Effects In Vascular Endothelial Cells Research Articles

Exosomes of endothelial progenitor cells repair injured vascular endothelial cells through the Bcl2/Bax/Caspase-3 pathway

The main objective of this study is to evaluate the influence of exosomes derived from endothelial progenitor cells (EPC-Exo) on neointimal formation induced by balloon injury in rats. Furthermore, the study aims to investigate the potential of EPC-Exo to promote proliferation, migration, and anti-apoptotic effects of vascular endothelial cells (VECs) in vitro. The underlying mechanisms responsible for these observed effects will also be thoroughly explored and analyzed. Endothelial progenitor cells (EPCs) was isolated aseptically from Sprague–Dawley (SD) rats and cultured in complete medium. The cells were then identified using immunofluorescence and flow cytometry. The EPC-Exo were isolated and confirmed the identities by western-blot, transmission electron microscope, and nanoparticle analysis. The effects of EPC-Exo on the rat carotid artery balloon injury (BI) were detected by hematoxylin and eosin (H&E) staining, ELISA, immunohistochemistry, immunofluorescence, western-blot and qPCR. LPS was used to establish an oxidative damage model of VECs. The mechanism of EPC-Exo repairing injured vascular endothelial cells was detected by measuring the proliferation, migration, and tube function of VECs, actin cytoskeleton staining, TUNEL staining, immunofluorescence, western-blot and qPCR. In vivo, EPC-Exo exhibit inhibitory effects on neointima formation following carotid artery injury and reduce the levels of inflammatory factors, including TNF-α and IL-6. Additionally, EPC-Exo downregulate the expression of adhesion molecules on the injured vascular wall. Notably, EPC-Exo can adhere to the injured vascular area, promoting enhanced endothelial function and inhibiting vascular endothelial hyperplasia Moreover, they regulate the expression of proteins and genes associated with apoptosis, including B-cell lymphoma-2 (Bcl2), Bcl2-associated x (Bax), and Caspase-3. In vitro, experiments further confirmed that EPC-Exo treatment significantly enhances the proliferation, migration, and tube formation of VECs. Furthermore, EPC-Exo effectively attenuate lipopolysaccharides (LPS)-induced apoptosis of VECs and regulate the Bcl2/Bax/Caspase-3 signaling pathway. This study demonstrates that exosomes derived from EPCs have the ability to inhibit excessive carotid intimal hyperplasia after BI, promote the repair of endothelial cells in the area of intimal injury, and enhance endothelial function. The underlying mechanism involves the suppression of inflammation and anti-apoptotic effects. The fundamental mechanism for this anti-apoptotic effect involves the regulation of the Bcl2/Bax/Caspase-3 signaling pathway.

Pericytes contribute to pulmonary vascular remodeling via HIF2α signaling

Vascular remodeling is the process of structural alteration and cell rearrangement of blood vessels in response to injury and is the cause of many of the world’s most afflicted cardiovascular conditions, including pulmonary arterial hypertension (PAH). Many studies have focused on the effects of vascular endothelial cells and smooth muscle cells (SMCs) during vascular remodeling, but pericytes, an indispensable cell population residing largely in capillaries, are ignored in this maladaptive process. Here, we report that hypoxia-inducible factor 2α (HIF2α) expression is increased in the lung tissues of PAH patients, and HIF2α overexpressed pericytes result in greater contractility and an impaired endothelial-pericyte interaction. Using single-cell RNAseq and hypoxia-induced pulmonary hypertension (PH) models, we show that HIF2α is a major molecular regulator for the transformation of pericytes into SMC-like cells. Pericyte-selective HIF2α overexpression in mice exacerbates PH and right ventricular hypertrophy. Temporal cellular lineage tracing shows that HIF2α overexpressing reporter NG2+ cells (pericyte-selective) relocate from capillaries to arterioles and co-express SMA. This novel insight into the crucial role of NG2+ pericytes in pulmonary vascular remodeling via HIF2α signaling suggests a potential drug target for PH.

ERRα attenuates vascular inflammation via enhanced NFκB degradation pathway.

