Rat Aortic Endothelial Cells Research Articles

Schisandrin C targets Keap1 and attenuates oxidative stress by activating Nrf2 pathway in Ang II-challenged vascular endothelium.

Vascular endothelial dysfunction plays a crucial role in the pathogenesis of cardiovascular diseases. Oxidative stress is a key pathophysiological mechanism underpinning endothelial dysfunction. Schisandrin C (Sch C), a dibenzocyclooctadiene derivative of Schisandra chinensis, has antioxidative properties. Here, we report the use of Sch C as a novel therapeutic for the treatment of angiotensin II (Ang II)-induced endothelial deficits and explore the underlying mechanisms and the target of Sch C. Our results demonstrated that Sch C treatment prevents aorta oxidative stress and improves relaxation in mice, challenged with subcutaneous infusion of Ang II. In addition, Sch C significantly ameliorates Ang II-induced oxidative stress in rat aortic endothelial cells. We then discovered that these antioxidative effects of Sch C are mediated through the induction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Using an expression plasmid and molecular docking, we identified that Kelch-like ECH-associated protein-1 (Keap1), a negative regulator of Nrf2, is a target of Sch C. These findings provide evidence for the potential use of Sch C as an antioxidative agent for treatment of vascular endothelial deficits.

Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress

Prior research has demonstrated cells exposed to silver nanoparticles (AgNPs) undergo endoplasmic reticulum (ER) stress leading to cellular apoptosis and toxicity, however, the fundamental mechanism underlying AgNP-induced ER stress is unknown. We hypothesize the biophysical interactions between AgNPs and adsorbed proteins lead to misfolded proteins to elicit an ER stress response. Our investigation examined rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high density lipoprotein (HDL) or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 nm and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not observed for the 100 nm AgNPs. Hyperspectral darkfield microscopy qualitatively confirmed that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the sub-cellular localization of AgNPs. Overall, these results suggest that AgNP size, surface area and BC formation governs the induction of ER stress and alterations in intracellular trafficking.

Formononetin induces vasorelaxation in rat thoracic aorta via regulation of the PI3K/PTEN/Akt signaling pathway.

BackgroundFormononetin (FMN) is an isoflavone that produces arterial vasodilation. However, the underlying molecular mechanisms are unclear.PurposeThe purpose of this study was to explore the vasorelaxant effect and the potential mechanism of FMN in vascular endothelium in isolated rat aorta.MethodsThe thoracic aortas of Sprague Dawley rats were isolated to test the arterial reactivity in the presence of FMN with or without inhibitors. Bioinformatics analyses, including a Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine and molecular docking methods, were performed to predict therapeutic targets responsible for the vascular protection produced by FMN. We used rat aortic endothelial cells (RAOECs) as an in vitro model to verify the potential mechanism through molecular biological analyses. The production of nitric oxide (NO) metabolites were evaluated via an NO assay kit according to the manufacturer’s instruction. The mRNA expression of eNOS was analyzed by polymerase chain reaction, and the protein levels of PTEN, phosphorylated Akt, and eNOS were measured by Western blot.ResultsWe found that FMN dilated rat aortic rings in a concentration-dependent manner, which was reduced by endothelium denudation and eNOS inhibition. The bioinformatics analyses indicated that FMN activity was associated with the PI3K/PTEN/Akt signaling pathway. Molecular biological studies demonstrated that FMN significantly elevated the levels of NO and eNOS mRNA and markedly increased the protein expression of phosphorylated Akt and eNOS in RAOECs, and decreased PTEN compared with a dimethyl sulfoxide group.ConclusionFMN performs vasorelaxation of the thoracic aorta through activating the PI3K/PTEN/Akt signaling pathway.

Schisandrin B protects against angiotensin II-induced endotheliocyte deficits by targeting Keap1 and activating Nrf2 pathway.

