VEGF,not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia

The purpose of this study was to determine the relationship between VEGF and mini-TyrRS/mini-TrpRS in angiogenesis in hypoxic culture and to begin to comprehend their mechanism in angiogenesis. We designed a VEGF gene silencing assay by using lentivirus vectors, and then western blotting was used to determine the protein expression of VEGF, VEGFR2 and pVEGFR2 in three groups in hypoxic culture at 3, 6, 12, or 24 h: (1) untransfected human umbilical vein endothelial cells (HUVECs) (Control); (2) pGCSIL-GFP lentivirus vector-transduced HUVECs (Mock); and (3) pGCSIL-shVEGF lentivirus vector-transduced HUVECs (Experimental). We also detected the effects of mini-TyrRS/mini-TrpRS peptides on HUVEC proliferation, migration and tube formation after lentivirus vector transfection and VEGFR2 antibody injection. The results indicated that expression of the mini-TyrRS protein was increased, whereas that of mini-TrpRS was specifically decreased in hypoxic culture both in control and mock groups. However, this trend in protein levels of mini-TyrRS and mini-TrpRS was lost in the experimental group after transduction with the pGCSIL-shVEGF lentivirus vector. The protein expression of VEGF was increased in hypoxic culture both in control and mock groups. After transduction with the pGCSIL-shVEGF lentivirus vector, the protein level of VEGF was noticeably decreased in the experimental group; however, for VEGFR2, the results showed no significant difference in VEGFR2 protein expression in any of the groups. For pVEGFR2, we found a distinct trend from that seen with VEGF. The protein expression of pVEGFR2 was sharply increased in hypoxic culture in the three groups. The addition of mini-TyrRS significantly promoted proliferation, migration and tube formation of HUVECs, while mini-TrpRS inhibited these processes in both control and mock groups in hypoxic culture. However, these effects disappeared after transduction with the pGCSIL-shVEGF lentivirus vector in the experimental group, but no significant difference was observed after VEGFR2 antibody injection. The protein expression of VEGF is similar to that of mini-TyrRS in hypoxic culture and plays an important role in the mini-TyrRS/mini-TrpRS-stimulated proliferation, migration and tube formation of HUVECs in hypoxia. These results also suggest that the change in mini-TyrRS and mini-TrpRS expression in hypoxic culture is not related to VEGFR2 and that some other possible mechanisms, are involved in the phosphorylation of VEGFR2.     

PGE(2) stimulates VEGF expression in endothelial cells via ERK2/JNK1 signaling pathways

Vascular endothelial growth factor (VEGF) plays an essential role in the initiation and regulation of angiogenesis-a crucial component of wound healing and cancer growth. Prostaglandins (PGs) stimulate angiogenesis but the precise mechanisms of their pro-angiogenic actions remain unexplained. We investigated whether prostaglandin E(2) (PGE(2)) can induce VEGF expression in rat gastric microvascular endothelial cells (RGMEC) and the signaling pathway(s) involved. We demonstrated that PGE(2) significantly increased ERK2 and JNK1 activation and VEGF mRNA and protein expression. Incubation of RGMEC with PD 98059 (MEK kinase inhibitor) significantly reduced PGE(2)-induced ERK2 activity, VEGF mRNA and protein expression. Furthermore, PD 98059 treatment almost completely abolished JNK1 activation. Our data suggest that PGE(2)-stimulates VEGF expression in RGMEC via transactivation of JNK1 by ERK2. One potential implication of this finding is that increased PG levels in cancers could facilitate tumor growth by stimulating VEGF synthesis and angiogenesis.     

Piperine,a dietary phytochemical,inhibits angiogenesis

Angiogenesis plays an important role in tumor progression. Piperine, a major alkaloid constituent of black pepper, has diverse physiological actions including killing of cancer cells; however, the effect of piperine on angiogenesis is not known. Here we show that piperine inhibited the proliferation and G₁/S transition of human umbilical vein endothelial cells (HUVECs) without causing cell death. Piperine also inhibited HUVEC migration and tubule formation in vitro, as well as collagen-induced angiogenic activity by rat aorta explants and breast cancer cell-induced angiogenesis in chick embryos. Although piperine binds to and activates the cation channel transient receptor potential vanilloid 1 (TRPV1), its effects on endothelial cells did not involve TRPV1 since the antiproliferative effect of piperine was not affected by TRPV1-selective antagonists, nor did HUVECs express detectable TRPV1 mRNA. Importantly, piperine inhibited phosphorylation of Ser 473 and Thr 308 residues of Akt (protein kinase B), which is a key regulator of endothelial cell function and angiogenesis. Consistent with Akt inhibition as the basis of piperine's action on HUVECs, inhibition of the phosphoinositide-3 kinase/Akt signaling pathway with LY-294002 also inhibited HUVEC proliferation and collagen-induced angiogenesis. Taken together, these data support the further investigation of piperine as an angiogenesis inhibitor for use in cancer treatment.     

