ABSTRACT: Molecular pathways regulating the development of arterial and venous endothelial cells (ECs) are now well-established, but control of parallel arterial-venous (A-V) alignment is unclear. We report that arterial-venous alignment in the skin is determined by apelin receptor (APJ) expression in venous ECs. We used microarrays to detail the global programme of gene expression in endothelial cells that has relationship with the deficient of APJ. Endothelial cells were marked and isolated by Fluorescence-activated cell sorting in Trizol.
Project description:The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues. A development or differentiation experiment design type assays events associated with development or differentiation or moving through a life cycle. Development applies to organism(s) acquiring a mature state, and differentiation applies to cells acquiring specialized functions. Using regression correlation
Project description:Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho Kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenousdependent requirement for protein prenylation in zebrafish and human endothelial cells.
Project description:Human ES or iPS Cells were differentiated into endothelial cells (ECs) defined by expression of CD31 (PECAM1) and CD144 (VE-Cadherin) on the cell surface. All ES or iPS derived ECs were greater than 90% double positive for these two markers. These in vitro derived ECs were compared to each other and to ECs from primary cell sources; arterial (aortic Ecs), venous (saphenous vein) and lymphatic. ES and iPS cells were differentiated into Ecs using a directed differentiation protocol using sequential addition of growth factors to recapitulate endogenous differentiation (FGF, BMP4, Activin, VEGF). These ECs were grown until 21 days after the start of differentiation. Primary cells were obtained from a commercial vendor and grown to passage 3 to minimize cellular senescence and in vitro artifacts. The RNA was cleaned and DNAse digested on RNeasy columns (Qiagen, Valencia, CA). RNA was amplified, fragmented and labeled using NuGEN Technologies Applause Plus kits and the manufacturer's instructions. Labeled samples were then hybridized to Affymetrix Human Gene 1.0 ST GeneChips. The GeneChips were then washed and stained on Affymetrix 450 Fluidics Stations and scanned on an Affymetrix 3000 Scanner according to standard protocols.
Project description:Etv2 transgene was expressed from ROSA26 locus by removing floxed STOP cassette by Tie-2 Cre transgene.VE-Cad+/CD45+ cells were sorted from control or tie-2-Etv2 E10.5 embryosYS(yolk sac) and compared for gene expression in duplicate. This study will reveal the effect of Etv2 transgene in E10.5 mouse embryo yolk sac. The effect of Etv2 overexpression in relevant mouse tissue will be important to understand its effect in comparison with in ES cells. Genes aberrantly regulated by Etv2 overexpression will help to understand the caveat when using Etv2 to induce endothelial and hematopoietic cells in vitro. Sample ID YS- ; (VECAD+CD45+;YS;Control) YS+12; (VECAD+CD45+;YS;Tg) YS+22 ; (VECAD+CD45+;YS;Tg)
Project description:Etv2 transgene was expressed from ROSA26 locus by removing floxed STOP cassette by Vav Cre transgene. KSL or Mac1+/Gr1+ cells were sorted from control or Vav-Etv2 bone marrow and compared for gene expression in duplicate. This study will reveal the effect of Etv2 transgene in adult hematopoietic cells. The effect of Etv2 overexpression in relevant mouse tissue will be important to understand its effect in comparison with in ES cells. Genes aberrantly regulated by Etv2 overexpression will help to understand the caveat when using Etv2 to induce endothelial and hematopoietic cells in vitro. Sample ID #1;KSL;Control #2;KSL;Control #3;KSL;Tg #4;KSL;Tg #5;Gr;Control #6;Gr;Control #7;Gr;Tg #8;Gr;Tg
Project description:Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous smooth muscle cells (SMCs) such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans. Overall design: Comparison of biomechanically stretched HUVEC treated with DMSO or Glycyrrhetinic acid
Project description:Phenotypic heterogeneity among arterial ECs is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in their atherosusceptibility. To explore EC heterogeneity, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary and iliac artery ECs. We demonstrate that in vivo the endothelial transcriptional profile of a coronary artery (the right coronary artery) is intrinsically different from that of a major conduit vessel (the external iliac artery), and that this difference is consistent with former vessel being more prone to atherosclerosis. Keywords: coronary atherosclerosis, endothelial heterogeneity, microarray, gene expression Endothelial cells were freshly harvest from right coronary, left and right iliac arteries from four pigs. RNA were isolated and expression profiles were obtained using olig microarrays.
