Endothelial Nitric Oxide Synthase Signaling Research Articles

Shexiang Tongxin Dropping Pill Promotes Angiogenesis through VEGF/eNOS Signaling Pathway on Diabetic Coronary Microcirculation Dysfunction.

To study the effect of Shexiang Tongxin Dropping Pill (STDP) on angiogenesis in diabetic cardiomyopathy mice with coronary microcirculation dysfunction (CMD). According to a random number table, 6 of 36 SPF male C57BL/6 mice were randomly selected as the control group, and the remaining 30 mice were injected with streptozotocin intraperitoneally to replicate the type 1 diabetes model. Mice successfully copied the diabetes model were randomly divided into the model group, STDP low-dose group [15 mg/(kg·d)], medium-dose group [30 mg/(kg·d)], high-dose group [60 mg/(kg·d)], and nicorandil group [15 mg/(kg·d)], 6 in each group. The drug was given by continuous gavage for 12 weeks. The cardiac function of mice in each group was detected at the end of the experiment, and coronary flow reserve (CFR) was detected by chest Doppler technique. Pathological changes of myocardium were observed by hematoxylin-eosin staining, collagen fiber deposition was detected by masson staining, the number of myocardial capillaries was detected by platelet endothelial cell adhesion molecule-1 staining, and the degree of myocardial hypertrophy was detected by wheat germ agglutinin staining. The expression of the vascular endothlial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) signaling pathway-related proteins in myocardial tissue was detected by Western blot. Compared with the model group, medium- and high-dose STDP significantly increased the left ventricular ejection fraction and left ventricular fraction shortening (P<0.01), obviously repaired the disordered cardiac muscle structure, reduced myocardial fibrosis, reduced myocardial cell area, increased capillary density, and increased CFR level (all P<0.01). Western blot showed that high-dose STDP could significantly increase the expression of VEGF and promote the phosphorylation of vascular endothelial growth factor receptor 2, phosphoinositide 3-kinase, protein kinase B, and eNOS (P<0.05 or P<0.01). STDP has a definite therapeutic effect on diabetic CMD, and its mechanism may be related to promoting angiogenesis through the VEGF/eNOS signaling pathway.

Integrated metabolomics analysis reveals mechanistic insights into variability in blood pressure response to thiazide diuretics and beta blockers.

Hypertensive patients with a higher proportion of genetic West African ancestry (%GWAA) have better blood pressure (BP) response to thiazide diuretics (TDs) and worse response to β-blockers (BBs) than those with lower %GWAA, associated with their lower plasma renin activity (PRA). TDs and BBs are suggested to reduce BP in the long term through vasodilation via incompletely understood mechanisms. This study aimed at identifying pathways underlying ancestral differences in PRA, which might reflect pathways underlying BP-lowering mechanisms of TDs and BBs. Among hypertensive participants enrolled in the Pharmacogenomics Evaluation of Antihypertensive Responses (PEAR) and PEAR-2 trials, we previously identified 8 metabolites associated with baseline PRA and 4 metabolic clusters (including 39 metabolites) that are different between those with GWAA <45% versus ≥45%. In the current study, using Ingenuity Pathway Analysis (IPA), we integrated these signals. Three overlapping metabolic signals within three significantly enriched pathways were identified as associated with both PRA and %GWAA: ceramide signaling, sphingosine 1- phosphate signaling, and endothelial nitric oxide synthase signaling. Literature indicates that the identified pathways are involved in the regulation of the Rho kinase cascade, production of the vasoactive agents nitric oxide, prostacyclin, thromboxane A2, and endothelin 1; the pathways proposed to underlie TD- and BB-induced vasodilatation. These findings may improve our understanding of the BP-lowering mechanisms of TDs and BBs. This might provide a possible step forward in personalizing antihypertensive therapy by identifying patients expected to have robust BP-lowering effects from these drugs.