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.     

Microvessel formation from mouse embryonic aortic explants is oxygen and VEGF dependent

To delineate the roles of O(2) and vascular endothelial growth factor (VEGF) in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O(2), but not room air, the addition of VEGF to explants stimulated the formation of CD31-positive, Flk-1-positive, Gs-IB(4)-positive structures in a concentration-dependent manner. Electron microscopy showed the structures to be capillary-like. VEGF-induced capillary-like structure formation was inhibited by sequestration of VEGF via addition of soluble Flt-1 fusion protein or anti-VEGF antibodies. Expression of Flk-1, but not Flt-1, was increased in embryonic aorta cultured under 5% O(2) relative to room air. Our data suggest that low O(2) upregulates Flk-1 expression in embryonic aorta in vitro and renders it more responsive to VEGF.     

Dopamine in vivo inhibits VEGF-induced phosphorylation of VEGFR-2, MAPK, and focal adhesion kinase in endothelial cells

Vascular permeability factor (VPF)/VEGF is a potent multifunctional cytokine and growth factor that has critical roles in vasculogenesis and in both physiological and pathological angiogenesis. Because it has been recently shown that the neurotransmitter dopamine at pharmacological dose can inhibit VEGF/VPF-mediated microvascular permeability, proliferation, and migration of endothelial cells in vitro, we therefore hypothesized that endogenous dopamine may regulate the actions of VPF/VEGF in vivo. We report that VPF/VEGF-induced phosphorylation of VEGF receptor 2, focal adhesion kinase, and MAPK in the endothelial cells is strikingly increased in both dopamine-depleted and dopamine D(2) receptor knockout mice compared with normal controls, thereby indicating that endogenous dopamine regulate these critical signaling cascades required for the in vivo endothelial functions of VPF/VEGF. Together, these observations provide new mechanistic insight into the dopamine-mediated inhibition of the activities of VPF/VEGF and suggest that endogenous neurotransmitter dopamine might be an important physiological regulator of VPF/VEGF activities in vivo.     

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases.

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.     

LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs

LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.     

Ferulic acid augments angiogenesis via VEGF,PDGF and HIF-1α

Therapeutic angiogenesis is critical to wound healing and ischemic diseases such as myocardial infarction and stroke. For development of therapeutic agents, a search for new angiogenic agents is the key. Ferulic acid, a phytochemical found in many fruits and vegetables, exhibits a broad range of therapeutic effects on human diseases, including diabetes and cancer. This study investigated the augmenting effect of ferulic acid on angiogenesis through functional modulation of endothelial cells. Through endothelial cell migration and tube formation assays, ferulic acid (10^?6–10^?4 M) was found to induce significant angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro without cytotoxicity. With chorioallantoic membrane assay, ferulic acid (10^?6–10^?5 M) was also found to promote neovascularization in vivo. Using Western blot analysis and quantitative real-time polymerase chain reaction, we found that ferulic acid increased vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression in HUVECs. Furthermore, the amounts of hypoxic-induced factor (HIF) 1α mRNA and protein, the major regulator of VEGF and PDGF, also showed up-regulation by ferulic acid. Electrophoretic migration shift assay showed that the binding activity of HIF-1α was also enhanced with ferulic acid treatment of HUVECs. Moreover, inhibitors of extracellular-signal-regulated kinase 1/2 and phosphoinositide-3 kinase (PI3K) abolished the binding activity of HIF-1α and the subsequent activation of VEGF and PDGF production by ferulic acid. Thus, both mitogen-activated protein kinase and PI3K pathways were involved in the angiogenic effects of ferulic acid. Taken together, ferulic acid serves as an angiogenic agent to augment angiogenesis both in vitro and in vivo. This effect might be observed through the modulation of VEGF, PDGF and HIF-1α.     

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.     

