p-eNOS Expression Research Articles

Electroacupuncture treatment ameliorated the long-term cognitive impairment via activating eNOS/NO pathway and related Aβ downregulation in sepsis-survivor mice

ObjectiveSepsis is a major challenge in intensive care unit worldwide and the septic survivors are left with long-term cognitive deficits. This work aims to explore the effects of electroacupuncture (EA) on long-term cognitive function and its underlying mechanism in sepsis-survivor mice. MethodsSepsis was induced by cecal ligation and puncture in C57BL/6 male mice. Seven days post-surgery, sepsis-survivor mice were treated with EA or nonacupoint EA for 17 days twice daily. Then, cognitive function was evaluated by Morris water maze task. The hippocampus tissue were collected from the mice at 30 days post-surgery. The level of nitric oxide and the expression of endothelial nitric oxide (eNOS), phospho-eNOS (p-eNOS), and amyloid β–peptide (Aβ) were measured. ResultsCompared with the sham-operated control, sepsis-survivors had significant cognitive deficits evidenced by the increased time of escape latency and reduced crossing number in Morris water maze task, as well as lower NO and p-eNOS level and higher Aβ level. EA treatment at GV20 and ST36 acupoints but not at a nonacupoint improved the cognitive function, increased the NO and p-eNOS level, and decreased Aβ generation; while eNOS inhibitor (l-NAME) undermined the efficacy of EA treatment. ConclusionIn conclusion, repeated EA treatment could ameliorate the long-term cognitive impairment via manipulating the expression of p-eNOS and related Aβ in sepsis-survivor mice.

Effects of Escitalopram on Endoplasmic Reticulum Stress and Oxidative Stress Induced by Tunicamycin.

Background: Major depressive disorder (MDD) was reported to be associated with endoplasmic reticulum stress (ERS) combined with oxidative stress (OS) (ERS/OS). Here, we aimed to investigate the effects of escitalopram (ESC) on blood-brain barrier (BBB) permeability and ERS/OS-related pathways in brain microvascular endothelial cells (bEnd.3 cells) induced by tunicamycin (TM).Methods: bEnd.3 cells were divided into four groups: control, TM, ESC, and ESC + TM groups. CCK-8 and flow cytometry were used to detect cell survival and apoptosis, respectively. The expression levels of proteins involved in cell permeability and ERS/OS-related pathways were assessed by western blot and immunofluorescence. Malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity were determined by commercial kits.Results: We revealed that TM-induced bEnd.3 cells exhibited remarkably decreased viability and increased apoptosis rate, while ESC treatment reversed these changes. Additionally, TM treatment resulted in markedly increased PERK, GRP78, ATF6, XBP1, and CHOP protein expression levels. On the contrary, the expression of PERK, GRP78, XBP1, and CHOP was obviously reduced in TM-induced bEnd.3 cells after ESC treatment. Moreover, TM significantly reduced the expression of p-eNOS and P-gp and increased the expression of CaMKII and MMP9 compared with the control group. However, ESC reversed these changes in TM-induced bEnd.3 cells. Furthermore, the expression of SOD was significantly decreased, while MDA was significantly increased by TM treatment. In contrast, the expression of SOD was dramatically increased, while MDA was remarkably decreased by ESC treatment.Conclusion: Our results demonstrated that ESC can inhibit ERS/OS and BBB permeability of TM-induced bEnd.3 cells. ESC may alleviate cognitive impairment and prevent comorbidities in MDD patients through ERS/OS.

Involvement of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) in endothelial dysfunction associated with pulmonary hypertension

AimsIncreased proliferation, inflammation, and endothelial microparticle (EMP) generation in the pulmonary vasculature lead to endothelial dysfunction in pulmonary hypertension (PH). Interestingly, MK2, a downstream of p38MAPK, is a central regulator of inflammation, proliferation, and EMP generation in cardiovascular diseases. However, the role of MK2 in pulmonary endothelial dysfunction remains unexplored. Main methodsThe Human Pulmonary Artery Endothelial cells (HPAECs) were exposed to hypoxia (1% O2) for 72 h, and MK2 inhibition was achieved by siRNA treatment. Western blotting, qualitative RT-PCR, immunocytochemistry, flow cytometry and enzyme-linked immunoassays were conducted to study pathological alterations and molecular mechanisms. Neoangiogenesis was studied using cell migration and tubule formation assays. For in vivo study, Male Sprague Dawley rats and MK2 knock-out mice with littermate control were treated with monocrotaline (MCT) 60 mg/kg and 600 mg/kg, respectively (s.c. once in rat and weekly in mice) to induce PH. MMI-0100 (40 μg/kg, i.p. daily for 35 days), was administered in rats to inhibit MK2. Key findingsMK2 inhibition significantly decreased inflammation, cell proliferation, apoptosis resistance, and improved mitochondrial functions in hypoxic HPAECs. Hypoxia promoted cell migration, VEGF expression, and angiogenesis in HPAECs, which were also reversed by MK2 siRNA. MK2 inhibition decreased EMP generation and increased the expression of p-eNOS in hypoxic HPAECs, a marker of endothelial function. Furthermore, MK2 deficiency and inhibition both reduced the EMP generation in mice and rats, respectively. SignificanceThese findings proved that MK2 is involved in endothelial dysfunction, and its inhibition may be beneficial for endothelial function in PH.

Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis

BackgroundEnhanced angiogenesis can promote diabetic wound healing. Mesenchymal stem cells (MSCs)-derived exosomes, which are cell-free therapeutics, are promising candidates for the treatment of diabetic wound healing. The present study aimed to investigate the effect of exosomes derived from MSCs pretreated with pioglitazone (PGZ-Exos) on diabetic wound healing.ResultsWe isolated PGZ-Exos from the supernatants of pioglitazone-treated BMSCs and found that PGZ-Exos significantly promote the cell viability and proliferation of Human Umbilical Vein Vascular Endothelial Cells (HUVECs) injured by high glucose (HG). PGZ-Exos enhanced the biological functions of HUVECs, including migration, tube formation, wound repair and VEGF expression in vitro. In addition, PGZ-Exos promoted the protein expression of p-AKT, p-PI3K and p-eNOS and suppressed that of PTEN. LY294002 inhibited the biological function of HUVECs through inhibition of the PI3K/AKT/eNOS pathway. In vivo modeling in diabetic rat wounds showed that pioglitazone pretreatment enhanced the therapeutic efficacy of MSCs-derived exosomes and accelerated diabetic wound healing via enhanced angiogenesis. In addition, PGZ-Exos promoted collagen deposition, ECM remodeling and VEGF and CD31 expression, indicating adequate angiogenesis in diabetic wound healing.ConclusionsPGZ-Exos accelerated diabetic wound healing by promoting the angiogenic function of HUVECs through activation of the PI3K/AKT/eNOS pathway. This offers a promising novel cell-free therapy for treating diabetic wound healing.Graphic abstract

Latent HIV‐1 Exosomes Induce Mitochondrial Hyperfusion due to Loss of Phosphorylated Dynamin‐related Protein 1 in Brain Endothelium

Background Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders (HAND). To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of either primary human brain microvascular endothelial cells (HBMVECs) in vitro or mice brain microvessels (MVs), ex vivo. Methods Exosomes were isolated by either ultracentrifugation from uninfected and latent HIV-1 infected T-cells (Jurkat Exo and J-Lat(9.2) Exo, respectively) and monocytes (U937 Exo and U1 Exo, respectively) or by using QIAGEN exoEasy Maxi kit from HIV-1 infected human peripheral blood mononuclear cells (HIV+PBMCs Exo). Exosomes were characterized by the qNano-IZON system. Initially, the assessment of mitochondrial characteristics was performed in primary HBMVECs, and this study was further extended in extracted cerebral MVs from mice brain expressing mito-Dendra2+ fluorescence protein (Green) in endothelial mitochondria. Results Quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. This was further supported by the detection of exosomal markers, e.g. CD63, CD81, Alix, TSG101 and the absence of large EV-related protein, GRP94. These exosomes were readily taken up by both primary HBMVECs and mice cerebral MVs. HIV-exosomes induced cellular and mitochondrial superoxide production but reduced mitochondrial membrane potential in HBMVECs. HIV-exosomes increased mitochondrial hyperfusion, possibly due to the loss of phosphorylated dynamin-related protein 1 (p-DRP1). We further confirmed that HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein accelerate brain endothelial dysfunction due to reduction in the expressions of (a) p-DRP1, (b) phosphorylated endothelial nitric oxide synthase (p-eNOS), and (c) blood brain barrier related tight junction proteins. Finally, HIV-1 infected primary human PBMCs produced more exosomes than uninfected controls, and exposure to HIV+PBMCs Exo also reduced both p-DRP1 and p-eNOS expressions in primary HBMVECs. Conclusions Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium. These mechanistic studies using HIV-exosomes may elucidate the etiology of HAND and provide novel therapeutic strategies.