We have previously reported that β-aminoisobutyric acid (BAIBA), a muscle-derived exercise mimetic, had anti-inflammatory and reactive oxygen species (ROS) scavenging effects in vascular endothelial cells through the enhanced expression of peroxisome proliferator-activated receptor gamma coactivator-1β (PGC-1β). Although BAIBA also increased the expression of estrogen-related receptor α (ERRα), the roles of ERRα in vascular endothelial cells have yet to be fully elucidated. Here, we found that human aortic endothelial cells (HAEC)-infected with ERRα-expressing adenovirus had significantly decreased mRNA levels of TNFα-stimulated pro-inflammatory molecules. However, ERRα overexpression had little effects on the mRNA levels of PGC-1β, peroxisome proliferator-activated receptors (PPAR), and almost all ROS scavenging molecules, except for superoxide dismutase 2 (SOD2). ERRα expression significantly decreased NFκB reporter activities in a dose-dependent manner with unaltered IκBα phosphorylation levels but with a significant increase in the mRNA levels of PDZ and LIM domain protein 2 (PDLIM2) and copper metabolism gene MURR1 domain-containing protein (COMMD1), which enhance the ubiquitination and degradation of NFκB. Also, PDLIM2 and COMMD1 mRNA levels were upregulated in BAIBA-treated HAEC. Finally, we identified the ERRα-response element in the COMMD1 promoter region (-283 to -29 bp). These results indicated that ERRα exerted anti-inflammatory effects in vascular endothelial cells through COMMD1-mediated attenuation of NFκB activity, which could be one of the atheroprotective mechanisms of physical exercise.

Angiotensin-(1-9) prevents angiotensin II-induced endothelial apoptosis through CNPY2/PERK pathway.

Endothelial apoptosis caused by activation of renin-angiotensin system (RAS) plays a vital part in the occurrence and progress of hypertension. Angiotensin-(1-9) (Ang-(1-9)) is a peptide of the counter-regulatory non-classical RAS with anti-hypertensive effects in vascular endothelial cells (ECs). However, the mechanism of action remains unclear. Considering that the endothelial apoptosis wascloselyrelatedto endoplasmic reticulum stress (ERS) and mitochondrial function. Herein, we aimed to elucidate the effects of Ang-(1-9) on endothelial apoptosis and the underlying molecular mechanism in angiotensin II (Ang II) induced hypertension. In human umbilical vascular endothelial cells (HUVECs), we observed Ang-(1-9) inhibited Ang II-induced ERS associated endothelial apoptosis. Mechanically, Ang-(1-9) inhibited endothelial apoptosis by blocking CNPY2/PERK mediated CaMKII/Drp1-dependent mitochondrial fission and eIF2α/CHOP signal. Consistent with above effects in HUVECs, in Ang II-induced hypertensive mice, we found administration of exogenous Ang-(1-9) attenuated endothelial apoptosis and arterial blood pressure, which were mediated by CNPY2/PERK signaling pathway. Our study indicated Ang-(1-9) inhibited Ang II-induced hypertension through CNPY2/PERK pathway. These findings may provide new insights for prevention and treatment of hypertension in future.

Protective effects of GHRH antagonists against hydrogen peroxide-induced lung endothelial barrier disruption.

PurposeGrowth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates growth hormone release from the anterior pituitary gland. GHRH antagonists (GHRHAnt) are anticancer agents, which also exert robust anti-inflammatory activities in malignancies. GHRHAnt exhibit anti-oxidative and anti-inflammatory effects in vascular endothelial cells, indicating their potential use against disorders related to barrier dysfunction (e.g. sepsis). Herein, we aim to investigate the effects of GHRHAnt against lung endothelial hyperpermeability.MethodsThe in vitro effects of GHRHAnt in H2O2-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Electric cell-substrate impedance sensing (ECIS) was utilized to measure transendothelial resistance, an indicator of barrier function.ResultsOur results demonstrate that GHRHAnt protect against H2O2-induced endothelial barrier disruption via P53 and cofilin modulation. Both proteins are crucial modulators of vascular integrity. Moreover, GHRHAnt prevent H2O2 – induced decrease in transendothelial resistance.ConclusionsGHRHAnt represent a promising therapeutic intervention towards diseases related to lung endothelial hyperpermeability, such as acute respiratory distress syndrome - related or not to COVID-19 - and sepsis. Targeted medicine for those potentially lethal disorders does not exist.

