Classical Angiogenic Factors Research Articles

Direct contribution of angiogenic factors to neurodevelopment: a focus on angiopoietins

Abstract Over the last two decades, it has become clear that classical molecules that regulate neurodevelopment also play an important role in directly regulating the development of the vascular system and vice versa. The prototypical angiogenic ligand vascular endothelial growth factor (VEGF) is by now also regarded as a molecular regulator of different neurodevelopmental processes, such as neuronal progenitor proliferation, migration and differentiation, dendritic and axonal branching and synaptogenesis. The direct effect of other classical angiogenic factors, such as angiopoietins and its receptor Tie2, on neurodevelopmental processes remains less defined. Recent work from our group indicates that the angiopoietin-Tie2 pathway does not only regulate blood vessel formation and stabilization but also simultaneously affect neuronal dendritogenesis in a cell-autonomous manner. In this mini-review, we will integrate our findings within the current understanding of the neurovascular link and within the previous knowledge of the potential effects of angiopoietins in the neuronal context.

Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton\u2019s jelly mesenchymal stem cells (WJ-MSC)

BackgroundAngiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context.MethodsMesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation.ResultsThe paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10–200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6β1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway.ConclusionsThus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.

Egfl7 Is Differentially Expressed in Arteries and Veins during Retinal Vascular Development

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Pilot Study of Angiogenic Response to Yttrium-90 Radioembolization with Resin Microspheres

PurposeTo investigate the impact of radioembolization with yttrium-90 resin microspheres on the regulation of angiogenesis through observation of serial changes in a spectrum of angiogenic markers and other cytokines after therapy. Materials and MethodsThis prospective pilot study enrolled 22 patients with liver-dominant disease deriving from biopsy-proven hepatocellular carcinoma (HCC) (n = 7) or metastatic colorectal carcinoma (mCRC) (n = 15). Circulating angiogenic markers were measured from serum samples drawn at baseline and at time points after therapy ranging from 6 hours to 120 days. Using multiplex enzyme-linked immunosorbent assay, several classic angiogenesis factors (vascular endothelial growth factor [VEGF], angiopoietin-2 [Ang-2], basic fibroblast growth factor [bFGF], platelet-derived growth factor subunit BB [PDGF-BB], thrombospondin-1 [Tsp-1]) and nonclassic factors (follistatin, leptin, interleukin [IL]-8) were evaluated. ResultsIncreases in cytokine levels ≥ 50% over baseline were observed in more than half of all patients studied for many cytokines, including classic angiogenic factors such as VEGF, Ang-2, and Tsp-1 as well as nonclassic factors IL-8 and follistatin (range, 36%–82% for all cytokines). Baseline cytokine levels in patients with overall survival (OS) < 6 months differed significantly from patients with longer survival for Ang-2 (P = .033) and IL-8 (P = .041). Patients with OS ≤ 6 months exhibited transient increases in VEGF and PDGF-BB after therapy compared with patients with OS > 6 months. ConclusionsRadioembolization is associated with early transient increases in many angiogenic cytokines. In this small sample size, some of these changes were associated with worse OS. This research has important implications for future studies of radioembolization with antiangiogenic therapy performed during and after the procedure.

Increased myocardial expression of angiopoietin-2 in patients undergoing urgent surgical revascularization for acute coronary syndromes

Myocardial ischemia triggers the expression of multiple angiogenic factors including vascular endothelial growth factor and its receptors. However, vascular endothelial growth factor does not act in isolation. To identify other genes important in the angiogenic response to clinically relevant myocardial ischemia. Paired intraoperative biopsies of ischemic and nonischemic myocardium were obtained from 12 patients with acute coronary syndromes (ACS) undergoing urgent coronary artery bypass graft surgery. Real-time polymerase chain reaction demonstrated significant upregulation of angiopoietin-2 (Ang-2) in ischemic myocardium, to a greater extent than other classical angiogenic factors. Microarray gene profiling identified Ang-2 to be among the top 10 differentially upregulated genes, in addition to genes involved in inflammation, cell signalling, remodelling and apoptosis. The present document is the first report of microarray analysis of patients with ACS, and supports an important role for Ang-2 in the angiogenic response to severe ischemia in the human heart. Common gene expression patterns in ACS may provide opportunities for targeted pharmacological and cellular intervention.

Erythropoietin, a hypoxia-regulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells

The ability of erythropoietin (EPO) to elicit a pro-angiogenic effect on human mesenchymal stem cells (hMSC) was tested. hMSC are currently under study as therapeutic delivery agents that target tumor vessels. Hypoxia favors the differentiation of hMSC towards a pro-angiogenic program. However, the classical angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, are not fully capable of restoring this effect. The hypoxia-regulated factor, EPO, induces angiogenesis in endothelial cells. Here, EPO's pro-angiogenic effect on hMSC was analyzed. hMSC were tested for EPO receptor expression by western blot, immunofluorescence, and flow cytometry assays. Downstream receptor signaling components JAK and STAT were measured by standard assays. Pro-angiogenesis effects mediated by EPO treatment of hMSC were measured by proliferation, cytokine, or pro-angiogenesis factor secretion, metalloprotease activation, migration, invasion, wound healing, and tubule formation assays. hMSC express the cognate EPO receptor and are capable of promoting angiogenesis following EPO treatment in all the angiogenesis assays tested. EPO-treated hMSC proliferate and secrete pro-angiogenesis factors more readily than untreated hMSC. EPO leads to increased hMSC chemotaxis, migration, and activation of matrix metalloprotease-2. This treatment causes greater recruitment of vessels as measured in an in vivo angiogenesis assay. EPO is capable of eliciting a pro-angiogenesis program in hMSC that instigates secretion of angiogenic factors and the subsequent recruitment of endothelium. This study defines a novel mechanism for tumor cell recruitment of blood vessels that is important to consider in the design of stem cell-based therapies.