Extracellular Endothelial Microvesicles Causative Agents in Obesity‐Related Endothelial Dysfunction

Obesity is associated with increased cardiovascular disease morbidity and mortality. Moreover, obese individuals demonstrate accelerated rates of atherosclerosis, as well as increased incidence of myocardial infarction and stroke. Extracellular microvesicles, particularly endothelial cell-derived microvesicles (EMVs), are seminal functional modulators of vascular health and disease. We have previously demonstrated that EMVs are elevated with obesity and associated with endothelial dysfunction, although the underlying mechanisms are unknown. The experimental aim of this study was to determine the effects of EMVs isolated from obese adults on endothelial cell inflammation, apoptosis and nitric oxide (NO) production. Twenty-four middle-aged adults were studied: 12 normal weight (8M/4F; BMI: 24.4+0.3 kg/m2; EMVs: 85+13 EMV/µL) and 12 obese (6M/6F; BMI: 31.1+0.4 kg/m2; EMVs: 160+18 EMV/µL). All subjects were sedentary, non-smokers, normotensive, normolipidemic, non-diabetic and free of overt coronary artery disease. EMVs (CD31+/CD42b-) were identified, enumerated, and isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with EMVs from either normal weight or obese adults. EMVs from obese adults induced significantly greater release of both IL-6 (108.2±2.3 vs 90.9±2.9 pg/mL) and IL-8 (74.5±2.9 vs 59.5±3.3 pg/mL) from endothelial cells vs EMVs from normal weight adults. Intracellular expression of total NF-kB p65 was not significantly different in cells treated with EMVs from the normal weight (78.2+9.3 AU) or obese (86.5+8.7 AU) subjects. However, EMVs from obese subjects induced ~25% higher expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (145.0+9.8 vs 114.5+8.8 AU). There were no significant differences in cellular expression of total p38-MAPK (190.6+18.4 vs 163.2+9.5 AU) and caspase-3 (250.9±20.9 vs 283.0±19.4 AU) in cells treated with EMVs from normal weight and obese adults. However, EMVs from obese adults induced significantly higher (~50%) expression of phospho-p38-MAPK(15.4+1.6 vs 9.2+0.7 AU) and active caspase-3 (168.2±18.9 vs 107.8±11.7 AU) than EMVs from normal weight adults. Total eNOS expression was not significantly different between cells treated with EMVs from normal weight (110.9+12.0 AU) and obese (112.8+14.4 AU) adults; however, p-eNOS (Ser1177) expression was ~50% lower (P<0.05) in the cells treated with EMVs from obese adults (18.4+3.4 vs 34.6+2.8 AU). Consistent with changes in p-eNOS, NO production was ~20% lower (6.9+0.4 vs 8.7+0.2 mmol/L) in cells treated with EMVs from obese adults. Increased inflammation, apoptosis susceptibility and decreased eNOS activity and NO production renders the vascular endothelium prone to atherosclerosis and thrombosis. Circulating EMVs likely contribute to the proatherogenic endothelial phenotype and, in turn, the increased incidence of cardiovascular disease with obesity.

Low Dose of Emodin Inhibits Hypercholesterolemia in a Rat Model of High Cholesterol.

BackgroundEmodin has been widely used in traditional Chinese medicine, but few studies have tried to understand the mechanism of its anti-hypercholesterolemic effect.Material/MethodsTo delineate the underlying pathways, high-cholesterol diet (HCD)-fed Sprague-Dawley rats were orally administrated emodin or the lipid-lowering medicine simvastatin. Emodin was administered at 10, 30, or 100 mg/kg, while simvastatin was administered at 10 mg/kg. Parameters measured included lipid profiles (serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, aorta endothelium-dependent vasorelaxation in response to acetylcholine, and nitric oxide (NO) production. RT-qPCR and western blotting were performed to evaluate aortic endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), and hepatic LDL receptor (LDLR). Indices of liver and serum oxidation were also measured.ResultsThe atherogenic index was increased by the HCD but significantly reduced in all treatment groups. The HCD-fed experimental group treated with emodin at 10 mg/kg had significantly lower serum total-C and LDL-C and improved aorta vasorelaxation and enhanced NO production. Also, emodin significantly attenuated the lipid profiles and restored endothelial function, as reflected by upregulated expression of hepatic LDLR and p-eNOS, respectively. Furthermore, emodin at 10 mg/kg significantly enhanced superoxide dismutase activity, lowered the malondialdehyde level in both liver and serum, and enhanced catalase activity in serum.ConclusionsThe ability of emodin to inhibit hypercholesterolemia in HCD-fed rats was associated with lower serum total-C and LDL-C, restoration of aortic endothelial function, and improved antioxidant capacity. Low-dose emodin showed better protection of aortic endothelium and better antioxidant activity than did higher doses.

