SUMOylation and coupling of eNOS mediated by PIAS1 contribute to maintenance of vascular homeostasis.

Endothelial dysfunction (ED) is commonly considered a crucial initiating step in the pathogenesis of numerous cardiovascular diseases. The coupling of endothelial nitric oxide synthase (eNOS) is important in maintaining normal endothelial functions. However, it still remains elusive whether and how eNOS SUMOylation affects the eNOS coupling. In the study, we investigate the roles and possible action mechanisms of protein inhibitor of activated STAT 1 (PIAS1) in ED. Human umbilical vein endothelial cells (HUVECs) treated with palmitate acid (PA) in vitro and ApoE-/- mice fed with high-fat diet (HFD) in vivo were constructed as the ED models. Our in vivo data show that PIAS1 alleviates the dysfunction of vascular endothelium by increasing nitric oxide (NO) level, reducing malondialdehyde (MDA) level, and activating the phosphatidylinositol 3-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-AKT-eNOS) signaling in ApoE-/- mice. Our in vitro data also show that PIAS1 can SUMOylate eNOS under endogenous conditions; moreover, it antagonizes the eNOS uncoupling induced by PA. The findings demonstrate that PIAS1 alleviates the dysfunction of vascular endothelium by promoting the SUMOylation and inhibiting the uncoupling of eNOS, suggesting that PIAS1 would become an early predictor of atherosclerosis and a new potential target of the hyperlipidemia-related cardiovascular diseases.

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein.

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/ß. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-ß, -7, -ß/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.

Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (rs1051740) and Enos (rs1799983).

Recurrent pregnancy loss (RPL) affects around 2% of women of reproductive age. Primary RPL is defined by ≥2 pregnancy losses and no normal birth delivery. In secondary RPL, the losses are after a normal pregnancy and delivery. Most cases have no clear aetiology, although primary cases are the most complex. Several gene single nucleotide polymorphisms (SNPs) have been associated with RPL. The frequency of some SNPs is increased in women suffering from RLP from Asian or Caucasian races; however, in admixed populations, the information on possible genetic links is scarce and contradictory. This study aimed to assess the frequency of two SNPs present in two different enzymes involved in medical conditions observed during pregnancy. It is a case-control study. Microsomal epoxy hydrolase (mEPH) is involved in detoxifying xenobiotics, is present in the ovaries, and is hormonally regulated. The endothelial nitric oxide synthase (NOS3) that forms nitric is involved in vascular tone. Two SNPs, rs1051740 (mEPH) and rs1799983 (NOS3), were assessed. The study included 50 controls and 63 primary RPL patients. The frequency of mutated alleles in both SNPs was significantly higher in patients (p < 0.05). Double-mutated homozygotes were encountered only in RPL patients (p < 0.05). Genetic polymorphisms rs1051740 and rs1799983 may be involved in primary RPL in the Venezuelan admix population. Genetic studies could provide crucial information on the aetiology of primary RPL.

ZFYVE21 promotes endothelial nitric oxide signaling and vascular barrier function in the kidney during aging.

ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.

Myoendothelial feedback in mouse mesenteric resistance arteries is similar between the sexes, dependent on nitric oxide synthase, and independent of TPRV4.