The effects of vascular endothelial cells on regulating post-irradiation microglia phenotype in irradiation-induced brain injury.

e14001 Background: The purpose was to elucidate the mechanism behind functional relevance between microglia and injured blood vessels after CRT and the role of fractalkine (FKN) for the dynamics in the microglia phenotype, hoping to provide a theoretical rationale for the therapeutic strategy of Radiation-induced brain injury (RIBI). Methods: The ultrastructural blood vessels in the hippocampus were examined by transmission electron microscopy. EMSA and dual-luciferase assays were used to confirm the activation of NF-κB induced by irradiation in. Migration and co-culture assays were used to investigate the the association between HUVEC and BV-2 cells after irradiation. We constructed CX3CR1-knockdown (receptor of FKN) lentiviral vector to produce CX3CR1-knockdown BV-2 cell lines. After cultured for 3h with/without FKN, BV-2 cells (Sh-NC/ Sh-CX3CR1) received 10-Gy irradiation. The CX3CR1wide-type/knockdownmice were used and subjected to lateral ventricular injection (ICV) of 5 μl FKN lentivirus or vector before CRT. The in vitro and in vivo phenotype transformation of microglia and its inflammatory factors release were detected by WB, real-time PCR, ELISA, and IF at different time-points after irradiation. Morris water maze test was used to test mice's cognitive function. Results: In the hippocampus of the CRT group, significant pathological changes were seen in ultrastructure of blood vessels.The NF-κB pathway was activated in vascular endothelial cells (ECs) after10-Gy irradiation. In vitro migration and Co-culture assays found that molecular signals, secreted by ECs via the NF-κB pathway, leading to the activation of microglia and their recruitment to blood vessels. ELISA and qRT-PCR revealed that 10-Gy irradiation promoted the secretion of FKN from the vascular ECs via activating the NF-κB pathway. Western blotting and Flow cytometry showed that pretreatment with exogenous FKN, before 10-Gy irradiation, promoted microglia M2 phenotype transformation, diminished radiation-induced pro-inflammatory factors, and enhanced phagocytosis capacity. In addition, in vivo experiments showed that up-regulation of FKN via FKN lentivirus promoted radiation-induced microglial M2 transformation in the hippocampus, and diminished the spatial memory injury of radiated mice. Lastly, while inhibiting the CX3CR1, which is exclusively expressed on microglia in the brain, the regulatory effect of FKN on microglia and cognitive ability of mice disappeared after radiation. Conclusions: FKN, mainly secreted by vascular ECs via the NF-κB pathway after radiation, may attenuate RIBI through microglia polarization toward M2 phenotype by binding to CX3CR1 on microglia. Our study has unveiled an important and novel role of FKN/CX3CR1 in RIBI, providing evidence to validate that promotion of the FKN/CX3CR1 axis could be a promising therapeutic strategy for the treatment of RIBI.

LCC-09, a Novel Salicylanilide Derivative, Exerts Anti-Inflammatory Effect in Vascular Endothelial Cells.

ObjectiveEndothelial cell (EC) activation facilitates leukocyte adhesion to vascular walls, which is implicated in a variety of cardiovascular diseases and is a target for prevention and treatment. Despite the development of anti-inflammatory medications, cost-effective therapies with significant anti-inflammatory effects and lower organ toxicity remain elusive. The goal of this study is to identify novel synthetic compounds that inhibit EC inflammatory response with minimal organ toxicity.Methods and ResultsIn this study, we discovered LCC-09, a salicylanilide derivative consisting of the functional fragment of magnolol, 2,4-difluorophenyl, and paeonol moiety of salicylate, as a novel anti-inflammatory compound in cultured ECs and zebrafish model. LCC-09 was shown to inhibit pro-inflammatory cytokine tumor necrosis factor-α (TNFα)-induced expression of adhesion molecules and inflammatory cytokines, leading to reduced leukocyte adhesion to ECs. Mechanistically, LCC-09 inhibits the phosphorylation of signal transducer and activator of transcription 1 (STAT1), TNFα-induced degradation of NF-κ-B Inhibitor-α (IκBα) and phosphorylation of NFκB p65, resulting in reduced NFκB transactivation activity and binding to E-selectin promoter. Additionally, LCC-09 attenuated TNFα-induced generation of reactive oxygen species in ECs. Molecular docking models suggest the binding of LCC-09 to NFκB essential modulator (NEMO) and Janus tyrosine kinase (JAK) may lead to dual inhibition of NFκB and STAT1. Furthermore, the anti-inflammatory effect of LCC-09 was validated in the lipopolysaccharides (LPS)-induced inflammation model in zebrafish. Our results demonstrated that LCC-09 significantly reduced the LPS-induced leukocyte recruitment and mortality of zebrafish embryos. Finally, LCC-09 was administered to cultured ECs and zebrafish embryos and showed minimal toxicities.ConclusionOur results support that LCC-09 inhibits EC inflammatory response but does not elicit significant toxicity.

