Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Opposing roles of STAT-1 and STAT-3 in regulating vascular endothelial growth factor expression in vascular smooth muscle cells.

Increased microvessel density in atherosclerotic plaques plays a major role in promoting plaque destabilization resulting in increased risk of stroke and myocardial infarction. Previously we have shown that expression of the inflammatory cytokine, Oncostatin-M (OSM), in human atherosclerotic plaques correlated with increased microvessel density, indicating a role for OSM in promoting plaque angiogenesis. The purpose of this study was to determine the mechanism by which OSM regulates Vascular Endothelial Growth Factor (VEGF) expression in human coronary artery smooth muscle cells. Using shRNA and overexpression studies, we have shown that the transcription factor, STAT-1 inhibited VEGF expression, while STAT-3 promoted the expression of VEGF. We further show that the mechanism by which STAT-1 and STAT-3 regulates VEGF expression is through modulation of Hypoxia Inducible Factor-1α (HIF-1α). STAT-1 suppresses HIF-1α expression, whereas STAT-3 positively regulates HIF-1α expression. These results provide evidence that activated STAT-1 and STAT-3 regulate VEGF expression indirectly, by modulating HIF-1α activity.

Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity.

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A(165) and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A(165) binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A(165) showed that heparin can also modulate the conformation and secondary structure of the protein.

Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens. Herein we report that VEGF binds to the heparin-II domain of Fn and that the cell-binding and VEGF-binding domains of Fn, when physically linked, are necessary and sufficient to promote VEGF-induced endothelial cell proliferation, migration, and Erk activation. Using recombinant Fn domains, the C-terminal heparin-II domain of Fn (type III repeats 13 to 14) was identified as a key VEGF-binding site. Mutation of the heparin-binding residues on FnIII(13-14) abolished VEGF binding, and peptides corresponding to the heparin-binding sequences in FnIII(13-14) inhibited VEGF binding to Fn. Fn fragments containing both the alpha5beta1 integrin-binding domain (III 9 to 10) and the VEGF-binding domain (III 13 to 14) significantly enhanced VEGF-induced EC migration and proliferation and induced strong phosphorylation of the VEGF receptor and Erk. Neither the cell-binding or VEGF-binding fragment of Fn alone had comparable VEGF-promoting effects. These results suggest that the mechanism of VEGF/Fn synergism is mediated extracellularly by the formation of a novel VEGF/Fn complex requiring both the cell-binding and VEGF-binding domains linked in a single molecular unit. These data also highlight a new function for the Fn C-terminal heparin-binding domain that may have important implications for angiogenesis and tumor growth.

Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity.

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, alpha5beta1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to alpha5beta1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality.

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells.

BACKGROUND: The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes. RESULTS: Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1. CONCLUSIONS: These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease.

Leg ulceration part 1: aetiology and pathophysiology.

In part one of this article, the underlying causes of chronic leg ulcers, professional issues and the anatomy and physiology of the lower limb are examined.

Modulation of vascular smooth muscle cell phenotype by STAT-1 and STAT-3.

OBJECTIVE: Smooth muscle cell (SMC) de-differentiation is a key step that leads to pathological narrowing of blood vessels. De-differentiation involves a reduction in the expression of the SMC contractile genes that are the hallmark of quiescent SMCs. While there is considerable evidence linking inflammation to vascular diseases, very little is known about the mechanisms by which inflammatory signals lead to SMC de-differentiation. Given that the Signal Transducers and Activators of Transcription (STAT) transcriptional factors are the key signaling molecules activated by many inflammatory cytokines and growth factors, the aim of the present study was to determine if STAT transcriptional factors play a role SMC de-differentiation. METHODS AND RESULTS: Using shRNA targeted to STAT-1 and STAT-3, we show by real time RT-PCR and Western immunoblots that STAT-1 significantly reduces SMC contractile gene expression. In contrast, STAT-3 promotes expression of SMC contractile genes. Over-expression studies of STAT-1 and STAT-3 confirmed our observation that STAT-1 down-regulates whereas STAT-3 promotes SMC contractile gene expression. Bioinformatics analysis shows that promoters of all SMC contractile genes contain STAT binding sites. Finally, using ChIP analysis, we show that both STAT-1 and STAT-3 associate with the calponin gene. CONCLUSION: These data indicate that the balance of STAT-1 and STAT-3 influences the differentiation status of SMCs. Increased levels of STAT-1 promote SMC de-differentiation, whereas high levels of STAT-3 drive SMC into a more mature phenotype. Thus, inhibition of STAT-1 may represent a novel target for therapeutic intervention in the control of vascular diseases such as atherosclerosis and restenosis.

Heparin modulates the conformation and signaling of platelet integrin αIIbß3.

