Proliferation Of Human Aortic Endothelial Cells Research Articles

Vascular endothelial dysfunction improvements in patients with uraemia using pentoxifylline-suppressing NLRP3 expressions and HMGB1 release.

This study aimed to investigate the protective effect of pentoxifylline (PTX) on vascular endothelial dysfunction in uraemia. The human aortic endothelial cells (HAECs) required for the experiments were all obtained from the National Collection of Authenticated Cell Cultures (Salisbury, UK). The permeability of HAECs was assessed. Each group had six samples. Compared with the healthy volunteer group, HAEC proliferation in the 20% uraemia group was significantly inhibited after 72 h (p < 0.001), co-localisation of nucleotide-binding domain, leucine-rich repeat-containing receptor family pyrin domain-containing 3 (NLRP3) and apoptosis-associated speck-like (ASC) protein induced by uremic serum was enhanced (p < 0.01) and high mobility group box 1 (HMGB1) release was increased (0.594 ± 0.057, p = 0.03). The co-immunoprecipitation of NLRP3, ASC and HMGB1 induced by uremic toxin was also enhanced (p < 0.01), and PTX inhibited this phenomenon. The expression of NLRP3 (0.810 ± 0.032, p = 0.02) and caspase-1 (0.580 ± 0.041, p = 0.03) was increased, whereas the expression of ZO-1 (0.255 ± 0.038, p = 0.03) and VE-cadherin (0.0546 ± 0.053, p = 0.02) was decreased in the uraemia group; compared with the healthy volunteer group, treated with PTX (NLRP3, 0.298 ± 0.042, p = 0.03; caspase-1, 0.310 ± 0.021, p = 0.03; ZO-1, 0.412 ± 0.028, p = 0.02; VE-cadherin, 0.150 ± 0.034, p = 0.02) and MCC950 (NLRP3, 0.432 ± 0.022, p = 0.03; caspase-1, 0.067 ± 0.031, p > 0.05; ZO-1, 0.457 ± 0.026, p = 0.03; VE-cadherin, 0.286 ± 0.017, p = 0.03) lessened this trend. Pentoxifylline promoted the HAEC permeability mediated by uremic toxins (1.507 ± 0.012, p = 0.02). In conclusion, PTX enhances the release of HMGB1, which is dependent on NLRP3 activation, and consequently exerts positive effects on interconnecting proteins, ultimately leading to an improvement in vascular permeability.

TXNRD1 knockdown inhibits the proliferation of endothelial cells subjected to oscillatory shear stress via activation of the endothelial nitric oxide synthase/apoptosis pathway

Atherosclerosis is the main cause of cardiovascular disease, and fluid shear stress is a key factor regulating its occurrence and development. Oscillatory shear stress (Oss) is an important pro-atherosclerosis factor. Oss mainly occurs in areas that are susceptible to atherosclerosis, but the exact mechanism of atherosclerosis induction remains unclear. Therefore, starting from the atheroprone phenotype that Oss stimulates abnormal vascular endothelial cell proliferation, this study aimed to reveal the underlying mechanism of Oss-induced atherosclerosis formation and to identify new targets for the prevention and treatment of atherosclerosis. In this study, the gene encoding thioredoxin reductase 1 (TXNRD1), which is closely related to atherosclerosis development and cell proliferation, was screened by analyzing the transcriptome sequencing data of static and Oss-treated human aortic endothelial cells (HAECs). Moreover, this study successfully verified that TXNRD1 mRNA and protein were significantly upregulated in Oss-treated HAECs. Oss significantly promoted the proliferation, migration, and tube formation of HAECs, whereas TXNRD1 knockdown impaired the proliferation, migration, and tube formation of Oss-treated HAECs, and this process was mainly achieved via activation of the apoptosis pathway. To further clarify whether Oss-sensitive TXNRD1 affects the apoptosis rate and proliferative ability of HAECs by regulating the endothelial nitric oxide synthase (eNOS) pathway, we used NG-nitro-L-arginine methyl ester (L-NAME) to inhibit eNOS activity and nitric oxide (NO) production. L-NAME significantly reversed the promoting effect of TXNRD1 knockdown on Oss-treated HAEC apoptosis, and it also abolished the inhibitory effect of TXNRD1 knockdown on the proliferation and S + G2 phase cell mass of Oss-treated HAECs. In conclusion, this study showed that TXNRD1 knockdown inhibited the proliferation of HAECs exposed to Oss by activating the eNOS/apoptosis pathway, revealing that TXNRD1 is involved in the dysregulation of Oss-induced endothelial cell proliferation. These findings provide new directions and insights into the prevention and treatment of atherosclerosis.