The C Loop of Fifth Epidermal Growth Factor-like Domain of Thrombomodulin Exerts Cytoprotective and Angiogenic Effects in Vitro and In Vivo

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerted cytoprotective and pro-angiogenic function in vitro and in vivo via G-protein coupled receptor 15 (GPR15). TME5 comprises three S-S bonds which divides TME5 into three loops: A (TME5A), B (TME5B) and C (TME5C). The present study aimed to identify the minimum structure of TME5 that produces favorable effects in vascular endothelial cells. The bromodeoxyuridine (BrdU) incorporation and in vivo Matrigel plug assays found that TME5C but not TME5A or TME5B stimulated proliferation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis, respectively. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs. Western blot analysis found that exposure of HUVECs to TME5C increased levels of anti-apoptotic myeloid cell leukemia-1 (Mcl-1) protein in association with activation of signal transduction pathways including extracellular signal-regulated kinase (ERK), AKT and mitogen-activated protein kinase p38. Importantly, TME5C did not affect thrombin clotting time in vitro. The cytoprotective function of TME5C was mediated by cell surface expressed GPR15, as TME5C was not able to protect vascular endothelial cells isolated from GPR15 knockout mice. Strikingly, TME5C successfully ameliorated hematopoietic stem cell transplantation (HSCT)-induced sinusoidal obstruction syndrome (SOS) in a murine model in association with counteracting reduction of sinusoidal ECs numbers, loss of integrity of sinusoidal wall and sinusoidal ECs detachment. Taken together, cytoprotective and the pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as SOS whose pathogenesis is based on the endothelial insults. DisclosuresNo relevant conflicts of interest to declare.

Uncovering a Distinct Gene Signature in Endothelial Cells Associated With Contrast Enhancement in Glioblastoma.

PurposeGlioblastoma (GBM) is the most aggressive and lethal type of brain tumors. Magnetic resonance imaging (MRI) has been commonly used for GBM diagnosis. Contrast enhancement (CE) on T1-weighted sequences are presented in nearly all GBM as a result of high vascular permeability in glioblastomas. Although several radiomics studies indicated that CE is associated with distinct molecular signatures in tumors, the effects of vascular endothelial cells, the key component of blood brain barrier (BBB) controlling vascular permeability, on CE have not been thoroughly analyzed.MethodsEndothelial cell enriched genes have been identified using transcriptome data from 128 patients by a systematic method based on correlation analysis. Distinct endothelial cell enriched genes associated with CE were identified by analyzing difference of correlation score between CE-high and CE–low GBM cases. Immunohistochemical staining was performed on in-house patient cohort to validate the selected genes associated with CE. Moreover, a survival analysis was conducted to uncover the relation between CE and patient survival.ResultsWe illustrated that CE is associated with distinct vascular molecular imprints characterized by up-regulation of pro-inflammatory genes and deregulation of BBB related genes. Among them, PLVAP is up-regulated, whereas TJP1 and ABCG2 are down-regulated in the vasculature of GBM with high CE. In addition, we found that the high CE is associated with poor prognosis and GBM mesenchymal subtype.ConclusionWe provide an additional insight to reveal the molecular trait for CE in MRI images with special focus on vascular endothelial cells, linking CE with BBB disruption in the molecular level. This study provides a potential new direction that may be applied for the treatment optimization based on MRI features.

S100A4 Is Involved in Stimulatory Effects Elicited by the FGF2/FGFR1 Signaling Pathway in Triple-Negative Breast Cancer (TNBC) Cells.

