Acetylcholine-induced Endothelium-dependent Relaxation Research Articles

In utero exposure to maternal diabetes exacerbates dietary sodium intake-induced endothelial dysfunction by activating cyclooxygenase 2-derived prostanoids.

Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2+/C96Y mice (Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E2 (PGE2) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE2. This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood.NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E2, and inflammation.

(247) A Rat Model for Bipolar Androgen Therapy is Evaluated for Its Effects on Erectile Dysfunction after Castration

Abstract Introduction Prostate cancer (PCa), an androgen-dependent tumor and one of the most prevalent cancers in males, has traditionally been treated with androgen suppression. In recent years, bipolar androgen treatment (BAT) has emerged as a potential therapeutic approach for men with metastatic castrate-resistant PCa. The control of testosterone (T) between castration and supraphysiological levels inhibits the proliferation of cancer cells and slows the progression of cancer. Furthermore, while there has been prior research between T and erectile function, there is no evidence linking BAT and erectile dysfunction (ED). Objective The purpose of the current investigation was to determine if BAT therapy was effective in treating ED in a castration-prone rat model. Methods Sprague-Dawley rats (n=30) were randomly divided into four groups: 1) Control, 2) Control rats-received BAT treatment, 3) castrated Rats, and 4) castrated rats-received BAT treatment. The rats were castrated by making bilateral scrotal incisions. A subcutaneous injection of testosterone propionate (250 mg/kg/week, intramuscular) was administered four weeks following castration. Four weeks after the initial dose, the second dose of the medication was given in the same manner. By stimulating the cavernous nerve, the intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio and total ICP were determined. Corpus cavernosum (CC) strips were used as in vitro preparations for evaluating the relaxant and contractile responses. Nitric oxide synthase (nNOS) and endothelial NOS (eNOS) protein expression was analyzed using Western blotting. Using Masson trichrome staining, the location of the smooth muscle in relation to the collagen was identified. Results Castrated rats' prostate weight was reduced when compared to uncastrated rats (P< 0.001). Compared to controls and castrated rats, BAT marginally boosted prostate weight reduction. The ICP/MAP ratio and total ICP for 7.5 V were lowest in castrated rats (P< 0.001 vs. controls). BAT enhanced ICP/MAP and total ICP responses (P<0.01 vs. castrated rats). In vitro, endothelium-dependent acetylcholine-induced relaxation at 1 nM (P 0.05 vs controls), endothelium-independent sodium nitroprusside-induced relaxation at 1 nM (P 0.01 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P<0.05 vs. controls) and sildenafil-induced relaxation at 10 M (P 0.05 vs controls) were all reduced in the CC strips from the castrated group. Additionally, the castrated group showed increased neurogenic contractile response at 40 Hz and the contractile response to phenylephrine at 1 mM (P 0.001 vs. controls). The variations in the relaxation and contractile responses were returned by BAT. The smooth muscle cell-to-collagen ratio and nNOS and eNOS protein expression were both markedly reduced in castrated rats, though BAT reversed these modifications. Conclusions The results of the study show that BAT improves neurogenic relaxation, endothelium relaxation, sildenafil-induced relaxing of the CC, and changes in penile morphology after castration. BAT might be a cutting-edge therapeutic approach for patients with castration-resistant prostate cancer who have an ED route. Disclosure No.

Evaluation of the component’s contribution in endothelium-dependent acetylcholine-induced relaxation of the rat aorta