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

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

Nicotinamide riboside relieves the severity of experimental necrotizing enterocolitis by regulating endothelial function via eNOS deacetylation

BackgroundNicotinamide adenine dinucleotide (NAD+) is involved in regulating oxidative stress. Although NAD+ is associated with various health issues, its role in the intestinal microcirculation in necrotizing enterocolitis (NEC) remains to be confirmed. In the current study, we explored whether nicotinamide riboside (NR), a natural NAD + precursor, ameliorates the severity of NEC through endothelial nitric oxide synthase(eNOS) signaling. MethodsA mouse experimental NEC model was induced by formula gavage and hypoxia in full-term mouse pups. Intestinal endothelial cells (MIMECs) were isolated and subjected to stress using tumor necrosis factor (TNF)-α. NR was administered to assess the intestinal microcirculation and lipid peroxidation levels and to explore the involved signaling pathways. ResultsNAD + levels were reduced after induction of NEC stress, which was associated with intestinal injury. NR administration promoted NAD + levels, attenuated oxidative stress and relieved the symptoms of experimental NEC, which were relevant to increased intestinal microcirculatory perfusion through the sirtuin (SIRT) 1 pathway in experimental NEC mice. However, this improvement was not found in eNOS-knockout mice. Consistently, MIMECs exposed to TNFα showed decreased SIRT1 activity associated with increased eNOS acetylation, which could bring about endothelial dysfunction due to limited nitric oxide production. NR administration increased the NAD + content and repressed the production of reactive oxygen species (ROS) in MIMECs under TNFα stress. NR also promoted SIRT1 activity and accordingly suppressed the eNOS acetylation levels under TNFα stress. ConclusionThe current data indicate that NR administration improves the survival of experimental NEC mice via SIRT1-associated eNOS acetylation/deacetylation modulation, which is implicated in endothelial dysfunction. Although NR is commonly found in the human diet, it may also be a promising strategy for NEC treatment because of its pathogenic association with NEC.

Shear stress-induced cellular senescence blunts liver regeneration through Notch-sirtuin 1-P21/P16 axis.

The mechanisms involved in liver regeneration after partial hepatectomy (pHx) are complicated. Cellular senescence, once linked to aging, plays a pivotal role in wound repair. However, the regulatory effects of cellular senescence on liver regeneration have not been fully elucidated. Mice subjected to pHx were analyzed 14 days after surgery. The incomplete remodeling of liver sinusoids affected shear stress-induced endothelial nitric oxide synthase (eNOS) signaling on day 14, resulting in the accumulation of senescent LSECs. Removing macrophages to augment LSEC senescence led to a malfunction of the regenerating liver. A dynamic fluctuation in Notch activity accompanied senescent LSEC accumulation during liver regeneration. Endothelial Notch activation by using Cdh5-CreERT NICeCA mice triggered LSEC senescence and senescence-associated secretory phenotype, which disrupted liver regeneration. Blocking the Notch by γ-secretase inhibitor (GSI) diminished senescence and promoted LSEC expansion. Mechanically, Notch-hairy and enhancer of split 1 signaling inhibited sirtuin 1 (Sirt1) transcription by binding to its promoter region. Activation of Sirt1 by SRT1720 neutralized the up-regulation of P53, P21, and P16 caused by Notch activation and eliminated Notch-driven LSEC senescence. Finally, Sirt1 activator promoted liver regeneration by abrogating LSEC senescence and improving sinusoid remodeling. Shear stress-induced LSEC senescence driven by Notch interferes with liver regeneration after pHx. Sirt1 inhibition accelerates liver regeneration by abrogating Notch-driven senescence, providing a potential opportunity to target senescent cells and facilitate liver repair after injury.

Nebivolol alleviates liver damage caused by methotrexate via AKT1/Hif1α/eNOS signaling

Despite the wide clinical indications, methotrexate (MTX) use is limited because of serious side effects including liver toxicity. MTX was shown to cause tissue damage by mainly oxidative stress and also inflammation and apoptosis. Thus, Nebivolol (NEB) which has antioxidant and antiapoptotic properties were thought to be effective against MTX-induced injury. This study aimed to evaluate the effects of NEB on MTX-induced liver toxicity via AKT/Hypoxia-Inducible Factor 1 Alpha (HIF1α)/Endothelial Nitric Oxide Synthase (eNOS) signaling pathways. Rats were divided into three groups as control, MTX, and NEB. A single dose of MTX (20 mg/kg intraperitoneally) was given to the rats on the first day of the experiment and NEB (10 mg/kg, daily by oral gavage) was given to the treatment group for a week. At the end of the experiment, bloods were taken for aspartate transaminase (AST), alanine aminotransferase (ALT), and total bilirubin (T-BIL) analyses. Liver tissues were harvested for biochemical (total oxidant status (TOS) and total antioxidant status (TAS), genetic (PCR analyses for AKT1, eNOS, and HIF1a), and histological (Hemotoxylin-Eosin, Masson Trichome, Periodic Acid Schiff-Asien Blue, reticulin for histological, and CD3 for immunohistochemical staining) analyses. MTX increased the levels of TOS values, AST, ALT, T-BIL levels and decreased the expressions of AKT/HIF1α/eNOS. NEB treatment reversed all these changes markedly via decreasing inflammation by nitric oxid (NO) production. In conclusion, NEB treatment significantly preserves the liver by decreasing oxidant levels and inflammatory parameters through HIF1α/eNOS signaling. Due to the antioxidant properties of NEB, it can be used in other liver injury models sharing the same pathway.

