV-induced abnormalities were seen in the absence of nicotine, these occurred earlier and were more severe with higher nicotine exposure. Thus, long-term vaping of e-cig can induce cardiovascular disease similar to TCS, and the severity of this toxicity increases with exposure duration and vape nicotine content.NEW & NOTEWORTHY A chronic mouse exposure model that mimics human e-cigarette vaping and allows comparison with tobacco cigarette smoking was developed and utilized to perform longitudinal studies of alterations in cardiovascular function. E-cigarette exposure led to the onset of cardiovascular disease similar to that with tobacco cigarette smoking. Impaired endothelium-dependent and endothelium-independent vasodilation with increased adrenergic vasoconstriction were observed, paralleling the onset of systemic hypertension and subsequent cardiac hypertrophy. This cardiovascular toxicity was dependent on exposure duration and nicotine dose.

Pleiotropic effects of erythropoietin on renal and aortic injury in sucrose-induced insulin resistant rats

We previously reported that erythropoietin inhibits renal and vascular dysfunction by activating PI3K/Akt pathway in animal models such as type 1 diabetes. The PI3K/Akt pathway is the central components in insulin signaling. Impairment of insulin signaling occurs not only in hepatic and muscle cells but also in vascular and renal cells, which are suggested to contribute to tissue injury. We investigated whether erythropoietin would inhibit glucose tolerance and vascular and renal injury. Rats were treated with 12%sucrose in drinking water and erythropoietin (150U/kg, 3 times/week) subcutaneously for 10 weeks. Despite our previous results, erythropoietin (150U/kg) induced hematopoiesis and hypertension. Maybe because of hypertension, impairment of endothelium-dependent vasodilation, medial thickening and proteinuria in insulin resistant rats were worsened by erythropoietin. While reducing the dose (75U/kg) and period (last 4 weeks of sucrose treatment) even caused mild hypertension, erythropoietin had no effects on endothelial function, medial thickness, or proteinuria. Oral glucose tolerance test and the value of HOMA-IR showed that erythropoietin improved insulin sensitivity. Erythropoietin reduced fibrotic collagen deposition in renal tubulointerstitial area and macrophage infiltration in aortic adventitia. In conclusion, erythropoietin might protect vascular and renal injury in insulin resistant state. Further investigation into insulin signaling and modification of treatment dose/period are needed.

Endothelial dysfunction and C-reactive protein predict the incidence of heart failure in hypertensive patients.

AimsEndothelial dysfunction and heart failure are associated, but no prospective studies demonstrated that impaired endothelium‐dependent vasodilation predicts incident heart failure. We designed this study to test whether endothelial dysfunction is associated with incident heart failure in a group of hypertensives.Methods and resultsWe enrolled 735 White never‐treated hypertensive outpatients free from heart failure, diabetes, chronic kidney disease, and previous cardiovascular events. Endothelium‐dependent vasodilation was investigated by intra‐arterial infusion of acetylcholine, and laboratory determinations were obtained by standard procedures. During the follow‐up [median 114 months (range 26–206)], there were 208 new cases of heart failure (3.1 events/100 patient‐years). Dividing the study population in progressors and non‐progressors, we observed that progressors were older, showed a higher prevalence of being female, and had a higher baseline heart rate, glucose, insulin, Homeostatic Model Assessment (HOMA), creatinine, and high‐sensitivity C‐reactive protein (hs‐CRP) mean values, while estimated glomerular filtration rate and maximal acetylcholine‐stimulated forearm blood flow were lower. In the multiple Cox regression analysis, female gender [hazard ratio (HR) = 1.454, 95% CI = 1.067–1.981], fasting glucose (HR = 1.186, 95% CI = 1.038–1.357), hs‐CRP (HR = 1.162, 95% CI = 1.072–1.259), HOMA (HR = 1.124, 95% CI = 1.037–1.219), acetylcholine‐stimulated forearm blood flow (HR = 0.779, 95% CI = 0.695–0.874), and estimated glomerular filtration rate (HR = 0.767, 95% CI = 0.693–0.849) maintained an independent association with the outcome. Successively, testing the interaction between forearm blood flow and hs‐CRP, we observed that patients who have hs‐CRP values above the median and forearm blood flow under the median show a higher risk of developing heart failure (HR = 7.699, 95% CI = 4.407–13.451).ConclusionsThe present data demonstrate that an impaired endothelium‐dependent vasodilation and hs‐CRP predict development of incident heart failure in hypertensives.

