Mouse Aortic Endothelial Cells Research Articles

Exercise-induced endothelial Mecp2 lactylation suppresses atherosclerosis via the Ereg/MAPK signalling pathway

Background and aimsLactylation, a recently identified post-translational modification (PTM), plays a central role in the regulation of multiple physiological and pathological processes. Exercise is known to provide protection against cardiovascular disease. However, whether exercise-generated lactate changes lactylation and is involved in the exercise-induced attenuation of atherosclerotic cardiovascular disease (ASCVD) remains unclear. The purpose of this study was to investigate the effects and mechanisms of exercise-induced lactylation on ASCVD. Methods and ResultsUsing the high-fat diet-induced apolipoprotein-deficient mouse model of ASCVD, we found that exercise training promoted Mecp2 lysine lactylation (Mecp2k271la); it also decreased the expression of vascular cell adhesion molecule 1 (Vcam-1), intercellular adhesion molecule 1 (Icam-1), monocyte chemoattractant protein 1 (Mcp-1), interleukin (IL)-1β, IL-6, and increased the level of endothelial nitric oxide synthase (Enos) in the aortic tissue of mice. To explore the underlying mechanisms, mouse aortic endothelial cells (MAECs) were subjected to RNA-sequencing and CHIP-qPCR, which confirmed that Mecp2k271la repressed the expression of epiregulin (Ereg) by binding to its chromatin, demonstrating Ereg as a key downstream molecule for Mecp2k271la. Furthermore, Ereg altered the mitogen-activated protein kinase (MAPK) signalling pathway through regulating the phosphorylation level of epidermal growth factor receptor, thereby affecting the expression of Vcam-1, Icam-1, Mcp-1, IL-1β, IL-6, and Enos in ECs, which in turn promoted the regression of atherosclerosis. In addition, increasing the level of Mecp2k271la by exogenous lactate administration in vivo also inhibits the expression of Ereg and the MAPK activity in ECs, resulting in repressed atherosclerotic progression. ConclusionsIn summary, this study provides a mechanistic link between exercise and lactylation modification, offering new insight into the anti-atherosclerotic effects of exercise-induced PTM.

Abstract 668: Identification And Functional Characterization Of Novel MicroRNAs Targeting Epsins For Angiogenesis And Vascular Remodeling

Objective: Epsin is evolutionarily conserved protein that involved in clathrin-mediated endocytosis. Our previous studies showed Epsins regulate VEGF signaling, which is critical for angiogenesis, and Epsins can also augment inflammatory signaling in endothelial cells (EC). However, how Epsins are regulated during inflammation mediated angiogenesis is not clear. We hypothesize that post-transcriptional regulation of Epsins by miRNA are critical for inflammation-induced angiogenesis. In this study, we aim to identify novel miRNAs that can bind to Epsin1/2 genes for regulation of inflammation mediated angiogenesis. Approach and Results: In vitro, we found Epsin1/2 are significantly induced by TNFa and oxidized LDL in mouse aortic endothelial cells (MAECs). To screen miRNAs that can regulate Epsin1/2 expressions, we performed small RNA-sequencing for the control and treated MAECs samples, and identified differentially expressed miRNAs that are predicted to bind to Epsin1/2. We identified 78 differentially expressed (DE) known miRNA and 464 DE novel miRNAs. Among them, 12 known miRNAs and 69 novel miRNAs were predicted to bind Epsin1/2. We validated 2 known miR (miR-6390 and miR-6539) and 3 novel miRNA candidates (miR105462, miR39474 and miR67284) by in vitro miRNA mimics experiments, and found that they all can robustly suppress Epsin1/2 expressions. We then generated miR105462-flox mouse lines by homologous recombination using CRISPR/Cas9. We cross the miR105462-flox mice with Cdh5-Cre ERT2 to generate inducible EC-specific deletion by tamoxifen (miR105562-iEKO). Interestingly, neonatally induced endothelial-specific loss of miR105462 lead to reduced microvascular density and fewer tip cells in the retina of postnatal day6 pups. Additionally, higher Epn1 expressions in retinal EC were detected in EC-specific miR105462 deficient mice. Conclusion: We identified novel miRNA that can regulate Epsin expression in EC. Using novel genetic models, we identified angiogenic defects in vivo when miR105462 is conditionally deleted in EC, implicating critical function elicit by these novel miRNAs. Future studies will be directed at defining the novel miRNA-dependent molecular mechanism for the angiogenesis.

