The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.

Vascular endothelial growth factor (VEGF)mRNA undergoes alternative splicing events that generate four different homodimeric isoforms, VEGF121, VEGF165, VEGF189, or VEGF206. VEGF121 is a nonheparin-binding acidic protein, which is freely diffusible. The longer forms, VEGF189 or VEGF206, are highly basic proteins tightly bound to extracellular heparin-containing proteoglycans. VEGF165 has intermediate properties. To determine the localization of VEGF isoforms, transfected human embryonic kidney CEN4 cells expressing VEGF165, VEGF189, or VEGF206 were stained by immunofluorescence with a specific monoclonal antibody. The staining was found in patches and streaks suggestive of extracellular matrix (ECM). VEGF165 was observed largely in Golgi apparatus-like structures. Immunogold labeling of cells expressing VEGF189 or VEGF206 revealed that the staining was localized to the subepithelial ECM. VEGF associated with the ECM was bioactive, because endothelial cells cultured on ECM derived from cells expressing VEGF189 or VEGF206 were markedly stimulated to proliferate. In addition, ECM-bound VEGF can be released into a soluble and bioactive form by heparin or plasmin. ECM-bound VEGF189 and VEGF206 have molecular masses consistent with the intact polypeptides. The ECM may represent an important source of VEGF and angiogenic potential.     

VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise.

VEGF-A contributes to muscle tissue angiogenesis following aerobic exercise training. The temporal response of the VEGF-A isoforms and their target receptors has not been comprehensively profiled in human skeletal muscle. We combined submaximal exercise with and without reduced leg blood flow to establish whether ischemia-induced metabolic stress was an important physiological stimuli responsible for regulating the VEGF-A system in humans. Nine healthy men performed two 45-min bouts of one-leg knee-extension exercise, with and without blood flow restriction. Muscle biopsies were obtained at rest and 2 and 6 h after exercise. Expression (mRNA) of the VEGF-A splice variants and related receptors [VEGF receptor (VEGFR)-1, VEGFR-2, and neuropilin-1] was determined by using qPCR. VEGF-A(total) expression increased more robustly after exercise with reduced blood flow, and initially this principally reflected an increase in VEGF-A(165). Six hours after exercise, there was a relatively greater increase in VEGF-A(189), and this response was not influenced by blood flow conditions. VEGFR-1 mRNA expression increased 2 h after exercise, and neuropilin-1 expression was transiently reduced, while all three receptors increased by 6 h. There was no evidence for the expression of the inhibitory VEGF-A(165B) variant in human skeletal muscle. Our study, reflecting both VEGF-A ligand and receptors, implicates metabolic perturbation as a regulator of human muscle angiogenesis and demonstrates that VEGF-A splice variants are distinctly regulated. Our findings also indicate that all three receptor genes exhibit different pretranslational regulation, in response to exercise in humans.     

Vascular endothelial growth factor from embryonic status to cardiovascular pathology

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF₁₂₁, VEGF₁₄₅, and VEGF₁₆₅ are secreted whereas VEGF_183, VEGF₁₈₉, and VEGF₂₀₆ are cell membrane-bound. VEGF₁₄₅ has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF₁₆₅ is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases. Key Words: Vascular endothelial growth factor (VEGF), Vascular pathogenesis     

Prostaglandin E2 induces degranulation-independent production of vascular endothelial growth factor by human mast cells