NOX5-induced uncoupling of endothelial NO synthase is a causal mechanism and theragnostic target of an age-related hypertension endotype.

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5-but not NOX1, NOX2, or NOX4-with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed-upon aging-severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

Effect of Anthocyanin-Rich Extract of Sour Cherry for Hyperglycemia-Induced Inflammatory Response and Impaired Endothelium-Dependent Vasodilation.

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/µL) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1α (IL-1α), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/µL anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

High-cholesterol diet during pregnancy induces maternal vascular dysfunction in mice: potential role for oxidized LDL-induced LOX-1 and AT1 receptor activation.

The lectin-like oxidized low-density-lipoprotein (oxLDL) receptor-1 (LOX-1) has been shown to induce angiotensin II (AngII) type 1 receptor (AT1) activation, contributing to vascular dysfunction. Preeclampsia is a pregnancy complication characterized by vascular dysfunction and increased LOX-1 and AT1 activation; however, whether LOX-1 and AT1 activity contributes to vascular dysfunction in preeclampsia is unknown. We hypothesized that increased oxLDL levels during pregnancy lead to LOX-1 activation and subsequent AT1 activation, resulting in vascular dysfunction. Pregnant wild-type (WT) and transgenic LOX-1 overexpressing (LOX-1tg) mice were fed a control diet (CD) or high-cholesterol diet (HCD, to impair vascular function) between gestational day (GD) 13.5-GD18.5. On GD18.5, AngII-induced vasoconstriction and methylcholine (MCh)-induced endothelium-dependent vasodilation responses were assessed in aortas and uterine arteries. HCD decreased fetal weight and increased circulating oxLDL/cholesterol levels in WT, but not in LOX-1tg mice. HCD did not alter AngII responsiveness or AT1 expression in both vascular beds; however, AngII responsiveness and AT1 expression were lower in aortas from LOX-1tg compared with WT mice. In aortas from WT-CD mice, acute oxLDL exposure induced AT1-mediated vasoconstriction via LOX-1. HCD impaired endothelium-dependent vasodilation and increased superoxide levels in WT aortas, but not uterine arteries. Moreover, in WT-CD mice oxLDL decreased MCh sensitivity in both vascular beds, partially via LOX-1. In summary, HCD impaired pregnancy outcomes and vascular function, and oxLDL-induced LOX-1 activation may contribute to vascular dysfunction via AT1. Our study suggests that LOX-1 could be a potential target to prevent adverse outcomes associated with vascular dysfunction in preeclampsia.

Abstract P099: Loss Of The Brown-Like Phenotype Of Aortic PVAT Is Associated With Mitochondrial Dysfunction In Obesity-Induced Endothelial Dysfunction In Female Rats

Endothelial dysfunction is a major complication of obesity and is an early contributor to hypertension. Perivascular adipose tissue (PVAT) surrounds most blood vessels and has different phenotypic properties based on its anatomical location. Thoracic aortic PVAT from humans and rodents is a brown-like adipose tissue and plays a vasculo-protective role under physiological conditions. In obesity, aortic PVAT expands, switches from a brown-like to a white-like phenotype and contributes to endothelial dysfunction. We hypothesized that loss of the brown-like phenotype of aortic PVAT in obesity is associated with mitochondrial dysfunction, resulting in PVAT and endothelial dysfunction. Eight-week-old female Wistar rats were randomized into two experimental groups: the Lean group (n=8) received a chow diet (5% fat, 48.7% carbohydrate [3.2% sucrose], 24.1% protein) and the Obese group (n=8) received a western diet (21% fat, 50% carbohydrate [34% sucrose], 20% protein), for 20 weeks. Increased body weight (340.57 vs. 265.37g leans, p<0.05) was confirmed in the obese group. At the experimental endpoint, thoracic aortas with intact (+PVAT) or removed PVAT (-PVAT) were obtained for analysis. Endothelial function was assessed in aortic rings +PVAT or -PVAT by performing concentration-response to acetylcholine using wire myography. The aortic ring (-PVAT) from the obese group exhibited impaired endothelium-dependent vasodilation (p<0.01). This effect was heightened in aortic rings (+PVAT) (p<0.05), showing a negative effect of PVAT on endothelial function during obesity. Mitochondrial dysfunction in PVAT from the obese group was characterized by decreased mitochondrial density (30% reduction, p<0.05), detected by quantification of Mitotracker fluorescence, and increased reactive oxygen species levels (4.34-fold increase, p<0.01), as evidenced by DHE staining. These effects were accompanied by decreased uncoupling protein-1 expression in the obese group (55% reduction, p<0.01). Moreover, Oil Red O staining showed larger lipid droplets in aortic PVAT from the obese group. Our results support that obesity-induced endothelial dysfunction is associated with a loss of the brown-like phenotype and mitochondrial dysfunction in PVAT.