The role of CNPY2 in endothelial injury and inflammation during the progress of atherosclerosis.

Vascular endothelial cells (VECs) injury is closely related to the occurrence and development of atherosclerosis. Canopy FGF signaling regulator 2 (CNPY2), a novel unfolded protein response promoter, has been reported to activate the PERK-CHOP pathway. This study aimed to explore whether CNPY2 is associated with atherosclerosis mediated by VEC injury. By establishing ApoE-/- mouse atherosclerosis model and oxidized low-density lipoprotein (ox-LDL) cell model, we found that CNPY2 was abnormally highly expressed in ApoE-/- mice and ox-LDL-induced mouse aortic endothelial cells (MAECs). Exogenous CNPY2 can significantly aggravate the activation, inflammation, and apoptosis of MAECs induced by ox-LDL and promote the activation of PERK/eIF2α/CHOP signal. The PERK inhibitor GSK2606414 can inhibit CNPY2-induced MAECs injury and PERK signal activation. In addition, in vivo animal experiments furtherly confirmed that CNPY2 could aggravate the process of atherosclerosis in ApoE-/- mice by activating PERK signaling. In conclusion, this study indicated that high level of CNPY2 induces VECs injury by activating PERK signaling and thus participating in the progress of atherosclerosis.

Methylglyoxal-Modified Albumin Effects on Endothelial Arginase Enzyme and Vascular Function.

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs' role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target.

Blocking of programmed cell death-ligand 1 (PD-L1) expressed on endothelial cells promoted the recruitment of CD8+IFN-γ+ T cells in atherosclerosis.

Programmed death ligand-1 (PD-L1) is involved in the negative regulation of immune responses in a variety of diseases. We evaluated the contribution of PD-L1 to the activation of immune cells that promote atherosclerotic lesion formation and inflammation. Compared to ApoE-/- mice that were provided a high-cholesterol diet in combination with anti-PD-L1 antibody developed a larger lipid burden with more abundant CD8+ T cells. The anti-PD-L1 antibody increased the abundance of CD3+PD-1+, CD8 + PD-1+,CD3+IFN-γ+ and CD8+IFN-γ+ T cell under high-cholesterol diet, as well as the serum tumor necrosis factor-α (TNF-a), IFN-γ, PF, GNLY, Gzms B and LTA. Interestingly, the anti-PD-L1 antibody increased the serum level of sPD-L1. In vitro, blocking of PD-L1 on the surface of mouse aortic endothelial cells with anti-PD-L1 antibody stimulated the activation and secretion of cytokines, including IFN-γ, PF, GNLY, Gzms B and LTA, from cytolytic CD8+IFN-γ+ T cell. However, the concentration of sPD-L1 was lower after treatment of the MAECs with anti-PD-L1 antibody. Our findings highlighted that blocking of PD-L1 promoted up-regulation of CD8 + IFN-γ + T cell-mediated immune responses, leading to the secretion of inflammatory cytokine that exacerbated the atherosclerotic burden and promoted inflammation. However, further studies are needed to gain insight into whether PD-L1 activation could be a novel immunotherapy strategy for atherosclerosis.

BASP1 promotes high glucose-induced endothelial apoptosis in diabetes via activation of EGFR signaling.

Diabetes mellitus is a common chronic disease of glucose metabolism. Endothelial dysfunction is an early event in diabetes complicated by cardiovascular disease. This study aimed to reveal the expression of BASP1 and its biological roles in endothelial cell dysfunction in diabetes complicated by cardiovascular disease. By analyzing the databases related to diabetes complicated with coronary heart disease, BASP1 was screened out as an upregulated gene. Human umbilical vein endothelial cells (HUVECs) and primary mouse aortic endothelial cells were treated with high glucose to establish cell models of diabetes-related endothelial dysfunction, and the expression changes of BASP1 were verified by RT-qPCR, western blot, and immunofluorescence. BASP1 was silenced or overexpressed by siRNA or overexpression plasmid, and its effects on cell migration, apoptosis, tube formation, inflammatory response, and ROS were detected. The possible signaling pathway of BASP1 was found and the mechanism of BASP1 on promoting the progression of endothelial dysfunction was explored using the EGFR inhibitor, gefitinib. Bioinformatics analysis indicated that the expression of BASP1 in patients with diabetes mellitus and concomitant coronary heart disease was increased. High glucose induced the upregulation of BASP1 expression in endothelial cells, and showed a time-dependent relationship. Silencing of BASP1 alleviated the damage of high glucose to endothelial cells. BASP1 regulated EGFR positively. The promoting effect of BASP1 on endothelial cell apoptosis may be achieved by regulating the EGFR pathway. BASP1 promotes endothelial cell injury induced by high glucose in patients with diabetes, which may be activated by activating the EGFR pathway.