IntroductionSchisandrin B (SchB), the main active constituent in Schisandra chinensis, has antioxidant activities. Endothelial dysfunction leads to various cardiovascular diseases. Oxidative stress is a crucial pathophysiological mechanism underpinning endothelial dysfunction.MethodsWe elucidated the role and underlying mechanisms of SchB in angiotensin II-induced rat aortic endothelial-cell deficits and explored targets of SchB through siRNA analysis and molecular docking. We measured apoptosis by TUNEL and oxidative stress by dihydroethidium (DHE) and 2’,7’ –dichlorofluorescin diacetate (DCF) staining.ResultsOur results demonstrated that SchB significantly ameliorated oxidative stress, mitochondrial membrane-potential depolarization and apoptosis in angiotensin II-challenged rat aortic endothelial cells. We further discovered that these antioxidative effects of SchB were mediated through induction of Nrf2. Importantly, using molecular docking and molecular dynamic simulation, we identified that Keap1, an adaptor for the degradation of Nrf2, was a target of SchB.ConclusionThese findings support the potential use of SchB as a Keap1 inhibitor for attenuating oxidative stress, and Keap1 might serve as a therapeutic target in the treatment of cardiovascular diseases.

ELUCIDATING THE MOLECULAR MECHANISMS OF MIR-26A ON THE PATHOGENESIS OF ABDOMINAL AORTIC ANEURYSM - IN VITRO STUDIES

Despite guideline recommendations for repair of Abdominal Aortic Aneurysm (AAA) when aortic diameter reaches 5.5 cm, the cumulative rates of aneurysm-related death remain high. Unfortunately, no therapies exist to fully prevent aneurysm growth. AAA is characterized by abnormal angiogenesis, impaired extracellular matrix synthesis and marked vessel inflammation -as such these are potential targets for preventative therapies. MicroRNA-26a (miR-26a) is a novel anti-angiogenic regulator of vascular smooth muscle cell (VSMC) function that is downregulated in AAA, and is a viable therapeutic target against AAA. We hypothesized that miR-26a can reverse the active molecular pathways in AAA. Cultured isolated rat aortic VSMCs and rat aortic endothelial cells (RAECs) were stimulated with the inflammatory interleukin-1β (IL-1β; 10 ng/ml) and then transfected with miR-26a, antimiR-26a and scrambled miR (siPORT™ NeoFX™ Transfection) to elucidate AAA cellular and molecular pathogenic pathways and the effects of miR-26a. Real-time qPCR (n=6 per group) and western blotting (n=6 per group) were performed to detect the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). Direct targets of miR-26a -phosphatase and tensin homolog (PTEN) and connective tissue growth factor (CTGF)- were quantified to assess miR-26a’s mechanism of action. Lastly, the angiogenic capacity of RAECs transfected with miR-26a or antimiR-26a were compared to control using the Matrigel™ Assay (n=6 per group). Upon IL-1β stimulation, miR-26a transfection reduced MMP-2 and MMP-9 mRNA levels back to baseline (p < 0.05) whereas antimiR-26a transfection showed no reduction. Stimulating rat VSMCs with IL-1β increased protein levels of active MMP-2 and MMP-9 in control and scramble miR-transfected groups. Transfection with miR-26a showed a reduction in levels of active and total MMP-2 and MMP-9 protein levels compared to control groups. Conversely, upon IL-1β stimulation, miR-26a transfection upregulated TIMP 1-4 levels (p < 0.05) whereas antimiR-26a transfection showed reduction back to baseline. TGFβ mRNA levels were downregulated after miR-26a transfection (p < 0.05) and upregulated after antimiR-26a transfection in VSMCs (p < 0.05). Lastly, overexpression of miR-26a led to reduced angiogenesis, with lower number of tubes and nodes, as compared to the control non-transfected RAECs (p < 0.0001) and antimiR-26a-transfected cells (p < 0.05). MiR-26a attenuates the inflammatory state of VSMCs through the downregulation of MMPs and upregulation of TIMPs and TGF-β, inhibits angiogenesis of RAECs and directly targets CTGF and PTEN, which are key molecules involved in AAA pathogenesis. This suggests that miR-26a is a viable therapeutic option for AAA.

Abstract P279: Regulation Of H 2 S Production By Cystathionine Gamma-lyase In Endothelial Cells.

