VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

C-reactive protein decreases expression of VEGF receptors and neuropilins and inhibits VEGF165-induced cell proliferation in human endothelial cells

C-reactive protein (CRP) is associated with cardiovascular disease. However, its biological functions for the vascular system are largely unknown. The objective of this study was to determine whether CRP could affect endothelial cell proliferation and expression of VEGF receptors (VEGFRs) and/or neuropilins. Human coronary artery endothelial cells (HCAECs) treated with CRP showed a significant reduction of mRNA levels of VEGFR-2, VEGFR-3, NRP-1, and NRP-2 by 34%, 63%, 41%, and 43%, respectively, as compared to untreated control cells (p < 0.05) by real-time PCR analysis. In addition, VEGF165-induced cell proliferation was determined by [3H]thymidine incorporation and MTS assay as well as capillary-like tube formation on Matrigel. HCAECs pretreated with CRP significantly decreased VEGF165-induced [3H]thymidine incorporation by 73%, MTS absorbance by 44%, and capillary-like tube formation by 54% as compared to CRP-untreated cells (p < 0.05). These data demonstrate that CRP significantly attenuates VEGF165-induced HCAEC proliferation and capillary-like tube formation through downregulation of expression of VEGFRs and NRPs. This study suggests a new molecular mechanism underlying the adverse effect of CRP on the vascular system.     

Cellular and soluble markers of tumor angiogenesis: From patient selection to the identification of the most appropriate postresistance therapy

Antiangiogenic drugs are now intensively used in clinical oncology, but some drawbacks still hamper their development. First, it is frequently unclear what patient subpopulation is likely to gain clinical benefit from these expensive therapies; second, there is evidence of (sometimes rapid) development of drug resistance in many patients; third, the results of some preclinical and clinical studies have suggested acceleration of malignant cell aggressiveness when some antiangiogenic therapies are terminated. Here we discuss the role of soluble molecules and cellular markers of neoplastic angiogenesis for patient selection and follow-up during treatment. These markers should help clinicians to decide the right therapy, advise them of the generation of mechanisms of drug resistance during antiangiogenic treatment, and finally suggest the most appropriate next line of therapy according to the new patterns of cancer vascularization induced by antiangiogenic therapies.     

Vascular galectins: Regulators of tumor progression and targets for cancer therapy

Galectins are a family of carbohydrate binding proteins with a broad range of cytokine and growth factor-like functions in multiple steps of cancer progression. They contribute to tumor cell transformation, promote tumor angiogenesis, hamper the anti-tumor immune response, and facilitate tumor metastasis. Consequently, galectins are considered as multifunctional targets for cancer therapy. Interestingly, many of the functions related to tumor progression can be linked to galectins expressed by endothelial cells in the tumor vascular bed. Since the tumor vasculature is an easily accessible target for cancer therapy, understanding how galectins in the tumor endothelium influence cancer progression is important for the translational development of galectin-targeting therapies.     

Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells

Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here, we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Functional symbiosis between endothelium and epithelial cells in glomeruli

In some capillary beds, pericytes regulate endothelial growth. Capillaries with high filtration capacity, such as those in renal glomeruli, lack pericytes. Glomerular endothelium lies adjacent to visceral epithelial cells (podocytes) that are anchored to and cover the anti-luminal surface of the basement membrane. We have tested the hypothesis that podocytes can function as endothelial supporting cells. Endothelial cells were outgrown from circulating endothelial progenitors of normal subjects and were extensively characterized. These blood outgrowth endothelial cells (BOECs) expressed endothelial markers, lacked stem cell markers, and expressed the angiopoietin-1 receptor, Tie-2, and the vascular endothelial growth factor (VEGF) receptor, Flk-1. Differentiated podocytes in culture expressed and secreted VEGF, which was upregulated 4.5-fold by high glucose. In complete medium, BOECs formed thin cell-cell connections and multicellular tubes on Matrigel, the in vitro correlate of angiogenesis. This was impaired in deficient media but rescued by co-incubation with Transwell Anopore inserts containing differentiated podocytes. To assess whether VEGF was the major podocyte-derived signal that rescued BOEC angiogenesis, we examined angiogenesis of control and Flk-1-deficient BOECs. Co-incubation with podocytes or addition of recombinant VEGF each rescued angiogenesis in control BOECs, but both failed to support maintenance and angiogenesis in Flk-1-deficient BOECs. Finally, co-culture with podocytes increased BOEC-proliferation. In concert, these findings suggest a model in which glomerular visceral epithelial cells function as pericyte-like endothelial supporting cells. Podocyte-derived VEGF is a required and sufficient regulator of vascular endothelial maintenance, and its upregulation in podocytes by high glucose may be the mechanism for the increased glomerular angiogenesis that is observed in vivo in early diabetic glomerular injury. These studies were supported by grants from the National Institutes of Health (NIH-NIDDK 63360) and the Juvenile Diabetes Research Foundation (JDRF-1-2004-78).     

Melanoma cell-derived vascular endothelial growth factor induces endothelial tubulogenesis within fibrin gels by a metalloproteinase-mediated mechanism

Angiogenesis is a process required not only for embryonal development but is encountered in wound healing and in pathological situations such as tumour growth. In vitro, formation of capillary-like structures can be induced by seeding human microvascular endothelial cells (HDMECs) on top of a fibrin matrix in the presence of phorbol 12-myristate 13-acetate (PMA) as a stimulating agent. In this study, we show that supernatants collected from high-invasive melanoma cells (BLM) induce the formation of tubular structures similar to PMA treatment whereas supernatants from low-invasive cells (WM164) did not. Analysis of proteins secreted into the supernatant of both melanoma cell lines identified differential expression of several pro-angiogenic proteins in high- and low-invasive melanoma cells. Vascular endothelial growth factor (VEGF) was strongly expressed by high- but not by low-invasive melanoma cells. Neutralisation of VEGF as well as inhibition of matrix metalloproteases (MMPs) using the broad spectrum MMP inhibitor 1,10-phenanthroline, both strongly reduced the melanoma-induced tube formation. PMA treatment of HDMECs on a fibrin matrix stimulated MT1-MMP synthesis, indicating that this protease is involved in PMA-induced angiogenesis. In addition, stimulation of HDMECs by supernatants of BLM melanoma cells resulted in a strong induction of ADAM-15, which is known to act as a metalloproteinase. In conclusion, these results show that VEGF released by melanoma cells is an important mediator of neo-vascularisation and that this process depends on the presence of metalloproteinases.     

Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation

We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.     

Neuropilins: structure, function and role in disease

NRPs (neuropilins) are co-receptors for class 3 semaphorins, polypeptides with key roles in axonal guidance, and for members of the VEGF (vascular endothelial growth factor) family of angiogenic cytokines. They lack a defined signalling role, but are thought to mediate functional responses as a result of complex formation with other receptors, such as plexins in the case of semaphorins and VEGF receptors (e.g. VEGFR2). Mutant mouse studies show that NRP1 is essential for neuronal and cardiovascular development, whereas NRP2 has a more restricted role in neuronal patterning and lymphangiogenesis, but recent findings indicate that NRPs may have additional biological roles in other physiological and disease-related settings. In particular, NRPs are highly expressed in diverse tumour cell lines and human neoplasms and have been implicated in tumour growth and vascularization in vivo. However, despite the wealth of information regarding the probable biological roles of these molecules, many aspects of the regulation of cellular function via NRPs remain uncertain, and little is known concerning the molecular mechanisms through which NRPs mediate the functions of their various ligands in different cell types.     

Sustained up-regulation of semaphorin 3A, Neuropilin1, and doublecortin expression in ischemic mouse brain during long-term recovery

Strategies to provide neuroprotection and to promote regenerative axonal outgrowth in the injured brain are thwarted by the plethora of axon growth inhibitors and the ligand promiscuity of some of their receptors. Especially, new neurons derived from ischemia-stimulated neurogenesis must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. Therefore, characterization of the expression of inhibitory molecules in long-term surviving ischemic brains following stroke is important for designing selective therapeutics. Here, we describe a long-term recovery mouse model for cerebral ischemia in which a brief transient occlusion of the middle cerebral artery (30 min) was followed by up to 30 days of long-term reperfusion. Significantly decreased grip strength motor function and increased expression of one of the major repulsive guidance cues, Semaphorin 3A (Sema3A) and its receptor Neuropilin1 (NRP1) occurred in brains of these mice. Interestingly, increased Doublecortin (DCX) expression occurred only in the lateral ventricular wall zone, but not in the dentate gyrus granule cell layer on the ischemic side of the brain. Importantly, no DCX positive cells were detected in the infarct core region after 30 d ischemic recovery. Collectively, these studies demonstrated the sustained elevation of Sema3A/NRP1 expression in the ischemic territory, which may contribute to the inhibitory microenvironment responsible for preventing new neurons from entering the infarct area. This model will be of use as a platform for testing anti-inhibitory therapies to stroke.     

MicroRNA signature and function in retinal neovascularization Agrawal S,Chaqour B简

Ischemic retinopathies are clinically well-defined chronic microvascular complications characterized by gradually progressive alterations in the retinal microvasculature and a compensatory aberrant neovascularization of the eye. The subsequent metabolic deficiencies result in structural and functional alterations in the retina which is highly susceptible to injurious stimuli such as diabetes, trauma, hyperoxia, inflammation, aging and dysplipidemia. Emerging evidence indicates that an effective therapy may require targeting multiple components of the angiogenic pathway. Conceptually, mircoRNA (miRNA)-based therapy provides the rationale basis for an effective antiangiogenic treatment. miRNAs are an evolutionarily conserved family of short RNAs, each regulating the expression of multiple protein-coding genes. The activity of specific miRNAs is important for vascular cell signaling and blood vessel formation and function. Recently, important progress has been made in mapping the miRNA-gene target network and miRNA-mediated gene expression control. Here we highlight the latest findings on angiogenic and antiangiogenic miRNAs and their targets as well as potential implications in ocular neovascular diseases. Emphasis is placed on how specific vascular-enriched miRNAs regulate cell responses to various cues by targeting several factors, receptors and/or signaling molecules in order to maintain either vascular function or dysfunction. Further improvement of our knowledge in not only miRNA specificity, turnover, and transport but also how miRNA sequences and functions can be altered will enhance the therapeutic utility of such molecules.     

MicroRNA signature and function in retinal neovascularization

Ischemic retinopathies are clinically well-defined chronic microvascular complications characterized by gradually progressive alterations in the retinal microvasculature and a compensatory aberrant neovascularization of the eye. The subsequent metabolic deficiencies result in structural and functional alterations in the retina which is highly susceptible to injurious stimuli such as diabe-tes, trauma, hyperoxia, inflammation, aging and dys-plipidemia. Emerging evidence indicates that an effec-tive therapy may require targeting multiple components of the angiogenic pathway. Conceptually, mircoRNA(miRNA)-based therapy provides the rationale basis for an effective antiangiogenic treatment. miRNAs are an evolutionarily conserved family of short RNAs, each regulating the expression of multiple protein-coding genes. The activity of specific miRNAs is important for vascular cell signaling and blood vessel formation and function. Recently, important progress has been made in mapping the miRNA-gene target network andmiRNA-mediated gene expression control. Here wehighlight the latest findings on angiogenic and antian-giogenic miRNAs and their targets as well as potentiaimplications in ocular neovascular diseases. Emphasis isplaced on how specific vascular-enriched miRNAs regu-late cell responses to various cues by targeting severafactors, receptors and/or signaling molecules in orderto maintain either vascular function or dysfunction. Fur-ther improvement of our knowledge in not only miRNAspecificity, turnover, and transport but also how miRNAsequences and functions can be altered will enhancethe therapeutic utility of such molecules.