Sitagliptin Ameliorates Creb5/lncRNA ENSMUST00000213271-Mediated Vascular Endothelial Dysfunction in Obese Mice.

Obesity is mediated by the changes in dyslipidemia, oxidative stress, and inflammation, leading to vascular endothelial dysfunction. Glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 inhibitors prevent the development of endothelial dysfunction. However, the underlying mechanism still remains largely unclear. Long non-coding RNAs (lncRNAs), one class of non-coding small RNAs, have been shown to exert a regulatory impact on the endothelial function in obesity. This study aimed to investigate whether the elevation of GLP-1 by a DPP-4 inhibitor sitagliptin improved vascular endothelial function by modulating lncRNAs in obese mice and to clarify the underlying molecular mechanism. Male C57BL/6J mice were fed a high-fat diet for 4 months to induce obesity and some obese mice were treated with sitagliptin for the last 1 month. Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and glucagon-like peptide-1 (GLP-1) in plasma were detected by ELISA. LncRNA expression profile was analyzed via microarray. Aortic relaxations were examined by myograph. Protein expressions and phosphorylations were determined using western blot. The differentially expressed lncRNAs were validated using qRT-PCR. Obese mice exhibited increased levels of TC and LDL, decreased concentrations of HDL and GLP-1 in plasma, and impaired aortic endothelium-dependent relaxations; such effects could be reversed by sitagliptin. Moreover, the altered expression profile of lncRNAs in the obese mouse aortae could be modulated by sitagliptin. Consistent with microarray analysis, qRT-PCR also revealed that lncRNA ENSMUST00000213271 was up-regulated in obese mouse aortae and aortic endothelial cells (ECs), which could be down-regulated by sitagliptin. Creb5 silencing reduced lncRNA ENSMUST00000213271 in obese mouse ECs. Knockdown of either Creb5 or lncRNA ENSMUST00000213271 restored the activation of AMPK/eNOS in obese mouse ECs. Furthermore, sitagliptin also suppressed Creb5 and lncRNA ENSMUST00000213271 and increased the phosphorylations of AMPK and eNOS in obese mice. Creb5/lncRNA ENSMUST00000213271 mediated vascular endothelial dysfunction through inhibiting AMPK/eNOS cascade in obesity. Elevation of GLP-1 by sitagliptin possibly improved endothelial function by suppressing Creb5/lncRNA ENSMUST00000213271 and subsequently restoring AMPK/eNOS activation in obese mice. This study will provide new evidence for the benefits of GLP-1 against vasculopathy in obesity.

PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging.

Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that binds to low-density lipoprotein receptors. Efferocytosis is the process by which phagocytes remove apoptotic cells. Both PCSK9 and efferocytosis play important roles in regulating redox biology and inflammation, the key factors contributing to vascular aging. This study was designed to investigate the impact of PCSK9 on efferocytosis in endothelial cells (ECs) and its implications in vascular aging. Methods and Results: Studies were performed in primary human aortic ECs (HAECs) and primary mouse aortic ECs (MAECs) isolated from male wild-type (WT) and PCSK9-/- mice, and in young and aged mice treated with saline or the PCSK9 inhibitor Pep2-8. Our findings include that recombinant PCSK9 protein induces defective efferocytosis and aging marker senescence-associated-β-galactosidase (SA-β-gal) expression in ECs, while PCSK9-/- restores efferocytosis and inhibits SA-β-gal activity. Further studies in aged mice showed that endothelial deficiency of MerTK, a critical receptor for efferocytosis that allows phagocytes to detect the presence of apoptotic cells, may be an indicator of vascular dysfunction in the aortic arch. Pep2-8 treatment markedly restored efferocytosis in endothelium from the aged mice. A proteomics study in the aortic arch from aged mice revealed that Pep2-8 administration significantly downregulates expression of NOX4, MAPK subunits, NF-κB, and secretion of pro-inflammatory cytokines, all known to promote vascular aging. Immunofluorescent staining showed that Pep2-8 administration upregulates expression of eNOS and downregulates expression of pro-IL-1β, NF-κB and p22phox compared to saline treated group. Conclusions: These findings provide initial evidence for the ability of aortic ECs to accomplish efferocytosis and argue for a role of PCSK9 in attenuating EC efferocytosis, thereby leading to vascular dysfunction and acceleration in vascular aging.