Staphylococcus aureus β-Toxin Exerts Anti-angiogenic Effects by Inhibiting Re-endothelialization and Neovessel Formation.

Staphylococcus aureus causes severe, life-threatening infections that often are complicated by severe local and systemic pathologies with non-healing lesions. A classic example is S. aureus infective endocarditis (IE), where the secreted hemolysin β-toxin potentiates the disease via its sphingomyelinase and biofilm ligase activities. Although these activities dysregulate human aortic endothelial cell activation, β-toxin effect on endothelial cell function in wound healing has not been addressed. With the use of the ex vivo rabbit aortic ring model, we provide evidence that β-toxin prevents branching microvessel formation, highlighting its ability to interfere with tissue re-vascularization and vascular repair. We show that β-toxin specifically targets both human aortic endothelial cell proliferation and cell migration and inhibits human umbilical vein endothelial cell rearrangement into capillary-like networks in vitro. Proteome arrays specific for angiogenesis-related molecules provided evidence that β-toxin promotes an inhibitory profile in endothelial cell monolayers, specifically targeting production of TIMP-1, TIMP-4, and IGFBP-3 to counter the effect of a pro-angiogenic environment. Dysregulation in the production of these molecules is known to result in sprouting defects (including deficient cell proliferation, migration, and survival), vessel instability and/or vascular regression. When endothelial cells are grown under re-endothelialization/wound healing conditions, β-toxin decreases the pro-angiogenic molecule MMP-8 and increases the anti-angiogenic molecule endostatin. Altogether, the data indicate that β-toxin is an anti-angiogenic virulence factor and highlight a mechanism where β-toxin exacerbates S. aureus invasive infections by interfering with tissue re-vascularization and vascular repair.

LincRNA-p21 alleviates atherosclerosis progression through regulating the miR-221/SIRT1/Pcsk9 axis.

Atherosclerosis (AS) is the main aetiology of coronary heart disease, cerebral infarction and peripheral vascular disease in humans. Long‐noncoding RNA (LincRNA)‐p21 has been reported to participate in the development of AS. Therefore, this study was designed to investigate the mechanism of LincRNA‐p21 on suppressing the development of AS. We fed ApoE−/− mice with a high‐fat diet to induce an AS mouse model where the lesion area of AS and the extent of lipid deposition were measured. The binding of LincRNA‐p21 and miR‐221 or miR‐221 and SIRT1 was measured using a dual luciferase reporter gene assay and RIP. Following loss‐ and gain‐ function assays, CCK8, EdU, Transwell assay and scratch test were performed to determine the biological processes of human aortic endothelial cells (HAECs). miR‐221 was highly expressed while SIRT1 was poorly expressed in AS. LincRNA‐p21 acted as a sponge for miR‐221. miR‐221 targeted and negatively regulated the expression of SIRT1. LincRNA‐p21 promoted the deacetylation of Pcsk9 by SIRT1 by competitively binding to miR‐221, whereby promoting HAEC proliferation, migration and tube formation. In conclusion, LincRNA‐p21 acted as a molecular sponge for miR‐221 to promote deacetylation of the promoter region of Pcsk9 by SIRT1, therefore preventing the development of AS.

Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells

The therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

MicroRNA-532-5p-programmed cell death protein 4 (PDCD4) axis regulates angiotensin II-induced human umbilical vein endothelial cell apoptosis and proliferation

BackgroundThis study was carried out to investigate the effect of microRNA miR-532-5p on the proliferation of hypertension endothelial cells. MethodsAngiotensin II (Ang II)-treated human umbilical vein endothelial cells (HUVECs) and primary human aortic endothelial cells (HAECs) were used as cell models to imitate the pathological changes in endothelial cells under hypertensive conditions. The expression levels of miR-532-5p and programmed cell death protein 4 (PDCD4) were detected by Quantitative Real-time PCR (qRT-PCR). The effects of miR-532-5p and PDCD4 on the proliferation of HUVECs and HAECs treated with Ang II were detected by Methyl Thiazolyl Tetrazolium (MTT) assay. The effects of miR-532-5p and PDCD4 on the apoptosis and cell cycle of HUVECs and HAECs treated with Ang II were detected by flow cytometry. Western blot was used to detect the expression levels of PDCD4, apoptosis-related proteins and cycle-related proteins in HUVECs and HAECs treated with Ang II. Bioinformatics analysis and Luciferase gene reporter assay were used to assess the relationship between miR-532-5p and PDCD4. ResultsThe expression levels of miR-532-5p were reduced, while the expression levels of PDCD4 were raised in Ang II-treated HUVECs and HAECs. MiR-532-5p mimic and si-PDCD4 restrained the apoptosis, promoted the proliferation of Ang II-treated HUVECs and HAECs and caused S-phase arrest of cells. PDCD4 was identified as a potential target for miR-532-5p. Knockdown of PDCD4 significantly affected apoptosis and proliferation of Ang II-treated HUVECs. MiR-532-5p regulates apoptosis and proliferation of Ang II-induced HUVECs and HAECs. In addition, overexpression of PDCD4 attenuated the effect of miR-532-5p on the proliferation of Ang II-treated HUVECs and HAECs. ConclusionMiR-532-5p inhibited the expression of PDCD4, thereby inhibiting apoptosis and promoting proliferation of Ang II-treated HUVECs and HAECs.

Ca2+-activated Cl− channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells

IntroductionCa2+-activated Cl− channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells. ObjectiveThis study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis. MethodsWhole-cell patch clamp techniques were used to record Ca2+-activated Cl− currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively. ResultsTMEM16A mediates the Ca2+-activated Cl− channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC50 of 0.1209 μM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased in vitro tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect. ConclusionThis study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.

NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR‑638/AKT/mTOR signaling in atherosclerosis.

Long non-coding RNAs (lncRNAs) have been validated to mediate the development of atherosclerosis (AS). In the present study, the molecular mechanisms and functions of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the advancement of human aortic endothelial cells (HAECs) were investigated. The levels of lncRNA-NEAT1 and miR-638 expression in clinical samples and cells were explored via quantitative reverse transcription polymerase chain reaction. Colony formation and CCK-8 assays were performed to determine the proliferative capacity of cells, and the apoptotic capacity of cells was analyzed on the basis of apoptotic cell proportion and caspase-3 activity. Then, the proportion of cells and correlations among phosphoglycerate kinase 1 (PGK1), NEAT1, and miR-638 were determined through RNA immunoprecipitation and luciferase assays and bioinformatics analysis. Moreover, the expression levels of Ki-67, proliferating cell nuclear antigen, PGK1, Bax, Bcl-2, (p)-mTOR, (p)-AKT, and β-catenin were analyzed via western blot analysis. In the serum of patients with AS and HAECs induced by oxidized low-density lipoprotein (ox-LDL), the expression level of miR-638 was decreased, whereas that of NEAT1 was increased. After ox-LDL therapy, NEAT1 knockdown suppressed HAEC proliferation and stimulated HAEC apoptosis, which could be reversed by the miR-638 inhibitor. NEAT1 inhibited miR-638 expression through direct mutual action. The following mechanical investigations revealed that PGK1 was a miR-638 target, whose expression was increased by NEAT1, a competing endogenous RNA of miR-638. Additionally, the miR-638 inhibitor contributed to proliferation and suppressed apoptosis through the activation of the AKT/mTOR signaling pathway in ox-LDL-induced HAECs. NEAT1 adjusted the AKT/mTOR signaling pathway via miR-638 in ox-LDL-induced HAECs to accelerate their proliferation and impede their apoptosis. This result revealed that NEAT1 may be valuable in the treatment of AS.