Phosphoinositide-3-Kinase/Akt–Endothelial Nitric Oxide Synthase Signaling Pathway Mediates the Neuroprotective Effect of Sevoflurane Postconditioning in a Rat Model of Hemorrhagic Shock and Resuscitation

Although extensive reports have demonstrated the neuroprotection of sevoflurane postconditioning in cases of focal and global cerebral ischemia/reperfusion, the underlying mechanisms are not completely elucidated. This study investigated whether this effect is related to endothelial nitric oxide synthase (eNOS) and mediated by the phosphoinositide-3-kinase pathway in a rat model of hemorrhagic shock and resuscitation. Adult male Sprague Dawley rats were subjected to hemorrhagic shock for 60 minutes and then resuscitation for 30 minutes in experimental groups. Sevoflurane postconditioning was performed at the beginning of resuscitation to completion. At 24 hours after resuscitation, the brain infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. The neuronal morphological changes and apoptosis were determined by hematoxylin and eosin staining and immunohistochemistry analysis, respectively. The activity of phosphorylated Akt and eNOS was evaluated by Western blot analysis. Brain injuries such as the cerebral infarct volume and pathological neuronal changes as well as cell apoptosis were observed in the hippocampus after hemorrhagic shock and resuscitation. Postconditioning with 2.4% sevoflurane significantly attenuated brain injuries. Wortmannin prevented the improvements of neuronal characteristics elicited by sevoflurane postconditioning as well as the hyperactivity of eNOS and phosphorylated Akt. Sevoflurane postconditioning could attenuate brain injury induced by hemorrhagic shock and resuscitation, and this neuroprotective effect may be partly by upregulation of eNOS through the phosphoinositide-3-kinase/Akt signaling pathway.

Acute Decompensated Heart Failure and the Kidney: Physiological, Histological and Transcriptomic Responses to Development and Recovery.

BACKGROUNDAcute decompensated heart failure (ADHF) is associated with deterioration in renal function—an important risk factor for poor outcomes. Whether ADHF results in permanent kidney damage/dysfunction is unknown.METHODS AND RESULTSWe investigated for the first time the renal responses to the development of, and recovery from, ADHF using an ovine model. ADHF development induced pronounced hemodynamic changes, neurohormonal activation, and decline in renal function, including decreased urine, sodium and urea excretion, and creatinine clearance. Following ADHF recovery (25 days), creatinine clearance reductions persisted. Kidney biopsies taken during ADHF and following recovery showed widespread mesangial cell prominence, early mild acute tubular injury, and medullary/interstitial fibrosis. Renal transcriptomes identified altered expression of 270 genes following ADHF development and 631 genes following recovery. A total of 47 genes remained altered post‐recovery. Pathway analysis suggested gene expression changes, driven by a network of inflammatory cytokines centered on IL‐1β (interleukin 1β), lead to repression of reno‐protective eNOS (endothelial nitric oxide synthase) signaling during ADHF development, and following recovery, activation of glomerulosclerosis and reno‐protective pathways and repression of proinflammatory/fibrotic pathways. A total of 31 dysregulated genes encoding proteins detectable in urine, serum, and plasma identified potential candidate markers for kidney repair (including CNGA3 [cyclic nucleotide gated channel subunit alpha 3] and OIT3 [oncoprotein induced transcript 3]) or long‐term renal impairment in ADHF (including ACTG2 [actin gamma 2, smooth muscle] and ANGPTL4 [angiopoietin like 4]).CONCLUSIONSIn an ovine model, we provide the first direct evidence that an episode of ADHF leads to an immediate decline in kidney function that failed to fully resolve after ≈4 weeks and is associated with persistent functional/structural kidney injury. We identified molecular pathways underlying kidney injury and repair in ADHF and highlighted 31 novel candidate biomarkers for acute kidney injury in this setting.