血管内皮细胞和心脏组织块的立体培养

本文旨在对比研究二维平面与三维立体培养模式下,内皮细胞和心脏组织形态学的差异。采用胶内、胶上、三明治模式、玻片培养小室模型等多种I型胶原立体培养模型,通过免疫荧光技术及显微形态学观察组织和细胞的生长情况。在二维平面培养中,原代心脏血管内皮细胞呈铺路石样排列;而在三维胶原培养模式中,内皮细胞呈长梭状形态,并迁入胶原培养介质中,和体内血管新生及血管生成过程中的内皮细胞活化表型相似。加入血管内皮生长因子(vascular endo- thelial growth factor VEGF)能增强内皮细胞管状结构的形成。在三维胶原中,心脏组织块生长良好,迁出的细胞将相邻组织块连接起来,组织块有自发的搏动。本工作表明,改进的薄层胶原培养、玻片培养小室模型和动脉条模型是较好的研究血管生成和血管新生的工具。在三维培养的情况下,内皮细胞通过空间增殖、迁移和锚定,可形成管状结构,比二维平面培养更适合用于血管新生的研究。不同的立体培养模型可用于不同目的的研究。     

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

Hepatocyte growth factor is a major mediator in heparin-induced angiogenesis

Heparin has a potent angiogenic effect in experimental animals and patients with ischemic diseases; however, the precise mechanism behind this angiogenesis remains to be clarified. The aim of this study was to determine whether the administration of heparin affects the levels of heparin-binding angiogenic factors in human plasma, and to identify the molecule responsible for heparin-induced angiogenesis. Plasma levels of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were measured before and after administration of 100 U, 3,000 U or 10,000 U of heparin in patients with coronary artery disease. Administration of 3,000 U or 10,000 U of heparin caused significant increases in plasma HGF (40- and 54-fold, respectively), in absence of obvious increases in bFGF and VEGF levels. Furthermore, compared with the serum collected before heparin administration, the serum collected after heparin administration had more prominent growth-promoting and vascular tube-inducing properties on endothelial cells, and these increased activities were completely inhibited by neutralization of HGF, whereas neutralization of bFGF and VEGF had no effect. These findings suggest that HGF plays a significant role in heparin-induced angiogenesis.     

Angiogenesis-inflammation cross-talk: vascular endothelial growth factor is secreted by activated T cells and induces Th1 polarization

Vascular endothelial growth factor (VEGF) and its receptors are critical in angiogenesis. The main player in the secretion and response to VEGF is the endothelial cell. We initiated this study to test whether T cells can secrete VEGF and are able to respond to it. Here we show that VEGF is secreted by T cells on stimulation by specific Ag or by IL-2 and by hypoxia; thus, activated T cells might enhance angiogenesis. Hypoxia also induced the expression in T cells of VEGFR2, suggesting that T cells might also respond to VEGF. Indeed, VEGF augmented IFN-gamma and inhibited IL-10 secretion by T cells responding to mitogen or Ag; thus, VEGF can enhance a Th1 phenotype. Encephalitogenic T cells stimulated in the presence of VEGF caused more severe and prolonged encephalomyelitis. Thus, T cells can play a role in angiogenesis by delivering VEGF to inflammatory sites, and VEGF can augment proinflammatory T cell differentiation.     

Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O(2), but not room air, the addition of FGF2 to explants stimulated the formation of Gs-IB(4-)positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% O(2) relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2.     

EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.     

Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibiting the matrix-degrading proteases

Flavonoids have been proposed to act as chemopreventive agents in numerous epidemiological studies and have been shown to inhibit angiogenesis and proliferation of tumor cells and endothelial cells in vitro. Angiogenesis requires tightly controlled extracellular matrix degradation mediated by extracellular proteolytic enzymes including matrix metalloproteinases (MMPs) and serine proteases, in particular, the urokinase-type plasminogen activator (uPA)-plasmin system. In this study, we have investigated the antiangiogenic mechanism of the flavonoids, genistein, apigenin, and 3-hydroxyflavone in a human umbilical vein endothelial cell (HUVEC) model. The stimulation of serum-starved HUVECs with vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF) caused marked increase in MMP-1 production and induced the pro-MMP-2 activation accompanied by the increase in MT1-MMP expression. However, pretreatment with flavonoids before VEGF/bFGF stimulation completely abolished the VEGF/bFGF-stimulated increase in MMP-1 and MT1-MMP expression and pro-MMP-2 activation. Genistein blocked VEGF/bFGF-stimulated increase in TIMP-1 expression and decrease in TIMP-2 expression. Apigenin and 3-hydroxyflavone further decreased TIMP-1 expression below basal level and completely abolished TIMP-2 expression. VEGF and bFGF stimulation also significantly induced uPA expression, most strikingly the level of 33 kDa uPA, and increased the expression of PA inhibitor (PAI)-1. Genistein, apigenin, and 3-hydroxyflavone effectively blocked the generation of 33 kDa uPA, and further decreased the activity of the 55 kDa uPA and the expression of PAI-1 below the basal level. In conclusion, these data suggest that genistein, apigenin, and 3-hydroxyflavone inhibit in vitro angiogenesis, in part via preventing VEGF/bFGF-induced MMP-1 and uPA expression and the activation of pro-MMP-2, and via modulating their inhibitors, TIMP-1 and -2, and PAI-1.