Hydrogen sulfide (H 2 S) is the most recently described endothelium-derived vasodilator. In the endothelium, H 2 S is predominantly produced by cystathionine γ-lyase (CSE). We and others have previously shown CSE inhibition diminishes acetylcholine (ACh)-induced dilation in aortic ring segments. However, the mechanism of CSE regulation is not well-defined. The goal of this study was to identify endogenous regulators of CSE in endothelial cells. Previous reports suggest that [Ca 2+ ] and phosphorylation regulate CSE activity. We previously reported that there was an increase in H 2 S production in rat aortic endothelial cells loaded with an H 2 S fluorescence indicator, sulfide fluor-7 acetoxymethylester (SF7-AM, 10 μM) after exposure to either ACh (10 μM) or an H 2 S donor (NaHS, 10 μM). Pretreatment with the CSE inhibitor, β-cyanoalanine (BCA, 100 μM) prevented the ACh-induced but not the NaHS-induced increases in SF 7 flourescence. Our current study tested the hypothesis that increasing intracellular calcium stimulates CSE-dependent H 2 S production. Cultured rat aortic endothelial cells were loaded with SF 7 and a nuclear dye (Nuclear-ID Red DNA stain, to correct for cell number) and fluorescence was measured at baseline and in response to a calcium ionophore (ionomycin, 100 nM). Ionomycin increased H 2 S production (Control: 0.8293, +ionomycin: 1.0365, p = 0.0001, n=3 of 7-15 wells per replicate). Pretreatment with BCA significantly decreased ionomycin-induced H 2 S production (+ionomycin: 1.0365, +BCA: 0.6600, p &lt; 0.0001, n=3 of 7-15 wells per replicate). Preliminary ongoing studies in pressurized mesenteric arteries suggest a tendency of CSE inhibition to reduce ACh-induced dilation (Control: 77.06%, +BCA: 42.04%, p=0.08, n=3-4 arteries per group). However, a larger sample size is needed. Taken together, these studies suggest that ACh elicits vasodilation through a Ca 2+ -dependent activation of CSE.

Abstract P144: Characterization of Vascular Endothelial and Smooth Muscle Exosomes and Their Role in Vascular SignalTransduction

Vascular homeostasis is predicated on the integral interaction between a myriad of cell types. Principle to homeostasis is the interaction between the endothelial cells (ECs) of the intima and the vascular smooth muscle cells (VSMCs) of the media. Disruption of the tightly controlled communication between these cell types can lead to development of pathophysiology. In the past decade, much study has been given to a novel means of intercellular communication in exosomes. Exosomes are nanovesicles derived from endosomal compartments capable of carrying functional proteins, small RNAs and lipids into extracellular space. Initially characterized in the field of immunology, little is known on exosomes within vascular biology. Therefore, we present an in vitro characterization of rat EC and VSMC exosomes and evidence of EC exosomes as initiators of signal transduction within VSMCs. Primary rat aortic EC and VSMC exosomes isolated in serum-free conditions displayed characteristic size, shape and protein content of exosomes as analyzed through nanoparticle tracking analysis, electron microscopy, and western blot. Interestingly, alpha smooth muscle actin was found to be cell-specific for VSMC exosomes. Proteomics identified differential expression between EC and VSMC exosomes suggesting altered cargo between the two populations. Incubation of EC exosomes with VSMC identified a dose- and time-dependent increase in the expression of vascular cell adhesion molecule 1 in VSMC with a maximum at 10 9 EC exosomes and 6 hours through immunoblot analysis. In addition, EC exosomes stimulated expression of early growth response protein 1 at 1-3 hours, suggesting activation of pro-inflammatory gene expression. EC exosomes stimulated a maximal rate of protein synthesis at 3 hours in VSMC (3.53 fold increase over control, p=0.016) determined through puromycin incorporation showing qualitative changes in cell function. Analysis of cell volume identified a hypertrophic response in VSMC after exosome incubation as well, displaying a role for exosomes in vascular hypertrophy. In conclusion, we present a broad characterization of exosomes in vascular biology and a functional crosstalk mechanism between RAEC and VSMC through exosome transfer.

Adipose-derived cells improve left ventricular diastolic function and increase microvascular perfusion in advanced age.