Salidroside inhibits endothelial‑mesenchymal transition via the KLF4/eNOS signaling pathway.

Homocysteine (Hcy) was discovered to be an independent risk factor for the development of atherosclerosis (AS). Moreover, endothelial-mesenchymal transition (EndMT) was found to be one of main mechanisms contributing to the pathogenesis of AS. Salidroside (SAL) has diverse pharmacological activities, including anti-inflammatory, anti-cancer, anti-oxidative and anti-fibrosis properties. However, whether SAL serves a beneficial role in Hcy-induced EndMT remains unknown. The present study aimed to investigate whether SAL exerted its effects on Hcy-induced EndMT via the Kruppel-like factor 4 (KLF4)/endothelial nitric oxide (NO) synthase (eNOS) signaling pathway. HUVECs were pretreated with high and low doses (10 or 50 µmol/l) of SAL for 2 h, followed by 1 mmol/l Hcy for 48 h to induce EndMT. Western blotting was used to analyze the protein expression levels of the endothelial marker, VE-cadherin, the mesenchymal cell marker, α-smooth muscle actin (SMA), and the nuclear transcription factors, KLF4 and eNOS. Wound healing assays were used to determine the cell migratory ability, and the levels of NO in the cell culture supernatants were measured using a nitrate reductase assay. Cellular immunofluorescence was used to analyze the expression and localization of KLF4. Small interfering (si)RNA targeting KLF4 (siKLF4) was used to knock down KLF4 expression in HUVECs. The results of the present study revealed that treatment with SAL upregulated the expression levels of VE-cadherin, downregulated the expression levels of α-SMA, reduced cell migration and activated the eNOS/NO signaling axis, as well as downregulated KLF4 expression and translocation to the nucleus. Compared with the SAL + siKLF4 co-administration group, no significant differences were observed in the expression levels of the phenotypic markers in the SAL or siKLF4 groups. In conclusion, the findings of the present study revealed that SAL may inhibit Hcy-induced EndMT via regulation of the KLF4/eNOS signaling pathway.

EFFECTS OF A GLUCAGON LIKE PEPTIDE-1 AGONIST AND RHO-KINASE INHIBITOR ON PULMONARY AND RIGHT VENTRICLE FUNCTION IN PULMONARY HYPERTENSIVE RATS WITH AND WITHOUT INSULIN RESISTANCE

Objective: Insulin resistance is now recognized as an important factor in the development of pulmonary hypertension (PH). Increased activation of RhoA/Rho-kinase (ROCK) inhibits the protective actions of endothelial nitric oxide (NO) synthase (eNOS) signaling in diabetes and PH. Glucagon like peptide-1 (GLP-1) agonists were shown to reverse these pathological changes. We investigated how liraglutide (GLP-1 agonist) and fasudil (ROCK inhibitor) treatments affect the progression of pulmonary and right ventricle (RV) dysfunction in PH rats with insulin resistance. Design and method: SU5416 (20 mg/kg) was administered to male Wistar and prediabetic Goto-Kakizaki (GK) rats followed by 3 weeks of chronic hypoxia (SuHx) or normoxia exposure. Osmotic pumps delivered a continuous infusion of liraglutide (1 mg/kg/d) or fasudil (100 mg/kg/d) for the final 3 weeks in some SuHx rats. Synchrotron microangiography of the left lung was performed in vivo. Results: During chronic hypoxia 30% mortality occurred due to RV failure associated with thrombosis in GK rats. Systolic RV pressure was 74 mm Hg and 128 mm Hg in GK and Wistar SuHx rats respectively. Prediabetes exacerbated pulmonary arteriole-artery rarefaction (two-fold occlusion increase) and pulmonary vasoconstriction, with acetylcholine (ACh)-mediated constriction at 3 weeks and loss of NO donor mediated dilation at 6 weeks SuHx. Wistar SuHx maintained ACh-mediated dilation in resistance vessels despite pronounced overall vasoconstriction of all branching orders. Neither strain differed in ROCK1/2 protein expression. Notably, liraglutide and fasudil improved pulmonary vasodilation, but only liraglutide halted vascular remodeling. Fasudil increased phosphorylation of eNOS at Ser1177 in the RV of GK SuHx. Liraglutide reduced RV systolic pressure and Fulton indices, while fasudil elevated RV pressure to 104 mm Hg in the GK SuHx. Conclusions: Reduced NO bioavailability contributes to exacerbated pulmonary arterial rarefaction and depressed RV contractility associated with prediabetes in SuHx rats. GLP-1 agonist treatment did not ameliorate endothelin-1/ROCK- mediated suppression of eNOS phosphorylation in the RV as observed with fasudil. While liraglutide intervention prevented pulmonary microvessel occlusion, inhibition of both ROCK1/2 isoforms with fasudil increased occlusion formation suggesting that ROCK1 might act to inhibit pulmonary microvessel muscularization.

