Endothelial Gene Expression Research Articles

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system.

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Investigating the Antiangiogenic, Anti-drug Resistance and Apoptotic Effects of Soy Isoflavone Extract Alone or in Combination with Docetaxel on Murine 4T1 Breast Tumor Model

ABSTRACTBackground: One major concern in the treatment of cancer patients during chemotherapy is drug resistance. Here we investigated the effects of soy isoflavone extracts alone or in combination with Docetaxel on the drug resistance, angiogenesis, apoptosis, and tumor volume in mouse 4T1 breast tumor model. Methods: Sixty female BALB/c mice were randomly divided into 4 groups: control, dietary soy isoflavone extract [Iso, 100 mg/kg diet (0.01%)], Docetaxel (10 mg/kg) injection, and the combination of dietary soy isoflavone extract and intravenous Docetaxel injection (Docetaxel + Iso). One week after the third injection, the breast tumors of eight mice from each group were excised to analyze NF-κBp65′ vascular endothelial growth factor receptor-2 (VEGFR2) and Pgp gene and protein expressions and the other seven mice were monitored for survival rate analysis until they died. Results: NF-κBp65 gene and protein expressions were significantly lower in the Docetaxel + Iso group in comparison with that of the Docetaxel group. VEGFR2 protein expression in the Docetaxel + Iso and Iso groups was significantly lower than that of the Docetaxel group. Conclusion: These findings may indicate that the combined use of isoflavone extracts together with chemotherapeutic agents has more efficient anti-carcinogenic effects than their individual use.

Abstract 008: Leptin Treatment Attenuates Vascular Dysfunction and Inflammation in Mouse Model(s) of Acquired Lipodystrophy via Reducing Nox1-derived Ros

Although extremely efficient at suppressing HIV replication, highly active antiretroviral therapy (HAART) induces lipodystrophy, a metabolic disorder characterized by an abnormal adipose tissue distribution, reduced leptin levels and vascular dysfunction. Leptin replacement therapy (LRT) is currently used to improve metabolic function in patients suffering from congenital lipodystrophy. Here, we analyzed whether LRT restores vascular function and inflammation in mice treated with the antiretroviral agent, ritonavir (Rit). Four weeks of Rit reduced body weight [control (C): 28.4±0.5 vs. Rit: 24.4±0.2g*, *P<0.05] and fat mass (C: 10±1 vs. Rit: 6.5±1 %*) confirming that it induces lipodystrophy. Rit impaired aortic endothelial function [Relaxation to acetylcholine: C: 74±4 vs. Rit: 21±15%*), increased ROS producing enzymes (NOX1 and NOXA1), induced vascular inflammation (increased IL-1β, MCP-1, GATA Binding Protein 3 and INF-γ gene expression) and increased TBARS levels. ROS scavenging via tempol or GKT137117, (Nox1/4 inhibitor) pre-incubation blunted endothelial dysfunction. LRT (10μg/day/7 days, osmotic mini-pump), at the end of the 3-week Rit, restored endothelial function, reduced Nox1 and NOXA1 gene expression and vascular inflammation. NOX1 deficiency in Nox1 KO mice protected mice from Rit-induced endothelial dysfunction and vascular inflammation. Increasing endothelial leptin sensitivity via specific deletion of protein tyrosine phosphatase 1B (Ptp1b) in endothelial cells ( Ptp1b-/-EC mice) protected mice from Rit-induced endothelial dysfunction and reduced Nox1 and NOXA1 gene expression, and vascular inflammation. To address the relevance of these observations to other forms of acquired lipodystrophy, experiments were repeated in mice in which lipodystrophy was induced at 8 week of age, by the deletion of Bscl2, a gene involved in adipocyte maturation. Bscl2 deletion reduced fat mass, and induced endothelial dysfunction via ROS-mediated mechanisms. Again, LRT reverted endothelial dysfunction by downregulating Nox1 expression. All together, these data presents leptin as a key regulator of endothelial oxidative stress level and as a potential avenue for the treatment vascular disease.