Myoendothelial feedback (MEF), the endothelium-dependent vasodilation following sympathetic vasoconstriction (mediated by smooth muscle to endothelium gap junction communication), has been well studied in resistance arteries of males, but not females. We hypothesized that MEF responses would be similar between the sexes, but different in the relative contribution of the underlying nitric oxide and hyperpolarization mechanisms, given that these mechanisms differ between the sexes in agonist-induced endothelium-dependent dilation. We measured MEF responses (diameter changes) of male and female first- to second-order mouse mesenteric arteries to phenylephrine (10 µM) over 30 min using isolated pressure myography ± blinded inhibition of nitric oxide synthase (NOS) using Nω-nitro-l-arginine methyl ester (l-NAME; 0.1-1.0 mM), hyperpolarization using 35 mM KCl, or transient receptor potential vanilloid 4 (TRPV4) channels using GSK219 (0.1-1.0 µM) or RN-1734 (30 µM). MEF was similar [%dilation (means ± SE): males = 26.7 ± 2.0 and females = 26.1 ± 1.9 at 15 min] and significantly inhibited by l-NAME (1.0 mM) at 15 min [%dilation (means ± SE): males = 8.2 ± 3.3, P < 0.01; females = 6.8 ± 1.9, P < 0.001] and over time (P < 0.01) in both sexes. l-NAME (0.1 mM) + 35 mM KCl nearly eliminated MEF in both sexes (P < 0.001-0.0001). Activation of TRPV4 with GSK101 (0.1-10 µM) induced similar dilation between the sexes. Inhibition of TRPV4, which is reportedly involved in the hyperpolarization mechanism, did not inhibit MEF in either sex. Similar expression of eNOS was found between the sexes with Western blot. Thus, MEF is prominent and similar in murine first- and second-order mesenteric resistance arteries of both sexes, and reliant primarily on NOS and secondarily on hyperpolarization, but not TRPV4.NEW & NOTEWORTHY We found that female mesenteric resistance arteries have similar postconstriction dilatory responses (i.e., myoendothelial feedback) to a sympathetic neurotransmitter analog as male arteries. Both sexes use nitric oxide synthase (NOS) and hyperpolarization, but not TRPV4, in this response. Moreover, the key protein involved in this pathway (eNOS) is similarly expressed in these arteries between the sexes. These similarities are surprising given that agonist-induced endothelium-dependent dilatory mechanisms differ in these arteries between the sexes.

Gestational intermittent hypoxia induces endothelial dysfunction and hypertension in pregnant rats: role of endothelin type B receptor†.

Obstructive sleep apnea is a recognized risk factor for gestational hypertension, yet the exact mechanism behind this association remains unclear. Here, we tested the hypothesis that intermittent hypoxia, a hallmark of obstructive sleep apnea, induces gestational hypertension through perturbed endothelin-1 signaling. Pregnant Sprague-Dawley rats were subjected to normoxia (control), mild intermittent hypoxia (10.5% O2), or severe intermittent hypoxia (6.5% O2) from gestational days 10-21. Blood pressure was monitored. Plasma was collected and mesenteric arteries were isolated for myograph and protein analyses. The mild and severe intermittent hypoxia groups demonstrated elevated blood pressure, reduced plasma nitrate/nitrite, and unchanged endothelin-1 levels compared to the control group. Western blot analysis revealed decreased expression of endothelin type B receptor and phosphorylated endothelial nitric oxide synthase, while the levels of endothelin type A receptor and total endothelial nitric oxide synthase remained unchanged following intermittent hypoxia exposure. The contractile responses to potassium chloride, phenylephrine, and endothelin-1 were unaffected in endothelium-denuded arteries from mild and severe intermittent hypoxia rats. However, mild and severe intermittent hypoxia rats exhibited impaired endothelium-dependent vasorelaxation responses to endothelin type B receptor agonist IRL-1620 and acetylcholine compared to controls. Endothelium denudation abolished IRL-1620-induced vasorelaxation, supporting the involvement of endothelium in endothelin type B receptor-mediated relaxation. Treatment with IRL-1620 during intermittent hypoxia exposure significantly attenuated intermittent hypoxia-induced hypertension in pregnant rats. This was associated with elevated circulating nitrate/nitrite levels, enhanced endothelin type B receptor expression, increased endothelial nitric oxide synthase activation, and improved vasodilation responses. Our data suggested that intermittent hypoxia exposure during gestation increases blood pressure in pregnant rats by suppressing endothelin type B receptor-mediated signaling, providing a molecular mechanism linking intermittent hypoxia and gestational hypertension.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored. METHODS: The expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured. RESULTS: Results showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1. CONCLUSIONS: NCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

New advances in the protective mechanisms of acidic pH after ischemia: Participation of NO.

BACKGROUND: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. OBJECTIVE: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. RESULTS: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. CONCLUSIONS: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

A low testosterone level impairs erectile function by increasing endocan expression in rat penile corpus cavernosum.