Tongxinluo attenuates atherosclerosis by inhibiting ROS/NLRP3/caspase-1-mediated endothelial cell pyroptosis

Ethnopharmacological relevanceTongxinluo (TXL) is one of the most common traditional Chinese medicines and plays a vital role in treating atherosclerosis (AS). Endothelial cell (EC) pyroptosis plays a crucial role in the development of AS. Previous research revealed the inhibitory function of TXL in EC apoptosis and autophagy. However, whether TXL can inhibit the pyroptosis of ECs has not been determined. Aim of the studyTo explore the influence of TXL on EC pyroptosis and determine its underlying mechanism of action in AS. Materials and methodsThe TXL components were determined by ultra-performance liquid chromatography coupled with a photodiode array detector. We used ApoE−/− mice to establish a disease model of AS. After treatment with TXL, we recorded pathological changes in the mice and performed immunofluorescence staining of mice aortas. We also measured protein and gene levels to explore the influence of TXL on pyroptosis in vivo. The model was established by stimulating mouse aortic endothelial cells (MAECs) with oxidized low-density lipoprotein (ox-LDL) and analyzing the effect of TXL on pyroptosis by Western blotting (WB), real-time PCR (RT-PCR), and flow cytometry (FCM). We also investigated the impact of TXL on reactive oxygen species (ROS) by FCM and WB. ResultsTen major components of TXL were detected. The vivo results showed that TXL inhibited the development of AS and decreased EC pyroptosis, the activation of caspase-1, and the release of inflammatory cytokines. The vitro experiments showed that TXL significantly reduced the extent of injury to MAECs by oxidized LDL (ox-LDL). TXL reversed the high expression of gasdermin D and other proteins induced by ox-LDL and had a significant synergistic effect with the caspase-1 inhibitor VX-765. We also confirmed that TXL decreased the accumulation of ROS and the expression levels of its essential regulatory proteins Cox2 and iNOS. When ROS accumulation was reduced, EC pyroptotic damage was reduced accordingly. ConclusionOur results indicated that TXL inhibited EC pyroptosis in AS. Reducing the accumulation of ROS may be the essential mechanism of AS inhibition by TXL.

Regenerative effects of mouse aortic endothelial cells in a murine model of critical limb ischemia

Critical limb ischemia of the is a serious disease that threatens a significant decrease in working ability and disability of patients. Cell therapy may be useful in correcting the endothelial dysfunction that accompanies this disorder. The aim of study was to evaluate the effectiveness of local transplantation of mouse aortic endothelial cells (MAECs) in a model of critical limb ischemia in mice. Materials and methods. Critical limb ischemia in FVB mice was modeled by femoral artery ligation. The primary culture of endothelial cells was obtained from the murine aortic intima. The endothelial phenotype of cells for the expression of CD31, CD38 and CD309 markers was confirmed by flow cytometry and 1•106 MAECs were transplanted intramuscularly into ischemic limb. Tissue perfusion was assessed by laser Doppler flowmetry as well as descriptive histology was used to analyze changes in ischemic muscle after cell transplantation compared to the control group. Results. After MAECs transplantation in animals with modeled critical limb ischemia, the skin of the foot kept pink color and the corresponding temperature of the healthy limb without signs of necrosis of the distal phalanges in contrast to animals of the control group. According to laser Doppler flowmetry data, a significant difference (p ≤ 0.05) in perfusion of ischemic and sham-operated limbs in animals of the control group remained at the level of Δ = 45.7 ± 13.1 %. In animals after MAECs transplantation, the difference of these indicators between limbs was only Δ = 14.0 ± 8.23 % and was not statistically significant. A histological examination of muscle tissue after MAECs transplantation demonstrated the signs of compensatory processes characterized by hyperplasia and hypertrophy of myocyte’s nuclei and lightening of the nucleoplasm with well-defined nucleoli in some myofibrils. In the cytoplasm of myocytes, intermediate Z-discs were clearly visualized, and the number of myofibrils in muscle fibers increased. Conclusion. In animals with model of critical limb ischemia, the transplantation of aorta-derived endothelial cells recover the perfusion of ischemic limbs and improve the histological indicators of muscle tissue.