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm2) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate, distance, and proliferation are induced by EndMT-derived activated fibroblasts. This model can be used to explore new therapeutics in a tumor microenvironment.

Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq

Identification of tissue-specific and developmentally active enhancers provides insights into mechanisms that control gene expression during embryogenesis. However, robust detection of these regulatory elements remains challenging, especially in vertebrate genomes. Here, we apply fluorescent-activated nuclei sorting (FANS) followed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify developmentally active endothelial enhancers in the zebrafish genome. ATAC-seq of nuclei from Tg(fli1a:egfp)y1 transgenic embryos revealed expected patterns of nucleosomal positioning at transcriptional start sites throughout the genome and association with active histone modifications. Comparison of ATAC-seq from GFP-positive and -negative nuclei identified more than 5,000 open elements specific to endothelial cells. These elements flanked genes functionally important for vascular development and that displayed endothelial-specific gene expression. Importantly, a majority of tested elements drove endothelial gene expression in zebrafish embryos. Thus, FANS-assisted ATAC-seq using transgenic zebrafish embryos provides a robust approach for genome-wide identification of active tissue-specific enhancer elements.

Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease

Background and aimsObesity is a risk factor for endothelial dysfunction and atherosclerosis. However, perivascular adipose tissue can release adipokines and other unknown adipose-derived relaxing factors. Therefore, we investigated the impact of obesity on vascular function and expression of genes in perivascular adipose tissue from internal mammary arteries of patients with coronary artery disease undergoing coronary artery bypass grafting. MethodsThe vessel function was compared between groups of patients with a body-mass index (BMI) between 25 and 30 kg/m2. The groups did not differ in age, gender (males), and ejection fraction. Vascular segments of internal mammary arteries were examined in a Mulvany myograph. Following preconstriction with noradrenaline, dose–response curves were assessed for relaxation with acetylcholine and sodium nitroprusside. ResultsMaximum contraction in response to potassium and noradrenaline was increased in obese patients with a BMI >30 kg/m2. EC50 of endothelium-dependent relaxation was impaired in patients with a BMI above 25, but below 30 kg/m2. Sodium nitroprusside-mediated maximal relaxation was not different between study groups. Integrin alpha X chain (ITGAX/CD11c) and macrophage mannose receptor (MRC1/CD206) expression was reduced in perivascular adipose tissue of patients with a BMI above 30 kg/m2, while adiponectin (ADPQ) expression was increased in the same tissue. ConclusionOur data suggest a partially reduced endothelial function in internal mammary arteries of adipose patients with a BMI between 25 and 30 kg/m2 undergoing coronary artery bypass grafting surgery. Increased adiponectin expression in perivascular tissue might contribute to maintenance of endothelial function in obese patients with a BMI above 30 kg/m2.

Abstract 600: Deposition of Endothelial Cell-derived Fibronectin by Alpha5beta1 Integrins Mediates Oxidized LDL-induced Proinflammatory Gene Expression and Early Atherosclerosis

Subendothelial basement membrane remodeling to a fibronectin-rich matrix precedes lesion development, and endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli. However, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. We now show that treating endothelial cells with oxidized LDL (oxLDL) potently induces endothelial fibronectin deposition through activation of the fibronectin-binding integrin α5β1, as inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely abrogates oxLDL-induced fibronectin deposition in vitro . Inducible endothelial-specific deletion of α5 integrins significantly blunts endothelial proinflammatory gene expression at atheroprone sites and diminishes atherosclerotic plaque formation in ApoE knockout mice, suggesting an important role for this integrin in early endothelial activation. However, endothelial α5 deletion surprisingly enhances fibronectin staining in the endothelial layer, potentially due to leak of plasma-derived fibronectin. Plasma fibronectin differs from cell-derived fibronectin due to the lack of two alternatively spliced domains, termed extradomain A (EIIIA) and extradomain B (EIIIB). Interestingly, loss of endothelial cell-derived fibronectin (fibronectin siRNA) completely inhibits oxLDL-induced VCAM-1 expression, and only rescue with cell-derived fibronectin, but not plasma fibronectin, reverses this inhibitory effect. This effect appears to be due to the presence of EIIIA and EIIIB domains, as endothelial cells isolated from EIIIA/EIIIB knockout mice also show reduced oxLDL-induced proinflammatory responses. However, it is unclear whether this response is due to specific EIIIA/EIIIB receptors or due to altered integrin signaling, as limiting the presence of cell-derived fibronectin prevents integrin α5 localization to focal adhesions and reduces fibronectin fibril length. Taken together, our data demonstrate that oxLDL stimulates α5 integrin-dependent deposition of endothelial-derived fibronectin into the subendothelial matrix and suggest that the source of fibronectin regulates the subsequent endothelial cell proinflammatory responses.