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype characterized by poor clinical outcome. In recent years, numerous advancements have been made to better understand the biological landscape of TNBC, though appropriate targets still remain to be determined. In the present study, we have determined that the expression levels of FGF2 and S100A4 are higher in TNBC with respect to non-TNBC patients when analyzing “The Invasive Breast Cancer Cohort of The Cancer Genome Atlas” (TCGA) dataset. In addition, we have found that the gene expression of FGF2 is positively correlated with S100A4 in TNBC samples. Performing quantitative PCR, Western blot, CRISPR/Cas9 genome editing, promoter studies, immunofluorescence analysis, subcellular fractionation studies, and ChIP assays, we have also demonstrated that FGF2 induces in TNBC cells the upregulation and secretion of S100A4 via FGFR1, along with the ERK1/2–AKT–c-Rel transduction signaling. Using conditioned medium from TNBC cells stimulated with FGF2, we have also ascertained that the paracrine activation of the S100A4/RAGE pathway triggers angiogenic effects in vascular endothelial cells (HUVECs) and promotes the migration of cancer-associated fibroblasts (CAFs). Collectively, our data provide novel insights into the action of the FGF2/FGFR1 axis through S100A4 toward stimulatory effects elicited in TNBC cells.

Autocrine and Paracrine Effects of Vascular Endothelial Cells Promote Cutaneous Wound Healing.

Background When vascular endothelial cells are subjected to external stimuli, paracrine hormones and cytokines act on adjacent cells. The regulation of the biological behaviour of cells is closely related to the maintenance of organ function and the occurrence and development of disease. However, it is unclear whether vascular endothelial cells affect the biological behaviour of cells involved in wound repair through autocrine and paracrine mechanisms and ultimately play a role in wound healing. We aimed to verify the effect of the autocrine and paracrine functions of vascular endothelial cells on wound healing. Materials and Methods ELISA was used to detect platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor in human umbilical vascular endothelial cell-conditioned medium (HUVEC-CM). Different concentrations of HUVEC-CM were used to treat different stem cells. CCK-8 and scratch assays were used to detect the proliferation and migration ability of each cell. A full-thickness dorsal skin defect model was established in mice, and skin wound healing was observed after the local injection of HUVEC-CM, endothelial cell medium (ECM), or normal saline. H&E staining and immunofluorescence were used to observe the gross morphology of the wound tissue, the epithelial cell migration distance, and the expression of CD3 and CD31. Results HUVEC-CM promotes the proliferation and migration of epidermal stem cells, skin fibroblasts, bone marrow mesenchymal stem cells, and HUVECs themselves. Furthermore, HUVEC-CM can promote angiogenesis in mouse skin wounds and granulation tissue formation and can accelerate wound surface epithelialization and collagen synthesis, thereby promoting wound healing. Conclusion Our results clearly suggest that it is practicable and effective to promote wound healing with cytokines secreted by vascular endothelial cells in a mouse model.

P71.01 The Effects of Vascular Endothelial Cells on Regulating Post-Irradiation Microglia Phenotype in Irradiation-Induced Brain Injury

P71.01 The Effects of Vascular Endothelial Cells on Regulating Post-Irradiation Microglia Phenotype in Irradiation-Induced Brain Injury

Gene Expression Profiles Induced by a Novel Selective Peroxisome Proliferator-Activated Receptor α Modulator (SPPARMα) Pemafibrate.

Pemafibrate is the first clinically-available selective peroxisome proliferator-activated receptor α modulator (SPPARMα) that has been shown to effectively improve hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C) levels. Global gene expression analysis reveals that the activation of PPARα by pemafibrate induces fatty acid (FA) uptake, binding, and mitochondrial or peroxisomal oxidation as well as ketogenesis in mouse liver. Pemafibrate most profoundly induces HMGCS2 and PDK4, which regulate the rate-limiting step of ketogenesis and glucose oxidation, respectively, compared to other fatty acid metabolic genes in human hepatocytes. This suggests that PPARα plays a crucial role in nutrient flux in the human liver. Additionally, pemafibrate induces clinically favorable genes, such as ABCA1, FGF21, and VLDLR. Furthermore, pemafibrate shows anti-inflammatory effects in vascular endothelial cells. Pemafibrate is predicted to exhibit beneficial effects in patients with atherogenic dyslipidemia and diabetic microvascular complications.

