Phosphorylation Of Synthase Research Articles

Fat-Specific Protein 27 Regulation of Vascular Function in Human Obesity.

BackgroundPathophysiological mechanisms that connect obesity to cardiovascular disease are incompletely understood. FSP27 (Fat‐specific protein 27) is a lipid droplet‐associated protein that regulates lipolysis and insulin sensitivity in adipocytes. We unexpectedly discovered extensive FSP27 expression in human endothelial cells that is downregulated in association with visceral obesity. We sought to examine the functional role of FSP27 in the control of vascular phenotype.Methods and ResultsWe biopsied paired subcutaneous and visceral fat depots from 61 obese individuals (body mass index 44±8 kg/m2, age 48±4 years) during planned bariatric surgery. We characterized depot‐specific FSP27 expression in relation to adipose tissue microvascular insulin resistance, endothelial function and angiogenesis, and examined differential effects of FSP27 modification on vascular function. We observed markedly reduced vasodilator and angiogenic capacity of microvessels isolated from the visceral compared with subcutaneous adipose depots. Recombinant FSP27 and/or adenoviral FSP27 overexpression in human tissue increased endothelial nitric oxide synthase phosphorylation and nitric oxide production, and rescued vasomotor and angiogenic dysfunction (P<0.05), while siRNA‐mediated FSP27 knockdown had opposite effects. Mechanistically, we observed that FSP27 interacts with vascular endothelial growth factor‐A and exerts robust regulatory control over its expression. Lastly, in a subset of subjects followed longitudinally for 12±3 months after their bariatric surgery, 30% weight loss improved metabolic parameters and increased angiogenic capacity that correlated positively with increased FSP27 expression (r=0.79, P<0.05).ConclusionsOur data strongly support a key role and functional significance of FSP27 as a critical endogenous modulator of human microvascular function that has not been previously described. FSP27 may serve as a previously unrecognized regulator of arteriolar vasomotor capacity and angiogenesis which are pivotal in the pathogenesis of cardiometabolic diseases linked to obesity.

Dysfunction of 67-kDa Laminin Receptor Disrupts BBB Integrity via Impaired Dystrophin/AQP4 Complex and p38 MAPK/VEGF Activation Following Status Epilepticus.

Status epilepticus (SE, a prolonged seizure activity) impairs brain-blood barrier (BBB) integrity, which results in secondary complications following SE. The non-integrin 67-kDa laminin receptor (67-kDa LR) plays a role in cell adherence to laminin (a major glycoprotein component in basement membrane), and participates laminin-mediated signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK). Thus, we investigated the role of 67-kDa LR in SE-induced vasogenic edema formation in the rat piriform cortex (PC). SE diminished 67-kDa LR expression, but increased laminin expression, in endothelial cells accompanied by the reduced SMI-71 (a rat BBB barrier marker) expression. Astroglial 67-kDa LR expression was also reduced in the PC due to massive astroglial loss. 67-kDa LR neutralization led to serum extravasation in the PC concomitant with the reduced SMI-71 expression. 67-kDa LR neutralization also decreased expressions of dystrophin and aquaporin-4 (AQP4). In addition, it increased p38 MAPK phosphorylation and expressions of vascular endothelial growth factor (VEGF), laminin and endothelial nitric oxide synthase (eNOS), which were abrogated by SB202190, a p38 MAPK inhibitor. Therefore, our findings indicate that 67-kDa LR dysfunction may disrupt dystrophin-AQP4 complex, which would evoke vasogenic edema formation and subsequent laminin over-expression via activating p38 MAPK/VEGF axis.

Arg-Leu-Tyr-Glu Suppresses Retinal Endothelial Permeability and Choroidal Neovascularization by Inhibiting the VEGF Receptor 2 Signaling Pathway.