Development of Poly(1,8-octanediol-co-citrate-co-ascorbate) Elastomers with Enhanced Ascorbate Performance for Use as a Graft Coating to Prevent Neointimal Hyperplasia.

Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg-1 cm-2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg-1 cm-2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young's modulus (2.15-2.61 MPa), elongation (189.5-214.6%) and ultimate tensile strength (2.73-3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.

Adiponectin inhibits proliferation of vascular endothelial cells induced by Ox-LDL by promoting dephosphorylation of Caveolin-1 and depolymerization of eNOS and up-regulating release of NO

Oxidized low density lipoprotein (ox-LDL) can induce the proliferation and differentiation of endothelial cells, which is one of the important mechanisms of ox-LDL atherosclerosis. Adiponectin is an endogenous bioactive polypeptide secreted by adipocytes, it participates in the metabolism of fat and glucose. It has the effect of reducing blood triglyceride and LDL content. Adiponectin also inhibits the abnormal proliferation and migration of endothelial cells, but its molecular mechanism is unclear. In this study, we used cell model of Ox-LDL-induced human aortic endothelial cells (HAECs) proliferation to analyze the molecular mechanism of APN inhibiting HAECs abnormal proliferation. The results showed that APN could inhibit the cell viability and DNA synthesis of HAECs after Ox-LDL treatment, up-regulate the apoptosis level and reduce the proportion of S + G2 phase cells. Further analysis showed that adiponectin could promote the dephosphorylation of Caveolin-1, which could dissociate eNOS and Caveolin-1, promote the phosphorylation of eNOS and enhance the synthesis of NO. NO increased expression levels of cleaved caspase 3 and p21 in the cells and inhibited the abnormal proliferation of HAECs. The regulation of phosphorylation and dephosphorylation of Caveolae-1 plays a key role in this process. Further study of the molecular mechanism of Caveolae-1 in the inhibition of HAECs abnormal proliferation by APN may reveal the potential of APN in the treatment of cardiovascular diseases.

A16523 Mapping of the angiotensin AT2 receptor-coupled signalling network by time-resolved quantitative phosphoproteomics in human aortic endothelial cells identified HDAC-1 and p53 to be involved in AT2 receptor-mediated anti-proliferation

Objectives: The angiotensin AT2-receptor (AT2R) is a main component of the protective arm of the renin-angiotensin system. In order to get new and deeper insights into AT2R mediated signalling, this study mapped the AT2R-coupled phosphoproteome in human aortic endothelial cells (HAEC) by quantitative phosphoproteomics. Methods: HAEC were stimulated with the AT2R agonist C21 (1 μM) for 1, 3, 5 or 20 minutes. Following protein digestion and TMT peptide labelling, samples were subjected to TiO2−SIMAC phospho-peptide enrichment and LC−MS/MS. Data were analysed using Proteome Discoverer and the human sequence libraries SwissProt and UniProt. For confirmation of phosphoproteomics results, expression of p53 and phosphorylation of HDAC-1 were determined by Western Blot. Cellular translocation of HDAC-1 and p53 was assessed by immunofluorescence and activity of HDAC-1 by deacetylation assay. HAEC proliferation was determined by resazurin assay and apoptosis by caspase 3/7 assay. Results: AT2R stimulation of HAEC changed the phosphorylation status of 265 proteins at 471 sites (281 phosphorylations and 190 dephosphorylations). Gene Ontology and STRING analysis revealed the predominant phospho-modification of proteins involved in anti-proliferation, cell differentiation and apoptosis including HDAC-1, p53, BCL2 or HSP90. AT2R-mediated inhibition of HDAC-1 and activation of p53 were confirmed by additional experiments demonstrating changes in cellular localisation of HDAC-1 and p53, in total expression of p53, and in enzymatic activity and phosphorylation of HDAC1. Conclusion: This study mapped the AT2R coupled phospho-signalling network. It identified dephosphorylation and deactivation of HDAC-1 as well as nuclear importation and increased expression of p53 as novel, potential mediators of AT2R-coupled anti-proliferation.