The revascular and transplanted effects of vascular endothelial cells on islets grafts

Objective To investigate the revascular and transplanted effects of islet grafts after co-transplantation with vascular endothelial cells (ECs) in diabetic rats. Methods The rat ECs and islet cells were isolated and purified, then subcutaneously co-transplanted to the inbred SD male rats with diabetes mellitus, the group of islets transplanted alone, group of phosphate buffered solution, and group of normal rats served as control. Islet grafts revascularization degree, islet cells activity and apoptosis were observed by immunohistochemical double staining of CD31, insulin and factor associated suicide (Fas) antibody. The results of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT), the blood glucose and insulin levels of the rats and the survival time of the islet grafts were compared. Results The blood glucose concentrations of IPGTT and IPITT, the blood glucose and insulin levels of rats analyzed by multivariate analysis in the ECs and islets transplantation group were better than those in the islets transplantation alone group (P<0.05). The islet grafts mean survival time of the ECs and islets transplantation group was longer than that of the islets transplantation alone group (P<0.05). On the 7th day after transplantation, mean microvascular density of islet grafts per square millimeter in the ECs and islets transplantation group was 26.4±6.1, significantly greater than that in the islets transplantation alone group 18.3±5.7 (P<0.05). In the ECs and islets transplantation group, insulin staining intensity was higher than that in the islets transplantation alone group (P<0.05), while factor associated suicide(Fas) staining intensity was lower than that in the islets transplantation alone group (P<0.05). Conclusion Co-transplantation with ECs could promote the revascularization of islet grafts and improve the effect of islet transplantation. (Chin J Endocrinol Metab, 2018, 34: 516-521) Key words: Islet transplantation; Endothelial cells; Revascularization; Diabetes mellitus

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Herein we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from Gpr15 knock-out (KO) mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal EC numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. The use of TME5C may be a promising treatment strategy to prevent or treat lethal complications, such as sinusoidal obstruction syndrome, whose pathogenesis is based on endothelial insults.

Abstract 707: Vascular Smooth Muscle Cell Tfeb Deletion Promotes Abdominal Aortic Aneurysms

Rationale: Abdominal aortic aneurysm (AAA) is a vascular disease with a very high mortality rate in the case of rupture. Vascular smooth muscle cells (VSMCs) are crucial to maintaining vascular integrity and function. Transcription Factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis in a variety of cell types via inducing the transcription of a coordinated lysosomal regulatory gene network. TFEB shows anti-inflammatory and anti-atherosclerotic effects in vascular endothelial cells and macrophages. However, the role of TFEB in vascular disease remains to be further explored. Objectives: To investigate the role of VSMC TFEB in AAA. Methods and Results: We found that TFEB was down-regulated in human aortic aneurysmal lesion compared with the non-lesion area by QRT-PCR and immunostaining. In human aortic smooth muscle cells (HASMCs), TFEB mRNA and protein abundance were decreased upon treatment with pro-inflammatory factors while adenovirus-mediated TFEB overexpression potently inhibits inflammation, apoptosis (caspase 3 cleavage and Annexin V staining) and matrix metalloproteinase activity (zymography). A consistent phenotype was observed in the TFEB knockdown HASMCs. Mechanistically, TFEB activates the PI3K-Akt pathway, and PI3K inhibitors (wortmannin and LY294002) abolished the anti-apoptotic effect of TFEB in HASMCs. Utilizing VSMC-specific TFEB deficiency mice (floxed-TFEB/myh11-ERT2 cre+), we determined the effect of TFEB on AAA formation in vivo. In the mouse aneurysm model induced by the combination of angiotensin II and 3-aminopropionitrile infusion, TFEB VSMC-deletion significantly increases aneurysm formation, rupture, and mortality (n =13-14 for each group, p&lt; 0.01). Conclusions: Our data reveal a critical protective role of TFEB in VSMC homeostasis, suggesting TFEB to be a potential target to treat aortic aneurysm.

Varenicline promotes endothelial cell migration by lowering vascular endothelial-cadherin levels via the activated α7 nicotinic acetylcholine receptor-mitogen activated protein kinase axis.