An early manifestation of coronary artery disease in advanced age is the development of microvascular dysfunction leading to deficits in diastolic function. Our lab has previously shown that epicardial treatment with adipose-derived stromal vascular fraction (SVF) preserves microvascular function following coronary ischemia in a young rodent model. Follow-up studies showed intravenous (i.v.) delivery of SVF allows the cells to migrate to the walls of small vessels and reset vasomotor tone. Therefore we tested the hypothesis that the i.v. cell injection of SVF would reverse the coronary microvascular dysfunction associated with aging in a rodent model. Fischer 344 rats were divided into 4 groups: young control (YC), old control (OC), old + rat aortic endothelial cells (O+EC) and old + GFP+ SVF cells (O+SVF). After four weeks, cardiac function and coronary flow reserve (CFR) were measured via echocardiography, and hearts were explanted either for histology or isolation of coronary arterioles for vessel reactivity studies. In a subgroup of animals, microspheres were injected during resting and dobutamine-stimulated conditions to measure coronary blood flow. GFP+ SVF cells engrafted and persisted in the myocardium and coronary vasculature four weeks following i.v. injection. Echocardiography showed age-related diastolic dysfunction without accompanying systolic dysfunction; diastolic function was improved in old rats after SVF treatment. Ultrasound and microsphere data both showed increased stimulated coronary blood flow in O+SVF rats compared to OC and O+EC, while isolated vessel reactivity was mostly unchanged. I.v.-injected SVF cells were capable of incorporating into the vasculature of the aging heart and are shown in this study to improve CFR and diastolic function in a model of advanced age. Importantly, SVF injection did not lead to arrhythmias or increased mortality in aged rats. SVF cells provide an autologous cell therapy option for treatment of microvascular and cardiac dysfunction in aged populations.

Improvement of vascular dysfunction by argirein through inhibiting endothelial cell apoptosis associated with ET-1/Nox4 signal pathway in diabetic rats

Endothelial cell apoptosis plays an important role in the pathophysiological mechanism of vascular complications in type 2 diabetes mellitus (T2DM). Argirein, a new synthetic compound was demonstrated to inactivate NADPH oxidase to alleviate cardiac dysfunction in T2DM. Here, we investigated whether argirein medication attenuated the vascular dysfunction in T2DM by inhibiting endothelial cell apoptosis which was associated with NADPH oxidase. The rat aortic endothelial cells (RAECs) were incubated with glucose (30 mM) for 48 hour in vitro. It was shown that high glucose significantly increased the protein expression of BAX (Bcl-2 Associated X protein) and Caspase-3 and decreased Bcl2 (B-Cell Leukemia/Lymphoma 2) protein level in RAECs, which was normalized by argirein medication. The annexin V-FITC bound cell percentage and DNA fragments in agarose electrophoresis were markedly suppressed by argirein to confirm the anti-apoptotic property of argirein in RAECs. Furthermore, we found that argirein blocked the endothelin (ET)-1/Nox4 signal-dependent superoxide (O2−.) generation, which regulated endothelial cell apoptosis in RAECs. In vivo, argirein intervention relieved the vasodilatory response to acetylcholine and restored the expressions of Nox4 and BAX in the aorta endothelium of high-fat diet (HFD)-fed rats following streptozocin (STZ) injection. For the first time, we demonstrated that argirein could inhibit vascular endothelial cell apoptosis, which was attributed to blocking ET-1/Nox4 signal-dependent O2− generation in RAECs. This current study revealed the therapeutic effects of argirein to prevent the vascular complication in T2DM through inhibiting endothelial cell apoptosis which was associated with the anti-oxidative property of argirein.

Overexpression of steroid receptor coactivators alleviates hyperglycemia-induced endothelial cell injury in rats through activating the PI3K/Akt pathway.