Abstract P240: Identifying the Angiotensin AT2-Receptor Coupled Phosphoproteome in Human Aortic Endothelial Cells by Time-Resolved, Quantitative Phosphoproteomics

Objectives: The angiotensin AT2-receptor (AT2R) is a main component of the protective arm of the renin-angiotensin system. In order to get new and deeper insights into AT2R mediated signalling, this study mapped the AT2R-coupled phosphoproteome in human aortic endothelial cells (HAEC) by quantitative phosphoproteomics. Methods: HAEC were stimulated with the AT2R agonist C21 (1 μM) for 1, 3, 5 or 20 minutes. Following protein digestion and TMT peptide labelling, samples were subjected to TiO2–SIMAC phospho-peptide enrichment and LC–MS/MS. Data were analysed using Proteome Discoverer and the human sequence libraries SwissProt and UniProt. For confirmation of phosphoproteomics results, expression of p53 and phosphorylation of HDAC-1 were determined by Western Blot. Cellular translocation of HDAC-1 and p53 was assessed by immunofluorescence and activity of HDAC-1 by deacetylation assay. HAEC proliferation was determined by resazurin assay and apoptosis by caspase 3/7 assay. Results: AT2R stimulation of HAEC changed the phosphorylation status of 265 proteins at 471 sites (281 phosphorylations and 190 dephosphorylations). Gene Ontology and STRING analysis revealed the predominant phospho-modification of proteins involved in anti-proliferation, cell differentiation and apoptosis including HDAC-1, p53, BCL2 or HSP90. AT2R-mediated inhibition of HDAC-1 and activation of p53 were confirmed by additional experiments demonstrating changes in cellular localisation of HDAC-1 and p53, in total expression of p53, and in enzymatic activity and phosphorylation of HDAC1. Conclusion: This study mapped the AT2R coupled phospho-signalling network. It identified dephosphorylation and deactivation of HDAC-1 as well as nuclear importation and increased expression of p53 as novel, potential mediators of AT2R-coupled anti-proliferation.

Abstract 286: Role of Glutathione in the Regulation of VEGFR2 in Human Aortic Endothelial Cells

Tissue ischemia due to blood vessel occlusion favors new blood vessel formation or angiogenesis. Although hypoxia is one of the important regulators of angiogenesis, angiogenesis is a rare event in many cardiovascular diseases (CVD). This decreased angiogenesis could be attributed to many factors including altered glutathione (GSH) redox state. Previously, our laboratory has shown an intricate relationship between decreases in GSH in ischemic tissues and angiogenesis. Therefore, we hypothesized that altered GSH redox state regulates angiogenesis by affecting the vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) pathway. We used human aortic endothelial cells (HAEC) and treated with diamide (DA), an oxidant that oxidizes GSH to GSSG, thereby altering GSH redox state. DA treatment decreased GSH and increased GSSG concentrations resulting in decreased GSH/GSSG. Using western blotting, we found that decreased GSH/GSSG activates VEGFR2. To identify the mechanism of VEGFR2 activation after DA treatment, we measured VEGF release into the media using ELISA. There was no difference in extracellular VEGF between the groups suggesting that DA activates VEGFR2 ligand-independently. We then investigated the role of protein tyrosine phosphatases (PTP) using a pan-PTP inhibitor, sodium orthovanadate, which did not alter VEGFR2 activation after DA treatment suggesting no role for PTPs in this pathway. We also studied the role of intracellular H 2 O 2 in DA-induced activation using PEG-catalase to quench H 2 O 2 and then treated with DA. PEG-catalase pretreatment did not affect VEGFR2 activation showing H 2 O 2 is not involved in DA-induced VEGFR2 activation. In order to validate the DA-induced receptor activation, we inhibited glutathione reductase using 2-AAPA to increase intracellular GSSG levels. 2-AAPA treatment alone activated VEGFR2, confirming receptor activation after altering GSH/GSSG. Lastly, altering GSH/GSSG decreased the proliferation of HAECs. Our results elucidate a novel role of glutathione especially GSSG in ligand-independent VEGFR2 activation and subsequent inhibition of cell proliferation. Based on our data, GSSG could be a possible mechanism for decreased angiogenesis in CVD.