Abstract 13254: Klotho Suppresses Endothelial-to-Mesenchymal Transition in the Aorta of Mice With Chronic Kidney Disease by Inhibition of Endothelial Senescent Activity

Background: Klotho is an anti-aging protein expressed primarily in the kidney and vasculature. Chronic kidney disease (CKD) results in hyperphosphatemia. We reported that high levels of phosphate downregulate Klotho expression in vascular cells. Endothelial-to-mesenchymal transition (EndMT) in arteries promotes the development of atherosclerosis, and CKD is a significant risk factor for atherosclerosis. However, the impact of CKD on EndMT in arteries is unclear and whether Klotho insufficiency plays a role in the mechanism underlying vascular EndMT remains unknown. This study tested the hypothesis that Klotho insufficiency associated with CKD induces senescence in arterial endothelial cells to upregulate EndMT. Methods and Results: We developed a CKD model in C57/BL6 mice (5-6 months old) on AIN-76A diet and treated with adenine and calcitriol. CKD was confirmed by markedly elevated blood levels of creatinine and phosphate, as well as by renal tubular damage. En face immunofluorescence staining of aorta and image quantification revealed that the levels of biomarkers of senescence (P21) and EndMT (vimentin) were greater, while Klotho levels were lower, in the aortic arch endothelial cells of CKD mice compared to sham control mice. P21 and vimentin were co-localized in a greater number of endothelial cells in the aortic arch of CKD mice. High phosphate treatment upregulated the levels of P21 and vimentin and reduced the levels of Klotho in both primary human and mouse aortic endothelial cells, and knockdown P21 attenuated EndMT induced by high phosphate. Recombinant Klotho suppressed both senescence and EndMT in primary human aortic endothelial cells. Further, prolonged treatment of CKD mice with recombinant Klotho attenuated endothelial senescence and EndMT in the aortic arch. Conclusion: Klotho insufficiency associated with CKD promotes endothelial senescence and EndMT in aorta. High phosphate is capable of inducing endothelial senescent change that provokes EndMT. Administration of recombinant Klotho or upregulation of Klotho expression may have therapeutic potential for suppressing pro-atherosclerosis activity in large arteries by prevention of endothelial senescence and resultant EndMT.

CircCRIM1 Ameliorates Endothelial Cell Angiogenesis in Aging through the miR-455-3p/Twist1/VEGFR2 Signaling Axis.

Background Aging leads to vascular endothelial cell senescence. Decreased expression of VEGFA and VEGFR2 plays a crucial role in impairing angiogenesis in senescent endothelial cells. Noncoding RNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs), regulate endothelial cell proliferation, differentiation, apoptosis, and migration and participate in the occurrence and development of vascular diseases. However, the mechanism of noncoding RNAs in age-related vascular endothelial dysfunction remains unclear. Here, we aimed to identify the circRNA that is associated with VEGF/VEGFR2 signaling pathway activation in angiogenesis. Methods Immunoblotting, quantitative reverse transcription-polymerase chain reaction (qRT–PCR), in vitro and in vivo experiments, luciferase assays, and chromatin immunoprecipitation followed by qRT–PCR (ChIP–qPCR) assays were performed to clarify the roles played by circCRIM1 in mouse aortic endothelial cell (MAEC) angiogenesis. Results CircCRIM1 expression was downregulated in both an aging mouse model of lower limb ischemia in vivo and aging MAECs in vitro. Overexpressing circCRIM1 mediated through a plasmid or adeno-associated virus (AAV) reversed the downregulation of angiogenesis-related phenotype acquisition during aging. MiR-455-3p was confirmed to be a potential target of circCRIM1 through luciferase assays followed by RNA fluorescence in situ hybridization (FISH), which revealed the colocalization of circCRIM1 and miR-455-3p. CircCRIM1 was found to be a competitive endogenous RNA that sponged miR-455-3p and regulated angiogenesis-related phenotypes in MAECs. Furthermore, Twist1 was found to be downstream of miR-455-3p. A ChIP–qPCR assay showed that Twist1 promoted VEGFR2 expression by binding to the promoter region, playing a vital role in angiogenesis. Conclusions Decreased expression of circCRIM1 impaired angiogenesis in aging via the miR-455-3p/Twist1/VEGFR2 axis. Our findings suggest that overexpression of circCRIM1 may be an effective therapeutic strategy for promoting ischemic lower limb blood flow recovery.