INTRODUCTION: The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbß3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbß3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. METHODS: We assessed conformational changes in αIIbß3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbß3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the ß3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbß3, and of recombinant fragments of αIIb and ß3, by surface plasmon resonance. RESULTS AND CONCLUSIONS: Heparin binding results in conformational changes in αIIbß3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin ß3 cytoplasmic domain, but the presence of heparin increased both ß3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant ß3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbß3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbß3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

Oncostatin M is expressed in atherosclerotic lesions: a role for Oncostatin M in the pathogenesis of atherosclerosis.

OBJECTIVE: Chronic inflammation plays a pivotal role in the development and progression of atherosclerosis. The inflammatory response is mediated by cytokines. The aim of this study was to determine if Oncostatin M (OSM), a monocyte and T-lymphocyte specific cytokine is present in atherosclerotic lesions. We also investigated the roles of signal transducer and activator of transcription (STAT)-1 and STAT-3 in regulating OSM-induced smooth muscle cell (SMC) proliferation, migration and cellular fibronectin (cFN) synthesis. METHODS AND RESULTS: Immunostaining of atherosclerotic lesions from human carotid plaques demonstrated the expression of OSM antigen in both macrophages and SMCs. Explanted SMCs from human carotid plaques expressed OSM mRNA and protein as determined by RT-PCR and Western blotting. Using the chow-fed ApoE(-/-) mouse model of atherosclerosis, we observed that OSM was initially expressed in the intima at 20 weeks of age. By 30 weeks, OSM was expressed in both the intima and media. In vitro studies show that OSM promotes SMC proliferation, migration and cFN synthesis. Lentivirus mediated-inhibition of STAT-1 and STAT-3 prevented OSM-induced SMC proliferation, migration and cellular fibronectin synthesis. CONCLUSIONS: These findings demonstrate that OSM is expressed in atherosclerotic lesions and may contribute to the progression of atherosclerosis by promoting SMC proliferation, migration and extracellular matrix protein synthesis through the STAT pathway.

Enhancement of capillary and cellular ingrowth in ePTFE implants with a proangiogenic recombinant construct derived from fibronectin.

Based on our discoveries of a unique, synergistic interplay between vascular endothelial growth factor (VEGF) and specific domains of the matrix protein fibronectin (FN), we used recombinant technology to create a new protein construct derived from the cell-binding and VEGF-binding domains of FN. We wished to test the hypothesis that this prototype recombinant FN (rFN) protein would enhance cellular and capillary ingrowth in vivo into expanded polytetrafluoroethylene (ePTFE) implants. ePTFE disks of high porosity (60 micron internodal distance) were embedded with fibrin gel and heparin, with/without mixtures of VEGF and rFN and were implanted subcutaneously in rats. Control implants embedded with fibrin glue and heparin alone showed an average of 8.5% (±0.51% standard error mean (SEM)) cellular ingrowth. The addition of either VEGF or rFN caused a modest but significant increase in cellular ingrowth (12.7 ± 1% and 11.8 ± 0.98%, respectively, p < 0.004). However, the combination of rFN/VEGF/heparin dramatically increased cellular ingrowth (27.6 ± 1.62%, p < 0.001), compared with all other treatments. Quantification of capillary ingrowth yielded the same pattern. These results suggest that the incorporation of such biological modulators into cardiovascular implants could offer new strategies for the design of a ready-made small diameter prosthetic graft with enhanced capacity for neovascularization and endothelialization.

Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells.

BACKGROUND: Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium. METHODS: Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured. RESULTS: As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration. CONCLUSIONS: VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Probing electric fields inside microfluidic channels during electroosmotic flow with fast-scan cyclic voltammetry.

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used in microfluidic channels. This method offers the advantage that it can resolve electroactive species not separated in the channel. In addition, this method provides a route to investigate the distribution of applied electrophoretic fields in microfluidic channels. To probe this, microelectrodes were inserted at various distances into channels and cyclic voltammograms recorded at 300 V/s were repeated at 0.1-s intervals. The use of a battery-powered laptop computer and potentiostat provided galvanic isolation between the applied electrophoretic field and the electrochemical measurements. In the absence of an external field, the peak potential for oxidation of the test solute, Ru(bpy)3(2+), was virtually unaltered by insertion of the microelectrode tip into the channel. When an electrophoretic field was applied, the peak potential for Ru(bpy)3(2+) oxidation shifted to more positive potentials in a manner that was directly proportional to the field in the channel. The shifts in peak potential observed with FSCV enabled direct compensation of the applied electrochemical potential. This approach was used to explore the electrophoretic field at the channel terminus. It was found to persist for more than 50 microm from the channel terminus. In addition, the degree of analyte dispersion was found to depend critically on the electrode position outside the channel.