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathologic ocular neovascularization and vascular leakage via activation of VEGF receptor 2 (VEGFR2). This study was undertaken to evaluate the therapeutic mechanisms and effects of the tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a VEGFR2 inhibitor, in the development of vascular permeability and choroidal neovascularization (CNV). In cultured human retinal microvascular endothelial cells (HRMECs), treatment with RLYE blocked VEGF-A-induced phosphorylation of VEGFR2, Akt, ERK, and endothelial nitric oxide synthase (eNOS), leading to suppression of VEGF-A-mediated hyper-production of NO. Treatment with RLYE also inhibited VEGF-A-stimulated angiogenic processes (migration, proliferation, and tube formation) and the hyperpermeability of HRMECs, in addition to attenuating VEGF-A-induced angiogenesis and vascular permeability in mice. The anti-vascular permeability activity of RLYE was correlated with enhanced stability and positioning of the junction proteins VE-cadherin, β-catenin, claudin-5, and ZO-1, critical components of the cortical actin ring structure and retinal endothelial barrier, at the boundary between HRMECs stimulated with VEGF-A. Furthermore, intravitreally injected RLYE bound to retinal microvascular endothelium and inhibited laser-induced CNV in mice. These findings suggest that RLYE has potential as a therapeutic drug for the treatment of CNV by preventing VEGFR2-mediated vascular leakage and angiogenesis.

Inhibition of acid sphingomyelinase activity ameliorates endothelial dysfunction in db/db mice.

Acid sphingomyelinase (aSMase) plays an important role in endothelial dysfunction. Here, we show that elevated aSMase activity and ceramide content were reduced by desipramine treatment in diabetic animals. The inhibitor of aSMase, desipramine, improved vascular dysfunction in db/db mice. High glucose (HG)-induced up-regulation of aSMase activity and ceramide levels were restored by treatment with aSMase siRNA or desipramine in endothelial cells. In addition, aSMase siRNA or desipramine treatment increased the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in diabetic mouse aortas and aortic endothelial cells with HG.These results indicate that inhibition of aSMase/ceramide pathway improves endothelium-dependent vascular relaxation (EDR) largely through regulating the eNOS/NO pathway in diabetic animals.

Pressure suppresses hepatocellular glycogen synthesis through activating the p53/Pten pathway.

Portal hypertension is the primary cause of complications in patients with chronic liver diseases, and markedly impacts metabolism within the nervous system. Until recently, the role of portal hypertension in hepatocellular metabolism was unclear. The present study demonstrated that an increase in extracellular pressure significantly decreased hepatocellular glycogen concentrations in HepG2 and HL-7702 cells. In addition, it reduced glycogen synthase activity, by inhibiting the phosphorylation of glycogen synthase 1. RNA-seq analysis revealed that mechanical pressure suppressed glycogen synthesis by activating the p53/phosphatase and tensin homolog pathway, further suppressing glycogen synthase activity. The present study revealed an association between mechanical pressure and hepatocellular glycogen metabolism, and identified the regulatory mechanism of glycogen synthesis under pressure.

Acupuncture reduces nicotine-induced norepinephrine release in the hypothalamus via the solitary NMDA receptor/NOS pathway

Noradrenergic projections from the nucleus tractus solitarius (NTS) to the hypothalamic paraventricular nucleus (PVN) are involved in nicotine (Nic) dependence. Nic induces hypothalamic norepinephrine (NE) release through N-methyl-d-aspartate receptors (NMDARs) and nitric oxide in the NTS. However, acupuncture attenuates Nic withdrawal-induced anxiety. Therefore, this study investigated the effects of acupuncture on Nic-induced hypothalamic NE release. Rats received an intravenous infusion of Nic (90 μg/kg, over 60 s) and extracellular NE levels in the PVN were determined by in vivo microdialysis. Immediately after Nic administration, the rats were bilaterally treated with acupuncture at acupoint HT7 (Shen-Men) or PC6 (Nei-Guan), or a non-acupoint (tail) for 60 s. Acupuncture at HT7, but not at PC6 or the tail, significantly reduced Nic-induced NE release. However, this was abolished by a post-acupuncture infusion of either NMDA or sodium nitroprusside into the NTS. Additionally, acupuncture at HT7, but not the control points, prevented Nic-induced plasma corticosterone secretion and inhibited Nic-induced increases in the phosphorylation of neuronal nitric oxide synthase (nNOS) and endothelial NOS in the NTS. These findings suggest that acupuncture at HT7 reduces Nic-induced NE release in the PVN via inhibition of the solitary NMDAR/NOS pathway.