BACKGROUND: The mechanism by which a state of low testosterone leads to erectile dysfunction (ED) has not been determined. Endocan is a novel marker of endothelial function. However, whether endocan is involved in the regulation of erectile function under low testosterone levels remains unclear. AIM: In this study we sought to determine whether a low-testosterone state inhibits erectile function by regulating endocan expression in the endothelial cells of the rat penile corpus cavernosum. METHODS: Thirty-six male Sprague-Dawley rats aged 8 weeks were randomly assigned to 6 groups (n = 6 per group) as follows: (1) control, (2) castration, (3) castration + testosterone treatment (treated with 3 mg/kg testosterone propionate per 2 days), (4) control + transfection (4 weeks after castration, injected with lentiviral vector (1 × 108 transduction units/mL, 10 µL), (5) castration + transfection, or (6) castration + empty transfection. One week after the injection, we measured the maximal intracavernous pressure/mean arterial pressure (ICPmax/MAP), serum testosterone and nitric oxide (NO) levels, and the expression of endocan, phospho-endothelial NO synthase (p-eNOS), eNOS, phospho-protein kinase B (p-AKT), and AKT in the rat penile corpus cavernosum. OUTCOMES: Under a low-androgen state, the expression of endocan in the rat penile corpus cavernosum was significantly increased, which inhibited the AKT/eNOS/NO signaling pathway and resulted in ED. RESULTS: In the castration group, the expression of endocan in the rat penile corpus cavernosum was significantly higher than that in the control group (P < .05). Additionally, the levels of p-AKT/AKT, p-eNOS/eNOS, and NO in the rat penile corpus cavernosum and ICPmax/MAP were significantly lower in the castration group than in the control group (P < .05). In the castration + transfection group compared with the castration group there was a significant decrease in the expression of endocan (P < .05) and an increase in the ratios of p-AKT/AKT, p-eNOS/eNOS, and ICPmax/MAP (P < .05) in the rat penile corpus cavernosum. CLINICAL IMPLICATIONS: Downregulating the expression of endocan in the penile corpus cavernosum may be a feasible approach for treating ED caused by hypoandrogenism. STRENGTHS AND LIMITATIONS: The results of this study indicte that endocan may affect NO levels and erectile function through multiple signaling pathways, but further experiments are needed to clarify the relationship between endocan and androgens. CONCLUSION: A low-testosterone state inhibits the AKT/eNOS/NO signaling pathway by increasing the expression of endocan in the rat penile corpus cavernosum and impairing erectile function in rats. Decreasing the expression of endocan in the penile corpus cavernosum can improve erectile function in rats with low testosterone levels.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Cannabidiol ameliorates lipopolysaccharide-induced cardiovascular toxicity by its antioxidant and anti-inflammatory activity via regulating IL-6, Hif1α, STAT3, eNOS pathway.

BACKGROUND: Systemic inflammation causes several organ damage by activating the intracellular signaling mechanisms. Heart and aorta tissues are the structures mostly affected by this situation. By examining underlying processes, this study sought to determine whether cannabidiol (CBD) may have protective effects against the cardiovascular damage brought on by lipopolysaccharide (LPS). MATERIALS AND METHODS: A total of 32 female rats were randomly allocated to one of four groups: control, lipopolysaccharide (LPS) (5 mg/kg, i.p., single dose), LPS + CBD (5 mg/kg, i.p., single dose), and CBD groups. The rats were killed six hours after receiving LPS, and tissues from the heart and aorta were taken. Histopathological and immunohistochemical analyzes were performed. Oxidative stress was evaluated biochemically by spectrophotometric method. Expression levels of genes were studied by RT-qPCR method. RESULTS: Histopathological analysis of the LPS group showed moderate hyperemia, hemorrhages, edema, inflammation, and myocardial cell damage. There was a slight to moderate increase in Cox-1, G-CSF, and IL-3 immunoexpressions, along with enhanced expressions of IL-6, Hif1α, and STAT3 genes, and decreased expressions of eNOS genes. Additionally, there were increased levels of TOS and decreased TAS levels observed biochemically. CBD treatment effectively reversed and improved all of these observed changes. CONCLUSIONS: CBD protects the heart and aorta against systemic inflammation through its antioxidant and anti-inflammatory activity via regulating IL-6, Hif1α, STAT3, and eNOS intracellular pathways.