The antihypertensive effect of MK on spontaneously hypertensive rats through the AMPK/Akt/eNOS/NO and ERK1/2/Cx43 signaling pathways

We investigated the antihypertensive effects of maximakinin (MK) on spontaneously hypertensive rats (SHRs). The effects of MK on arterial blood pressure in SHRs were observed, and flow cytometry and 4,5-diaminofluorescein-2 staining were used to examine MK-induced nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). Western blotting was used to analyze the effects of MK on the expression of AMP-activated protein kinase (AMPK), Akt, Connexin 43, ERK1/2, p38, and p-eNOS in HUVECs. The results showed that MK induced a more significant antihypertensive effect on SHRs than bradykinin (BK). MK induced significant increases in endothelial nitric oxide synthase (eNOS) phosphorylation and NO release in HUVECs. MK also significantly increased the phosphorylation of Akt and AMPK in HUVECs. The AMPK inhibitor compound C blocked the effect of MK on the generation of NO. MK induced the phosphorylation of ERK1/2, p38, and Connexin 43. The expression of p-Connexin 43 was significantly decreased in the presence of the ERK1/2 inhibitor U0126 but not the p38 inhibitor SB203580. The effects of MK on the phosphorylation of AMPK and ERK1/2 were significantly decreased by the BK B2 receptor inhibitor HOE-140. In summary, MK can significantly reduce blood pressure in SHRs. The antihypertensive effect might be mediated through the activation of the BK B2 receptor, while the downstream AMPK/PI3K/Akt/eNOS/NO and ERK1/2/Connexin 43 signaling pathways play additional roles.

Latent HIV-Exosomes Induce Mitochondrial Hyperfusion Due to Loss of Phosphorylated Dynamin-Related Protein 1 in Brain Endothelium

Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.

Trehalose protects the endothelium from cadmium-induced dysfunction.

The objective of the present study is to identify the possible regulatory role of trehalose (Tre) against cadmium chloride (CdCl2 )-induced endothelial cell dysfunction. To screen the dose-dependent effect of both Tre and CdCl2 , amethylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was performed. Interestingly, MTT assay results have shown that co-incubation of Tre (1 mM) with CdCl2 significantly decreased the CdCl2 (5 µM) cytotoxicity. Nitric oxide (NO) measurement using Griess assay and 4-amino-5-methylamino-2',7'-difluorofluoresceinfluorescence probe results have shown that CdCl2 decreases NO production in endothelial cells. Western blotting analysis results showed that CdCl2 decreases endothelial nitric oxide synthase (eNOS) and phospho endothelial nitric oxide synthase (peNOS) expression. The present study results have also observed that CdCl2 treatment increases reactive oxygen species (ROS) production. However, combination treatment (Tre + CdCl2 ) could restore the NO production in CdCl2 -treated cells. In addition, combination treatment could also restore eNOS and peNOS expression in endothelial cells. Moreover, Tre treatment was found to decrease CdCl2 -induced ROS production. Collectively, the present study results demonstrate that Tre possesses a significant protective action against CdCl2 -mediated endothelial dysfunction by increasing NO production, eNOSand peNOS expression, and by decreasing oxidative stress.

PDGF-BB/SA/Dex injectable hydrogels accelerate BMSC-mediated functional full thickness skin wound repair by promoting angiogenesis.

Wound healing is a well-orchestrated dynamic and interactive process, which needs a favorable microenvironment and suitable angiogenesis. Platelet derived growth factor-BB (PDGF-BB) plays a crucial role in wound healing. However, the short half-life of PDGF-BB limits its efficacy. In the present study, we successfully synthesized an injectable hydrogel with sodium alginate (SA) and dextran (Dex) as a delivery system to simultaneously deliver PDGF-BB and bone marrow-derived mesenchymal stem cells (BMSCs) in the wound. Our work demonstrates that the PDGF-BB protein enhanced the survival, migration and endothelial cell (EC) differentiation of BMSCs in vitro. The PDGF-BB/SA/Dex hydrogels could sustainably release PDGF-BB with excellent biocompatibility in vitro and in vivo. Besides, these composite hydrogels loaded with BMSCs could accelerate wound healing by improving epithelialization and collagen deposition. In addition, the PDGF-BB/SA/Dex hydrogels promoted the EC-differentiation of transplanted BMSCs and proliferation of hair follicle stem cells in the wound. Furthermore, the expressions of angiogenesis-specific markers, PDGFR-β, p-PI3K, p-Akt, and p-eNOS, were obviously increased in the PDGF-BB/SA/Dex/BMSCs group. In conclusion, the PDGF-BB/SA/Dex injectable hydrogels could accelerate BMSC-mediated skin wound healing by promoting angiogenesis via the activation of the PDGF-BB/PDGFR-β-mediated PI3K/Akt/eNOS pathway, which may provide a new therapeutic strategy for stem cell therapy in wound healing.