Inhibition of miR‑34a prevents endothelial cell apoptosis by directly targeting HDAC1 in the setting of atherosclerosis.

Despite recent medical advances, atherosclerosis is a global burden accounting for numerous mortalities and hospital admissions. MicroRNAs (miRNAs/miRs) regulate cardiovascular biology and disease, but the role of microRNA‑34a in atherosclerosis remains unclear. In the present study, it was demonstrated that miR‑34a was highly expressed in atherosclerotic lesions and oxidized low‑density lipoprotein (Ox‑LDL)‑treated human aortic endothelial cells (HAECs) (atherosclerotic cell model) using reverse transcription‑quantitative polymerase chain reaction. The expression of histone deacetylase (HDAC) 1 was reduced in atherosclerotic lesions and Ox‑LDL treated HAECs. TargetScan predicted that HDAC1 is the potential target of miR‑34a and the double‑luciferase reporter assay confirmed that HDAC1 was directly targeted by miR‑34a. Furthermore, miR‑34a inhibitor significantly enhanced the cell viability of HAECs and the cell apoptosis was suppressed. In addition, the expression of apoptotic‑related proteins was detected by western blotting. The results showed that miR‑34a inhibitor significantly upregulated B‑cell lymphoma 2, procaspase‑3, procaspase‑9 and proto‑oncogene c‑Myc protein expression, and downregulated the expression of p21. In contrast, co‑transfection of HDAC1‑small interfering RNA and miR‑34a inhibitor eliminated the effects of miR‑34a on HAECs. This indicated that miR‑34a inhibitor promoted cell viability and prevented cell apoptosis of HAECs through regulating HDAC1. In conclusion, it was demonstrated that miR‑34a promoted atherosclerotic formation by modulating the proliferation and apoptosis of HAECs, and regulating the expression of apoptosis‑related proteins by targeting HDAC1.

OxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

Cholesterol loading rescues OxLDL‐induced activation of endothelial Rho/ROCK/Akt/p27kip1 cascade

Oxidized modifications of LDL (oxLDL) are known to activate multiple signaling pathways that lead to impairment and disbalance of endothelial function impairment of the barrier function and including abnormal proliferation. It is generally believed that oxLDL exerts its effects on endothelial function via enhanced loading of cholesterol. Our studies, however, show that this is not the case. In contrast, we show that oxLDL and cholesterol loading have opposite effects on several key signaling events in human aortic endothelial cells (HAECs). Moreover, we show that cholesterol loading may rescue oxLDL‐induced effects on EC signaling. We also we provide comparative analysis of the effects of two major types of oxLDL, copper oxidized LDL (Cu‐oxLDL) and Lipoxygenase oxidized LDL (LPO‐oxLDL). Specifically, we show that both, Cu‐oxLDL or LPO‐oxLDL significantly increase Akt phosphorylation whereas inhibiting Akt with a specific inhibitor MK2206 blocks oxLDL‐induced increase in the proliferation of HAECs. Furthermore, blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibits oxLDL‐induced Akt phosphorylation and cell proliferation induced by both Cu‐ and LPO‐oxLDL. In contrast, blocking Rac1 with MSC inhibitor has no effect neither on oxLDL‐induced Akt phosphorylation or cell proliferation of HAECs. Further evidence shows that oxLDL‐induced Rho/Akt pathway leads to downregulation of p27kip1, a well‐known negative regulator of cell proliferation. Most importantly, we also show that preloading cells with cholesterol prevents oxLDL‐induced activation of RhoA and oxLDL‐induced Akt phosphorylation. Furthermore, cholesterol pre‐loading also rescues oxLDL‐induced loss of p27kip1 expression. We propose that oxLDL impairs endothelial function by increasing the cellular content of oxysterols which disrupt the structure of endothelial membrane, an effect that can be repaired by an increase in cellular cholesterol.Support or Funding InformationNIH grants HL‐073965 and HL‐083298 to I.L.