Varenicline is a widely used and effective drug for smoking cessation. Despite its efficacy, varenicline increases the risk of cardiovascular disease. We previously demonstrated that varenicline aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice. However, little is known about its effects in vascular endothelial cells. Therefore, we examined whether varenicline promotes migration of human umbilical vein endothelial cells (HUVECs) using the Boyden chamber assay. Varenicline (100μM) markedly promoted migration of HUVECs and decreased expression of vascular endothelial (VE)-cadherin, an endothelial adhesion molecule. Extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) signaling were markedly activated by varenicline. Methyllycaconitine (MLA; 100nM), an α7 nicotinic acetylcholine receptor (nAChR) antagonist, but not dihydro-β-erythroidine hydrobromide (DHβE; 20μM) blocked varenicline-stimulated migration and varenicline-activated ERK, p38 and JNK signaling in HUVECs. MLA (100nM), PD98059 (an ERK inhibitor; 20μM), SB203580 (a p38 inhibitor; 20μM) and SP600125 (a JNK inhibitor; 20μM) also blocked cell migration and varenicline-induced downregulation of VE-cadherin expression in HUVECs. These findings suggest that varenicline promotes HUVEC migration by lowering VE-cadherin expression due to activated ERK/p38/JNK signaling through α7 nAChR. These processes probably contribute to varenicline-aggravated atherosclerotic plaque. Hence, an increased risk of cardiovascular events upon varenicline treatment might occur and must be considered in patients with cardiovascular diseases.

Compartmentalized Regulation of Vascular Endothelial Cell Responses to Fluid Shear Stress by Phosphodiesterase 4D7 (PDE4D7)

While fast flowing blood in linear arterial segments exposes arterial vascular endothelial cells (VECs) to high levels of laminar fluid shear stress (FSS, 15–20 dyne/cm2), slow or oscillatory blood flow, such as is present at arterial bifurcations, exposes VECs to modest levels of non‐linear FSS (1–3 dyne/cm2). These hemodynamic features make arterial bifurcations, arches and branch points especially prone to atherosclerosis. Exposing VECs to high laminar FSS promotes establishment of an adaptive (anti‐thrombotic, anti‐atherogenic and barrier forming) phenotype, an effect that is reversed following exposure of these cells to low / non‐linear levels of FSS. Previously we reported that a cAMP‐signalosome, composed of exchange protein activated by cAMP1 (EPAC1), Rap1 and a phosphodiesterase 4D (PDE4D) allowed cAMP to promote barrier functions in static VEC cultures. This signalosome was shown to associate with components of VEC adherens junctions, including VE‐cadherin (VECAD) and β‐catenin. Recently, we reported that activation of this same AJ‐associated signalosome allowed cAMP, via EPAC1‐mediated activation of Rap1, to promote expression of an adaptive phenotype even in VECs exposed to low levels of FSS. Indeed, siRNA‐mediated knockdown of EPAC1, Rap1 or PDE4D reduced the adaptive responses of VECs exposed to FSS and increased their pro‐thrombotic and pro‐angiogenic potential. Interestingly, PDE4D knockdown reduced responses in these cells to levels equivalent to those detected in cells in which EPAC1 was knocked down. We reported that this effect was due to the fact that PDE4D acts as a tether to localize EPAC1 within this important cAMP‐signalosome. Recently we identified phosphodiesterase 4D7 (PDE4D7) as the PDE4D variant that populates this VEC cAMP‐signalosome. Notably, VEC alignment in the direction of flow was reduced by knockdown of either pan‐PDE4D, selective PDE4D7 or EPAC1 when subjected to low FSS. Both Pan‐PDE4D or selective PDE4D7 siRNA‐mediated knockdown markedly reduced the ability of VECs to respond adaptively to FSS, and promoted loss of EPAC1 from the cAMP‐signalosome. In contrast, PDE4 inhibitors only modestly altered these responses in VECs exposed to various levels of FSS. These findings, together with our earlier work, are consistent with the idea that the tethering actions of PDE4D7 are dominant in this complex in VECs. Using telomerase‐immortalized human aortic endothelial cells, we created a cell‐based system in which the separate catalytic and tethering actions of PDE4D7 in controlling VEC responses to FSS could be measured. Overall, our findings are consistent with the novel idea that the EPAC1 tethering function of PDE4D7 is dominant in regulating EPAC1/Rap1‐mediated effects in VECs and that the cAMP hydrolytic function of PDE4D7 has a hyperlocalized impact within the AJ‐delimited nanodomain. Our findings pave the way for the development of displacing peptide (DP)‐based tools through which to regulate cAMP‐mediated function in VECs exposed to mal‐adaptive levels of FSS.Support or Funding InformationFunding provided by the CIHR.