Gualou-Xiebai-Banxia decoction protects against type II diabetes with acute myocardial ischemia by attenuating oxidative stress and apoptosis via PI3K/Akt/eNOS signaling.

Gualou-Xiebai-Banxia decoction has a long history of medical use for treating cardiovascular diseases in China. In this study, we investigated the protective effect and underlying mechanisms GXB in type II diabetes with acute myocardial ischemia (T2DM-AMI) rats. We hypothesized that GXB may display its protective effect on T2DM-AMI by reducing endothelial progenitor cells (EPCs) apoptosisviaactivating PI3K (phosphatidyl inositol 3-kinase)/Akt (serine/threonine protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling. Rats were challenged with a high-fat diet and intraperitoneal injection of streptozotocin to induce a model of type II diabetes mellitus (T2DM) and coronary ligation to induce acute myocardial infarction (AMI). Changes in metabolites were assessed via enzyme-linked immunoassay and biochemical examination. The number and apoptosis rate of EPCs in peripheral blood were detected by flow cytometry. Target mRNAs and proteins in EPCs were analyzed by RT-PCR and Western blot analysis. The results demonstrated that GXB treatment decreased T2DM-AMI-associated changes in plasma fasting blood glucose, muscular enzymes, and blood lipids, and reduced oxidative stress. Furthermore, EPC apoptosis was increased in T2DM-AMI rats and was associated with decreased mRNA and protein levels of PI3K, Akt, and eNOS compared to the controls. Conversely, T2DM-AMI rats treated with GXB exhibited more circulating EPCs and downregulated levels of cell apoptosis, combined with increased mRNA and protein levels of PI3K, Akt, and eNOS compared to those of untreated T2DM-AMI rats. Our study showed that GXB treatment mitigated EPC apoptosis and promoted PI3K/Akt/eNOS signaling in T2DM-AMI rats.

Promotion of Aerobic Exercise Induced Angiogenesis Is Associated With Decline in Blood Pressure in Hypertension: Result of EXCAVATION-CHN1.

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Physiologic Consequences of Caveolin-1 Ablation in Conventional Outflow Endothelia.

PurposePolymorphisms at the caveolin-1/2 locus are associated with glaucoma and IOP risk and deletion of caveolin-1 (Cav1) in mice elevates IOP and reduces outflow facility. However, the specific location/cell type responsible for Cav1-dependent regulation of IOP is unclear. We hypothesized that endothelial Cav1 in the conventional outflow (CO) pathway regulate IOP via endothelial nitric oxide synthase (eNOS) signaling.MethodsWe created a mouse with targeted deletion of Cav1 in endothelial cells (Cav1ΔEC) and evaluated IOP, outflow facility, outflow pathway distal vascular morphology, eNOS phosphorylation, and tyrosine nitration of iridocorneal angle tissues by Western blotting.ResultsEndothelial deletion of Cav1 resulted in significantly elevated IOP versus wild-type mice but not a concomitant decrease in outflow facility. Endothelial Cav1 deficiency did not alter the trabecular meshwork or Schlemm's canal morphology, suggesting that the effects observed were not due to developmental deformities. Endothelial Cav1 deletion resulted in eNOS hyperactivity, modestly increased protein nitration, and significant enlargement of the drainage vessels distal to Schlemm's canal. L-Nitro-arginine methyl ester treatment reduced outflow in Cav1ΔEC but not wild-type mice and had no effect on the size of drainage vessels. Endothelin-1 treatment decrease the outflow and drainage vessel size in both wild-type and Cav1ΔEC mice.ConclusionsOur results suggest that hyperactive eNOS signaling in the CO pathway of both Cav1ΔEC and global Cav1 knockout mice results in chronic dilation of distal CO vessels and protein nitration, but that Cav1 expression in the trabecular meshwork is sufficient to rescue CO defects reported in global Cav1 knockout mice.