Ghrelin inhibits in vitro angiogenic activity of rat brain microvascular endothelial cells

Ghrelin, a 28-amino acid peptide originally isolated from rat stomach, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Evidence has been provided that ghrelin and GHS-Rs are highly expressed in the cardiovascular system, including endothelial cells (ECs), of which they regulate the growth in vitro. It, therefore, seemed worthwhile to investigate the effect of ghrelin on in vitro angiogenesis, using cultures of rat ECs derived from brain microvessels (neuromicrovascular ECs, NECs). ECs, when cultured on a supportive matrix, form a network of tubule-like structures, and such process is enhanced by the classic angiogenic factors, including fibroblast growth factor-2 (FGF-2). After seeding on Matrigel-coated wells, NECs formed within 18 h a meshwork of capillary-like structures; vinblastine (2 x 10(-12) M) disrupted the meshwork, while FGF-2 (50 ng/ml) increased its density. Ghrelin (10(-8) M) exerted a vinblastine-like effect and counteracted the stimulatory action of FGF-2. Computerized image-analysis confirmed these observations. FGF-2 enhanced the proliferation rate and lowered the apoptotic rate of NECs cultured on plastic wells, and ghrelin exerted opposite effects and completely reversed the proliferogenic and antiapoptotic actions of FGF-2. In contrast to vinblastine, ghrelin did not increase lactate dehydrogenase release from cultured NECs, thereby ruling out the possibility that its effects may ensue from an aspecific cytotoxic action. FGF-2 enhanced tyrosine kinase (TK) and mitogen activated protein kinase (MAPK) p42/p44 activities of NECs. Ghrelin significantly decreased TK and MAPK p42/p44 activities and effectively counteracted the effect of FGF-2. Taken together, the present findings indicate that ghrelin exerts a marked in vitro antiangiogenic action, and that the mechanism underlying this effect involves the inhibition of TK/MAPK-dependent cascades.

Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth.

We have found through ex vivo and in vivo angiogenesis models that the adrenomedullin gene-related peptide, proadrenomedullin NH2-terminal 20 peptide (PAMP), exhibits a potent angiogenic potential at femtomolar concentrations, whereas classic angiogenic factors such as vascular endothelial growth factor and adrenomedullin mediate a comparable effect at nanomolar concentrations. We found that human microvascular endothelial cells express PAMP receptors and respond to exogenous addition of PAMP by increasing migration and cord formation. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. In addition, the peptide fragment PAMP(12-20) inhibits tumor cell-induced angiogenesis in vivo and reduces tumor growth in xenograft models. Together, our data demonstrate PAMP to be an extremely potent angiogenic factor and implicate this peptide as an attractive molecular target for angiogenesis-based antitumor therapy.

Role of chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction, a key regulator of endothelial cell responses.

Chemokines are small proteins that act as cell attractants via the activation of G protein-coupled receptors. Chemokines play an important role in several pathophysiological processes such as inflammation and immunity. Many proinflammatory chemokines also support the development of vascular blood supply at the site of inflammation. Similarly, tumor-generated chemokines can contribute to tumor growth by promoting angiogenesis. Recently, significant advances have been made in understanding the contribution of chemokines to the angiogenesis process. This review will discuss first the evidence supporting the direct contribution of different chemokine subfamily members, including CC, CXC, and CX3C chemokines, as positive or negative regulators of the angiogenesis process based on the expression of their cognate receptors on endothelial cells. Additionally, the relationship between classic angiogenic factors and chemokine receptor expression on endothelial cells, and the implications of chemokine production by cancer cells will be analyzed with particular emphasis on the CXCL12/stromal-cell derived factor-1 interaction with CXCR4.

Oncostatin M induces angiogenesis in vitro and in vivo.

Neovascularization of the atherosclerotic plaque is responsible for its weakening and consequently for the complications of vascular disease. Macrophages are a source of growth factors that can modulate angiogenesis. In this study, we analyzed the effect of oncostatin M (OSM) on angiogenesis, as it could be involved in the development of atherosclerosis. The effect of OSM was compared with those of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). On human dermal microvasculature endothelial cells (HMEC-1s), OSM (22.5 to 112.5 pmol/L) induced a dose-dependent increase in cell proliferation greater than that induced by the classic angiogenic factors vascular endothelial growth factor (VEGF; 543 pmol/L) and basic fibroblast growth factor (bFGF; 1.1 nmol/L). LIF (19 to 475 pmol/L) induced only a 30% increase in cell proliferation, and IL-6 had no effect. Furthermore, in a modified Boyden-chamber model, OSM, LIF, and IL-6 were chemoattractant for HMEC-1s. In a tridimensional gel of fibrin, OSM increased tube formation and tube length, which were already noticeable by day 3. LIF and IL-6 induced a weaker effect that was only obvious by day 10. The angiogenic effect of OSM was also demonstrated in vivo in a rabbit corneal model: OSM was more potent than LIF, the length of the neovessels being longer with OSM than with LIF, whereas IL-6 was without effect. We tested factors that could be involved in the proliferative effect of OSM on HMEC-1s. OSM induced only a slight increase in the urokinase receptor and a 60% increase in VEGF secretion, whereas it does not modify IL-8 secretion or bFGF levels. The effect of OSM seems to depend on endothelial cell origin and cell species: OSM (up to 112.5 pmol/L) did not induce human umbilical vein endothelial cell proliferation and even had a small inhibitory effect (17%) on calf pulmonary artery endothelial cells. In conclusion, OSM induces an angiogenic effect on capillary endothelial cells, which could be, at least in part, implicated in pathological processes such as atherosclerosis or tumor growth.