LncRNA as a Therapeutic Target for Angiogenesis.

Out of 3 billion base pairs in human genome only ~2% code for proteins; and out of 180,000 transcripts in human cells, about 20,000 code for protein, remaining 160,000 are non-coding transcripts. Most of these transcripts are more than 200 base pairs and constitute a group of long non-coding RNA (lncRNA). Many of the lncRNA have its own promoter, and are well conserved in mammals. Accumulating evidence indicates that lncRNAs act as molecular switches in cellular differentiation, movement, apoptosis, and in the reprogramming of cell states by altering gene expression patterns. However, the role of this important group of molecules in angiogenesis is not well understood. Angiogenesis is a complex process and depends on precise regulation of gene expression. Dysregulation of transcription during this process may lead to several diseases including various cancers. As angiogenesis is an important process in cancer pathogenesis and treatment, lncRNA may be playing an important role in angiogenesis. In support of this, lncRNA microvascular invasion in hepatocellular carcinoma (MVIH) has been shown to activate angiogenesis. Furthermore, lncRNA-Meg3-knockout mouse showed increased expression of vascular endothelial growth factor pathway genes and increased cortical microvessel density. Overall, there is strong evidence that lncRNA is an important class of regulatory molecule, and a number of studies have demonstrated that these can be targeted to change cellular physiology and functions. In this review, we have attempted to summarize these studies and elucidate the potential of this novel regulatory molecule as a therapeutic target.

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression.

NOSTRIN regulates gene signatures, pleiotropic functions and NFkB‐TRAF6 signaling axis of endothelial cells: Implications in intrauterine growth restriction.

Endothelial cell functions, including angiogenesis and vascular permeability are known to be regulated by endothelial nitric oxide synthase (eNOS) and its bioactive product nitric oxide (NO). Pharmacological inhibition or genetic disruption of eNOS attenuated angiogenesis during tissue repair, resulting in delayed wound closure, which was reversed by addition of NO donors. VEGF mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. These observations emphasize the ability of eNOS‐derived NO to promote angiogenesis. NOSTRIN is classically known to sequester eNOS, thereby attenuating NO production. We show here that NOSTRIN affects endothelial cells by down regulating several genes, related to invasion and angiogenesis. Interestingly, this effect of NOSTRIN on endothelial cell gene expression is independent of eNOS activity. NOSTRIN also affects the expression of secreted cytokines involved in inflammatory responses. Ectopic over‐expression of NOSTRIN is inhibitory to endothelial cell proliferation, migration, invasion, VEGF‐induced capillary tube formation as well as adhesion. Furthermore, NOSTRIN directly interacts with TRAF6 and inhibits NFkB activity. Interestingly, TNF‐α induced NFkB pathway activation is overturned by NOSTRIN. These results have widespread biological implications as over expression of NOSTRIN leading to down regulation of NFkB pathway and consequent triggering anti‐angiogenic cascade might inhibit tumorigeneis and cancer progression as well as in patho‐physiological conditions such as intra‐uterine growth restriction (IUGR). IUGR is the second leading cause of peri‐natal mortality and 40% of total still‐births world‐wide is contributed by IUGR. Interestingly, there was massive up regulation of NOSTRIN transcript as well as protein in the mesometrial compartment of implantation site in the IUGR rats as compared to the controls. NOSTRIN up regulation was associated with down regulation in mesometrial compartment of angiogenesis and invasion related genes that are known to be regulated by NOSTRIN. These include receptor tyrosine kinases, and a pro‐angiogenic ligand, adhesion molecules, proteases. Furthermore, NOSTRIN elevation during IUGR was also associated with down regulation of several cytokines, such as, IL6, Ccl2, Cxcl1 and Cxcl2 in the mesometrial uterus. As expected, elevated levels of NOSTRIN was associated with down regulation of NFkB signalling pathway. In line with its up regulation in IUGR, NOSTRIN was found to be negatively regulated by HIF1α and HIF‐1α decreased significantly in the mesometrial uterus in IUGR. Taken together, our data establish cellular function of NOSTRIN and demonstrate the involvement of NOSTRIN‐NFkB signalling pathway in IUGR.Support or Funding InformationThis work was supported by grants from Indian Council of Medical Research (2012‐0524 to RA); Department of Biotechnology (BT/PR9113/MED/97/134/2013 to RA); Shreeta Chakraborty is a Senior Research Fellow funded by University Grant Commission, India.

