Angiogenic Response Of Endothelial Cells Research Articles

The role of cytochrome P450 epoxygenases in retinal angiogenesis.

The purpose of this study was to investigate the role(s) of cytochrome P450 epoxygenases (CYPs) and their products, the epoxyeicosatrienoic acids (EETs), in hypoxia-induced VEGF production and pathologic retinal angiogenesis. Human retinal astrocytes, Müller cells, and retinal microvascular endothelial cells (HRMEC) were exposed to hypoxia, and relative CYP2C expression was measured by RT-PCR. Astrocyte and Müller cell VEGF production was measured by ELISA after exposure to hypoxia and treatment with the general CYP inhibitor, SKF-525a. Human retinal microvascular endothelial cells were treated with the CYP product, 11,12-epoxyeicosatrienoic acid [EET], or SKF-525a in the presence or absence of VEGF. Proliferation of HRMEC and tube formation were assayed. Oxygen-induced retinopathy (OIR) was induced in newborn rats. Retinal CYP2C11 and CYP2C23 expression were measured by RT-PCR. The OIR rats received SKF-525a by intravitreal injection and preretinal neovascularization (NV) was quantified. Retinal VEGF protein levels were measured by ELISA. Human retinal astrocytes were the only cells to exhibit significant induction of CYP2C8 and CYP2C9 mRNA expression by hypoxia. Astrocytes, but not Müller cells, exhibited reduced hypoxia-induced VEGF production when treated with SKF-525a. 11,12-EET induced HRMEC proliferation and tube formation, and SKF-525a inhibited VEGF-induced proliferation. Oxygen-induced retinopathy induced expression of CYP2C23, but had no effect on CYP2C11. SKF-525a inhibited retinal NV and reduced retinal VEGF levels in OIR rats. The CYP-derived 11,12-EET may exhibit a proangiogenic biological function in the retina following stimulation by hypoxia in astrocytes. Inhibition of CYP may provide a rational therapy against retinal NV, because it can reduce VEGF production and VEGF-induced angiogenic responses in endothelial cells.

AB0115 Non-Canonical Nf-κB Signaling Enhances Angiogenesis in A Novel 3D Model of Rheumatoid Arthritis Synovial Inflammation

BackgroundAngiogenesis plays a crucial role in the progression of rheumatoid arthritis (RA) and is considered a switch from acute to chronic inflammation. Previously, we have demonstrated that NF-κB inducing kinase...

A1.21 Non-canonical NF-κB signalling enhances angiogenesis in a novel 3D model of rheumatoid arthritis synovial inflammation

BackgroundAngiogenesis plays a crucial role in the progression of rheumatoid arthritis (RA) and is considered a switch from acute to chronic inflammation. Previously, we have demonstrated that NF-κB inducing kinase...

Protein engineering to develop a redox insensitive endothelial nitric oxide synthase

The zinc tetrathiolate (ZnS4) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H2O2). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H2O2. The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.

EXPRESSION OF THE PROANGIOGENIC PROTEIN HEPATOMA-DERIVED GROWTH FACTOR IN NEOVESSELS IN AN ARTERIAL VENOUS LOOP-BASED TISSUE ENGINEERING CHAMBER

Boosting angiogenesis is a crucial process to enhance tissue growth in tissue engineering (TE). Hepatoma-derived growth factor (HDGF) has been identified as an angiogenic factor, but its involvement in angiogenesis in an arteriovenous loop-based TE chamber developed by the laboratory is unclear. In this study, the authors first examined the effects of HDGF on angiogenic responses in endothelial cells and in a corneal model of neovascularization, and then characterized the expression of HDGF in the TE chamber. HDGF (1–500 ng/mL) induced concentration-dependent angiogenic responses in human endothelial cells in vitro (proliferation, migration, and tube formation). Local application of HDGF stimulated neovascularization in a rat model of corneal angiogenesis. In the TE chamber, there was an increase in blood vessel volume from day 3 to day 14. Immunofluorescence microscopy revealed that HDGF is highly expressed in the neovessels in the chamber. Peak expression of HDGF (day 3) coincided with the infiltration of inflammatory cells, and the mRNA level of endogenous HDGF correlated with that of tumor necrosis factor α (TNFα). In vitro, TNFα stimulated HDGF expression in endothelial cells. The data suggest that HDGF may be involved in angiogenic responses in the TE chamber and the proinflammatory cytokine TNFα may have a pivotal role in stimulating HDGF expression. Enhancing HDGF signaling may be a new approach to extend vascularization for TE.

Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis.