Low HDL cholesterol and the eNOS Glu298Asp polymorphism are associated with inducible myocardial ischemia in patients with suspected stable coronary artery disease.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) gene deficiency is known to cause impaired coronary vasodilating capability in animal models. In the general clinical population, the eNOS gene polymorphisms, able to affect eNOS activity, were associated with cardiometabolic risk features and prevalence of coronary artery disease (CAD). AIM: To investigate the association of eNOS Glu298Asp gene polymorphism, cardiometabolic profile, obstructive CAD and inducible myocardial ischemia in patients with suspected stable CAD. METHODS: A total of 506 patients (314 males; mean age 62 ± 9 years) referred for suspected CAD was enrolled. Among these, 325 patients underwent stress ECG or cardiac imaging to assess the presence of inducible myocardial ischemia and 436 patients underwent non-invasive computerized tomography or invasive coronary angiography to assess the presence of obstructive CAD. Clinical characteristics and blood samples were collected for each patient. RESULTS: In the whole population, 49.6% of patients were homozygous for the Glu298 genotype (Glu/Glu), 40.9% heterozygotes (Glu/Asp) and 9.5% homozygous for the 298Asp genotype (Asp/Asp). Obstructive CAD was documented in 178/436 (40.8%) patients undergoing coronary angiography while myocardial ischemia in 160/325 (49.2%) patients undergoing stress testing. Patients with eNOS Asp genotype (Glu/Asp + Asp/Asp) had no significant differences in clinical risk factors and in circulating markers. Independent predictors of obstructive CAD were age, gender, obesity, and low HDL-C. Independent predictors of myocardial ischemia were gender, obesity, low HDL-C and Asp genotype. In the subpopulation in which both stress tests and coronary angiography were performed, the Asp genotype remained associated with increased myocardial ischemia risk after adjustment for obstructive CAD. CONCLUSION: In this population, low-HDL cholesterol was the only cardiometabolic risk determinant of obstructive CAD. The eNOS Glu298Asp gene polymorphism was significantly associated with inducible myocardial ischemia independently of other risk factors and presence of obstructive CAD.

Role of thrombospondin-1 in high-salt-induced mesenteric artery endothelial impairment in rats.

The matrix glycoprotein thrombospondin-1 (THBS1) modulates nitric oxide (NO) signaling in endothelial cells. A high-salt diet induces deficiencies of NO production and bioavailability, thereby leading to endothelial dysfunction. In this study we investigated the changes of THBS1 expression and its pathological role in the dysfunction of mesenteric artery endothelial cells (MAECs) induced by a high-salt diet. Wild-type rats, and wild-type and Thbs1-/- mice were fed chow containing 8% w/w NaCl for 4 weeks. We showed that a high salt diet significantly increased THBS1 expression and secretion in plasma and MAECs, and damaged endothelium-dependent vasodilation of mesenteric resistance arteries in wild-type animals, but not in Thbs1-/- mice. In rat MAECs, we demonstrated that a high salt environment (10-40 mM) dose-dependently increased THBS1 expression accompanied by suppressed endothelial nitric oxide synthase (eNOS) and phospho-eNOS S1177 production as well as NO release. Blockade of transforming growth factor-ß1 (TGF-ß1) activity by a TGF-ß1 inhibitor SB 431542 reversed THBS1 up-regulation, rescued the eNOS decrease, enhanced phospho-eNOS S1177 expression, and inhibited Smad4 translocation to the nucleus. By conducting dual-luciferase reporter experiments in HEK293T cells, we demonstrated that Smad4, a transcription promoter, upregulated Thbs1 transcription. We conclude that THBS1 contributes to endothelial dysfunction in a high-salt environment and may be a potential target for treatment of high-salt-induced endothelium dysfunction.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Azelnidipine protects HL-1 cardiomyocytes from hypoxia/reoxygenation injury by enhancement of NO production independently of effects on gene expression.

It remains to be elucidated whether Ca2+ antagonists induce pharmacological preconditioning to protect the heart against ischemia/reperfusion injury. The aim of this study was to determine whether and how pretreatment with a Ca2+ antagonist, azelnidipine, could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury in vitro. Using HL-1 cardiomyocytes, we studied effects of azelnidipine on NO synthase (NOS) expression, NO production, cell death and apoptosis during H/R. Action potential durations (APDs) were determined by the whole-cell patch-clamp technique. Azelnidipine enhanced endothelial NOS phosphorylation and NO production in HL-1 cells under normoxia, which was abolished by a heat shock protein 90 inhibitor, geldanamycin, and an antioxidant, N-acetylcysteine. Pretreatment with azelnidipine reduced cell death and shortened APDs during H/R. These effects of azelnidipine were diminished by a NOS inhibitor, L-NAME, but were influenced by neither a T-type Ca2+ channel inhibitor, NiCl2, nor a N-type Ca2+ channel inhibitor, ω-conotoxin. The azelnidipine-induced reduction in cell death was not significantly enhanced by either additional azelnidipine treatment during H/R or increasing extracellular Ca2+ concentrations. RNA sequence (RNA-seq) data indicated that azelnidipine-induced attenuation of cell death, which depended on enhanced NO production, did not involve any significant modifications of gene expression responsible for the NO/cGMP/PKG pathway. We conclude that pretreatment with azelnidipine protects HL-1 cardiomyocytes against H/R injury via NO-dependent APD shortening and L-type Ca2+ channel blockade independently of effects on gene expression.

Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.

BACKGROUND: Alamandine is a newly characterized peptide of renin angiotensin system. Our study aims to investigate the osteo-preservative effects of alamandine, explore underlying mechanism and bring a potential preventive strategy for postmenopausal osteoporosis in the future. METHODS: An ovariectomy (OVX)-induced rat osteoporosis model was established for in vivo experiments. Micro-computed tomography and three-point bending test were used to evaluate bone strength. Histological femur slices were processed for immunohistochemistry (IHC). Bone turnover markers and nitric oxide (NO) concentrations in serum were determined with enzyme-linked immunosorbent assay (ELISA). The mouse embryo osteoblast precursor (MC3T3-E1) cells were used for in vitro experiments. The cell viability was analysed with a Cell Counting Kit­8. We performed Alizarin Red S staining and alkaline phosphatase (ALP) activity assay to observe the differentiation status of osteoblasts. Western blotting was adopted to detect the expression of osteogenesis related proteins and AMP-activated protein kinase/endothelial nitric oxide synthase (AMPK/eNOS) in osteoblasts. DAF-FM diacetate was used for semi-quantitation of intracellular NO. RESULTS: In OVX rats, alamandine alleviated osteoporosis and maintained bone strength. The IHC showed alamandine increased osteocalcin and collagen type I α1 (COL1A1) expression. The ELISA revealed alamandine decreased bone turnover markers and restored NO level in serum. In MC3T3-E1 cells, alamandine promoted osteogenic differentiation. Western blotting demonstrated that alamandine upregulated the expression of osteopontin, Runt-related transcription factor 2 and COL1A1. The intracellular NO was also raised by alamandine. Additionally, the activation of AMPK/eNOS axis mediated the effects of alamandine on MC3T3-E1 cells and bone tissue. PD123319 and dorsomorphin could repress the regulating effect of alamandine on bone metabolism. CONCLUSION: Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.

Flavonoid Compounds in Hippophae rhamnoides L. Protect Endothelial Cells from Oxidative Damage Through the PI3K/AKT-eNOS Pathway.