Mast cells accumulate in large numbers at angiogenic sites, where they have been shown to express a number of proangiogenic factors, including vascular endothelial growth factor (VEGF-A). PGE(2) is known to strongly promote angiogenesis and is found in increased levels at sites of chronic inflammation and around solid tumors. The expression pattern of VEGF and the regulation of VEGF-A by PGE(2) were examined in cord blood-derived human mast cells (CBMC). CBMC expressed mRNA for five isoforms of VEGF-A and other members of the VEGF family (VEGF-B, VEGF-C, and VEGF-D) with strong expression of the most potent secretory isoforms. PGE(2) was a very strong inducer of VEGF-A(121/165) production by CBMC and also elevated VEGF-A mRNA expression. The amount of VEGF-A(121/165) protein production induced by PGE(2) was 4-fold greater than that induced by IgE-mediated activation of CBMC. Moreover, the response to PGE(2) as well as to other cAMP-elevating agents such as forskolin and salbutamol was observed under conditions that were not associated with mast cell degranulation. CBMC expressed substantial levels of the EP(2) receptor, but not the EP(4) receptor, when examined by flow cytometry. In contrast to other reported PGE(2)-mediated effects on mast cells, VEGF-A(121/165) production occurred via activation of the EP(2) receptor. These data suggest a role for human mast cells as a potent source of VEGF(121/165) in the absence of degranulation, and may provide new opportunities to regulate angiogenesis at mast cell-rich sites.     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones that overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knock down NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared with V189shCtl, under stress conditions. Unexpectedly, NRP1 knockdown had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity toward NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.Key words: VEGF isoforms, survival, apoptosis, NRP-1, breast cancer cells     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.     

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.     

Human lactiferous mammary gland cells produce vascular endothelial growth factor (VEGF) and express the VEGF receptors,Flt-1 AND KDR/Flk-1

Human milk contains a variety of growth factors. Recently, it was reported that vascular endothelial growth factor (VEGF) was one of them. We investigated milk VEGF isoforms, their functions, and VEGF receptors on mammary gland epithelial cells (MEC). The VEGF concentration in human milk was 74.3+/-34.9ng/ml on the first day after delivery, and rapidly decreased in a couple of days to 6.2+/-2.3ng/ml on the fifth day, and matured milk maintained about 4ng/ml. In an MTT assay, human milk accelerated HUVEC proliferation and MV303, a neutralizing antibody of VEGF, blocked 17.3 % of the effect. Immunoprecipitation and Western blotting showed that VEGF121 and VEGF165 were contained in human colostrums, and RT-PCR of human MEC confirmed that VEGF121, VEGF165 and VEGF189 were present. By immunostaining of human breast tissues, RT-PCR of MEC from human colostrum and measurement of the VEGF concentrations of conditioned media of cultured human MEC, it was confirmed that VEGF was produced by MEC. MEC was also expressed VEGF receptors, flt-1 and Flk-1/KDR. These results speculate us that the existence of autocrine or paracrine system within breast tissue via VEGF receptors on MEC and have a role in lactation.     

Effect of acute exercise and exercise training on VEGF splice variants in human skeletal muscle

The present study investigated the effect of an acute exercise bout on the mRNA response of vascular endothelial growth factor (VEGF) splice variants in untrained and trained human skeletal muscle. Seven habitually active young men performed one-legged knee-extensor exercise training at an intensity corresponding to approximately 70% of the maximal workload in an incremental test five times/week for 4 wk. Biopsies were obtained from the vastus lateralis muscle of the trained and untrained leg 40 h after the last training session. The subjects then performed 3 h of two-legged knee-extensor exercise, and biopsies were obtained from both legs after 0, 2, 6, and 24 h of recovery. Real-time PCR was used to examine the expression of VEGF mRNA containing exon 1 and 2 (all VEGF isoforms), exon 6 or exon 7, and VEGF(165) mRNA. Acute exercise induced an increase (P < 0.05) in total VEGF mRNA levels as well as VEGF(165) and VEGF splice variants containing exon 7 at 0, 2, and 6 h of recovery. The increase in VEGF mRNA was higher in the untrained than in the trained leg (P < 0.05). The results suggest that in human skeletal muscle, acute exercise increases total VEGF mRNA, an increase that appears to be explained mainly by an increase in VEGF(165) mRNA. Furthermore, 4 wk of training attenuated the exercise-induced response in skeletal muscle VEGF(165) mRNA.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.     

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.