Mathematical Model Predicts Effective Strategies to Inhibit VEGF-eNOS Signaling.

The endothelial nitric oxide synthase (eNOS) signaling pathway in endothelial cells has multiple physiological significances. It produces nitric oxide (NO), an important vasodilator, and enables a long-term proliferative response, contributing to angiogenesis. This signaling pathway is mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic species that is often targeted to inhibit tumor angiogenesis. However, inhibiting VEGF-mediated eNOS signaling can lead to complications such as hypertension. Therefore, it is important to understand the dynamics of eNOS signaling in the context of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important angiogenic inhibitor that, through interaction with its receptor CD47, has been shown to redundantly inhibit eNOS signaling. However, the exact mechanisms of TSP1′s inhibitory effects on this pathway remain unclear. To address this knowledge gap, we established a molecular-detailed mechanistic model to describe VEGF-mediated eNOS signaling, and we used the model to identify the potential intracellular targets of TSP1. In addition, we applied the predictive model to investigate the effects of several approaches to selectively target eNOS signaling in cells experiencing high VEGF levels present in the tumor microenvironment. This work generates insights for pharmacologic targets and therapeutic strategies to inhibit tumor angiogenesis signaling while avoiding potential side effects in normal vasoregulation.

Shixiang Plaster, a Traditional Chinese Medicine, Promotes Healing in a Rat Model of Diabetic Ulcer Through the receptor for Advanced Glycation End Products (RAGE)/Nuclear Factor kappa B (NF-κB) and Vascular Endothelial Growth Factor (VEGF)/Vascular Cell Adhesion Molecule-1 (VCAM-1)/Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathways.

BackgroundShixiang plaster is a traditional Chinese medicine has been used to treat chronic ulcers, including diabetic ulcers. Aminoguanidine is a hydrazine derivative that inhibits the formation of advanced glycosylation end products (AGEs). This study aimed to investigate the effects of shixiang plaster and aminoguanidine on wound healing in the streptozotocin-induced rat model of diabetes and the molecular mechanisms involved.Material/MethodsSprague-Dawley rats treated with intraperitoneal streptozotocin and given surgical wounds were divided into the untreated chronic ulcer group (n=10), the aminoguanidine group (n=10), the shixiang plaster group (n=10), and the control group with sham surgery (n=10). Granulation tissue samples underwent light microscopy to evaluate angiogenesis and immunohistochemistry to identify AGE, vascular endothelial growth factor (VEGF), and CD34 expression. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot measured mRNA and protein expression of receptor for advanced glycation end products (RAGE), vascular cell adhesion molecule-1 (VCAM-1), nuclear factor kappa B (NF-κB) and endothelial nitric oxide synthase (eNOS).ResultsThe shixiang plaster group showed a significant increase in angiogenesis in ulcer granulation tissue, significantly reduced expression of AGEs and increased expression of VEGF and CD34 expression in granulation tissue compared with the untreated chronic ulcer group (p<0.05). The shixiang plaster group showed significantly down-regulated expression of RAGE and VCAM-1 compared with the untreated chronic ulcer group (p<0.05). Shixiang plaster promoted angiogenesis by activating the NF-κB p65 associated pathway and eNOS activation.ConclusionsShixiang plaster promoted healing in a rat model of diabetic ulcer through the RAGE/NF-κB and VEGF/VCAM-1/eNOS signaling pathways.

A Role of Agrin in Maintaining the Stability of Vascular Endothelial Growth Factor Receptor-2 during Tumor Angiogenesis

Endothelial cell (EC) recruitment is central to the vascularization of tumors. Although several proteoglycans have been implicated in cancer and angiogenesis, their roles in EC recruitment and vascularization during tumorigenesis remain poorly understood. Here, we reveal that Agrin, which is secreted in liver cancer, promotes angiogenesis by recruiting ECs within tumors and metastatic lesions and facilitates adhesion of cancer cells to ECs. In ECs, Agrin-induced angiogenesis and adherence to cancer cells are mediated by Integrin-β1, Lrp4-MuSK pathways involving focal adhesion kinase. Mechanistically, we uncover that Agrin regulates VEGFR2 levels that sustain the angiogenic property of ECs and adherence to cancer cells. Agrin attributes an ECM stiffness-based stabilization of VEGFR2 by enhancing interactions with Integrin-β1-Lrp4 and additionally stimulates endothelial nitric-oxide synthase (e-NOS) signaling. Therefore, we propose that cross-talk between Agrin-expressing cancer and ECs favor angiogenesis by sustaining the VEGFR2 pathway.