Ulinastatin Inhibits the Proliferation, Invasion and Phenotypic Switching of PDGF-BB-Induced VSMCs via Akt/eNOS/NO/cGMP Signaling Pathway.

BackgroundAtherosclerosis is a chronic inflammatory disease responsible for thrombosis, blood supply disorders, myocardial infarction and strokes, eventually leading to increased deaths and reduced quality of life. As inflammation plays a vital role in the development of this disease, the present study aims to investigate whether urinary trypsin inhibitor (UTI) with anti-inflammatory property can inhibit the proliferation, invasion and phenotypic switching of PDGF-BB-induced vascular smooth muscle cells (VSMCs) and probe its potential mechanism.MethodsWestern blot was used to detect the expressions of the proteins related to the Akt/eNOS/NO/cGMP signaling pathway, phenotypic switching and proliferation. CCK-8 assay and EdU staining were used to detect cell proliferation of VSMCs. Transwell and wound healing assays were respectively conducted to measure the invasion and migration of VSMCs. The concentration of NO was evaluated by NO detection kit. ELISA assay analyzed the expression of cyclic GMP (cGMP).ResultsThe expressions of p-Akt and p-eNOS were elevated by UTI treatment. Furthermore, UTI inhibited the proliferation, migration and invasion of VSMCs. UTI also increased the expressions of proteins related to phenotypic switching. The amount of NO and expression of cGMP were both elevated under UTI treatment.ConclusionUTI inhibits the proliferation, invasion and phenotypic switching of PDGF-BB-induced VSMCs via Akt/eNOS/NO/cGMP signaling pathway, which might provide a theoretical basis for the UTI treatment of atherosclerosis.

Contribution of peptidyl prolyl isomerase (Pin1) to development of pulmonary hypertension via pulmonary vascular endothelial cell dysfunction

Abstract Background Endothelial dysfunction is thought to be a major contributor to overall pathogenesis of vasculopathy seen in pulmonary hypertension (PH), which is manifested by the impaired release of nitric oxide (NO) generated through endothelial nitric oxide synthase (eNOS) in endothelial cells. Activation of human eNOS is regulated by phosphorylation at multiple sites including Thr33 and Ser114, which residues are followed by Pro. The peptidyl isomerase Pin1 specifically isomerizes the phospho-protein having Ser/Thr-Pro bond and regulates their activity. Pin1 is involved in proliferation, cell cycle, and apoptosis in cancer, by isomerizing some functional molecules such as JNK, JUN, cyclin D, BAX, etc. However, it is controversial whether direct interaction of Pin1 with eNOS and how eNOS activity is altered by Pin1, especially in PH. Purpose We aimed to clarify whether Pin1 contributes to the PH development using Pin1 knockout mice and Pin1 affects the expression of phosphorylated eNOS (p-eNOS) molecule and pulmonary arterial endothelial cell (PAEC) apoptosis. Methods and results Wild (WT) and Pin1-deficient mice (KO) were exposed to hypoxia (10% O2) or normoxia for 3 weeks to generate hypoxia-induced PH. Hypoxia-inducible factor (HIF1α) expression in lungs was significantly enhanced in WT-hypoxia (WH, n=6) and KO-hypoxia (KH, n=6), suggesting that hypoxic response was certainly occurred in these mice. Pulmonary arterial pressure did not elevate in KH compared with KO-normoxia (KN, n=6) and WT-normoxia (WN, n=6), it was significantly increased only in WH (P&amp;lt;0.01), indicating that KO did not develop PH by hypoxia. The gain of RV weight was parallel to the increase of pulmonary arterial pressure. Western blot showed that p-eNOS expression in lungs was significantly decreased in WH compared to WN, however, the expression was not different between KH and KN. It suggests that Pin1 plays a regulatory role in p-eNOS expression in hypoxic response. In cultured PAECs, the expression of p-eNOS and eNOS was markedly increased by siRNA-mediated Pin1 knockdown. Immunoprecipitation study showed the possibility of Pin1 binding to p-eNOS molecule. Apoptosis evaluated by caspase-3/7 activity by fluorescent assay and cleaved caspase-3 expression by Western blot was significantly increased by Pin1 overexpression in PAECs; however, it was significantly decreased by Pin1 knockdown. Moreover, the exaggeration of apoptosis induced by doxorubicin was markedly increased by Pin1 overexpression compared with control in PAECs; however, it was clearly suppressed by Pin1 knockdown. Conclusion This study suggests that endogenous Pin1 contributes to the development of PH partly via the dysfunction of PAECs, that is, by the interference with p-eNOS expression and by the increase of apoptosis inducibility to external stimuli. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Myricitrin pretreatment ameliorates mouse liver ischemia reperfusion injury