Role of thioredoxin-interacting protein in mediating endothelial dysfunction in hypertension

Excessive oxidative stress is a major causative factor of endothelial dysfunction in hypertension. As an endogenous pro-oxidant, thioredoxin-interacting protein (TXNIP) contributes to oxidative damage in various tissues. The present study aimed to investigate the role of TXNIP in mediating endothelial dysfunction in hypertension. In vivo, an experimental model of acquired hypertension was established with two-kidney, one-clip (2K1C) surgery. The expression of TXNIP in the vascular endothelial cells of multiple vessels was significantly increased in hypertensive rats compared with sham-operated rats. Resveratrol, a TXNIP inhibitor, suppressed vascular oxidative damage and increased the expression and activity of eNOS in the aorta of hypertensive rats. Notably, impaired endothelium-dependent vasodilation was effectively improved by TXNIP inhibition in hypertensive rats. In vitro, we observed that Ang II increased the expression of TXNIP in primary human aortic endothelial cells (HAECs) and that TXNIP knockdown by RNA interference alleviated cellular oxidative stress damage and mitigated the impaired eNOS activation and intracellular nitric oxide (NO) production observed in Ang II-treated HAECs. However, inhibiting thioredoxin (TRX) with PX-12 completely blunted the protective effect of silencing TXNIP. In addition, TXNIP knockdown facilitated TRX expression and promoted TRX nuclear translocation to further activate AP1 and REF1. TRX overexpression exhibited favorable effects on eNOS/NO homeostasis in Ang II-treated HAECs. Thus, TXNIP contributes to oxidative stress and endothelial dysfunction in hypertension, and these effects are dependent on the antioxidant capacity of TRX, suggesting that targeting TXNIP may be a novel strategy for antihypertensive therapy.

Disorders of microcirculation in the mechanism of bisphosphonate osteonecrosis: preliminary study in rats.

We investigated the possibilities of angioprotection and the reduction of osteonecrosis in rats that had been given bisphosphonates. In our experiment, 27 rats were divided into three groups: Group 1 was injected with saline; Group 2 was given zoledronic acid for six weeks; and Group 3 was given zoledronic acid for six weeks, with added doses of sulodexide after three weeks. After that we constructed a model of how the teeth should be extracted. The velocity of linear blood flow in the periodontal area of an extracted tooth in rats was studied using laser and high-frequency Doppler ultrasound (with the application of the vasoactive substance acetylcholine 3% for 1min). We evaluated changes in the structure of the bony tissues of the head using computed tomography, comparing the control group with the saline group. A rapid reduction in microcirculation was detected during the use of zoledronic acid for six weeks. A smaller reduction in microcirculation was detected after three weeks of treatment with sulodexide and zoledronic acid. There was a reduction in blood flow in the mucous membranes and, to a greater extent, in bony tissue. Zoledronic acid causes significant impairment of the periosteal blood flow to the mucous membranes because of a complex of disorders, which includes both the cellular component (impaired endothelium-dependent vasodilation of the mucous membrane vessels) and by reducing the intensity of microcirculation in the mucous membranes and bony tissues. Sulodexide, however, improves the restoration of blood flow and reduces the severity of osteonecrosis.