Aerobic Training-induced Increase In Follistatin-like 1 Secretion Is Associated With eNOS Phosphorylation In Diabetic Rats.

INTRODUCTIN: Aerobic exercise training reduces arterial stiffness via elevation of nitric oxide (NO) production in type 2 diabetes. Follistatin-like 1 (FSTL1) is a muscle-derived myokine and stimulates protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signaling via binding to receptor (DIP2A) in endothelial cells. However, it is unclear whether FSTL1 secretion induced by aerobic exercise training is related to a reduction of arterial stiffness in type 2 diabetic rats. PURPOSE: This study aimed to investigate whether FSTL1 secretion induced by aerobic exercise training is related to a reduction of arterial stiffness with activation of aortic eNOS phosphorylation in type 2 diabetic rats. METHODS: Twenty-week-old male type 2 diabetic (OLETF) rats were randomly divided into sedentary control (OLETF-Con: n=7) and aerobic exercise training (OLETF-Ex: treadmill running for 60min at 25m/min, 5days/week: n=7) groups. In addition, 7 male LETO rats were used as healthy sedentary control. After 8-week experiment, we measured carotid-femoral pulse wave velocity (cfPWV: as an index of arterial stiffness), and thoracic aorta was isolated after collection of blood. RESULTS: Fasting blood glucose and cfPWV significantly increased (p<0.05) and aortic Akt and eNOS phosphorylation levels, plasma nitrate/nitrite (NOx) level, and serum FSTL1 protein level significantly decreased (p<0.05) in OLETF-Con as compared to LETO. OLETF-Ex showed significant decrease in fasting blood glucose and cfPWV (p<0.05), and significant increase in aortic Akt and eNOS phosphorylation, plasma NOx, and serum FSTL1 protein levels (p<0.05) as compared to OLETF-Con. However, no significant difference in aortic DIP2A protein level among three groups was seen. Serum FSTL1 protein level was positively correlated with aortic eNOS phosphorylation level (p<0.05, r=0.630), and plasma NOx level was negatively correlated with cfPWV (p<0.05, r=-0.546). CONCLUSION: The results suggest that the increase in FSTL1 secretion induced by aerobic exercise training is associated with the reduction of arterial stiffness via activation of arterial NO production-related signaling pathway in type 2 diabetic rats. Supported by Grants-in-Aid for Scientific Research (#17H02182, #16K13059, M. Iemitsu)

Effect of tyrosol on myocardial ischemia-reperfusion injury in diabetic rats and the role of SIRT1/AMPK/eNOS signaling pathway

Objective To evaluate the effect of tyrosol on myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the role of silent mating-type information regulation 1 (SIRT1)/adenosine monophosphate-activated protein kinase (AMPK)/endothelial nitric oxide synthase (eNOS) signaling pathway. Methods SPF healthy adult male Sprague-Dawley rats, weighing 200-220 g, were intraperitoneally injected with streptozotocin 60 mg/kg to establish the model of diabetes mellitus.Fifty-six diabetic rats were divided into 4 groups (n=14 each) using a random number table method: sham operation group (S group), myocardial I/R group(I/R group), myocardial I/R plus tyrosol group (I/R+ T group), and myocardial I/R plus tyrosol plus SIRT1 inhibitor EX527 group (I/R+ T+ E group). In I/R+ T and I/R+ T+ E groups, tyrosol 20 mg·kg-1·d-1 was given by gavage for 45 consecutive days, and the equal volume of normal saline was given in the other two groups.In I/R+ T+ E group, EX527 5 mg·kg-1·d-1 was intraperitoneally injected for 3 consecutive days before ischemia, and EX527 5 mg/kg was intraperitoneally injected at 20 min before reperfusion.Myocardial I/R was induced by ligation of the left anterior descending branch of coronary artery for 30 min followed by 2-h reperfusion.The myocardial infarct volume was measured by TTC staining.The levels of creatine kinase-MB (CK-MB), lactic dehydrogenase (LDH) and 5-F2t-isoprostane in serum and superoxide dismutase (SOD) in myocardial tissues were detected by enzyme-linked immunosorbent assay.The expression of SIRT1, AMPK, phosphorylated AMPK (p-AMPK), eNOS and p-eNOS was detected by Western blot. Results Compared with S group, the levels of serum CK-MB, LDH and 15-F2t-Isoprostane and myocardial infarction volume were significantly increased, the SOD activity was decreased, and the SIRT1 expression was down-regulated in I/R group, and the levels of serum CK-MB, LDH and 15-F2t-Isoprostane and myocardial infarction volume were significantly increased, the SOD activity was decreased, the SIRT1 expression was down-regulated, and the expression of p-AMPK and p-eNOS was up-regulated in I/R+ T and I/R+ T+ E groups (P 0.05). Compared with I/R+ T group, the levels of CK-MB, LDH and 15-F2t-isoprostane in serum and myocardial infarct volume were significantly increased, the SOD activity was increased, and the expression of SIRT1, p-AMPK and p-eNOS was down-regulated in I/R+ T+ E group (P<0.05). Conclusion Tyrosol can mitigate myocardial I/R injury, and the mechanism may be related to activating SIRT1/AMPK/eNOS signaling pathway and inhibiting oxidative stress response in diabetic rats. Key words: Antioxidants; Diabetes mellitus; Myocardial reperfusion injury; Sirtuin 1; Protein-serine-threonine kinases; Nitric oxide synthase