BackgroundMyricitrin has been reported to exert protective effects on liver diseases, but the protective effects of myricitrin against liver ischemia reperfusion (I/R) injury and the underlying mechanisms remain unexplored. This study aimed to investigate the effects of myricitrin on liver I/R injury and elucidate the underlying mechanisms. MethodsMice were pretreated with myricitrin before liver I/R injury modeling. The mice were pretreated with either myricitrin or vehicle prior to liver ischemia. Some mice were further pretreated with the PI3K inhibitor LY294002. Liver tissues and blood samples were collected after 6 h of reperfusion.The degree of liver damage was determined by the serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and lactic dehydrogenase (LDH) and histological examinations. The tumour necrosis factor-α (TNF-α), interleukin--1β (IL-1β), IL-4 and IL-10 expression levels were assessed by qRT-PCR and enzyme-linked immunosorbent assays (ELISAs). Serum superoxide dismutase (SOD) activity, catalase (CAT) activity, and contents of malondialdehyde (MDA), glutathione (GSH) and nitric oxide (NO) contents were measured. Western blotting and caspase-3 activity were conducted to determine the effect of myricitrin on apoptosis. The expression levels of proliferation related genes (Cyclin D1 and Cyclin E1) were determined by qRT-PCR and western-blotting. The expression of p-Akt, p-mTOR and p-eNOS in liver tissue were investigated by western-blotting. ResultsMyricitrin not only significantly decreased the ALT, AST and LDH levels but also reduced the necrotic areas in the liver tissue compared with liver I/R injury group. In addition, myricitrin pretreatment alleviated liver injury by inhibiting the inflammatory response and suppressing oxidative stress. Western blotting and caspase-3 activity revealed that myricitrin inhibited liver I/R induced-apoptosis. Myricitrin promoted hepatocyte proliferation following liver I/R injury by upregulating the expression levels of Cyclin D1 and Cyclin E1. Further experiments indicated that the myricitrin pretreatment increased nitric oxide (NO) production by activating the PI3K/Akt signaling pathway. However, myricitrin triggered the hepatocyte proliferation and NO synthase activation was blocked by LY294002. ConclusionThese results demonstrate that myricitrin alleviates liver I/R injury by suppressing oxidative stress, the inflammatory response, and apoptosis, improving liver proliferation and upregulating p-eNOS expression.

Ac2-26 Alleviates Brain Injury after Cardiac Arrest and Cardiopulmonary Resuscitation in Rats via the eNOS Pathway.

Background Brain injury is the leading cause of death following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Ac2-26 and endothelial nitric oxide synthase (eNOS) have been shown to reduce neuroinflammation. This study is aimed at determining the mechanism by which Ac2-26 protects against inflammation during brain injury following CA and CPR. Methods Sixty-four rats were randomized into sham, saline, Ac2-26, and Ac2-26+L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor) groups. Rats received Ac2-26, Ac2-26+L-NIO, or saline after CPR. Neurologic function was assessed at baseline, 24, and 72 hours after CPR. At 72 hours after resuscitation, serum and brain tissues were collected. Results Blood-brain barrier (BBB) permeability increased, and the number of surviving neurons and neurological function decreased in the saline group compared to the sham group. Anti-inflammatory and proinflammatory factors, neuron-specific enolase (NSE) levels, and the expression of eNOS, phosphorylated (p)-eNOS, inducible nitric oxide synthase (iNOS), and oxidative stress-related factors in the three CA groups significantly increased (P < 0.05). BBB permeability decreased, and the number of surviving neurons and neurological function increased in the Ac2-26 group compared to the saline group (P < 0.05). Ac2-26 increased anti-inflammatory and reduced proinflammatory markers, raised NSE levels, increased the expression of eNOS and p-eNOS, and reduced the expression of iNOS and oxidative stress-related factors compared to the saline group (P < 0.05). The effect of Ac2-26 on brain injury was reversed by L-NIO (P < 0.05). Conclusions Ac2-26 reduced brain injury after CPR by inhibiting oxidative stress and neuroinflammation and protecting the BBB. The therapeutic effect of Ac2-26 on brain injury was largely dependent on the eNOS pathway.