MiR-351 promotes atherosclerosis in diabetes by inhibiting the ITGB3/PIK3R1/Akt pathway and induces endothelial cell injury and lipid accumulation

BackgroundThe miR-351 gene is significantly upregulated in diabetic mice with atherosclerosis. However, the mechanism by which its presence is important for the overall disease has not been elucidated. Therefore, this study will investigate the mechanism of miR-351 in the process of diabetes mellitus with atherosclerosis through miR-351 gene knockout mice.MethodsIn this study, miR-351−/− C57BL/6 mice were first induced to form a type 2 diabetes mellitus model with atherosclerosis by STZ injection and a high-fat diet. Pathological tests (oil red O, HE, and Masson staining) combined with biochemical indices (TC, TG, LDL-C, HDL-C, TNF-α, hs-CRP, NO, SOD, MDA, CAT, and GSH-Px) were performed to evaluate the pathological degree of atherosclerosis in each group. Mouse aortic endothelial cells were treated with oxidized low-density lipoprotein (ox-LDL) and 30 mM glucose to establish a diabetic atherosclerosis cell model. Combined with cell oil red O staining and flow cytometry, the effects of silencing miR-351 on lipid accumulation and cell apoptosis in the diabetic atherosclerosis cell model were determined. Fluorescence in situ hybridization was used to detect the localization and transcription levels of miR-351 in cells. The target genes of miR-351 were predicted by bioinformatics and verified by dual-luciferase activity reporting. Western blotting was used to detect the expression levels of phosphorylated inosine 3-kinase regulatory subunit 1 (PIK3R1)/serine/threonine kinase 1 (Akt) and apoptosis-related proteins after transfection with integrin subunit β3 (ITGB3) small interfering ribonucleic acid (siRNA).ResultsThe expression of the miR-351 gene was significantly increased in the high-fat wild-type (HWT) group, and its expression was significantly decreased in the knockout mice. Silencing miR-351 effectively alleviated atherosclerosis in mice. The levels of miR-351 expression, apoptosis, lipid accumulation, and oxidative stress in ox-LDL + high glucose-induced endothelial cells were significantly increased. These phenomena were effectively inhibited in lentivirus-infected miR-351-silenced cell lines. Bioinformatics predicted that miR-351-5p could directly target the ITGB3 gene. Transfection of ITGB3 siRNA reversed the downregulation of apoptosis, decreased oil accumulation, and decreased oxidative stress levels induced by miR-351 silencing. In addition, it inhibited the activation of the PIK3R1/Akt pathway.ConclusionSilencing miR-351 upregulates ITGB3 and activates the PIK3R1/Akt pathway, thereby exerting anti-apoptosis and protective effects on endothelial cells.

VEGF receptor heterodimers and homodimers are differentially expressed in neuronal and endothelial cell types.

PurposeWe have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells.MethodsPC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/μL) or VEGF-B (50 ng/μL) or “vehicle” (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells.ResultsTIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells.ConclusionsDifferences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Impairment of Ceramide-Mediated Endothelial Instant Membrane Resealing During Diabetes Mellitus.

Recent studies have indicated that instant cell membrane resealing (ICMR) controls the activation of NOD-like receptor pyrin domain containing 3 (Nlrp3) inflammasomes in endothelial cells, thereby initiating and promoting vascular inflammation. It remains unknown whether this impaired ICMR occurs under diabetic condition or hyperglycemia contributing to endothelial dysfunction leading to vascular inflammation, a hallmark of diabetic vascular injury. The present study aims to examine whether ICMR occurs during in control and diabetic mice and to explore related molecular mechanisms associated with acid sphingomyelinase (ASM)-mediated ceramide production. Using confocal microscopy, we demonstrated that mouse aortic endothelial cells (MAECs) exposed to high glucose levels exhibited much more retarded ICMR after laser-induced membrane injury, compared to that in control cells. The high glucose-induced impairment of membrane resealing in MAECs was prevented when these cells were pretreated with sphingomyelin or C24-ceramide. Mechanistically, high glucose treatment decreased association of membrane ceramide with annexin A5, an essential element of membrane repair machinery. Consistently, the association of ceramide with annexin A5 was significantly reduced in the coronary arterial endothelium of mice with streptozotocin-induced diabetes mellitus compared to that in non-diabetic control mice. Moreover, a marked reduction of the association of ceramide with annexin A5 was observed in coronary arterial endothelium of ASM knockout mice regardless of their diabetic status. Lastly, high glucose treatment or ASM gene deletion substantially impaired ICMR in coronary arterial endothelium of mice receiving membrane puncturing agents. Collectively, our data suggest that ceramide-mediated ICMR in vascular endothelial cells is impaired during diabetes mellitus due to dissociation of ceramide with annexin A5 and ASM play a critical role in this ICMR.