MicroRNA-26a Regulates Pathological and Physiological Angiogenesis by Targeting BMP/SMAD1 Signaling

The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3'-untranslated region, an effect that decreased expression of Id1 and increased p21(WAF/CIP) and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti-miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Toll-Like Receptor 2–Blocking Antibodies Promote Angiogenesis and Induce ERK1/2 and AKT Signaling via CXCR4 in Endothelial Cells

Toll-like receptor 2 (TLR2) inhibition by function blocking antibodies (ABs) is associated with enhanced preservation of endothelial cell function during vascular disease. In the present study, we investigated the capacity of TLR2-blocking ABs to modulate the angiogenic response of endothelial cells in vitro and in vivo. Incubation of endothelial cells with mono- or polyclonal anti-TLR2 ABs resulted in increased tube formation, sprouting, and migration of endothelial cells compared with controls. In a mouse model of hindlimb ischemia, using TLR2-deficient or anti-TLR2 AB-treated wild-type mice resulted in increased new capillary formation and enhanced reperfusion. The effects of anti-TLR2 ABs were similar to those exerted by stromal cell-derived factor-1, and we show that anti-TLR2 ABs yet not TLR2 ligands lead to comparable activation of extracellular signal-regulated kinase1/2 and AKT but not p38 mitogen-activated protein kinase as activation of the CXCR4 canonical signal transduction pathways by stromal cell-derived factor-1. Immunoprecipitation of TLR2 revealed that anti-TLR2 ABs initiate an association of TLR2 with CXCR4 and mitogen-activated protein kinase activation. The proangiogenic properties of anti-TLR2 ABs were abolished by both G-protein inhibition and CXCR4 knockdown in endothelial cells. Our results provide evidence for a proangiogenic effect of TLR2-blocking ABs on endothelial cells in vitro and in vivo. They identify a novel molecular mechanism linking TLR2 to angiogenic processes that is independent from the activation of inflammatory cascades and further support the concept of a beneficial effect of TLR2 inhibition for endothelial cell function in vascular disease.

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.

Supression of SIRT1 in angiogenesis of endothelial cells induced by HCMV infection

Background Human cytomegalovirus (HCMV) is closely related to diseases including atherosclerosis, transplanted vascular sclerosis and arterial restenosis. It has been proved that angiogenesis induced by HCMV infection could result in vascular diseases. This article aims to investigate the mechanism of angiogenesis induced by HCMV infection in endothelial cells. Methods Endothelial cells (EA.hy926 cells) were divided into HCMV infected group and mock infected group. The cells were collected at 2, 6, 12 and 24 h after infection. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze SIRT1 mRNA and protein levels. Endothelial cells were incubated respectively by Resveratrol and SIRT1 small interference RNA (siRNA) for 2 h before infection. Twenty⁃four hours after infection, the proliferation, migration and tubule formation of cells were assessed by CCK-8, migration assay and tubule formation assay to detect the angiogenic response of endothelial cells. Results Compared to mock infected group, the expression of SIRT1 mRNA in HCMV infected group remained unchanged (F = 1.395, P = 0.304), but the expression of SIRT1 protein decreased gradually ( F = 23.927, P = 0.000). Under the treatment of Resveratrol and SIRT1 siRNA, migration ( P = 0.008, 0.003) and tubule formation ( P = 0.012, 0.008) of endothelial cells increased or reduced. The proliferation, however, remained unchanged (P = 0.969, 0.948). Conclusion HCMV infection promotes proliferation, migration and tubule formation of endothelial cells, and its mechanism may be related to the supression of SIRT1.

Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells

Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for understanding how the hypoxic matrix may regulate angiogenesis in tumors.

Involvement of COX‐2/PGE2 signalling in hypoxia‐induced angiogenic response in endothelial cells

To evaluate the impact of hypoxia on the angiogenic capability of endothelial cells (ECs), and further investigate whether the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signalling is involved in the angiogenic response of ECs to hypoxia. We explored the impact of various periods (1, 3, 6, 12, 24 hrs) of hypoxia (2% O2) on human umbilical vein endothelial cells (HUVECs) in vitro. We observed cell viability, migration, tube formation, analysed COX-2, vascular endothelial growth factor (VEGF), AQP1 mRNA transcription, protein expression and measured PGE2, VEGF protein concentration in cell supernatants. Then we treated HUVECs with COX-2 selective inhibitor NS398, EP1/2 combined antagonist AH6809 and exogenous PGE2 to investigate the role of COX-2/PGE2 signalling in the angiogenic response of ECs to hypoxia. The results demonstrated that short-term hypoxic treatment enhanced HUVECs proliferation, migration, tube formation, significantly up-regulated COX-2, VEGF, AQP1 mRNA level, protein expression and promoted PGE2, VEGF release. The pharmacological inhibition study revealed that exposure of HUVEC to NS398 and AH6809 under hypoxia impaired the biological responses of ECs to hypoxia. Exogenous PGE2 augments the effects of hypoxia on HUVECs, and partially reversed the inhibitory effects of NS398 on HUVECs proliferation and angiogenic capability. Short-term hypoxic treatment enhanced angiogenic capability of ECs, and COX-2/PGE2 signalling may play a critical role in the biological response of ECs to hypoxia.

Cross‐talk between endothelial and breast cancer cells regulates reciprocal expression of angiogenic factors in vitro

Reciprocal growth factor exchange between endothelial and malignant cells within the tumor microenvironment may directly stimulate neovascularization; however, the role of host vasculature in regulating tumor cell activity is not well understood. While previous studies have examined the angiogenic response of endothelial cells to tumor-secreted factors, few have explored tumor response to endothelial cells. Using an in vitro co-culture system, we investigated the influence of endothelial cells on the angiogenic phenotype of breast cancer cells. Specifically, VEGF, ANG1, and ANG2 gene and protein expression were assessed. When co-cultured with microvascular endothelial cells (HMEC-1), breast cancer cells (MDA-MB-231) significantly increased expression of ANG2 mRNA (20-fold relative to MDA-MB-231 monoculture). Moreover, MDA-MB-231/HMEC-1 co-cultures produced significantly increased levels of ANG2 (up to 580 pg/ml) and VEGF protein (up to 38,400 pg/ml) while ANG1 protein expression was decreased relative to MDA-MB-231 monocultures. Thus, the ratio of ANG1:ANG2 protein, a critical indicator of neovascularization, shifted in favor of ANG2, a phenomenon known to correlate with vessel destabilization and sprouting in vivo. This angiogenic response was not observed in nonmalignant breast epithelial cells (MCF-10A), where absolute protein levels of MCF-10A/HMEC-1 co-cultures were an order of magnitude less than that of the MDA-MB-231/HMEC-1 co-cultures. Results were further verified with a functional angiogenesis assay demonstrating well-defined microvascular endothelial cell (TIME) tube formation when cultured in media collected from MDA-MB-231/HMEC-1 co-cultures. This study demonstrates that the angiogenic activity of malignant mammary epithelial cells is significantly enhanced by the presence of endothelial cells.

The HIF1 target geneNOX2promotes angiogenesis through urotensin-II

Urotensin-II (U-II) has been considered as one of the most potent vasoactive peptides, although its physiological and pathophysiological role is still not finally resolved. Recent evidence suggests that it promotes angiogenic responses in endothelial cells, although the underlying signalling mechanisms are unclear. Reactive oxygen species derived from NADPH oxidases are major signalling molecules in the vasculature. Because NOX2 is functional in endothelial cells, we investigated the role of the NOX2-containing NADPH oxidase in U-II-induced angiogenesis and elucidated a possible contribution of hypoxia-inducible factor-1 (HIF-1), the master regulator of hypoxic angiogenesis, in the response to U-II. We found that U-II increases angiogenesis in vitro and in vivo, and these responses were prevented by antioxidants, NOX2 knockdown and in Nox2(-/-) mice. In addition, U-II-induced angiogenesis was dependent on HIF-1. Interestingly, U-II increased NOX2 transcription involving HIF-1, and chromatin immunoprecipitation confirmed NOX2 as a target gene of HIF-1. In support, NOX2 levels were greatly diminished in U-II-stimulated isolated vessels derived from mice deficient in endothelial HIF-1. Conversely, reactive oxygen species derived from NOX2 were required for U-II activation of HIF and upregulation of HIF-1. In line with this, U-II-induced upregulation of HIF-1 was absent in Nox2(-/-) vessels. Collectively, these findings identified HIF-1 and NOX2 as partners acting in concert to promote angiogenesis in response to U-II. Because U-II has been found to be elevated in cardiovascular disorders and in tumour tissues, this feed-forward mechanism could be an interesting anti-angiogenic therapeutic option in these disorders.

Propanolol and angiogenesis inhibition in hereditary haemorrhagic telangiectasia

Editorial on Albinana et al. Thromb Haemost 2012; 108: 41-53. Hereditary haemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular disorder, which occurs at a frequency of about one in 5,000 people (1–3). Vascular malformations in HHT patients range from regions of capillary dilation (telangiectases) in the nasal septum, oral mucosa, skin, or gastrointestinal tract, to large arterial-venous malformations (AVM) in organs such as lung, brain, or liver. The lesions can give rise to spontaneous recurrent nasal or gastrointestinal bleeding, leading to significant blood loss. Therapeutic options include surgery, or manipulation of coagulation and fibrinolytic pathways (2). However, these treatments are insufficient, and novel therapies are needed. The most frequent cases of HHT are caused by mutations in the Endoglin (ENG) or ALK1 genes. Endoglin and Alk1 are cell surface receptors for members of the TGF-β family of cytokines. The biology of TGF-β is complex and many of the observed activities are dependent on the biological context (4); notably, TGF-β can either stimulate or inhibit angiogenesis. Yet, genetic studies in mice have provided clear evidence for a role of Endoglin and Alk1 in vascular development and malformation. Mice carrying heterozygous mutations in the ENG or ALK1 genes develop HHT-like vascular phenotypes. The current hypothesis suggests haploinsufficiency of these genes as the major cause of HHT in men (1, 3). However, the development of the vascular lesions in HHT has not only been attributed to decreased TGF-β signalling but also to increased activity of the pro-angiogenic VEGF pathway. Treatments that result in elevated TGF-β signalling or in angiogenesis inhibition might reduce pathological endothelial cell activation in HHT lesions and appear promising. Various candidate drugs have been proposed for the treatment of HHT on the basis of these considerations. For example, estrogen therapy using raloxifene has been shown to increase the expression of ENG and ALK1 in cultured endothelial cells, and to improve endothelial cell functions like tubulogenesis and migration. This suggests that raloxifene counteracts the haploinsufficiency of ENG and ALK1 and may be beneficial for epistaxis treatment in HHT menopausal women (5). Inhibitors of VEGF, a key regulator of physiological and pathological angiogenesis, are obvious candidates for angiogenesis inhibition. The VEGF-specific antibody bevacizumab, which is approved for the treatment of several malignancies, can be beneficial for the treatment of epistaxis and anaemia in HHT patients (6). However, VEGF has important functions in the healthy adult organism, and inhibiting its activity can have undesired effects. Cancer patients who received bevacizumab therapy suffered from fever, headache, rash, or chills; notably, also epistaxis occurred (7). Although these events were generally mild to moderate in severity, caution is necessary. This is also true for thalidomide. This drug was originally used to treat nausea during pregnancy, but was removed from the market after severe congenital defects became overt, which were attributed to its anti-angiogenic activity resulting in defective vascular development (8). However, thalidomide enjoyed a renaissance in medicine recently. It was reported to inhibit gastrointestinal bleeding in a HHT patient who received the drug as anti-cancer therapy (9), and to reduce the frequency of nosebleeds in HHT patients (8). Although these effects might be attributed to thalidomide s anti-angiogenic activity, a recent study revealed an alternative potential mechanism of action which may guide the development of novel therapeutics for HHT (8). Thalidomide was shown to promote vessel maturation in ENG+/mice which develop HHT-like lesions, as well as in nasal mucosal biopsies of HHT patients. Vessel maturation involved enhanced pericyte and vascular smooth muscle cell coverage in a PDGF-B dependent manner. Ideally, drugs for anti-angiogenesis or normalisation of the endothelium in HHT lesions should be in clinical use without severe side effects. In this issue of Thrombosis and Haemostasis, Albinana et al. propose the use of the β-blocker propanolol for the treatment of HHT (10). This non-selective β-adrenergic receptor antagonist inhibits β1 and β2 adrenergic receptors. It causes vasoconstriction and has been used for the treatment of cardiovascular disorders like hypertension, angina, or cardiac arrhythmias. Propanolol has in addition antiangiogenic properties: it can reduce the expression of VEGF and inhibits typical angiogenic responses in endothelial cells, such as VEGFR-2 phosphorylation, cell proliferation and migration, as well as tube formation (11). Recently, propanolol has been reported to improve capillary haemangiomas of infancy (12). These vascular tumors are formed by endothelial cells that proliferate under the influence of elevated VEGF levels. The question arises whether propanolol might be useful for the treatment of HHT as well. Albinana and colleagues address this question by studying endothelial responses in vitro. Apart from confirming the antiangiogenic activity of pro-

IL-8-mediated angiogenic responses of endothelial cells to lipid antigen activation of iNKT cells depend on EGFR transactivation

iNKT cells are a unique T cell subset, which is CD1d-restricted and specific for glycolipid antigens. In advanced atherosclerotic plaques, focal collections of inflammatory cells correlate with areas of intraplaque neovascularization. We reported recently that iNKT cells might facilitate intraplaque neovascularization by enhancing EC migration and sprouting in an IL-8-dependent manner. This study investigated the participating effector mechanisms. In ECs, CM, derived from antigen-stimulated human iNKT cells (CM+), induced up-regulation of IL-8R CXCR2 and the phosphorylation of EGFR and of multiple intracellular signaling effectors, including FAK, Src, Erk, Jnk, p38-MAPK, and STAT1 and -3. We found that a cascade of events, which were IL-8-dependent and involved EGFR activation, was responsible for signaling through FAK and Src kinases and necessary for acquisition of angiogenic morphology, migration in a two-dimensional wound assay, and sprout outgrowth in a three-dimensional model of angiogenesis in vitro. The data support that IL-8-dependent activation of angiogenic behavior in ECs, in response to activated iNKT, involves CXCR2, transactivation of EGFR, and subsequent FAK/Src signaling. We found too that activated iNKT increased VEGFR2 expression in ECs. Functional studies confirmed that EGF is the motogenic-enhancing factor in CM+ and is necessary, together with an exogenous source of VEGF, for iNKT-promoted sprout formation. EGFR inhibition may represent a novel therapeutic modality aimed at plaque stabilization through control of neovascularization within developing atherosclerotic plaques.

Neoangiogenesis contributes to the development of hemophilic synovitis

AbstractJoint arthropathy secondary to recurrent hemarthroses remains a debilitating complication of hemophilia despite the use of prophylactic factor concentrates. Increased vascularity and neoangiogenesis have been implicated in the progression of musculoskeletal disorders and tumor growth. We hypothesized that de novo blood vessel formation could play a major role in the pathogenesis of hemophilic joint disease (HJD). We observed a 4-fold elevation in proangiogenic factors (vascular endothelial growth factor-A [VEGF-A], stromal cell–derived factor-1, and matrix metalloprotease-9) and proangiogenic macrophage/monocyte cells (VEGF+/CD68+ and VEGFR1+/CD11b+) in the synovium and peripheral blood of HJD subjects along with significantly increased numbers of VEGFR2+/AC133+ endothelial progenitor cells and CD34+/VEGFR1+ hematopoietic progenitor cells. Sera from HJD subjects induced an angiogenic response in endothelial cells that was abrogated by blocking VEGF, whereas peripheral blood mononuclear cells from HJD subjects stimulated synovial cell proliferation, which was blocked by a humanized anti-VEGF antibody (bevacizumab). Human synovial cells, when incubated with HJD sera, could elicit up-regulation of HIF-1α mRNA with HIF-1α expression in the synovium of HJD subjects, implicating hypoxia in the neoangiogenesis process. Our results provide evidence of local and systemic angiogenic response in hemophilic subjects with recurrent hemarthroses suggesting a potential to develop surrogate biologic markers to identify the onset and progression of hemophilic synovitis.

MiR-200b Targets Ets-1 and Is Down-regulated by Hypoxia to Induce Angiogenic Response of Endothelial Cells

The miR-200 family plays a crucial role in epithelial to mesenchymal transition via controlling cell migration and polarity. We hypothesized that miR-200b, one miR-200 family member, could regulate angiogenic responses via modulating endothelial cell migration. Delivery of the miR-200b mimic in human microvascular endothelial cells (HMECs) suppressed the angiogenic response, whereas miR-200b-depleted HMECs exhibited elevated angiogenesis in vitro, as evidenced by Matrigel® tube formation and cell migration. Using in silico studies, miR target reporter assay, and Western blot analysis revealed that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), a crucial angiogenesis-related transcription factor, serves as a novel direct target of miR-200b. Knocking down endogenous Ets-1 simulated an anti-angiogenic response of the miR-200b mimic-transfected cells. Certain Ets-1-associated genes, namely matrix metalloproteinase 1 and vascular endothelial growth factor receptor 2, were negatively regulated by miR-200b. Overexpression of Ets-1 rescued miR-200b-dependent impairment in angiogenic response and suppression of Ets-1-associated gene expression. Both hypoxia as well as HIF-1α stabilization inhibited miR-200b expression and elevated Ets-1 expression. Experiments to identify how miR-200b modulates angiogenesis under a low oxygen environment illustrated that hypoxia-induced miR-200b down-regulation de-repressed Ets-1 expression to promote angiogenesis. This study provides the first evidence that hypoxia-sensitive miR-200b is involved in induction of angiogenesis via directly targeting Ets-1 in HMECs.

Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.