Genomic cell lineage analysis uncovers novel endothelial gene expression programs for metabolic and immune regulation

Endothelial cells (ECs) take on a variety of cellular phenotypes that depend on their niches, making it difficult to discern lineage‐wide traits. Therefore, we sought to identify novel aspects of EC biology by defining a core EC transcriptional signature shared amongst ECs. We accomplished this by condensing multiple sources of EC transcriptomic and genomic data sets using multivariate statistics and phylogenetic analyses. Our comparative multi‐tissue analyses confirmed expected gene expression patterns such as Flt1, Pecam1, Cdh5, Tek, and Ptprb as core elements of ECs. We also observed strong statistical associations for a variety of genes without prominent roles in ECs, such as the oncostatin M receptor (Osmr), cysteine and tyrosine rich 1 (Cyyr1) and an uncharacterized cDNA sequence (BC028528) that were always positively associated with ECs compared to other cell types. This implied that associations with tightly regulated genes can be obscured by EC variability but may be important for fundamental EC biology. Strikingly, hallmark interferon alpha and gamma gene sets were associated with all EC subtypes and ECs showed highest enrichment in an interferon alpha activation gene signature (p<1e‐15). More broadly, glycolytic and mitochondrial metabolic gene sets were strongly down‐regulated in resting ECs while regulatory transcriptional and translational program gene sets were up‐regulated in these cells. Together, these findings support the existence of key metabolic and immune regulators that are necessary for constitutive EC function in all lineages that can be carefully scrutinized by gene modulation in experimental systems.Support or Funding InformationNIH T32

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

SummaryIn vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

Effect of calcitonin gene-related peptide on osteoporotic fracture healing in vitro

Objective To explore the effects of calcitonin gene-related peptide (CGRP) on proliferation of vascular endothelial cells (VECs) and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) of osteoporotic rats in vitro, and cell/molecular mechanism of promoting osteoporotic fracture healing. Methods 10 3-month-old SD rats were employed to make osteoporosis model by taking bilateral ovariectomy. Micro-CT analysis was used to determine the success of modeling at 6 months of postoperation. BMSCs and VECs were isolated from osteoporotic rats by blood adherence method. 10-7-10-10 mol/L CGRP were added to the culture medium. CCK-8 method was used to examine the cell proliferation. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR. Results BMSCs and VECs of osteoporotic rats were isolated successfully.The optical density (OD) values of CCK-8 method increased in a dose-dependent manner, for BMSCs at 7 d, the control group of OD value was 0.58±0.02, the CGRP group 10-7-10-10 mol/L was to 0.80±0.03, 0.79±0.03, 0.74±0.03, 0.69±0.03, difference was significant; for VECs, the control group of OD value was 2.20±0.01, the CGRP group 10-7-10-10 mol/L was to 4.12±0.04, 3.90±0.02, 3.56±0.04, 3.12±0.04, difference was significant. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR, difference between the control group and CGRP group were all significant. Conclusion 10-7 mol/L, 10-10 mol/L of CGRP can directly promote the proliferation of the BMSCs and VECs of osteoporotic rats, promote the osteogenic differentiation of BMSCs, improve the expression of VEGF gene and protein secretion, promote angiogenesis. Key words: Calcitonin gene-related peptide; Osteoporosis; Fractures, bone