The regulation of rat aorta vascular tone involves various factors, including endothelium-derived hyperpolarization factor (EDHF), nitric oxide (NO), prostaglandins, and sensory nerves. While these elements can function independently, their pathways intersect at various points, complicating the assessment of their individual contributions. The aim of this study was to establish the numerical contributions of EDHF, NO, prostaglandins, and also the effect of the sensory nerve on acetylcholine-induced relaxation on the background of phenylephrine preconstriction using contraction and relaxation measurements in Wistar rat thoracic aorta. EDHF, whose action is mediated through potassium channels, emerges as a crucial regulator. Blockage of inward rectifier potassium (KIR) channels integral to EDHF significantly abolishes 50% of the relaxation amplitude in comparison to control conditions. Endothelial TRPV4 channel, exhibiting a fine-tuning role, contributes to a 25% reduction in the amplitude of acetylcholine-induced relaxation in comparison to control relaxation. NO demonstrates its vasodilatory prowess, with NO blockage eliminating 77% of the residual relaxation effect after KIR blockage. Blockage of prostaglandin functions, modulated by cyclooxygenase 1, reduces relaxation by 44% in comparison to control relaxation. Desensitization of sensory nerves with capsaicin, shows a minor yet significant role, in the reduction of acetylcholine-induced relaxation amplitude by 10%. In conclusion, we established that the main element of acetylcholine-induced relaxation is EDHF with approximately 50% of relaxation amplitude depending on it.

Portulaca Oleracea L. (Purslane) Extract Protects Endothelial Function by Reducing Endoplasmic Reticulum Stress and Oxidative Stress through AMPK Activation in Diabetic Obese Mice.

Portulaca oleracea L. (purslane) is a food and a traditional drug worldwide. It exhibits anti-inflammatory, anti-oxidative, anti-tumor, and anti-diabetic bioactivities; but its activity on diabetic-associated endothelial dysfunction is unknown. This study aimed to investigate the effect of purslane on endothelial function and the underlying mechanisms. Male C57BL/6 mice had 14-week ad libitum access to a high-fat rodent diet containing 60% kcal% fat to induce obesity and diabetes whereas purslane extract (200 mg/kg/day) was administered during the last 4 weeks via intragastric gavage. Primary rat aortic endothelial cells and isolated mouse aortas were cultured with a risk factor, high glucose or tunicamycin, together with purslane extract. By ESI-QTOF-MS/MS, flavonoids and their glycoside products were identified in the purslane extract. Exposure to high glucose or tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations in aortas and induced endoplasmic reticulum (ER) stress and oxidative stress with the downregulation of 5' AMP-activated protein kinase (AMPK)/ endothelial nitric oxide synthase (eNOS) signaling. Co-incubation with purslane significantly ameliorated these impairments. The effects of purslane were abolished by Compound C (AMPK inhibitor). Four-week purslane treatment ameliorated aortic relaxations, ER stress, and oxidative stress in diabetic obese mice. This study supported that purslane protected endothelial function, and inhibited ER stress and oxidative stress in vasculature through AMPK/eNOS activation, revealing its therapeutic potential against vascular complications in diabetes.

Hyperandrogenism Protects Against High Blood Pressure by Nongenomic Mechanisms and Obesity Causes Hypertension in Females with Polycystic Ovary Syndrome

ABSTRACT Background Androgens induce vasorelaxation and reduce blood pressure in different mammals, including humans. Most women with polycystic ovary syndrome (PCOS), with hyperandrogenism, are obese and exhibit hypertension; thus, the fact that androgens increase blood pressure (BP) is controversial. Our aim was to determine whether hypertension is produced by androgen excess and/or obesity. Methods Experiments were performed in dehydroepiandrosterone; (DHEA, s.c)-induced PCOS model. BP from nonobese and obese rats with PCOS (fed a normal or high-fat diet, respectively) was evaluated weekly for 10 weeks by plethysmography and compared between them. We determined whether androgen receptors are responsible for androgen action on BP in rats with PCOS; a group of DHEA-treated rats was implanted with pellets of an antiandrogen and was compared with nonobese rats with PCOS. Isometric tension from aortas of nonobese and obese rats was recorded and compared to explore the integrity of the vascular endothelium when acetylcholine-induced endothelium-dependent vascular relaxation on phenylephrine contraction. Additionally, BP was obtained from 30 women diagnosed with PCOS: nonobese (BMI ≤25) and obese women (BMI ≥35) and compared with healthy counterparts; 15 obese and 15 nonobese women. Results Nonobese rats and women with PCOS showed hypotension, while obese rats and women with PCOS displayed hypertension. Healthy obese women were hypertensive and nonobese women remained normotensive. Antiandrogen did not modify the BP values in nonobese rats with PCOS, and obese rats with PCOS revealed marked endothelial dysfunction. Conclusions Our findings show that obesity is responsible for hypertension in PCOS and partial endothelial damage was observed, which may contribute to elevated BP. Remarkably, hyperandrogenism is capable of regulating BP to low values that are androgen receptor-independent.