Sea buckthorn, a traditional medicinal plant, has been used for several years in China for the prevention and treatment of various diseases, a practice closely associated with its significant antioxidant activity. The aim of this study was to investigate the protective effects of sea buckthorn flavonoids on vascular endothelial cells in an oxidative stress environment. We isolated and extracted active compounds from sea buckthorn and investigated their impact on endothelial nitric oxide synthase (eNOS) activity through the PI3K/AKT-eNOS signaling pathway through a combination of network pharmacology and cellular experiments, elucidating the regulatory effects of these compounds on endothelial cell functions. Three flavonoids, named Fr.4-2-1, Fr.4-2-2 and Fr.4-2-3, were obtained from sea buckthorn. The results of network pharmacology indicated that they might exert their effects by regulating the PI3K-AKT signaling pathway. In vitro results showed that all three flavonoids were effective in alleviating the degree of oxidative stress in cells, among which Fr.4-2-1 exerted its antioxidant effects by modulating the PI3K/AKT-eNOS pathway. Flavonoids in sea buckthorn can effectively inhibit oxidative stress-induced cellular damage, preserving the integrity and functionality of endothelial cells, which is crucial for maintaining vascular health and function.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

The Impact of LY487379 or CDPPB on eNOS Expression in the Mouse Brain and the Effect of Joint Administration of Compounds with NO• Releasers on MK-801- or Scopolamine-Driven Cognitive Dysfunction in Mice.

The role of endothelial nitric oxide synthase (eNOS) in the regulation of a variety of biological processes is well established, and its dysfunction contributes to brain pathologies, including schizophrenia or Alzheimer's disease (AD). Positive allosteric modulators (PAMs) of metabotropic glutamate (mGlu) receptors were shown to be effective procognitive compounds, but little is known about their impact on eNOS expression and stability. Here, we investigated the influence of the acute and chronic administration of LY487379 or CDPPB (mGlu2 and mGlu5 PAMs), on eNOS expression in the mouse brain and the effect of the joint administration of the ligands with nitric oxide (NO) releasers, spermineNONOate or DETANONOate, in different combinations of doses, on MK-801- or scopolamine-induced amnesia in the novel object recognition (NOR) test. Our results indicate that both compounds provoked eNOS monomer formation, and CDPPB at a dose of 5 mg/kg exaggerated the effect of MK-801 or scopolamine. The coadministration of spermineNONOate or DETANONOate enhanced the antiamnesic effect of CDPPB or LY487379. The best activity was observed for ineffective or moderate dose combinations. The results indicate that treatment with mGluR2 and mGluR5 PAMs may be burdened with the risk of promoting eNOS uncoupling through the induction of dimer dissociation. Administration of the lowest possible doses of the compounds with NO• donors, which themselves have procognitive efficacy, may be proposed for the treatment of schizophrenia or AD.

Effects of dipeptidyl peptidase 4 inhibition on the endothelial control of the vascular tone.

Dipeptidyl peptidase 4 (DPP4) is a serine protease known to cleave incretin hormones, which stimulate insulin secretion after food intake, a fact that supported the development of its inhibitors (DPP4i or gliptins) for the treatment of type 2 diabetes mellitus. In addition to their glucose-lowering effects, DPP4i show benefits for the cardiovascular system that could be related, at least in part, to their protective action on vascular endothelium. DPP4i have been associated with the reversal of endothelial dysfunction, an important predictor of cardiovascular events and a hallmark of diseases such as atherosclerosis, diabetes mellitus, hypertension, and heart failure. In animal models of these diseases, DPP4i increase nitric oxide bioavailability and limits oxidative stress, thereby improving the endothelium-dependent relaxation. Similar effects on flow-mediated dilation and attenuation of endothelial dysfunction have also been noted in human studies, suggesting a value for gliptins in the clinical scenario, despite the variability of the results regarding the DPP4i used, treatment duration, and presence of comorbidities. In this mini-review, we discuss the advances in our comprehension of the DPP4i effects on endothelial regulation of vascular tone. Understanding the role of DPP4 and its involvement in the signaling mechanisms leading to endothelial dysfunction will pave the way for a broader use of DPP4i in conditions that endothelial dysfunction is a pivotal pathophysiological player.