Vegf signaling promotes vascular endothelial differentiation by modulating etv2 expression

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis.

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication.

Abstract TMP114: Interrogation of Brain Endothelial Cell Gene Expression Reveals Cyclooxygenase-2 as a Therapeutic Target After Subarachnoid Hemorrhage

Introduction: The pathophysiology of subarachnoid hemorrhage (SAH) is complex and includes disruption of the blood-brain barrier (BBB). We have previously optimized methods to freshly isolate BBB endothelial cells (BECs) and performed genome-wide expression profiling to uncover new therapeutic targets in an unbiased manner. In this study we validated 2 of the most highly upregulated targets, cyclooxygenase-2 (Cox2) and angiopoietin-2 (Angpt2). Methods: The prechiasmatic blood injection mouse model of SAH was used with experimental end-points at 24h. BBB disruption was assessed using intraperitoneal-injected cadaverine-based fluorescent dye. Global neurobehavioral assessments utilized the modified Garcia score. BECs were purified either by magnetic-based sorting for CD45-CD31+ cells or by fluorescence-activated cell sorting for Tie2+Pdgfrb- cells. Microarray results from RNA extracted from BECs were validated with real-time PCR. Immunofluorescence of coronal brain sections and enzyme-linked immunosorbent assays of brain homogenates were performed to determine protein expression. Intraperitoneal injections of the Cox2 inhibitor celecoxib (10mg/kg) were administered 30min and 12h after SAH induction. Results: CD45-CD31+ BECs derived from SAH mice demonstrated 6.6-fold and 5.4-fold increase in Cox2 and Angpt2 gene expression, respectively (n=4 per group, p<0.05, p<0.01). Tie2+Pdgfrb- BECs similarly showed 5.8-fold and 5.3-fold increase in Cox2 and Angpt2 gene expression after SAH, respectively (n=4 per group, p<0.05, p<0.05), which demonstrates the robustness of these results. We observed increased Cox2 protein expression in intraparenchymal vessels and increased Angpt2 protein expression in brain homogenates after SAH. Celecoxib treatment resulted in modest reduction in BBB disruption and improved neurobehavioral outcomes. Celecoxib treatment abrogated SAH-induced upregulation of Cox2 expression, but not Angpt2, in BECs. Conclusions: In summary, we identified Cox2 as a potential therapeutic target in SAH. The observed beneficial effects of celecoxib treatment suggest that Cox2 may be critical to BBB disruption after SAH and its mechanism appears to be independent of Angpt2.

Dietary restriction and aerobic exercise attenuate obesity‐induced lymphatic dysfunction

Abstract Obesity is a deleterious condition characterized by excess adipose tissue deposition and a body mass index greater than 30.0. It is a major risk factor for many diseases, including coronary heart disease and non-insulin dependent diabetes mellitus. Obesity leads to systemic low-grade inflammation caused by cytokines and adipokines. Although obesity has effects on several bodily systems, the lymphatic system is especially important since its relationship with obesity is bidirectional. Obesity is the major contributing factor to the progression and development of lymphedema and impaired lymphatic function is thought to regulate the pathology of obesity in organ systems via modulation of the differentiation and migration of inflammatory cells. It has been shown obesity leads to lymphatic dysfunction which is characterized by impaired lymphatic vessel pumping and density, increased lymphatic vessel leakiness, and altered lymphatic endothelial cell gene expression (Savetsky et al. 2014). Despite the clinical importance of lymphatic physiology in obese patients, an effective therapeutic strategy to regulate lymphatic function remains to be determined. This article is protected by copyright. All rights reserved.

Downregulation of transgelin blocks interleukin-8 utilization and suppresses vasculogenic mimicry in breast cancer cells.

Vasculogenic mimicry (VM) is a non-classical mechanism recently described in many tumors, whereby cancer cells, rather than endothelial cells, form blood vessels. Transgelin is an actin-binding protein that has been implicated in multiple stages of cancer development. In this study, we investigated the role of transgelin in VM and assessed its effect on the expression of endothelial and angiogenesis-related genes during VM in MDA-MB-231 breast cancer cells. We confirmed the ability of MDA-MB-231 cells to undergo VM through a tube formation assay. Flow cytometry analysis revealed an increase in the expression of the endothelial-related markers VE-cadherin and CD34 in cells that underwent VM, compared with those growing in a monolayer, which was confirmed by immunocytochemistry. We employed siRNA to silence transgelin, and knockdown efficiency was determined by western blot analyses. Downregulation of transgelin suppressed cell proliferation and tube formation, but increased IL-8 levels in Matrigel cultures. RT-PCR analyses revealed that the expression of IL-8, VE-cadherin, and CD34 was unaffected by transgelin knockdown, indicating that increased IL-8 expression was not due to enhanced transcriptional activity. More importantly, the inhibition of IL-8/CXCR2 signaling also resulted in suppression of VM with increased IL-8 levels, confirming that increased IL-8 levels after transgelin knockdown was due to inhibition of IL-8 uptake. Our findings indicate that transgelin regulates VM by enhancing IL uptake. These observations are relevant to the future development of efficient antivascular agents. Impact statement Vasculogenic mimicry (VM) is an angiogenic-independent mechanism of blood vessel formation whereby aggressive tumor cells undergo formation of capillary-like structures. Thus, interventions aimed at angiogenesis might not target the entire tumor vasculature. A more holistic approach is therefore needed in the development of improved antivascular agents. Transgelin, an actin-binding protein, has been associated with multiple stages of cancer development such as proliferation, migration and invasion, but little is known about its role in vasculogenic mimicry. We present here, an additional mechanism by which transgelin promotes malignancy by way of its association with the occurrence of VM. Although transgelin knockdown did not affect the transcript levels of most of the angiogenesis-related genes in this study, it was associated with the inhibition of the uptake of IL-8, accompanied by suppressed VM, indicating that transgelin is required for VM. These observations are relevant to the future development of efficient antivascular agents.

Gene Expression, Oxidative Stress, and Senescence of Primary Coronary Endothelial Cells Exposed to Postprandial Serum of Healthy Adult and Elderly Volunteers after Oven-Cooked Meat Meals.

Epidemiological studies have linked high consumption of meat with major age-related diseases including cardiovascular diseases. Abnormal postprandial increases in plasma lipids after a meat meal have been hypothesized among the pathogenetic mechanisms. However, it is still unknown if the postprandial serum derived after a normal meat meal is able to affect endothelial function, and if the type of meat and the age of the donors are critical factors. Here, we show the effects of postprandial sera derived from healthy adults and elderly volunteers who consumed meat meals on human coronary artery endothelial cell (HCAEC) oxidative stress, gene expression, DNA damage, and cellular senescence. We observed that a single exposure to postprandial serum induces a slight increase in ROS that is associated with modulation of gene expression pathways related to oxidative stress response and metabolism. The postprandial-induced increase in ROS is not associated with a measurable DNA oxidative damage. However, repeated exposure to postprandial serum induces an acceleration of cellular senescence. Taking into account the deleterious role of cellular senescence in age-related vascular diseases, the results suggest a new mechanism by which excessive meat consumption and time spent in postprandial state may affect health status during aging.