Protective Effects of Allicin on ISO-Induced Rat Model of Myocardial Infarction via JNK Signaling Pathway

Objective: This research was aimed to explore protective effects of allicin on rat model of myocardial infarction via JNK signaling pathway. Methods: Rat myocardial ischemia model was established with subcutaneous injection of isoproterenol (ISO). Seventy-five rats were randomly divided into 5 groups (n = 15): sham group, ISO group, low-dose group (1.2 mg/kg/days for 7 days), medium-dose group (1.8 mg/kg/days for 7 days), and high-dose group (3.6 mg/kg/days for 7 days). Routine HE staining and Masson staining were performed to observe myocardial histopathology. The expression of oxidative stress-related indicators, heart tissue apoptosis-related proteins, and JNK and p-JNK proteins were measured for different groups. Results: Compared with the sham group, the T wave value of the ISO group was significantly increased (p < 0.01). When allicin was administered, the T wave values at different time points in all groups were all decreased. Compared with the sham group, the ratio of eNOS, Bcl-2/Bax was significantly decreased, and p-eNOS, iNOS, caspase-3, caspase-9, and Cyt-c were significantly elevated in the ISO group (p < 0.05). After allicin was administered, significant changes in these proteins were observed in the medium- and high-dose groups. There was no significant change in the expression of JNK protein in the ISO group compared with the sham group; however, the expression of eNOS and p-JNK protein were significantly upregulated (p < 0.01) and the expression of p-eNOS and iNOS were significantly downregulated (p < 0.01). When allicin was administered, expression of p-JNK protein was significantly downregulated. Conclusion: Allicin can reduce oxidative stress damage and cardiomyocyte apoptosis in rat model of myocardial infarction and can significantly regulate JNK signaling pathway.

Diabetes Upregulates Oxidative Stress and Downregulates Cardiac Protection to Exacerbate Myocardial Ischemia/Reperfusion Injury in Rats.

Diabetes exacerbates myocardial ischemia/reperfusion (IR) injury by incompletely understood mechanisms. We explored whether diabetes diminished BAG3/Bcl-2/Nrf-2/HO-1-mediated cardioprotection and overproduced oxidative stress contributing to exaggerated IR injury. Streptozotocin-induced diabetes enhanced hyperglycemia, cardiac NADPH oxidase p22/p67 expression, malondialdehyde amount and leukocyte infiltration, altered the mesenteric expression of 4-HNE, CaSR, p-eNOS and BAG3 and impaired microvascular reactivity to the vasoconstrictor/vasodilator by a wire myography. In response to myocardial IR, diabetes further depressed BAG3/Bcl-2/Nrf-2/HO-1 expression, increased cleaved-caspase 3/poly(ADP-ribose) polymerase (PARP)/TUNEL-mediated apoptosis and exacerbated IR-induced left ventricular dysfunction characterized by further depressed microcirculation, heart rate, left ventricular systolic pressure and peak rate of pressure increase/decrease (±dp/dt) and elevated left ventricular end-diastolic pressure (LVEDP) and Evans blue-2,3,5-triphenyltetrazolium chloride-stained infarct size in diabetic hearts. Our results implicated diabetes exacerbated IR-induced myocardial dysfunction through downregulated BAG3/Bcl-2/Nrf-2/HO-1 expression, increased p22/p67/caspase 3/PARP/apoptosis-mediated oxidative injury and impaired microvascular reactivity.

(-)-Epigallocatechin-3-Gallate Inhibits eNOS Uncoupling and Alleviates High Glucose-Induced Dysfunction and Apoptosis of Human Umbilical Vein Endothelial Cells by PI3K/AKT/eNOS Pathway.

IntroductionDiabetes can increase the risk of cardiovascular disease. This study aimed to explore the effect of (-)-epigallocatechin-3-gallate (EGCG) on high glucose (HG)-induced dysfunction and apoptosis of vascular endothelial cells.Materials and MethodsThe viability of human umbilical vein endothelial cells (HUVECs) treated with different concentrations and times of EGCG was detected by CCK-8 assay. The expression levels of ROS, NO and BH4 in HUVECs after treatment were detected by respective ELISA kits. The expression of p-eNOS, eNOS, NOX4, bcl2, bax, cleaved-caspase3, caspase3, p-PI3K, p-AKT, PI3K and AKT in HUVECs was detected by Western blot analysis. The apoptosis of HUVECs after treatment was analyzed by TUNEL assay.ResultsThe viability of HUVECs was not obviously changed when treated with different concentrations and times of EGCG. The expression of ROS, NOX4 and eNOS (monomer) was increased, while the expression of NO, p-eNOS, eNOS, BH4 and eNOS (dimer) was decreased in HUVECs of HG group. EGCG could gradually reverse the effect of high glucose on HG-treated HUVECs from 10 μM to 50 μM. The apoptosis of HUVECs was also increased in HG group and EGCG decreased the apoptosis of HUVECs. PI3K/AKT signaling pathway was suppressed in HG-treated HUVECs while activated by EGCG treatment. When the PI3K/AKT signaling pathway was inhibited by LY294002 (AKT inhibitor), the protective effect of EGCG on HG-treated HUVECs was weakened.ConclusionEGCG could inhibit eNOS uncoupling and alleviate endothelial dysfunction and apoptosis of HG-treated HUVECs by activating the PI3K/AKT/eNOS pathway.