Nitric Oxide Stimulates Matrix Synthesis and Deposition by Adult Human Aortic Smooth Muscle Cells Within Three-Dimensional Cocultures

Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.

The Effect of Type 1 IFN on Human Aortic Endothelial Cell Function In Vitro: Relevance to Systemic Lupus Erythematosus

Cardiovascular disease (CVD) is an important cause of morbidity and mortality in patients with systemic lupus erythematosus. The etiopathogenesis of premature CVD is not fully understood, but recently interferon-alpha (IFNα) has been implicated as a contributing factor. Since IFNα has been associated with both disease activity and endothelial dysfunction in lupus patients, we aimed to determine whether IFNα has direct effects on human aortic endothelial cell (HAoEC) function in vitro. We studied the function of IFNα2b-treated HAoECs in terms of cell proliferation, capillary-like network formation, and nitric oxide (NO) generation. Changes in gene expression were also analyzed using an exon gene array. IFNα2b regulated the expression of 198 genes, including recognized interferon-stimulated genes (ISGs). Gene ontology analysis showed over-representation of genes involved in antigen presentation and host response to virus but no significant changes in clusters of genes recognized as important in endothelial cell activation or dysfunction. HAoEC proliferation, tubule formation, and NO bioavailability were unchanged, suggesting that IFNα in isolation does not have a direct impact on aortic endothelial cell function.

Stimulatory effects of the ionic products from Ca–Mg–Si bioceramics on both osteogenesis and angiogenesis in vitro

Ideal biomaterials for bone tissue engineering should have the capability to guide the osteogenic differentiation of mesenchymal stem cells and, at the same time, to stimulate angiogenesis of endothelia cells. In this study it was found that three Ca–Mg–Si-containing bioceramics (bredigite Ca7MgSi4O16, akermanite Ca2MgSi2O7 and diopside CaMgSi2O6) had osteogenic and angiogenic potential. The effects of three silicate ceramics on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the angiogenesis of human aortic endothelial cells (HAECs) were explored in comparison with β-tricalcium phosphate (β-TCP) bioceramics. The proliferation, alkaline phosphatase (ALPase) activity and bone-related gene expression (COL1, ALPase, OP, BSP and OC) of hBMSCs were significantly enhanced upon stimulation with ionic extracts of these silicate bioceramics. In addition, the results showed that extracts from the three silicate bioceramics also stimulated HAEC proliferation and in vitro angiogenesis with improved NO synthesis and angiogenic gene expression (KDR, FGFR1, ACVRL1 and NOS3). Among the three silicate ceramics bredigite showed the highest osteogenic and angiogenic potential and with the highest extract Si (possibly Si(OH)3O−) concentration, while diopside had the lowest osteogenic and angiogenic potential with the lowest extract Si concentration. Furthermore, it was found that the concentration of Si ions in extracts of the three silicate bioceramics was obviously higher than that of β-TCP ceramics, indicating an important role of Si ions in stimulating cell proliferation, osteogenic differentiation and angiogenesis. The results suggest that the silicate-based akermanite and bredigite ceramics might be good scaffold biomaterials for bone tissue engineering applications due to their distinctive dual functions of osteogenesis/angiogenesis stimulation.

Osteosarcoma cells induce endothelial cell proliferation during neo‐angiogenesis

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase-3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy.