Transcription factor Ap-1 mediates proangiogenic MIF expression in human endothelial cells exposed to Angiotensin II

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.     

Schwann cells promote endothelial cell migration

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor

Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.     

The novel antioxidant 3-O-caffeoyl-1-methylquinic acid induces Nrf2-dependent phase II detoxifying genes and alters intracellular glutathione redox

Induction of detoxifying phase II genes by chemopreventive agents represents a coordinated protective response against oxidative stress and neoplastic effects of carcinogens. We have earlier shown that a novel antioxidant from the bamboo leaves constituent 3-O-caffeoyl-1-methylquinic acid (MCGA3) induces heme oxygenase-1 (HO-1) and protects endothelial cells from ROS-induced endothelial injury. The purpose of this study was to elucidate the induction mechanism of HO-1 and other phase II genes by MCGA3 in human umbilical vascular endothelial cells (HUVECs). Using Northern blotting and RT-PCR, we found that treatment of HUVECs with MCGA3 increased, in a dose and time-dependent manner, steady-state mRNA levels of the selected phase II genes including HO-1, ferritin, gamma-glutamylcysteine lygase, glutathione reductase, and glutathione transferase, which were dependent on Nrf2 nuclear translocation. The observed phase II gene induction by MCGA3 was found to be associated with MCGA3-mediated cytoprotective activity, ROS-scavenging potency, and the increase in the cellular levels of both reduced (GSH) and oxidized glutathione (GSSG). Interestingly, exposure to MCGA3 resulted in a decreased ratio of GSH/GSSG, which was negatively related with mRNA level of phase II genes. By employing N-acetylcysteine and GSH biosynthetic enzyme inhibitors as well as prooxidants, hemin and H(2)O(2), we show that a decreased intracellular GSH/GSSG homeostasis, at least in part, may be involved in the MCGA3-mediated phase II gene induction and Nrf2 translocation, although the attenuation of HO-1 expression with SP 600125 supports a partial involvement of JNK signaling.     

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.     

Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis

Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.     

Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.