Can Racial Differences In Endothelial Dysfunction Be Explained By Mirnas?

MicroRNAs (miRs) are short noncoding RNAs that regulate gene expression post-transcriptionally. It is well documented that exercise improves endothelial function, possibly, by modifying flow-responsive miR expression (e.g. miRs-126*, -92-a and -21). MiR-126* is an anti-atherogenic miR that regulates vascular integrity, angiogenesis, and inflammation. Whereas, miR-92a and -21 are pro-atherogenic miRs that result in a reduction in endothelial nitic oxide synthase (eNOS) activity. MiR-92a targets the transcription factor Kruppel-like factor 4 (KLF4) leading to lower eNOS expression. Whereas miR-21 targets phosphatase and tensin homologue (PTEN) to inhibit activation of the PI3K/Akt signaling pathway involved in activating eNOS. We have shown that African Americans (AA) exhibit a low grade of chronic systemic inflammation, exaggerated response to inflammatory cytokines, and a higher prevalence of endothelial dysfunction compared to Caucasians (CA). PURPOSE: To investigate potential racial differences in miRs expression and downstream target proteins in activated endothelial cells (EC). METHODS: Human Umbilical Vein Endothelial Cells (n=6 cell lines; 3 CA & 3 AA) were cultured under two conditions: Control or TNF-α (10 ng/mL) for 4 hours. Protein expression was measured for PTEN, KLF4, p-eNOSser-1117 and eNOS. Total RNA was harvested to measure miR-21, miR-126*, and miR92a. A two-way ANOVA was used to assess the effects of race and condition. RESULTS: No racial differences were found in protein expression of PTEN, KLF4, and eNOS at baseline or with TNF-α stimulation. AA had lower p-eNOS expression (p=0.008) compared to CA ECs under both conditions. No racial differences were found in miR-126 and miR-21 in either condition. However, AA were trending towards higher miR-92a expression (p=0.07) compared to CA ECs under both conditions. CONCLUSIONS: Although we show significantly lower levels of p-eNOS and higher miR-92a expression in activated ECs of AA compared to CA, KLF4 protein levels were not significantly different. Therefore, the specific miRs studied do not explain the racial differences observed in endothelial dysfunction in an inflammation model. Future research should investigate potential racial differences in how miRs respond to high laminar shear stress, as an exercise memetic.

Differences in Post-ischemic Motor Recovery and Angiogenesis of MCAO Rats Following Electroacupuncture at Different Acupoints.

Electroacupuncture (EA) can promote nerve and vascular regeneration, confer neuroprotection, inhibit apoptosis and inflammatory reactions, reduce oxidative stress injury, regulate neurochemicals and inhibit the formation of brain oedema in cerebral ischemic. However, the precise site of EA stimulation in the treatment of cerebral ischemic is unclear. In the present study, we investigated the effect of EA at the acupoints of different meridians in motor function recovery and the involvement of Vascular Endothelial Growth Factor (VEGF), phosphorylated Protein Kinase B (P-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS) and Platelet Endothelial Cell Adhesion Molecule-1(CD31) were examined in the peri-infarction cortex of rats. The Middle cerebral artery occlusion (MCAO) model or sham surgery was performed in a total of Ninety male Sprague-Dawley rats. Rats were randomly divided into five groups: a sham group, a middle cerebral artery occlusion (MCAO) group, a Yang meridian group, a Yin meridian group and a combined Yang and Yin meridian group. EA stimulus was given during the middle cerebral artery occlusion. The neurobehavioural function was measured using Modified Neurological Severity Scores (mNSS), the rotarod test and the ladder rung walking test, and the protein expression of VEGF, P-Akt, p-eNOS in the peri-infarction cortex was detected by Western blot. Immunofluorescence was used to measure the vascular density of the peri-infarction cortex. EA at different meridian acupoints has no effect on the infarction volume, while EA at Yin meridian acupoints significantly promoted neurobehavioural functional recovery, increased the vascular density and enhanced protein kinase B/Endothelial nitric oxide synthase (Akt/eNOS) phosphorylation and VEGF expression. In the early stage of stroke, EA at Yin meridian acupoints can improve neurobehavioural functional recovery and the mechanism of this effect may be related to the enhanced expression of VEGF, P-Akt and p-eNOS in the peri-infarction cortex of rats.