Gene Expression Profile Of Endothelial Cells Research Articles

Semaphorins: From Angiogenesis to Inflammation in Rheumatoid Arthritis.

To study the potential role of semaphorins in the pathogenesis of rheumatoid arthritis (RA). Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in RA endothelial cells (ECs) and control ECs derived from circulating progenitors. Expression of class 3 and class 4 semaphorins and their receptors in the serum of RA patients and healthy controls was assessed by immunohistochemical analysis in synovial tissue and by enzyme-linked immunosorbent assay. Microarray analysis revealed differential expression of class 3 and class 4 semaphorins and their receptors in RA ECs. Semaphorin 4A (SEMA4A), plexin D1, and neuropilin 1 messenger RNA (mRNA) levels were markedly increased in RA ECs by 1.75-, 2.21-, and 1.68-fold, respectively. Stimulation with tumor necrosis factor (TNF) led to a 2-fold increase in SEMA4A mRNA levels in RA ECs, and deficient SEMA4A expression modified RA EC angiogenic properties. Class 3 and class 4 semaphorins as well as their receptors were overexpressed in RA synovial tissue. A respective 1.30-fold increase and 1.54-fold increase in SEMA4A and SEMA3E, as well as a 24% decrease in SEMA3A, was observed in the serum of RA patients. Serum levels of SEMA4A, SEMA4D, and SEMA3A correlated with levels of inflammation and proangiogenic markers. In 2 independent cohorts of patients with low disease activity or with RA in remission, the presence of SEMA4A identified patients with residual disease activity. Gene expression profiling of ECs identified class 3 and class 4 semaphorins as potential biomarkers and therapeutic candidates in RA, with confirmed overexpression in ECs, synovial vessels, and serum, and correlation with validated markers of inflammation and angiogenesis. Thus, semaphorins might be novel and appealing EC-derived inflammatory and proangiogenic targets in RA.

The biological responses and mechanisms of endothelial cells to magnesium alloy.

Due to its good biocompatibility and degradability, magnesium alloy (Mg alloy) has shown great promise in cardiovascular stent applications. Rapid stent re-endothelialization is derived from migrated and adhered endothelial cells (ECs), which is an effective way to reduce late thrombosis and inhibit hyperplasia. However, fundamental questions regarding Mg alloy affecting migration and adhesion of ECs are not fully understood. Here, we evaluated the effects of Mg alloy on the ECs proliferation, adhesion and migration. A global gene expression profiling of ECs co-culturing with Mg alloy was conducted, and the adhesion- and migration-related genes were examined. We found that Mg alloy had no adverse effects on ECs viability but significantly affected ECs migration and adhesion. Co-cultured with Mg alloy extract, ECs showed contractive adhesion morphology and decreased motility, which was supported by the down-regulation of adhesion-related genes (Paxillin and Vinculin) and migration-related genes (RAC 1, Rho A and CDC 42). Accordingly, the re-endothelialization of Mg alloy stent was inhibited in vivo. Our results may provide new inspiration for improving the broad application of Mg alloy stents.

Autotaxin-Lysophosphatidic Acid Axis Acts Downstream of Apoprotein B Lipoproteins in Endothelial Cells.

As they travel through the blood stream, plasma lipoproteins interact continuously with endothelial cells (ECs). Although the focus of research has mostly been guided by the importance of lipoproteins as risk factors for atherosclerosis, thrombosis, and other cardiovascular diseases, little is known about the mechanisms linking lipoproteins and angiogenesis under physiological conditions, and particularly, during embryonic development. In this work, we performed global mRNA expression profiling of endothelial cells from hypo-, and hyperlipidemic zebrafish embryos with the goal of uncovering novel mediators of lipoprotein signaling in the endothelium. Microarray analysis was conducted on fluorescence-activated cell sorting-isolated fli1:EGFP(+) ECs from normal, hypo-, and hyperlipidemic zebrafish embryos. We found that opposed levels of apoprotein B lipoproteins result in differential expression of the secreted enzyme autotaxin in ECs, which in turn affects EC sprouting and angiogenesis. We further demonstrate that the effects of autotaxin in vivo are mediated by lysophosphatidic acid (LPA)-a well-known autotaxin activity product-and that LPA and LPA receptors participate as well in the response of ECs to lipoprotein levels. Our findings provide the first in vivo gene expression profiling of ECs facing different levels of plasma apoprotein B lipoproteins and uncover a novel lipoprotein-autotaxin-LPA axis as regulator of EC behavior. These results highlight new roles for lipoproteins as signaling molecules, which are independent of their canonical function as cholesterol transporters.

Endothelial cell adhesion to soluble vascular endothelial growth factor receptor‐1 triggers a cell dynamic and angiogenic phenotype

The aim of this study was to identify the molecular signals produced in human endothelial cells (ECs) by the interaction of α5β1 integrin with soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) present in the extracellular matrix. We generated a gene expression profile of ECs adhering to sVEGFR-1 or to fibronectin, the classic extracellular matrix ligand for α5β1 integrin or in a nonadhering condition. Several biological pathways were differently modulated, 3 protein kinase C substrates [adducin, myristoylated alanine-rich protein kinase C substrate (MARCKS), and radixin] were differently expressed and phosphorylated when cells adhering to sVEGFR-1 were compared with those adhering to fibronectin. Rac1 activation and Gα13 protein involvement through the interaction with radixin were also detected after attachment to sVEGFR-1, and these responses depended on active VEGFR-2 signaling. On sVEGFR-1, ECs exhibited a motile phenotype that was consistent with the abundant presence of MARCKS, a stabilizer of dynamic adhesions. Moreover, ECs silenced for radixin expression no longer responded to the proangiogenic VEGFR-1-derived peptide 12. We propose that the presence of sVEGFR-1 in the EC microenvironment directs α5β1 integrin signaling to generate a dynamic, motile phenotype. Our findings also provide new insights into the mechanism of action of proangiogenic peptide 12, relevant to a therapeutic perspective.

Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs

IntroductionA major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.MethodsMSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.ResultsThe results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.ConclusionsThis study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.

Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium

BackgroundTargeting tumor angiogenesis and vasculature is a promising strategy for the inhibition of tumor growth and dissemination. Evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. Our efforts focused on the molecular characterization of endothelial cells (EC) in the search for selective markers of tumor vasculature that might be helpful for the development of effective therapeutic approaches.ResultsWe investigated by microarray analysis the gene expression profiles of EC purified and cultured from tumor (ovarian carcinoma [HOC-EC]) and normal (human adrenal gland [HA-EC]) tissue specimens. We found distinct transcriptional features characterizing the EC of different origin, and identified 158 transcripts highly expressed by HOC-EC. We analyzed four of these genes, ADAM23, FAP, GPNMB and PRSS3, which were not previously known to be expressed by endothelium. In vitro experiments confirmed the higher expression of the selected genes in tumor-derived endothelium with no expression in tumor cells. In vivo investigation by in situ hybridization established that ADAM23, GPNMB and PRSS3 expression is localized on blood vessels of human cancer specimens.ConclusionThese findings elucidate some of the molecular features of the tumor endothelium. Comparative transcriptomic analysis allowed us to determine molecular differences of tumor and normal tissue-derived endothelium and to identify novel markers that might be exploited to selectively target tumor vasculature.

Hypoxia-induced increase in NO production and eNOS expression by murine pulmonary microvascular endothelial cells

Morphology, function and gene-expression profile of endothelial cells (ECs) are specific for particular organs, vascular beds or microenvironments. ECs lining different segments along the pulmonary arterial bed vary in the expression of surface molecules. Histochemical labeling of living murine lung sections with Bandeiraea simplicifolia lectin revealed a preferential staining of ECs coating the capillary network of alveolar septa. These ECs were isolated employing magnetic beads coated with Bandeiraea simplicifolia lectin, expanded and characterized. After propagation up to passage 20 the ECs remained their capacity to bind Bandeiraea simplicifolia lectin and were able to take up DiI-Ac-LDL, indicating their microvascular origin. Endothelial nitric oxide synthase (eNOS) is widely expressed within endothelial cells (ECs) lining the vasculature and plays a pivotal role in regulation of the vascular tone. NO released by ECs diffuses into the underlying smooth muscle cells causing them to relax. Additionally, NO is related to non-contractile functions such as regulation of gene expression. Immunohistochemical analysis of the isolated microvascular ECs demonstrated a predominantly nuclear localization of eNOS. Exposure to hypoxia (90min) elevated NO production as detected by enhanced DAF-2T fluorescence which also dominated in the nucleus. Increased expression of eNOS mRNA was found after 6h of hypoxia, and enhanced protein levels after 15h of hypoxia. Thus, NO released from pulmonary microvascular ECs appears to be involved in short-term and subacute adaptation of the alveolar region to hypoxia.

Mediators of endothelial cell injury in low perfusion conditions

Abstract Introduction: During free tissue transfer operations, endothelial cells (EC) are exposed to a period of ischemia that can affect outcome. We compared gene expression profiles of ECs in normal culture conditions to serum-deprived conditions as an in vitro model of tissue stress. Methods: ECs from multiple body sites were grown in EGM-2MV with 5% fetal bovine serum (FCS). For serum-deprived samples, ECs were switched to EGM-2MV with 0.25% FCS. After 24h, ECs were harvested and mRNA was extracted. Gene expression patterns were compared between the normal and serum-deprived samples using cDNA microarray. Specific gene expression was then confirmed by quantitative PCR. Results: There are 1083 genes that show more than a four fold difference in expression when comparing normal versus serum-deprived growth conditions. ECs exposed to serum deprivation up regulate inflammatory and coagulation factor genes while cell cycle genes are down regulated. Up regulated inflammatory mediators included MCP-1, interferon-alpha, TGF-alpha, colony stimulating factor 2, TNFC, and TNF receptor-associated factor 5. Coagulation factors included thrombin and the thrombin receptor. The down regulated cell cycle genes included cyclin B2 and c-myc binding protein. Conclusions: Our data suggest that serum deprivation induces gene expression of inflammatory mediators and coagulation factors that can lead to poor outcome during tissue transfer. Understanding the mechanism of EC injury may lead to the development of rationally designed therapies aimed at minimizing complications and improving outcomes.

Uncovering the complexities of Kaposi's sarcoma through genome-wide expression analysis

Gene-expression profiling of endothelial cells infected with Kaposi's sarcoma-associated herpesvirus has led to a greater understanding of the histogenesis and pathogenesis of Kaposi's sarcoma.