Acute canagliflozin treatment protects against in vivo myocardial ischemia\u2013reperfusion injury in non-diabetic male rats and enhances endothelium-dependent vasorelaxation

BackgroundThe sodium–glucose cotransporter-2 (SGLT2) inhibitor canagliflozin has been shown to reduce major cardiovascular events in type 2 diabetic patients, with a pronounced decrease in hospitalization for heart failure (HF) especially in those with HF at baseline. These might indicate a potent direct cardioprotective effect, which is currently incompletely understood. We sought to characterize the cardiovascular effects of acute canagliflozin treatment in healthy and infarcted rat hearts.MethodsNon-diabetic male rats were subjected to sham operation or coronary artery occlusion for 30 min, followed by 120 min reperfusion in vivo. Vehicle or canagliflozin (3 µg/kg bodyweight) was administered as an intravenous bolus 5 min after the onset of ischemia. Rats underwent either infarct size determination with serum troponin-T measurement, or functional assessment using left ventricular (LV) pressure–volume analysis. Protein, mRNA expressions, and 4-hydroxynonenal (HNE) content of myocardial samples from sham-operated and infarcted rats were investigated. In vitro organ bath experiments with aortic rings from healthy rats were performed to characterize a possible effect of canagliflozin on vascular function.ResultsAcute treatment with canagliflozin significantly reduced myocardial infarct size compared to vehicle (42.5 ± 2.9% vs. 59.3 ± 4.2%, P = 0.006), as well as serum troponin-T levels. Canagliflozin therapy alleviated LV systolic and diastolic dysfunction following myocardial ischemia–reperfusion injury (IRI), and preserved LV mechanoenergetics. Western blot analysis revealed an increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric-oxide synthase (eNOS), which were not disease-specific effects. Canagliflozin elevated the phosphorylation of Akt only in infarcted hearts. Furthermore, canagliflozin reduced the expression of apoptotic markers (Bax/Bcl-2 ratio) and that of genes related to myocardial nitro-oxidative stress. In addition, treated hearts showed significantly lower HNE positivity. Organ bath experiments with aortic rings revealed that preincubation with canagliflozin significantly enhanced endothelium-dependent vasodilation in vitro, which might explain the slight LV afterload reducing effect of canagliflozin in healthy rats in vivo.ConclusionsAcute intravenous administration of canagliflozin after the onset of ischemia protects against myocardial IRI. The medication enhances endothelium dependent vasodilation independently of antidiabetic action. These findings might further contribute to our understanding of the cardiovascular protective effects of canagliflozin reported in clinical trials.

SAT-LB011 Role of Endothelium Epithelial Sodium Channel in Arterial Stiffness

Excessive epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Our recent data has shown that activation of EnNaC mediates aortic endoplasmic reticulum stress, redox oxidative stress, expression of pro-inflammatory cytokines, and aortic remodeling. These abnormalities are potentially related to abnormal expression and activation of sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) in metabolic disorders such as obesity. We hypothesized that EnNaC mediates Western diet (WD)-induced endothelium and aortic stiffness by increasing aortic oxidative stress and associated oxidative stress related inhibition of Sirt1 and AMPK expression and activation. Accordingly, sixteen to twenty week-old EnNaC-/- and wild type littermate female mice were fed a mouse chow or an obesogenic WD containing excess fat (46%) and fructose (17.5%) for 16 weeks. EnNaC activation mediated WD-induced increase of Na+ currents in isolated lung endothelial cells, reduction of phosphorylation of endothelial nitric oxide synthase (eNOS), as well as impairment of aortic endothelium-dependent relaxation to acetylcholine (10-9-10-4 mol/L) in a wire myograph. The expression and activation of Sirt1 and AMPK were inhibited in WD EnNaC+/+ mice and these abnormalities were prevented in EnNaC-/- mice. Importantly, EnNaC specific KO prevented WD induced aortic oxidative stress and related endothelium stiffness and impairment of endothelium (NO)-dependent relaxation. These data suggest that endothelial specific EnNaC activation mediates WD-induced aortic oxidative stress, decreased expression and activation of Sirt1, AMPK, eNOS, as well as endothelial and aortic vessel stiffness. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Sigma‐1 receptor agonists elicit arteriolar dilation via Akt/eNOS activation, and also have endothelial barrier protective properties

Sigma‐1 receptors (σ1) have previously gained considerable attention pertaining to neuroprotection through its actions on calcium homeostasis, and the mitochondria‐associated ER membrane domain. Our laboratory recently showed that the σ1 agonist afobazole causes an endothelial NO‐dependent decrease in contraction amplitude. However, little is known about the mechanisms and functions of σ1 receptor in endothelial cells. In the current study, we investigated how σ1 agonists impact brain arteriolar tone and the Akt/eNOS pathway. Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. The nonselective σ agonist afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM in the presence or absence of the σ1 antagonist BD1047. The selective σ1 agonist, PRE‐084 was used to stimulate human umbilical vein endothelial cells (HUVEC), in which phosphorylation of endothelial NO synthase (eNOS) or Akt phosphorylation on their activation sites (S1177 and S473, respectively) were evaluated by western blotting. NO production was measured by fluorescence microscopy in cells loaded with the indicator dye DAF‐FM. Trans‐endothelial electrical resistance (TER) was used to determine the ability of PRE‐084 to attenuate disruption of endothelial barrier function caused by the the mitochondrial function inhibitors CCCP and antimycin‐A, or the inflammatory cytokine IL‐1β. The results show that afobazole significantly dilated rat brain arterioles, which was inhibited by the σ1 antagonist BD‐1047. Selective activation of σ1 with PRE‐084 significantly increased eNOS phosphorylation on S1177 and this was diminished in the presence of BD‐1047. Akt phosphorylation on S473 following PRE‐084 treatment was also enhanced. In addition, afobazole and PRE‐084 enhanced NO production in endothelial cells. PRE‐084 significantly attenuated decreases in TER caused by IL‐1β or CCCP, but not antimycin‐A, suggesting a potential barrier‐protective role for σ1 receptor, with some limitations that may be related to the source of mitochondrial dysfunction during inflammation. Collectively, our results suggest that σ1 receptor activation can cause vasodilation via the Akt/eNOS pathway in endothelial cells, and that σ1 may enhance endothelial barrier function under inflammatory conditions.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paeo‐tang attenuates disharmony between the heart and kidney in L‐NAME‐induced hypertension

Hypertension has been approved to cause disharmony between the heart and kidney such as cardiac hypertrophy and kidney dysfunction. In traditional oriental medicine Paeo‐tang (PET) has been shown to have effects on blood circulation improvement. However, the beneficial effect of PET on hypertension remains unknown. In this study, we investigated that PET attenuates blood pressure and suppresses cardiac hypertrophy in NG‐nitro‐L‐arginine methylester (L‐NAME) rat model. Hypertensive rat models were induced by the administration of L‐NAME (40 mg/kg/day) and then PET (50 or 100 mg/kg/day) or Olmetec was treated for 2 weeks. PET treatment significantly suppressed the systolic blood pressure and decreased intima‐media thickness in the thoracic aorta. PET restored the L‐NAME‐induced decrease in aortic relaxation response to acetylcholine and atrial natriuretic peptide (ANP). However, PET didn't restore the aortic relaxation response to sodium nitroprusside. PET markedly restored the phosphorylation of endothelial nitric oxide synthase in aortic tissue. Heart weight and ANP mRNA expression in heart tissue were decreased by PET in a dose‐dependent manner. PET ameliorated the functional decline in the kidney such as albumin, blood urea nitrogen, creatinine. These results demonstrated that PET possesses vascular protective effects against L‐NAME‐induced hypertension. In addition, PET regulates harmony between the heart and kidney via inhibition of cardiac hypertrophy and renal dysfunction.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adiponectin Attenuates Cyclophilin A Induced Vascular Remodeling in Response to Mechanical Stretch and Angiotensin‐II

Background and aimsThe mechanisms that contribute to the development of hypertension leading to cardiovascular diseases (CVD) have not been fully‐elucidated. Increased vascular pressure is associated with the formation of reactive oxygen species (ROS) in vascular smooth muscle cells. ROS has been related to the secretion of Cyclophlin A (CyPA). Moreover, studies have shown the incorporation of CyPA in CVD. Obesity is associated with decrease in adiponectin (APN) levels. Several studies have revealed that APN preserves the normal physiology of the heart. In this study, we aimed to investigate whether hypertension/mechanical stretch enhances the overexpression of CyPA protein and to explore the molecular mechanisms that mediates stretch‐induced CyPA expression. We will also assess the different possible pathways of CyPA induced vascular remodeling. We will focus on the protective effects of APN and its effects on CyPA protein expression.MethodsRat portal veins (RPV) organ culture with or without stretching along with aorta organ culture with or without angiotensin‐II (Ang‐II) were performed to study CyPA protein expression. CyPA treatment was also done on aorta and RPV. Western blot was used to study the expression of various proteins. Immunohistochemistry was performed to study ROS, CyPA, adiponectin and leptin expressions.ResultsThis study has shown that CyPA expression increases in response to stretch in RPV and Ang‐II in aorta. Moreover, CyPA was able to increase extracellular signal‐regulated kinases 1/2 (ERK1/2) activation and to decrease 5′‐AMP‐activated protein kinase (AMPK) and endothelial nitric oxide synthase (e‐NOS) phosphorylation. CyPA was also able to increase ROS and leptin expression and to attenuate APN expression. Furthermore, we have shown that APN decreases CyPA expression. This study also demonstrated that RhoA/ROCK inhibition with Y‐27632 and ROS inhibition with apocynin inhibits CyPA expression.ConclusionBased on the obtained results, we showed that mechanical stretch and Ang‐II upregulates CyPA protein expression via activation of ROS and RhoA/ROCK pathway. We also demonstrated that CyPA induces vascular remodeling by phosphorylation/activation of ERK 1/2, by inhibition of AMPK and e‐NOS phosphorylation, by activation of leptin and downregulation of adiponectin. Moreover, the protective effect of adiponectin was also studied and we demonstrated that APN anti‐hypertrophic effect was in part due to inhibition of CyPA activity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular Endothelial Growth Factor Receptor 3 Regulates Endothelial Function Through β-Arrestin 1.

Receptor signaling is central to vascular endothelial function and is dysregulated in vascular diseases such as atherosclerosis and pulmonary arterial hypertension (PAH). Signaling pathways involved in endothelial function include vascular endothelial growth factor receptors (VEGFRs) and G protein-coupled receptors, which classically activate distinct intracellular signaling pathways and responses. The mechanisms that regulate these signaling pathways have not been fully elucidated and it is unclear what nodes for cross talk exist between these diverse signaling pathways. For example, multifunctional β-arrestin (ARRB) adapter proteins are best known as regulators of G protein-coupled receptor signaling, but their role at other receptors and their physiological importance in the setting of vascular disease are unclear. We used a combination of human samples from PAH, human microvascular endothelial cells from lung, and Arrb knockout mice to determine the role of ARRB1 in endothelial VEGFR3 signaling. In addition, a number of biochemical analyses were performed to determine the interaction between ARRB1 and VEGFR3, signaling mediators downstream of VEGFR3, and the internalization of VEGFR3. Expression of ARRB1 and VEGFR3 was reduced in human PAH, and the deletion of Arrb1 in mice exposed to hypoxia led to worse PAH with a loss of VEGFR3 signaling. Knockdown of ARRB1 inhibited VEGF-C-induced endothelial cell proliferation, migration, and tube formation, along with reduced VEGFR3, Akt, and endothelial nitric oxide synthase phosphorylation. This regulation was mediated by direct ARRB1 binding to the VEGFR3 kinase domain and resulted in decreased VEGFR3 internalization. Our results demonstrate a novel role for ARRB1 in VEGFR regulation and suggest a mechanism for cross talk between G protein-coupled receptors and VEGFRs in PAH. These findings also suggest that strategies to promote ARRB1-mediated VEGFR3 signaling could be useful in the treatment of pulmonary hypertension and other vascular disease.

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

Matrine attenuates high-fat diet-induced in vivo and ox-LDL-induced in vitro vascular injury by regulating the PKCα/eNOS and PI3K/Akt/eNOS pathways.

Lipid metabolism disorders lead to vascular endothelial injury. Matrine is an alkaloid that has been used to improve obesity and diabetes and for the treatment of hepatitis B. However, its effect on lipid metabolism disorders and vascular injury is unclear. Here, we investigated the effect of matrine on high‐fat diet fed mice and oxidized low‐density lipoprotein (ox‐LDL)‐induced human umbilical vein endothelial cells (HUVECs). Computational virtual docking analyses, phosphoinositide 3‐kinase (PI3K) and protein kinase C‐α (PKCα) inhibitors were used to localize matrine in vascular injuries. The results showed that matrine‐treated mice were more resistant to abnormal lipid metabolism and inflammation than vehicle‐treated mice and exhibited significantly alleviated ox‐LDL‐stimulated dysfunction of HUVECs, restored diminished nitric oxide release, decreased reactive oxygen species generation and increased expression phosphorylation of AKT‐Ser473 and endothelial nitric oxide synthase (eNOS)‐Ser1177. Matrine not only up‐regulates eNOS‐Ser1177 but also down‐regulates eNOS‐Thr495, a PKCα‐controlled negative regulator of eNOS. Using computational virtual docking analyses and biochemical assays, matrine was also shown to influence eNOS/NO via PKCα inhibition. Moreover, the protective effects of matrine were significantly abolished by the simultaneous application of PKCα and the PI3K inhibitor. Matrine may thus be potentially employed as a novel therapeutic strategy against high‐fat diet‐induced vascular injury.

Inositol 1,4,5-Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation.

BackgroundEndothelial NO synthase plays a central role in regulating vasodilation and blood pressure. Intracellular Ca2+ mobilization is a critical modulator of endothelial NO synthase function, and increased cytosolic Ca2+ concentration in endothelial cells is able to induce endothelial NO synthase phosphorylation. Ca2+ release mediated by 3 subtypes of inositol 1,4,5‐trisphosphate receptors (IP 3Rs) from the endoplasmic reticulum and subsequent Ca2+ entry after endoplasmic reticulum Ca2+ store depletion has been proposed to be the major pathway to mobilize Ca2+ in endothelial cells. However, the physiological role of IP 3Rs in regulating blood pressure remains largely unclear.Methods and ResultsTo investigate the role of endothelial IP 3Rs in blood pressure regulation, we first generated an inducible endothelial cell–specific IP 3R1 knockout mouse model and found that deletion of IP 3R1 in adult endothelial cells did not affect vasodilation and blood pressure. Considering all 3 subtypes of IP 3Rs are expressed in mouse endothelial cells, we further generated inducible endothelial cell–specific IP 3R triple knockout mice and found that deletion of all 3 IP 3R subtypes decreased plasma NO concentration and increased basal blood pressure. Furthermore, IP 3R deficiency reduced acetylcholine‐induced vasodilation and endothelial NO synthase phosphorylation at Ser1177.ConclusionsOur results reveal that IP 3R‐mediated Ca2+ release in vascular endothelial cells plays an important role in regulating vasodilation and physiological blood pressure.

Magnesium lithospermate B ameliorates microcirculation perfusion in rats by promoting vascular NO production via activating the PI3K/AKT pathway.

Microcirculation morphologically refers to the blood flow in vessels of less than 150 μm in diameter, including arterioles, capillaries and venules, which provides nutrients and removes metabolic byproducts within tissues. Microcirculation dysfunction is involved in the pathological progress of many diseases, such as obesity, hypertension, and insulin resistance. In this study we investigated the effects of magnesium lithospermate B (MLB), an active compound of the traditional Chinese medicine Slavia miltiorrhiza, on the microcirculation dysfunction in rats and the underlying molecular mechanisms. The effects of MLB on microcirculation were assessed in vivo by measuring the hindlimb blood perfusion in dextran-induced microcirculation dysfunction rats and mesentery blood flow in anesthetized rats. We demonstrated that administration of MLB restored the impaired rat hindlimb blood flow and promoted the mesenteric micoperfusion in vivo. We further revealed in these two animal models that MLB treatment significantly increased the production of total nitrite in vascular tissues (mesentery, aorta, and heart), which was confirmed in human microvascular endothelial cells (HMEC-1) treated with MLB in vitro. Moreover, we showed that MLB treatment significantly increased the phosphorylation of endothelium nitric oxide synthase (eNOS) via inducing AKT phosphorylation in vivo and in vitro. Co-administration of the eNOS inhibitor L-NAME (20 mg/kg) abolished the protective effects of MLB against dextran-induced microcirculation dysfunction in rats, whereas pretreatment with PI3K inhibitor LY294002 (10 μM) prevented eNOS activation in MLB-treated HMEC-1 cells. Our results suggest that MLB can restore the microcirculation dysfunction via activating eNOS, and in turn enhancing the vascular nitric oxide production, which is medicated by MLB-caused activation of the PI3K/AKT pathway.

Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

Metformin prevents vascular damage in hypertension through the AMPK/ER stress pathway.

Metformin is an antidiabetic drug. However, the pleiotropic beneficial effects of metformin in nondiabetic models still need to be defined. The objective of this study is to investigate the effect of metformin on angiotensin II (Ang II)-induced hypertension and cardiovascular diseases. Mice were infused with Ang II (400 ng/kg per min) with or without metformin for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure associated with enhanced vascular endoplasmic reticulum (ER) stress markers, which were blunted after metformin treatment. Moreover, hypertension-induced reduction in phosphorylated AMPK, endothelial nitric oxide synthase (eNOs) phosphorylation, and endothelium-dependent relaxation (EDR) in mesenteric resistance arteries (MRA) were rescued after metformin treatment. Infusion of ER stress inducer (tunicamycin, Tun) in control mice induced ER stress in MRA and reduced phosphorylation of AMPK, eNOS synthase phosphorylation, and EDR in MRA without affecting systolic blood pressure (SBP). All these factors were reversed subsequently with metformin treatment. ER stress inhibition by metformin improves vascular function in hypertension. Therefore, metformin could be a potential therapy for cardiovascular diseases in hypertension independent of its effects on diabetes.

Ursolic acid prevents doxorubicin-induced cardiac toxicity in mice through eNOS activation and inhibition of eNOS uncoupling.

In addition to the known antitumour effects of ursolic acid (UA), increasing evidence indicates that this molecule plays a role in cardiac protection. In this study, the effects of ursolic acid on the heart in mice treated with doxorubicin (DOX) were assessed. The results showed that ursolic acid improved left ventrical fractional shortening (LVFS) and left ventrical ejection fraction (LVEF) of the heart, increased nitrogen oxide (NO) levels, inhibited reactive oxygen species (ROS) production and decreased cardiac apoptosis in mice treated with doxorubicin. Mechanistically, ursolic acid increased AKT and endothelial nitric‐oxide synthase (eNOS) phosphorylation levels, and enhanced eNOS expression, while inhibiting doxorubicin induced eNOS uncoupling through NADPH oxidase 4 (NOX4) down‐regulation. These effects of ursolic acid resulted in heart protection from doxorubicin‐induced injury. Therefore, ursolic acid may be considered a potential therapeutic agent for doxorubicin‐associated cardiac toxicity in clinical practice.

Docosahexaenoic acid reduces adenosine triphosphate-induced calcium influx via inhibition of store-operated calcium channels and enhances baseline endothelial nitric oxide synthase phosphorylation in human endothelial cells.

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, 100 µM) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to 50 µM. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated Ca2+-transient. This effect was preserved in the presence of BAPTA (10 and 20 µM) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane (75 µM) to inhibit store-operated calcium channel or thapsigargin (2 µM) to delete calcium store. In addition, DHA (12 µM) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a Ca2+ activated mode to a preferentially controlled phosphorylation mode.