Impaired Endothelial Caveolin‐1‐TRPV4 Channel Signaling Contributes to Endothelial Dysfunction in Pulmonary Hypertension

Impaired endothelium‐dependent vasodilation is a key contributor to elevated pulmonary arterial pressure (PAP) in pulmonary hypertension (PH). While the mechanisms of pathogenesis of PH remain elusive, recent studies show that spatially localized Ca2+ influx via endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels promotes vasodilation in resistance‐sized pulmonary arteries (PAs) by activating endothelial nitric oxide synthase (eNOS) and releasing nitric oxide (NO). Caveolin‐1 (Cav‐1EC) is an essential structural protein within the pulmonary endothelium, where it has been found to associate with TRPV4EC channels in cultured endothelial cells. We hypothesized that Cav‐1EC regulates TRPV4EC channel activity and PAP, and that impaired Cav‐1EC‐TRPV4EC signaling contributes to elevated PAP in PH. To test these hypotheses, we used right ventricular systolic pressure (RVSP) and PAP measurements in normoxic or chronic hypoxic Cav‐1EC−/− and TRPV4EC−/− mice, and pressure myography and high‐speed Ca2+ imaging studies in PAs from these mice. TRPV4EC channel activity and TRPV4EC‐mediated vasodilation were significantly impaired in PAs from Cav‐1EC−/− mice, suggesting that Cav‐1EC enhances TRPV4EC channel activity in PAs. Purinergic signaling plays an important role in pulmonary circulation, and we recently identified adenosine triphosphate (ATP) as a novel physiological activator of TRPV4EC channels in PAs. ATP activation of TRPV4EC channels was abolished by protein kinase C (PKC) inhibition, and was absent in the arteries from Cav‐1EC−/− mice, supporting a Cav‐1EC‐PKC‐TRPV4EC signaling that enables ATP activation of TRPV4‐EC channels. Resting RVSP and PAP were elevated in Cav‐1EC−/− and TRPV4EC−/− mice supporting the concept that Cav‐1EC‐TRPV4EC signaling lowers resting PAP. Moreover, exposure to chronic hypoxia (CH, 3 weeks) resulted in higher increases in RVSPs in Cav‐1EC−/− and TRPV4EC−/− mice when compared to the respective wild‐type control mice, suggesting that Cav‐1EC‐TRPV4EC channel signaling is protective against CH‐induced PH. We further used two well‐established models of PH— CH and sugen 5416 injection + CH. TRPV4EC channel activity, and ATP‐ and PKC‐activation of TRPV4EC channels were impaired in PAs from both the models of PH, suggesting that Cav‐1EC‐PKC‐TRPV4EC signaling may be impaired in PH. Taken together, these results suggest that Cav‐1EC‐PKC‐TRPV4EC signaling lowers resting PAP, and is impaired in PH. Deciphering the mechanisms that impair the Cav‐1EC‐PKC‐TRPV4EC signaling in PH may lead to novel therapeutic strategies to restore endothelium‐dependent vasodilation in PH.Support or Funding InformationHL121484‐01, HL‐138496

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Regulation of ion channels in the microcirculation by mineralocorticoid receptor activation.

The mineralocorticoid receptor (MR) has classically been studied in the renal epithelium for its role in regulating sodium and water balance and, subsequently, blood pressure. However, the MR also plays a critical role in the microvasculature by regulating ion channel expression and function. Activation of the MR by its endogenous agonist aldosterone results in translocation of the MR into the nucleus, where it can act as a transcription factor. Although most of the actions of the aldosterone can be attributed to its genomic activity though MR activation, it can also act by nongenomic mechanisms. Activation of this ubiquitous receptor increases the expression of epithelial sodium channels (ENaC) in both the endothelium and smooth muscle cells of peripheral and cerebral vessels. MR activation also regulates activity of calcium channels, calcium-activated potassium channels, and various transient receptor potential (TRP) channels. Modification of these ion channels results in a myriad of negative consequences, including impaired endothelium-dependent vasodilation, alterations in generation of myogenic tone, and increased inflammation and oxidative stress. Taken together, these studies demonstrate the importance of studying the impact of the MR on ion channel function in the vasculature. While research in this area has made advances in recent years, there are still many large gaps in knowledge that need to be filled. Crucial future directions of study include defining the molecular mechanisms involved in this interaction, as well as elucidating the potential sex differences that may exist, as these areas of understanding are currently lacking.