Heparin's Anti‐Inflammatory Effects in Vascular Endothelial Cells are Mediated Through Transmembrane Receptor 184A

The glycosaminoglycan heparin has been used as an anti‐coagulant in the treatment of cardiovascular diseases for decades. The mechanism of heparin binding to enzyme inhibitor antithrombin III in the coagulation cascade has been well‐documented. Heparin has also been shown to illicit anti‐proliferative and anti‐inflammatory responses in vascular cells, though mechanism(s) remain unclear. Recently we have used antibodies that mimic heparin effects in vascular smooth muscle cells to identify transmembrane protein 184A (TMEM184A) as a putative heparin receptor. The objective of this study was to investigate TMEM184A expression and function in vascular endothelial cells. We observed TMEM184A expression in cultured endothelial cells and in the zebrafish caudal fin vasculature through immunofluorescence using commercial antibodies against a region near the N‐terminus and a region of 20 amino acids of an internal region. To determine whether heparin effects on inflammatory responses were mediated through TMEM184A, rat aortic endothelial cells (RAOECs) were electroporated with control or TMEM184A shRNA. Control and knockdown RAOECs were treated with 200 μg/ml heparin for 20 min, followed by 25 ng/mL TNFα. Quantitative immunofluorescence of activated JNK or p38 showed that heparin treatment prior to TNFα significantly decreased JNK and p38 activation in control shRNA cells compared to cells treated with TNFα alone. However, in TMEM184A knockdown cells, heparin pre‐treatment did not decrease JNK or p38 activation. Heparin induction of DUSP1 was found to be critical for these effects. In control ECs, heparin pre‐treatment decreased TNFα‐induced stress fiber formation. To investigate whether TMEM184A was also necessary for these effects, stress fibers were quantified through immunofluorescence. In TMEM184A knockdown RAOECs, heparin pre‐treatment did not decrease TNFα‐induced actin stress fiber formation. In conclusion, our results show that TMEM184A is expressed specifically in the vasculature in vitro and in vivo. We provide functional evidence that anti‐inflammatory effects of heparin are dependent at least in part on TMEM184A. Further investigation of heparin's anti‐inflammatory effects through TMEM184A in the vasculature is crucial for developing and improving therapeutic strategies in vascular disease.Support or Funding InformationThis work was supported by NIH grant HL54269 to LLK

The dipeptidyl peptidase-IV inhibitor inhibits the expression of vascular adhesion molecules and inflammatory cytokines in HUVECs via Akt- and AMPK-dependent mechanisms

Recently, dipeptidyl peptidase-IV (DPP-IV) inhibitor, a major anti-hyperglycemic agent, has received substantial attention as a possible therapeutic target for inflammatory diseases such as atherosclerosis. However, the direct molecular mechanisms through which DPP-IV inhibitor mediates anti-inflammatory effects in vascular endothelial cells have not been clarified. The effects of the DPP-IV inhibitor, gemigliptin, were analyzed in human umbilical vein endothelial cells (HUVECs) and THP-1 cells. Using Western blotting, we demonstrated that gemigliptin efficiently increased the level of AMP-activated protein kinase (AMPK) and Akt phosphorylation in a dose-dependent manner. The levels of lipopolysaccharide (LPS)-mediated phosphorylated nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) were significantly decreased after gemigliptin treatment. Furthermore, gemigliptin reduced LPS-induced expression of adhesion molecules and inflammatory cytokines such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), and IL-6 in HUVECs. In macrophage-like THP-1 cells, gemigliptin effectively inhibited LPS- and low-density lipoprotein (LDL)-induced foam cell formation. However, these anti-inflammatory and anti-atherosclerotic effects of gemigliptin in HUVECs and THP-1 cells were significantly reduced after treatment with an AMPK or an Akt inhibitor. Our results suggest that gemigliptin efficiently inhibited LPS-induced pro-inflammatory effects in vascular endothelial cells by attenuating NF-κB and JNK signaling via Akt/AMPK-dependent mechanisms. Therefore, the DPP-IV inhibitor, gemigliptin, may directly protect the vascular endothelium against inflammatory diseases such as atherosclerosis.