Recombinant EGFL7 Mitigated Pressure Overload-Induced Cardiac Remodeling by Blocking PI3K /AKT/ Signaling in Macrophages.

Inflammation and endothelial dysfunction play an essential role in heart failure (HF). Epidermal growth factor-like protein 7 (EGFL7) is upregulated during pathological hypoxia and exerts a protective role. However, it is unclear whether there is a link between abnormal EGFL7 expression and inflammation in overload stress-induced heart failure. Our results showed that EGFL7 transiently increased during the early 4 weeks of TAC and in hypertensive patients without heart failure. However, it decreased to the basal line in the heart tissue 8 weeks post-transverse aortic constriction (TAC) or hypertensive patients with heart failure. Knockdown of EGFL7 with siRNA in vivo accelerated cardiac dysfunction, fibrosis, and macrophage infiltration 4 weeks after TAC. Deletion of macrophages in siRNA-EGFL7-TAC mice rescued that pathological phenotype. In vitro research revealed the mechanism. PI3K/AKT/N signaling in macrophages was activated by the supernatant from endothelial cells stimulated by siRNA-EGFL7+phenylephrine. More macrophages adhered to endothelial cells, but pretreatment of macrophages with PI3Kγ inhibitors decreased the adhesion of macrophages to endothelial cells. Ultimately, treatment with recombinant rmEGFL7 rescued cardiac dysfunction and macrophage infiltration in siRNA-EGFL7-TAC mice. In conclusion, EGFL7 is a potential inhibitor of macrophage adhesion to mouse aortic endothelial cells. The downregulation of EGFL7 combined with increased macrophage infiltration further promoted cardiac dysfunction under pressure overload stress. Mechanistically, EGFL7 reduced endothelial cell adhesion molecule expression and inhibited the PI3K/AKT/NFB signaling pathway in macrophages.

Targeting Peptide, Fluorescent Reagent Modified Magnetic Liposomes Coated with Rapamycin Target Early Atherosclerotic Plaque and Therapy.

Atherosclerosis is the leading cause of global morbidity and mortality. Its therapy requires research in several areas, such as diagnosis of early arteriosclerosis, improvement of the pharmacokinetics and bioavailability of rapamycin as its therapeutic agents. Here, we used the targeting peptide VHPKQHR (VHP) (or fluorescent reagent) to modify the phospholipid molecules to target vascular cell adhesion molecule-1 (VCAM-1) and loaded ultrasmall paramagnetic iron oxide (USPIO/Fe3O4) plus rapamycin (Rap) to Rap/Fe3O4@VHP-Lipo (VHPKQHR-modified magnetic liposomes coated with Rap). This nanoparticle can be used for both the diagnosis and therapy of early atherosclerosis. We designed both an ex vivo system with mouse aortic endothelial cells (MAECs) and an in vivo system with ApoE knockout mice to test the labeling and delivering potential of Rap/Fe3O4@VHP-Lipo with fluorescent microscopy, flow cytometry and MRI. Our results of MRI imaging and fluorescence imaging showed that the T2 relaxation time of the Rap/Fe3O4@VHP-Lipo group was reduced by 2.7 times and 1.5 times, and the fluorescence intensity increased by 3.4 times and 2.5 times, respectively, compared with the normal saline group and the control liposome treatment group. It showed that Rap/Fe3O4@VHP-Lipo realized the diagnosis of early AS. Additionally, our results showed that, compared with the normal saline and control liposomes treatment group, the aortic fluorescence intensity of the Rap/Fe3O4@VHP-Lipo treatment group was significantly weaker, and the T2 relaxation time was prolonged by 8.9 times and 2.0 times, indicating that the targeted diagnostic agent detected the least plaques in the Rap/Fe3O4@VHP-Lipo treatment group. Based on our results, the synthesized theragnostic Rap/Fe3O4@VHP-Lipo serves as a great label for both MRI and fluorescence bimodal imaging of atherosclerosis. It also has therapeutic effects for the early treatment of atherosclerosis, and it has great potential for early diagnosis and can achieve the same level of therapy with a lower dose of Rap.