MicroRNA-615-5p Regulates Angiogenesis and Tissue Repair by Targeting AKT/eNOS (Protein Kinase B/Endothelial Nitric Oxide Synthase) Signaling in Endothelial Cells.

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

The GLP‐1 agonist liraglutide lowers blood pressure in a placental ischemic model of preeclampsia

Preeclampsia is a multi‐system disorder of pregnancy characterized by widespread vascular dysfunction. Though the precise etiology of preeclampsia remains largely enigmatic, emerging evidence suggests that an ischemic placenta releases circulating vasoactive factors all of which culminate in endothelial dysfunction. The incretin hormone glucagon‐like peptide 1 (GLP‐1) has been extensively studied for its therapeutic benefit in diabetes mellitus, where it acts to enhance endogenous insulin secretion. In addition to its role in glucose homeostasis, GLP‐1 receptors are present on endothelial cells and activate the endothelial nitric oxide synthase (eNOS) signaling axis. In turn, GLP‐1 receptor agonists have been demonstrated to improve endothelial dysfunction. Therefore, we hypothesized that administration of a GLP‐1 receptor agonist would attenuate endothelial dysfunction and improve blood pressure in our model of preeclampsia.In order to test this hypothesis, timed pregnant CD‐001 rats (an outbred sub‐strain of Sprague‐Dawley rats) underwent surgery on gestation day 15 to place silver clips on the aorta and ovarian‐uterine arterial anastomoses. This procedure restricts blood flow to the fetal‐placental unit by 60%, producing a model of reduced uterine perfusion pressure (RUPP) that mimics several characteristics of human preeclampsia. From gestation day 15 to 20, animals received daily subcutaneous injections of 0.3 mg/kg liraglutide or vehicle. Animals were instrumented with carotid catheters on gestation day 19 and blood pressure determined on day 20 while the animals were conscious and restrained. Daily weight, food intake, and pup outcome were collected.We found that blood pressure was significantly lower in RUPP animals treated with liraglutide (107.8 ±2.5 vs 117.6 ±4.0 mmHg, n = 5 and 8 respectively, p&lt;0.05). Though food intake was diminished in the liraglutide‐treated group immediately following surgery (2.0 ±0.6 vs. 10.8 ±1.6 grams/day on gestation day 15), it rapidly recovered (16.3 ±1.6 vs. 14.5 ±1.8 grams/day on gestation day 19), and there was no significant difference between body weight (259±10 vs 287 ±14 grams on gestation day 20), litter size (8 ±2 vs 10 ±1 live pups), or pup weight (2.07 ±0.05 vs 2.15 ±0.11 grams).This data shows that GLP‐1 receptor agonism effectively lowers blood pressure in a placental ischemic model of preeclampsia. Although the mechanisms contributing to the improved blood pressure remain unclear, these data suggest that the GLP‐1 system represents a new potential therapeutic target in preeclampsia. Importantly, given its role in diabetes, several GLP‐1 receptor agonists are already FDA approved. Future studies will examine vascular mechanisms by which GLP‐1 receptor agonism protects against the development of placental ischemia‐induced hypertension.Support or Funding Information18PRE34050024HL136684P20GM104357This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.