Project description:Background: Venous hypertension is often present in advanced and in acute decompensated heart failure (HF). However, it is unclear whether high intravenous pressure can cause alterations in homeostasis by promoting inflammation and endothelial cell (EC) activation. We used an experimental model of acute, local venous hypertension to study the changes in circulating inflammatory mediators and EC phenotype that occur in response to biomechanical stress. Methods and Results: Twenty-four healthy subjects (14 men, age 35±2 years) were studied. Venous arm pressure was increased to ~30 mmHg above baseline level by inflating a tourniquet cuff around the dominant arm (test arm). Blood and endothelial cells (ECs) were sampled from test and control arm (lacking an inflated cuff) before and after 75 minutes of venous hypertension, using angiocatheters and endovascular wires. Magnetic beads coated with EC specific antibodies were used for EC separation; amplified mRNA was analyzed by Affymetrix HG-U133 2.0 Microarray. Plasma endothelin-1 (ET-1), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1) and chemokine (C-X-C motif) ligand 2 (CXCL2) were significantly increased in the congested arm. 5,332 probe sets were differentially expressed in venous ECs before vs. after testing. Among the 143 probe sets that exhibited a significant absolute fold change >2, we identified several inflammatory mediators including ET-1, VCAM-1, and CXCL2. Conclusions: Acute experimental venous hypertension is sufficient to cause local increase in circulating inflammatory mediators and to activate venous ECs in healthy human subjects. Additional work is needed to determine the effect of venous hypertension in patients with established HF. 24 samples were analyzed from 12 patients. Each patient contributed 2 samples (1 prior to intervention and 1 after intervention). The pre-intervention sample serves as the control.
Project description:In patients with severe kidney disease AVF are surgically placed in order to create good access for hemodialysis. In these dialysis patients the failure rates of AVF can be as high as 24% within 6 months after surgery, causing ineffective dialysis and necessitating additional clinical interventions. The pathological processes known to lead to AVF failure are beginning to be unravelled include the formation of venous neointimal hyperplasia (VNIH), thrombosis (Chang et al. PMID 16105066), and venous stenosis (Kanterman et al. PMID7892454, Tang et al. 1998), resulting in a reduced blood flow through the fistula. We established a rat model for AVF failure in human kidney dialysis patients. The characterization of this model has been previously described (Globerman et al. 2011, PubmedID:22002501). In this model the AVFs are surgically constructed in the right leg by connecting the superficial epigastric vein SEV to the common femoral artery (CFA), resulting in exposure of the SEV to arterial pressure with pulsatile and low resistant flow patterns (Globerman et al. 2011, PMID22002501). In the present study we utilized this AVF model in order to assess the effects of arterialized flow, with consequent pathological changes of the vessel wall due to surgical AVF instalment, on the transcriptome of endothelium from the SEV. Within the SEV these pathologies of the vessel wall include the formation of NIH in the main branch, and stenosis in the side branches. By employing this rat model we assessed the changes of the endothelial transcriptome in relation to these pathologies in order to gain mechanistic understanding of the potential roles of venous endothelium in AVF failure, as well as to identify potential biomarkers preceding AVF failure. AVF surgery was performed on N=6 rats, and 13-14 days after surgery the rats were sacrificed. AVFs were extracted from the rats, and microarray transcriptome analyses were performed on luminal endothelial cells that isolated from four different sites of the AVF by employing Laser Capture Microdissection (LCM). These sites included (i) N=5 AVF sections of the superficial epigastric vein (SEV) with neointimal hyperplasia (NIH), (ii) N=3 AVF sections of SEV side branches with luminal stenosis, (iii), N=6 sections of the SEV located distally from a ligation thereby omitting exposure to arterialized flow and consequently preventing the development of NIH or stenosis, and (iv) N=3 sections of the AVF common femoral artery without signs of NIH or stenosis.
Project description:Circulating microRNAs (miRNAs) presented in venous plasma have recently been demonstrated as powerful biomarkers for the diagnosis and prognostic prediction of complex diseases like cancer. Nevertheless, those presented in arterial plasma have been ignored based on the assumption that the miRNA profiles in arterial and venous plasma would be identical. Here, we disputed this intuitive assumption by comparing arterial and venous plasma miRNA expression profiles from male rats using microarray technique. Though the microRNA profiles were largely similar, a considerable number of miRNAs showed significant differential expression, including 10 arterial highly expressed miRNAs and 14 venous highly expressed miRNAs. The differentially expressed miRNAs were validated by qRT-PCR. We performed computational analysis of the function enrichment and disease association of these miRNAs and their targets. Our analysis also suggested significant correlations between plasma miRNA expression and tissue miRNA expression. Four arterial highly expressed miRNAs showed enriched expression in specific tissues and thus could serve as novel biomarker candidates. Overall design: Three healthy male rats were prepared. For each rat, one arterial plasma sample and one venous plasma sample were extracted. MiRNA expression profiles in three matched pairs of arterial/venous plasma samples were measured by using microarray.