Hyperglycemia is a major factor in vascular endothelial injury that finally leads to a cardiovascular event. Steroid receptor coactivators (SRCs) are a group of non-DNA binding proteins that induce structural changes in steroid receptors (nuclear receptors) critical for transcriptional activation. SRCs, namely, SRC-1, SRC-2, and SRC-3, are implicated in the regulation of vascular homeostasis. In this study we investigate the role of SRCs in hyperglycemia-induced endothelial injury. Aortic endothelial cells were prepared from normal and diabetic rats, respectively. Diabetic rats were prepared by injection of streptozotocin (50 mg/kg, i.p.). The expression levels of SRC-1 and SRC-3 were significantly decreased in endothelial cells from the diabetic rats. Similar phenomenon was also observed in aortic endothelial cells from the normal rats treated with a high glucose (25 mM) for 4 h or 8 h. The expression levels of SRC-2 were little affected by hyperglycemia. Overexpression of SRC-1 and SRC-3 in high glucose-treated endothelial cells significantly increased the cell viability, suspended cell senescence, and inhibited cell apoptosis compared with the control cells. We further showed that overexpression of SRC-1 and SRC-3 markedly suppressed endothelial injury through restoring nitric oxide production, upregulating the expression of antioxidant enzymes (SOD, GPX, and CAT), and activating the PI3K/Akt pathway. The beneficial effects of SRC-1 and SRC-3 overexpression were blocked by treatment with the PI3K inhibitor LY294002 (10 mM) or with the Akt inhibitor MK-2206 (100 nM). In conclusion, hyperglycemia decreased SRC-1 and SRC-3 expression levels in rat aortic endothelial cells. SRC-1 and SRC-3 overexpression might protect against endothelial injury via inhibition of oxidative stress and activation of PI3K/Akt pathway.

Acid Sphingomyelinase Down-regulation Alleviates Vascular Endothelial Insulin Resistance in Diabetic Rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in patients with type 2 diabetes. Acid sphingomyelinase (ASM) is a soluble glycoprotein which plays a vital role in the development and progression of various diseases such as cardiovascular and metabolic diseases. However, it remains unknown if ASM regulates insulin resistance in vascular endothelial cells in type 2 diabetes. ASM down-regulation with gene silencing and selective inhibitor amitriptyline was used in the rat aortic endothelial cells (RAECs) treated with palmitic acid (PA), a common saturated free fatty acid, which is thought to be the major cause of insulin resistance. It was shown that ASM down-regulation increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of pIRS-1-ser307 and AKT-ser473 via ceramide, consequently resulting in the decrease of the production of endothelial nitric oxide synthase (eNOS) and nitric oxide in PA-induced RAECs. We further found that ASM down-regulation blocked the Nox2- and Nox4-dependent superoxide (O2 -· ) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vivo, amitriptyline relieved the vasodilatory response to acetylcholine and restored the level of ceramide, Nox2 and Nox4 in the aorta endothelium of high-fat diet-fed rats following an injection of streptozotocin. Taken together, these results suggest that ASM down-regulation can improve endothelial insulin resistance which is attributed to inhibiting redox signalling in RAECs. Thus, these data support the idea that ASM is a promising clinical biomarker and potential therapeutic target for diabetic vascular complication.

Astragaloside IV Improves Vasodilatation Function by Regulating the PI3K/Akt/eNOS Signaling Pathway in Rat Aorta Endothelial Cells

Coronary heart disease (CHD) remains a major public health burden. Endothelial-dependent coronary artery vasoreactivity is a significant indicator of vascular function. Endothelial dysfunction is characterized by decreased nitric oxide (NO) bioavailability and predicts late cardiovascular events. Astragaloside IV (AGIV) is the main active component of the herb Astragalus membranaceus. Although it shows a significant protective effect against vascular endothelial dysfunction, the mechanisms of AGIV promoting the vascular dilation have not been elucidated. This study investigated the vasodilator effect of AGIV on rat aortic rings and the underlying effect of AGIV via the PI3K/Akt/eNOS signaling pathway. We measured the relaxation of isolated RARs after different concentrations of AGIV treatment. Rat aorta endothelial cells were cultured with different doses of AGIV, dimethylsulfoxide, and NG-nitro L-arginine methyl ester. The expression of phosphorylated (p)-Akt and -endothelial nitric oxide synthase (p-eNOS) were tested by Western blot analysis. The messenger (m)RNA expression of eNOS was quantified by real-time polymerase chain reaction. AGIV exerted a vasodilator effect on the aortic rings and increased the NO content in a concentration-dependent manner. The vasorelaxation was suppressed by an eNOS inhibitor. AGIV regulated the PI3K/Akt/eNOS signaling pathway via phosphorylation of Akt at Ser⁴⁷³ and dephosphorylation of eNOS at Thr⁴⁹⁵. The mRNA expression of eNOS was remarkably upregulated by AGIV. AGIV significantly induced the dilation of the aortic rings, leading to the vasodilator response by enhancing the eNOS release via the PI3K/Akt/eNOS signaling pathway.

Angiotensin (1–7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor

Angiotensin (1–7) has been reported to be a ligand for the GPCR MAS1. Small molecule MAS1 modulators have also been recently characterized. Aside from convincing evidence for MAS1 activation of Gq signaling, little is known about MAS1 mediated signaling pathways initiated by these ligands, especially Ang (1–7). We performed a comprehensive characterization of recombinant MAS1 signaling induced by Ang (1–7) and small molecule ligands through numerous G protein-dependent and independent pathways, and in a signaling pathway agnostic approach. We find that small molecule ligands modulate numerous G protein-dependent and independent pathways through MAS1, including Gq and Gi pathways, GTPγS binding, β-arrestin recruitment, Erk1/2 and Akt phosphorylation, arachidonic acid release, and receptor internalization. Moreover, in dynamic mass redistribution (DMR) assays that provide a pathway-agnostic readout of cellular responses, small molecule agonists produced robust responses. In contrast, Ang (1–7) failed to induce or block signaling in any of these assay platforms. We detected specific binding of radiolabeled Ang (1–7) to rat aortic endothelial cell (RAEC) membranes, but not to recombinant MAS1. Biphasic, concentration-dependent biased signaling responses to Ang II were detected in RAEC. These phases were associated with vastly different DMR characteristics and this likely provides a molecular basis for previously observed concentration-dependent divergent physiological actions of Ang II. Both phases of Ang II signaling in RAECs were potently inhibited by Ang (1–7), providing a plausible molecular mechanism for Ang (1–7) as counter regulator of the Ang II- AT1 axis, responsible at least in part for Ang (1–7) physiological activities.

Notoginsenoside Fc attenuates high glucose-induced vascular endothelial cell injury via upregulation of PPAR-γ in diabetic Sprague–Dawley rats

Endothelial injury from high glucose (HG) plays a dominant role in atherosclerosis, diabetes-induced vasculopathy, and vascular remodeling. Notoginsenoside Fc (Fc), a novel saponin isolated from P. notoginseng, has been shown to exhibit properties that counteract platelet aggregation. However, the potential roles and molecular mechanisms of Fc in preventing cardiovascular injury have yet to be explored. In this study, we present novel data that show the ability of Fc to prevent early atherosclerosis of diabetic Sprague–Dawley (SD) rats in vivo and to attenuate endothelial cell injury in vitro. Our results indicate that Fc protects rat aortic endothelial cells (RAOECs) from HG-induced injury by inhibiting apoptosis and promoting proliferation as well as by reducing endothelial cell production of pro-inflammatory cytokines: TNF-α, IL-1β, IL-6, ICAM-1. Furthermore, the downregulation of peroxisome proliferator-activated receptor-γ (PPAR-γ) in HG-challenged endothelial cells was prevented by Fc. Inhibition of PPAR-γ abrogated the effects of Fc on HG-induced pro-inflammatory cytokine production in RAOECs. These results indicate that Fc has a preventative effect on HG-induced endothelial cell injury partly through a PPARγ-mediated pathway, suggesting that Fc might provide a potential new therapeutic option for the treatment of diabetic vascular complications.

Ticagrelor protects against AngII-induced endothelial dysfunction by alleviating endoplasmic reticulum stress

Ticagrelor has been reported to decrease cardiovascular mortality compared with clopidogrel. This benefit cannot be fully explained by the more efficient platelet inhibition. Many studies demonstrated that ticagrelor improved endothelial function, leaving the mechanism elusive though. The present study aims to investigate whether ticagrelor protects against endothelial dysfunction induced by angiotensinII (AngII) through alleviating endoplasmic reticulum (ER) stress. Male Sprague Dawley rats were infused with AngII or vehicle and administrated with ticagrelor or vehicle for 14 days. Reactive oxygen species (ROS) was detected. Aortas from normal mice were incubated with endoplasmic reticulum stress inducer tunicamycin with or without ticagrelor. Vasorecactivity was measured on wire myography. Rat aortic endothelial cells (RAECs) were pretreated with ticagrelor followed by AngII or tunicamycin. Endothelial nitric oxide synthase (eNOS) phosphorylation and ER stress markers were determined by western blotting. Impaired endothelial function, induction of ER stress, reduced eNOS phosphorylation and elevated ROS generation was restored by ticagrelor treatment in vivo. In addition, tunicamycin induced endothelial dysfunction was improved by ticagrelor. In vitro, the induction of ER stress and inhibited eNOS phosphorylation in REACs exposed to AngII as well as tunicamycin was reversed by co-culturing with ticagrelor. In conclusion, ticagrelor protects against AngII-induced endothelial dysfunction via alleviating ER stress.

Assessment of HIF-1\u03b1 expression and release following endothelial injury in-vitro and in-vivo

BackgroundEndothelial injury is an early and enduring feature of cardiovascular disease. Inflammation and hypoxia may be responsible for this, and are often associated with the up-regulation of several transcriptional factors that include Hypoxia Inducible Factor-1 (HIF-1). Although it has been reported that HIF-1α is detectable in plasma, it is known to be unstable. Our aim was to optimize an assay for HIF-1α to be applied to in vitro and in vivo applications, and to use this assay to assess the release kinetics of HIF-1α following endothelial injury.MethodsAn ELISA for the measurement of HIF-1α in cell-culture medium and plasma was optimized, and the assay was used to determine the best conditions for sample collection and storage. The results of the ELISA were validated using Western blotting and immunohistochemistry (IHC). In vitro, a standardized injury was produced in a monolayer of rat aortic endothelial cells (RAECs) and intracellular HIF-1α was measured at intervals over 24 h. In vivo, a rat angioplasty model was used. The right carotid artery was injured using a 2F Fogarty balloon catheter. HIF-1α was measured in the plasma and in the arterial tissue (0, 1, 2, 3 and 5 days post injury).ResultsThe HIF-1α ELISA had a limit of detection of 2.7 pg/mL and was linear up to 1000 pg/ mL. Between and within-assay, the coefficient of variation values were less than 15%. HIF-1α was unstable in cell lysates and plasma, and it was necessary to add a protease inhibitor immediately after collection, and to store samples at -80 °C prior to analysis. The dynamics of HIF-1α release were different for the in vitro and in vivo models. In vitro, HIF-1α reached maximum concentrations approximately 2 h post injury, whereas peak values in plasma and tissues occurred approximately 2 days post injury, in the balloon injury model.ConclusionHIF-1α can be measured in plasma, but this requires careful sample collection and storage. The carotid artery balloon injury model is associated with the transient release of HIF-1α into the circulation that probably reflects the hypoxia induced in the artery wall.

Argirein alleviates vascular endothelial insulin resistance through suppressing the activation of Nox4-dependent O2- production in diabetic rats.

Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in type 2 diabetes mellitus (T2DM). Therefore, there are great potential clinical implications in developing pharmacological interventions targeting endothelial insulin resistance. Our previous studies indicated that argirein which was developed by combining rhein with L-arginine by a hydrogen bond, could substantially relieved stress related exacerbation of cardiac failure and alleviated cardiac dysfunction in T2DM, which was associated with suppressing NADPH oxidase activity. However, it is unclear whether argirein treatment attenuates the vascular lesion and dysfunction in T2DM and its underlying mechanisms. The rat aortic endothelial cells (RAECs) were used to treat with palmitic acid (PA), a most common saturated free fatty acid, which could induce insulin resistance. It was showed that argirein increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of IRS-1-ser307 and AKT-ser473, consequently resulting in the increase of the production of eNOS and NO in PA-induced RAECs. We further found that argirein blocked the Nox4-dependent superoxide (O2-.) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vitro, argirein increased the release of endothelial NO to relieve the vasodilatory response to acetylcholine and insulin, and restored the expression of Nox4 and pIRS-1-ser307 in the aorta endothelium of high-fat diet (HFD)-fed rats following an injection of streptozocin (STZ). These results suggested that argirein could improve endothelial insulin resistance which was attributed to inhibiting Nox4-dependent redox signaling in RAECs. These studies thus revealed the novel effect of argirein to prevent the vascular complication in T2DM.

Exogenous SERP1 attenuates restenosis by restoring GLP-1 receptor activity in diabetic rats following vascular injury

The activity of glucagon-like peptide 1 (GLP-1R) is essential for preventing restenosis following vascular injury; however, the mechanism of dysfunctional GLP-1R glycosylation and ways to enhance the activity of GLP-1R on vascular surfaces in diabetic patients are poorly understood. In the present study, we investigated the N-glycosylation level and role of stress-associated endoplasmic reticulum protein 1 (SERP1) in preventing restenosis following carotid injury in diabetic rats. Our results showed that N-glycosylation levels in both rat aortic endothelial cells (RAOECs) and rat vascular smooth muscle cells (VSMCs) decreased gradually following glucose treatment in a concentration dependant manner. Furthermore, co-immunoprecipitation (Co-IP) analyses indicated that SERP1 could interact with GLP-1R in RAOECs and VSMCs. Moreover, SERP1 enhanced GLP-1R N-glycosylation and increased the production of phosphorylated endothelial nitric oxide synthase (eNOS) as well as proliferation of RAOECs. SERP1 also increased phosphorylated adenosine monophosphate activated protein kinase (AMPK) and decreased the migration of VSMCs. Importantly, intima media thickness (IMT) and neointimal hyperplasia were alleviated in the carotid artery of diabetic rats injected with SERP1 following balloon injury. We also found an increase in re-endothelialization and a decrease in VSMC proliferation in the carotid artery of diabetic rats injected with SERP1. In summary, the remarkable effects of SERP1 on reducing restenosis following vascular injury may contribute to future advancements in the treatment of diabetic vascular complications.

Cell alignment guided by nano/micro oriented collagen fibers and the synergistic vascularization for nervous cell functional expression

The majority of tissues within human body are constituted of designated structures to enable specific functions. Much effort has been done to engineer artificial fabric cell-laden scaffolds which are widely used for a great diversity of linear tissue constructs. For this purpose, collagen microfibers are of great concern among diverse materials while the control of cell-laden fiber formation and orientated structure is still unsolvable. Here, we developed a novel microfluidic-based strategy for continuous fabrication and assembly of three-dimensional (3D) cell-laden oriented collagen hydrogel microfibers. Inspired by the flow-introduced shear force in a microfluidic chip, collagen hydrogel microfibers obtained the oriented fabric structure which could guide rat pheochromocytoma cells (PC12) oriented spreading and enhance relative cellular functional expression. Rat aortic endothelial cells (RAOECs) were introduced to construct a co-cultured microfiber model, which further facilitated the functional expression of neural cells due to the synergistic effect of both vascularized-like cells and neural-like cells. Moreover, the ability of assembling collagen microfibers into larger constructs will benefit a variety of applications in tissue engineering and biomedical research.

Neuregulin-1 attenuates stress-induced vascular senescence.

Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence. Senescence was induced in cultured rat aortic endothelial cells (ECs) and aortic smooth muscle cells (SMCs) by 2 h exposure to 30 µM hydrogen peroxide (H2O2). Cellular senescence was confirmed after 72 h using senescence-associated-β-galactosidase staining (SA-β-gal), cell surface area, and western blot analyses of SA pathways (acetyl-p53, p21). Recombinant human NRG-1 (rhNRG-1, 20 ng/mL) significantly reduced H2O2-induced senescence, as shown by a lower number of SA-β-gal positive cells, smaller surface area and lower expression of acetyl-p53. In C57BL/6 male mice rendered diabetic with streptozotocin (STZ), rhNRG-1 attenuated cellular senescence in aortic ECs and SMCs. Next, we created mice with SMC-specific knockdown of the NRG-1 receptor ErbB4. Aortic SMCs isolated from SMC-specific ErbB4 deficient mice (ErbB4f/+ SM22α-Cre+) showed earlier cellular senescence in vitro compared with wild-type (ErbB4+/+ SM22α-Cre+) SMCs. Furthermore, when rendered diabetic with STZ, ErbB4f/+ SM22α-Cre+ male mice showed significantly more vascular senescence than their diabetic wild-type littermates and had increased mortality. This study is the first to explore the role of NRG-1 in vascular senescence. Our data demonstrate that NRG-1 markedly inhibits stress-induced premature senescence in vascular cells in vitro and in the aorta of diabetic mice in vivo. Consistently, deficiency in the NRG-1 receptor ErbB4 provokes cellular senescence in vitro as well as in vivo.