Ghrelin improves endothelial function and reduces blood pressure in Ang II-induced hypertensive mice: Role of AMPK

ABSTRACT Background Endothelial dysfunction is a major pathophysiology observed in hypertension. Ghrelin, a key regulator of metabolism, has been shown to play protective roles in cardiovascular system. However, whether it has the effect of improving endothelial function and lowering blood pressure in Ang II-induced hypertensive mice remains unclear. Methods In this study, hypertension was induced by continuous infusion of Ang II with a subcutaneous osmotic pumps and ghrelin (30 μg/kg/day) was intraperitoneal injection for 4 weeks. Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph and superoxide production in mouse aortae was assessed by fluorescence imaging. Results We found that ghrelin had protective effects on Ang II-induced hypertension by inhibiting oxidative stress, increasing NO production, improving endothelial function, and lowering blood pressure. Furthermore, ghrelin activated AMPK signaling in Ang II-induced hypertension, leading to inhibition of oxidative stress. Compound C, a specific inhibitor of AMPK, reversed the protective effects of ghrelin on the reduction of oxidative stress, the improvement of endothelial function and the reduction of blood pressure. Conclusions our findings indicated that ghrelin protected against Ang II-induced hypertension by improving endothelial function and lowering blood pressure partly through activating AMPK signaling. Thus, ghrelin may be a valuable therapeutic strategy for hypertension.

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.

17α-ethinylestradiol modulates endothelial function in ovariectomized rat carotid arteries

17α-ethinylestradiol (EE2), a derivative of 17β-estradiol (E2), is a potent estrogenic substance that is used as the estrogenic component of oral contraceptives (OCPs). However, women who take OCPs have an increased risk of cardiovascular events. Since few studies have examined EE2 endothelial effects, we explored the effects of EE2 on endothelial function in ovariectomized and isoflavone-free rats. After ovariectomy, 12-week-old female Sprague-Dawley rats were assigned to EE2, E2 or control groups. After 16 weeks, the EE2 and E2 groups were orally administered EE2 (8.3 μg/day) and E2 (12.6 μg/day) for 4 weeks, respectively. At 18 weeks, endothelial denudation of the left common carotid arteries was performed, and they were harvested at 20 weeks. The rats in the EE2 and E2 groups exhibited significantly decreased body weights and significantly increased uterine weights, respectively, but no differences were observed between the EE2 and E2 groups. The EE2 and E2 groups showed significantly enhanced acetylcholine-induced endothelium-dependent relaxation, with apamin plus charybdotoxin inhibiting only the EE2 group. Endothelial nitric oxide (NO) synthase expression was significantly higher in the EE2 group than in the control, but lower than in the E2 group. The intima-to-media ratio of denuded arteries was significantly lower in the E2 group than in the other groups, suggesting that NO decreased in the EE2 group compared to the E2 group. We conclude that EE2 has a weaker ability than E2 to produce NO and, for the first time, we demonstrate the ability of EE2 to enhance the activity of endothelial-derived hyperpolarizing factor.

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.

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.

Overexpression of miR-210 ameliorates endothelial dysfunction in type 2 diabetic mice

Abstract Background MicroRNA (miR)-210 plays a protective role in many cardiometabolic diseases, and its levels are reduced in whole blood, erythrocytes, and plasma in type 2 diabetes mellitus (T2DM). Our recent study demonstrated that miR-210 is downregulated in carotid artery plaques from patients with T2DM compared to non-diabetic patients. However, the functional role of miR-210 in endothelial dysfunction is not fully understood. Purpose This study aimed to investigate the potential therapeutic value of miR-210 overexpression against T2DM-associated endothelial dysfunction. Methods miR-210 transgenic mice (TG) at age of 8 weeks were subjected to Western Diet (WD) for 12 weeks. During the last 10 days of the diet regime, doxycycline or vehicle dissolved in drinking water was given to switch on miR-210 (miR-210/on) expression or keep it off (miR-210/off), respectively. Age-matched wild-type (WT) control mice received normal chow through the same period. Additionally, WT and T2DM db/db mice at age of 15–20 weeks received tail vein injections of miR-210 mimic or miR-210 scramble oligonucleotides. All animals were euthanized at the end of the treatment or 72h after the i.v. injections for determination of endothelial function by acetylcholine-induced endothelium-dependent relaxation (EDR) of isolated aortic segments from all groups of mice using wire myographs. The expression of protein tyrosine phosphatase 1B (PTP1B; a miR-210 target protein) and the levels of 4-hydroxynonenal (4-HNE; an oxidative stress marker) were measured in aortic segments by immunohistochemistry. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results EDR in aortic segments from miR-210/off TG mice fed WD and T2DM db/db mice injected with miR-210 scramble was significantly impaired compared to vessels from WT controls fed with regular chow (Fig. 1A, B). Of note, EDR was markedly improved or even restored in aortae from miR-210/on TG mice treated with WD and T2DM db/db mice injected with miR-210 mimic (Fig. 1A, B). Furthermore, the expression of PTP1B and the levels of 4-HNE, which is formed by lipid peroxidation, were significantly elevated in the aortae from miR-210/off TG mice treated with WD when compared to WT controls (Fig. 2A–D). The expression was attenuated in aortae of miR-210/on TG mice treated with WD compared to miR-210/off TG mice (Fig. 2A–D). There was a significant increase in the expression of PTP1B and a trend to increased 4-HNE levels in aortae of db/db mice injected with miR-210 scramble vs. control mice (Fig. 2E–H). A significant reduction in PTP1B but not 4-HNE was observed in db/db mice injected with miR-210 mimic (Fig. 2E–H). Conclusion Genetic and pharmacological overexpression of miR-210 ameliorates endothelial dysfunction in mice fed with WD and db/db mice. Increasing miR-210 levels may become a potential treatment strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation

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.

3,4',5-Trimethoxy-trans-stilbene Alleviates Endothelial Dysfunction in Diabetic and Obese Mice via Activation of the AMPK/SIRT1/eNOS Pathway.

3,4′,5-trimethoxy-trans-stilbene (TMS) is a methoxylated derivative of resveratrol. Previous studies showed the vaso-protective effects of resveratrol; nevertheless, research on this derivative is scarce. The current study aimed to explore whether TMS can alleviate endothelial dysfunction in diabetic and obese mice, along with the underlying mechanisms. Thoracic aortas isolated from male C57BL/6J mice and primary cultures of rat aortic endothelial cells were treated with high glucose with or without TMS. High glucose exposure impaired acetylcholine-induced endothelium-dependent relaxations, down-regulated NO bioavailability and the AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1)/endothelial nitric oxide synthase (eNOS) pathway, increased endoplasmic reticulum (ER) stress and oxidative stress, which were reversed by TMS treatment. Moreover, the protective effects of TMS were abolished by Compound C (AMPK inhibitor), and EX527 (SIRT1 inhibitor). The mice were fed with high-fat diet (60% kcal% fat) for 14 weeks to establish a diabetic and obese model, and were orally administered TMS (10 mg/kg/day) in the last 4 weeks. Chronic TMS treatment alleviated endothelial dysfunction via enhancing the AMPK/SIRT1/eNOS pathway and attenuated oxidative stress and ER stress in aortas of diet-induced obese mice. In summary, our study reveals the potent vaso-protective effect of TMS and its therapeutic potential against endothelial dysfunction in metabolic disorders.

Loss of Endothelial Cytochrome P450 Reductase Induces Vascular Dysfunction in Mice.

POR (cytochrome P450 reductase) provides electrons for the catalytic activity of the CYP (cytochrome P450) monooxygenases. CYPs are dual-function enzymes as they generate protective vasoactive mediators derived from polyunsaturated fatty acids but also reactive oxygen species. It is not known in which conditions the endothelial POR/CYP system is beneficial versus deleterious. Here, the activity of all CYP enzymes was eliminated in the vascular endothelium to examine its impact on vascular function. An endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-) was generated. Vascular function was studied by organ chamber experiments. eNOS (endothelial nitric oxide synthase) activity was accessed by heavy arginine/citrulline LC-MS/MS detection and phosphorylation of serine1177 in aortic rings. CYP-derived epoxyeicosatrienoic acids and prostanoids were measured by LC-MS/MS. Gene expression of aorta and endothelial cells was profiled by RNA sequencing. Blood pressure was measured by telemetry. Acetylcholine-induced endothelium-dependent relaxation was attenuated in isolated vessels of ecPOR-/- as compared with control mice. Additionally, ecPOR-/- mice had attenuated eNOS activity and eNOS/AKT phosphorylation. POR deletion reduced endothelial stores of CYP-derived epoxyeicosatrienoic acids but increased vascular prostanoids. This phenomenon was paralleled by the induction of genes implicated in eicosanoid generation. In response to Ang II (angiotensin II) infusion, blood pressure increased significantly more in ecPOR-/- mice. Importantly, the cyclooxygenase inhibitor Naproxen selectively lowered the Ang II-induced hypertension in ecPOR-/- mice. POR expression in endothelial cells maintains eNOS activity and its loss results in an overactivation of the vasoconstrictor prostanoid system. Through these mechanisms, loss of endothelial POR induces vascular dysfunction and hypertension.

TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

The role of the Ca2+-permeable ion channel TRPC5 in regulating vasocontraction in obesity is poorly understood. Here, we investigated whether TRPC5 contributes to vascular dysfunction in obesity by promoting endothelium-dependent contraction via activation of cytosolic phospholipase A2 (cPLA2) in the aortic endothelial cells of obese mice. Acetylcholine-induced endothelium-dependent relaxation and contraction in the aorta were measured using wire myography. PLA2 activity was measured by the fluorogenic PLA2 substrate Bis-BODIPY™ FL C11-PC. The intracellular Ca2+ level in response to acetylcholine was measured by Fluo-4 fluorescence. Endothelium-derived contracting factors were assessed by enzyme immunoassay. Diet-induced obesity (DIO) attenuated endothelium-dependent vasodilation, enhanced endothelium-dependent contraction (EDC), and increased the expression of TRPC5 in the mouse aorta. Activation of TRPC5 promoted EDC in the wild-type mouse aorta, whereas pharmacological inhibition and genetic knockout of TRPC5 decreased EDC in the DIO mouse aorta. Moreover, cPLA2 phosphorylation and activity were higher in aortic endothelial cells from DIO mice, and this was attenuated by inhibition and knockout of TRPC5. Cyclooxygenase 2 (COX-2) expression was increased in DIO mouse endothelium and was decreased by a TRPC5 inhibitor and knockout of TRPC5. Release of prostaglandins F2α (PGF2α) and E2 (PGE2) was involved in TRPC5-regulated EDC in DIO mice. This study demonstrated that TRPC5 contributes to endothelial and vascular dysfunction and is involved in EDC through activation of cPLA2 and enhanced COX-2-PGF2α/PGE2 levels in DIO mice.

Del Nido cardioplegia better preserves cardiac diastolic function but histidine-tryptophan-ketoglutarate is better for endothelial function.

The effectiveness of myocardial protection of cardioplegia has been a matter of debate for decades. This study was designed to compare cardiac and endothelial protection of 3 clinically used cardioplegias: del Nido cardioplegia (DNC), histidine-tryptophan-ketoglutarate (HTK) and blood cardioplegia (BC) followed by HTK (BC + HTK) in a rat model of ischaemia/reperfusion (I/R). Sixty male Wistar rats were subjected to either 120 min of global ischaemia at 4°C followed by 90 min of reperfusion (I/R) at 37°C or no I/R (control) in a Langendorff apparatus and were randomly allocated to 5 groups: control, I/R, DNC, HTK and BC + HTK. Cold cardioplegia solutions were administered at doses of 20 ml/kg for DNC and HTK or 10 ml/kg for BC followed by HTK. Haemodynamic parameters were continuously recorded using an intraventricular balloon. The endothelium-dependent relaxation to acetylcholine was measured in the left anterior descending artery using a myograph. Protein expression of cardiac troponin T (cTnT) and creatine kinase MB was determined by western blot. During reperfusion, HTK had higher left ventricular systolic pressure whereas DNC had lower left ventricular end-diastolic pressure, better left ventricular developed pressure and best +dp/dtmax and -dp/dtmax than the other 2 groups but the differences disappeared at the end of the reperfusion. HTK or BC + HTK preserves the acetylcholine-induced endothelium-dependent relaxation better than DNC (Emax = 48.2 ± 8.0% in DNC vs 75.0 ± 8.0% in HTK, P < 0.05; vs 96.9 ± 3.5% in BC + HTK, P < 0.001). The protein levels of cTnT and creatine kinase MB were downregulated in the 3 groups. All 3 cardioplegias prevented myocardial damage against I/R injury at the end of reperfusion. DNC demonstrated better preserved diastolic function of the left ventricle whereas HTK or BC + HTK showed better preserved coronary endothelial function. These findings may suggest that currently no 'perfect' cardioplegia exists and that exploration for the 'perfect' cardioplegia is needed.

COMP (Cartilage Oligomeric Matrix Protein), a Novel PIEZO1 Regulator That Controls Blood Pressure.

Vascular endothelial cells are critical for maintaining blood pressure (BP) by releasing biologically active molecules, such as nitric oxide. A non-endothelial cell resident matricellular protein, COMP (cartilage oligomeric matrix protein), plays a pivotal role in maintaining cardiovascular homeostasis, but little is known about its regulatory effect on BP. Mice were infused with AngII (angiotensin II; 450 ng/kg per minute) for 3 days via an osmotic minipump, and BP was monitored by a tail-cuff system. Second-order mesenteric arteries were isolated from mice for microvascular tension measurement. Nitric oxide was detected by an electron paramagnetic resonance technique. Small-interfering RNA transfection, co-immunoprecipitation, bioluminescence resonance energy transfer assays, and patch-clamp electrophysiology experiments were used for further detailed mechanism investigation. COMP-/- mice displayed elevated BP and impaired acetylcholine-induced endothelium-dependent relaxation compared with wild-type mice with or without AngII. Inhibition of eNOS (endothelial nitric oxide synthase) abolished the difference in endothelium-dependent relaxation between wild-type and COMP-/- mice. Furthermore, COMP directly interacted with the C-terminus of Piezo1 via its C-terminus and activated the endogenous Piezo1 currents, which induced intracellular Ca2+ influx, Ca2+/calmodulin-dependent protein kinase type II and eNOS activation, and nitric oxide production. The Piezo1 activator, Yoda1, reduced the difference in endothelium-dependent relaxation and BP in wild-type and COMP-/- mice. Moreover, COMP overexpression increased eNOS activation and improved endothelium-dependent relaxation and BP. Our study demonstrated that COMP is a novel Piezo1 regulator that plays a protective role in BP regulation by increasing cellular Ca2+ influx, eNOS activity, and nitric oxide production.

Vitamin D deficiency attenuates endothelial function by reducing antioxidant activity and vascular eNOS expression in the rat microcirculation.

This study examined the effects of vitamin D deficiency on vascular function and tissue oxidative status in the microcirculation; and whether or not these effects can be ameliorated with calcitriol, the active vitamin D metabolite. Three groups (n=10 each) of male Sprague Dawley rats were fed for 10weeks with control diet (CR), vitamin D-deficient diet without (DR), or with oral calcitriol supplementation (0.15μg/kg) for the last four weeks (DSR). After 10weeks, rats were sacrificed; mesenteric arterial rings were studied using wire myograph. Oxidative stress biomarkers malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were measured in the mesenteric arterial tissue. Vascular protein expression of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Acetylcholine-induced endothelium-dependent relaxation of DR was lower than CR. eNOS expression and SOD activity were lower in mesenteric arterial tissue of DR compared to CR. Calcitriol supplementation to DSR did not ameliorate the above parameters; in fact, augmented endothelium-dependent contraction was observed. Serum calcium was higher in DSR compared to CR and DR. In conclusion, vitamin D deficiency impaired microvascular vasodilation, associated with eNOS downregulation and reduced antioxidant activity. Calcitriol supplementation to vitamin D-deficient rats at the dosage used augmented endothelium-dependent contraction, possibly due to hypercalcaemia.

Panax Notoginseng Protects against Diabetes-Associated Endothelial Dysfunction: Comparison between Ethanolic Extract and Total Saponin.

Previous studies revealed a cardioprotective potential of Panax notoginseng to relieve acute myocardial infarction and focal cerebral ischemia-reperfusion. However, whether P. notoginseng protects endothelial function in diabetes and the underlying mechanisms remain to be explored. P. notoginseng contains several chemical components including saponins, which are commonly believed as the major bioactive ingredients. The present study was aimed to examine and compare the vaso-protective effects of the ethanolic extract of P. notoginseng (PNE) and total saponin (PNS). Both aortas and carotid arteries were isolated from male C57BL/6J mice for ex vivo treatment with risk factors (high glucose or tunicamycin) with and without the presence of PNS and PNE. Diabetic model was established by feeding the mice with a high-fat diet (45% kcal% fat) for 12 weeks, while PNS and PNE were administrated by oral gavage at 20 mg/kg/day for another 4 weeks. Ex vivo exposure to high glucose impaired acetylcholine-induced endothelium-dependent relaxations in mouse aortas, decreased phosphorylation of AMPK and eNOS, and induced endoplasmic reticulum (ER) stress and oxidative stress. These effects were reversed by cotreatment of PNS and PNE with PNS being more potent. Furthermore, the vaso-protective effects were abolished by Compound C (AMPK inhibitor). Chronic treatment with PNS and PNE improved endothelium-dependent relaxations and alleviated ER stress and oxidative stress in aortas from high-fat diet-induced obese mice. PNE was more effective to improve glucose sensitivity and normalize blood pressure in diabetic mice. The present results showed that PNS and PNE reduced ER stress and oxidative stress and, subsequently, improved endothelial function in diabetes through AMPK activation. This study provides new inspiration on the therapeutic potential of P. notoginseng extract against vascular diseases associated with metabolic disorders.

Abstract MP16: Perivascular Adipose Tissue Inflammation Impairs Microvascular Endothelial Function In People Living With HIV

Introduction and hypothesis: We had reported that people living with HIV (PLWH) have microvascular endothelial dysfunction and increased ROS. We now tested the hypothesis that perivascular adipose tissue (PVAT) could enhance oxidative stress and inflammation and impair the function of subcutaneous microarterioles (SMAs) in PLWH. Methods: SMAs were obtained from young, virally-suppressed HIV-infected subjects (n=11) or matched controls (n=11). Subjects were without associated CVD risk factors. Microvascular reactivity and PVAT function were accessed by myograph from isolated subcutaneous vessels with or without PVAT of skin biopsy. Results: The HIV-infected group had significantly (P&lt;0.05) increased adipose MDA, TNFα, IL-1α, leptin and reduced adiponectin. The PVAT-denuded SMAs from the HIV group had significantly (P&lt;0.05) impaired acetylcholine-induced endothelium-dependent relaxation factor (EDRF, 26±4 vs 38±3%) and NO activity (0.35±0.03 vs 0.58± 0.07 Δfluoresence unit) and significantly (P&lt;0.05) increased contraction to U-46,619 (200±8 vs 141±7%) and endothelin 1 (ET1, 167±12 vs 118±17%) and ROS generation (0.32 ± 0.06 vs 0.1 ± 0.03 (E/DHE fluoresce unit). PVAT enhanced EDRF (50±4 vs 38±3 %) and NO (0.84 ±0.1 vs 0.58±0.07 Δfluoresence unit ) only in controls (P&lt;0.05). The reduction of U46,619 anti-contractivity by PVAT is decreased in HIV (48±7 vs 85±9%, P&lt;0.05). Conclusion: HIV-infected individuals have intrinsic vascular defects from ROS augmented by extrinsic vascular defects from adipose inflammation that impairs the beneficial microvascular PVAT signaling. Therefore, targets for potential prevention of cardiovascular morbidity in PLWH should include the elimination of ROS and inflammation in both microvessels themselves and the surrounding extravascular PVAT.