Abstract 489: Glutathione Regulates Endothelial Cell Function And Vegfr2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P<0.001). MAECs were isolated and relative migration rates were determined using under-agarose migration assays. Het MAECs were 1.25 fold faster than WT (n=4 p<0.001) while KO MAECs were slower, 0.90 fold (n=3, p<0.05). Cell proliferation assays are currently underway. Concurrently, we inhibited glutathione reductase in primary human aortic endothelial cells using 2-AAPA. 2-AAPA (75 μm) alone didn’t activate VEGFR2 (determined by western blot for pY1175 VEGFR2). 2-AAPA in combination with H 2 O 2 (100 μM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2-AAPA with H 2 O 2 induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p<0.01, n=6), This effect was attenuated by the addition of DTT. Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2-AAPA + H 2 O 2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H 2 O 2 mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H 2 O 2 activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H 2 O 2 on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.

Glutathione Regulates Endothelial Cell Function and VEGFR2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized window of reactive oxygen species. Mice heterozygous for the Glutamate cysteine ligase modifier subunit knockout have augmented ischemic remodeling. During occlusion the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling.MethodsTo test this hypothesis, we first preformed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype controls (WT). VEGF stimulated chemotaxis was determined using the under‐agarose migration assay. Primary human aortic endothelial cells (HAEC) were purchased from Lonza and cultured following suppliers’ recomendations. Phospho‐Y1175 VEGFR2/total VEGFR2 and phospho‐Y416 proto‐oncogene c‐Src (Src)/total Src were determined by western blotting.ResultsOutgrowth area in the Het group was significantly larger than WT and KO at day 7 (2.15 fold, n=4, P<0.001). Het MAECs were 1.25 fold faster (n=4 p<0.001) while KO MAECs were 0.90 fold slower than WT (n=3, p<0.05) migrating towards VEGF‐A. Concurrently, we inhibited glutathione reductase in HAECs using 2‐acetylamino‐3‐[4‐(2‐acetylamino‐2‐carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2‐AAPA). 2‐AAPA (75 µM) alone didn’t significantly activate VEGFR2. However, 2‐AAPA (75 µM) in combination with H2O2 (100 µM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2‐AAPA both with and without H2O2 induced a significant increase in active Src, 5.3 and 9.0 fold (p<0.05, p<0.01, n=6) respectively. This effect was attenuated by the addition of Dithiothreitol (1mM). Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2‐AAPA + H2O2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2‐AAPA + H2O2 mediated activation of Src. Glutathione S‐transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 µM) abrogated the effects of 2‐AAPA + H2O2 on VEGFR2.ConclusionCombined these data suggest Src mediates the 2‐AAPA + H2O2 activation of VEGFR2. And that glutathionylation is necessary for this effect, co‐immunoprecipitation experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.

Enhanced expression of lectin‐like oxidized LDL receptor‐1 is implicated in the sodium arsenite‐induced increase in foam cells from murine macrophages

Arsenic is a naturally occurring toxic element that is present in the air, food, soil, and water of many countries, including the U.S. The U.S. Geological Survey reported that about 44 million people in the U.S. use private wells for water and estimated that over 2 million of those people drink from wells with high concentrations (>10 ug/L) of arsenic. Epidemiological studies have shown that chronic arsenic exposure is a significant risk factor for cardiovascular diseases, including atherosclerosis. However, the cellular and molecular mechanisms connecting arsenic exposure to atherosclerosis are not known. It has been established that Macrophage‐derived, lipid‐loaded, foam cells are a key component of atherosclerotic plaques and are essential for their formation. Furthermore scavenger receptors, such as lectin‐like oxLDL receptor 1 (LOX‐1), CD36, and scavenger receptor type A (SR‐A), regulate the lipid uptake of macrophages and are likely key to the transformation of these cells to foam cells. Our previous study showed that in mouse aortic endothelial cells, arsenic exposure stimulated the uptake of oxidized low‐density lipoprotein (oxLDL) by upregulating the expression of LOX‐1. To see if LOX‐1 might play a key role in foam cell formation, in this study, we used Oil‐red‐O assay to show that sodium arsenite (SA) exposure dose‐dependently enhanced the uptake of oxLDL in murine RAW 264.7 macrophages with a commensurate increase in foam cell count. In addition, while we showed that SA treatment caused a significant increase in both LOX‐1 mRNA and protein levels, only slight or no significant increases were observed in CD36 or SR‐A mRNA expression. Our results indicate that in arsenic induced atherosclerosis, LOX‐1 may play a critical and perhaps unique role among scavenger receptors in the production of foam cells. Further studies, including in vivo and in vitro, are warranted to verify and clarify the role of Lox‐1 in a disease model and to identify other cells and components of the disease pathway.