Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

Signaling via vascular endothelial growth factor receptors

Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1-3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1.     

Immunolocalization of vascular endothelial growth factor in the GH3 cell line

The question of whether vascular endothelial growth factor (VEGF) is expressed in the GH3 cell line was investigated using immunocytochemistry, immunoelectron microscopy, and Western blotting. Using immunocytochemistry, VEGF was demonstrated in approximately 90% of the cells. Immunopositivity was localized mainly in the paranuclear Golgi region. In a small minority of cells, diffuse cytoplasmic immunostaining was also noted. By immunoelectron microscopy VEGF was evident in the secretory granules, cytoplasmic vesicles, rough endoplasmic reticulum, and the Golgi apparatus. Western blotting confirmed the results of the morphologic studies. It can be concluded that VEGF, which is know to induce angiogenesis and to increase vascular permeability, is produced in the prolactin- and growth hormone (GH)-secreting GH3 cell line. The functional role of VEGF in the GH3 cells is unknown. It is possible that this growth factor affects endocrine activity of GH3 cells by a paracrine mechanism.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Immunolocalization of vascular endothelial growth factor in rat condylar cartilage during postnatal development

It is well known that angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. To address angiogenesis of endochondral ossification in the condyle, we examined the appearance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in condylar cartilage of the growing rat. The early expression of VEGF at various sites during condylar cartilage development indicates that VEGF plays a role in the regulation of angiogenesis at each site of bone formation. From the findings of Flt-1 immunoreactivity, the VEGF produced by the chondrocytes of the hypertrophic zone should contribute to the promotion of endothelial cell proliferation and to stimulate migration and activation of osteoclasts in condylar cartilage, resulting in the invasion of these cells into the mineralized zone.Junko Aoyama and Eiji Tanaka contributed equally to this work     

Signal transduction by vascular endothelial growth factor receptors

VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Characterization of the receptors for vascular endothelial growth factor

Vascular endothelial growth factor (vEGF) is a recently discovered mitogen for endothelial cells. It is also a potent angiogenic factor. We have characterized the vEGF receptors of endothelial cells using both binding and cross-linking techniques. Scatchard analysis of equilibrium binding experiments revealed two types of high-affinity binding sites on the cell surfaces of bovine endothelial cells. One of the sites has a dissociation constant of 10(-12) M and is present at a density of 3 x 10(3) receptors/cell. The other has a dissociation constant of 10(-11) M, with 4 x 10(4) receptors/cell. A high molecular weight complex containing 125I-vEGF is formed when 125I-vEGF is cross-linked to bovine endothelial cells. This complex has an apparent molecular mass of 225 kDa. Two other faintly labeled complexes with apparent molecular masses of 170 and 195 kDa also are detected. Reduction in the presence of dithiothreitol causes a substantial increase in the labeling intensity of the 170- and 195-kDa complexes, suggesting that these complexes are derived from the 225-kDa complex by reduction of disulfide bonds. The labeling of the vEGF receptors was inhibited by an excess of unlabeled vEGF but not by high concentrations of several other growth factors. Suramin and protamine, as well as several species of lectins, inhibited the binding. The expression of functional vEGF receptors was inhibited when the cells were preincubated with tunicamycin, indicating that glycosylation of the receptor is important for the expression of functional vEGF receptors. Pretreatment with swainsonine on the other hand, did not prevent formation of functional receptors. However, the mass of the 225-kDa complex is decreased by 20 kDa when 125I-vEGF is cross-linked to swainsonine-treated endothelial cells.     

Transforming growth factor-beta(1), -beta(2), -beta(3), basic fibroblast growth factor and vascular endothelial growth factor expression in keratinocytes of burn scars

Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.     

Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.

Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we investigated the role of nitric oxide (NO) for the expression of vascular endothelial growth factor (VEGF), which represents the most important angiogenic factor during the proliferative phase of skin repair. Since keratinocytes are the major source of VEGF production during this process, we used cultured keratinocytes (HaCaT cell line) as an in vitro model to investigate NO action on growth factor- and cytokine-stimulated VEGF expression. Exogenously added NO enhanced transforming growth factor-beta1-, keratinocyte growth factor-, interleukin-1beta-, tumor necrosis factor-alpha-, and interferon-gamma-induced VEGF mRNA and protein synthesis in keratinocytes. We could demonstrate that high-level expression of cytokine-induced VEGF mRNA in keratinocytes is dependent on endogenously produced NO, as inhibition of the coinduced iNOS by N(G)-monomethyl-L-arginine (L-NMMA) markedly decreased cytokine-triggered VEGF mRNA levels in the cells. We also established an in vivo model in mice to investigate the role of NO during wound healing. During excisional wound repair, mice were treated with L-N(6)-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared to control mice, L-NIL-treated animals were characterized by markedly reduced VEGF mRNA levels during the inflammatory phase of repair. Immunohistochemistry demonstrated reduced VEGF protein expression and a completely disorganized pattern of VEGF-expressing keratinocytes within the hyperproliferative epithelium at the wound edge in L-NIL-treated mice. We demonstrate that triggering of VEGF expression is a crucial molecular mechanism underlying NO function during wound healing.     

Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor receptors in lung cancer

OBJECTIVE: To determine the expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor receptors (FGFR), in non-small cell lung carcinoma (NSCLC) in relation to tumor stage (TN0, TN1, TN2) and in association with the expression of p53 protein, a potential suppressor of tumor angiogenesis. STUDY DESIGN: The immunohistochemical (IHC) expression of VEGF and FGFR was examined in paraffin sections of 56 NSCLC in relation to the presence of lymph node metastases and p53 expression. Nodal status of NSCLC determined: 27 tumors, N0; 16, N1; and 13, N2 stage. Semiquantitative analysis with a score corresponding to IHC staining intensity and percentage of positive cells was used. Statistical analysis was performed with the chi 2 test. RESULTS: A significant association was noted between VEGF and FGFR expression in NSCLC. No relation was found between VEGF, FGFR expression and lymph node metastasis or p53 expression. CONCLUSION: We assume that VEGF and FGFR act in a synergistic manner in NSCLC and that their expression is not related to lymph node metastases. Angiogenesis is a very complex phenomenon and heterogeneous within tumors. Also, it is affected by microenviromental factors.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Vasoactive intestinal peptide (VIP) increases vascular endothelial growth factor (VEGF) expression and secretion in human breast cancer cells

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.     

Effect of heme and heme oxygenase-1 on vascular endothelial growth factor synthesis and angiogenic potency of human keratinocytes

BACKGROUND: Skin injury leads to the release of heme, a potent prooxidant which is degraded by heme oxygenase-1 (HO-1) to carbon monoxide, iron, and biliverdin, subsequently reduced to bilirubin. Recently the involvement of HO-1 in angiogenesis has been shown; however, the role of heme and HO-1 in wound healing angiogenesis has not been yet investigated. RESULTS: Treatment of HaCaT keratinocytes with hemin (heme chloride) induced HO-1 expression and activity. The effect of heme on vascular endothelial growth factor (VEGF) synthesis is variable: induction is significant after a short, 6 h treatment with heme, while longer stimulation may attenuate its production. The involvement of HO-1 in VEGF synthesis was confirmed by inhibition of VEGF expression by SnPPIX, a blocker of HO activity and by attenuation of HO-1 mRNA expression with specific siRNA. Importantly, induction of HO-1 by hemin was able to overcome the inhibitory effect of high glucose on VEGF synthesis. Moreover, HO-1 expression was also induced in keratinocytes cultured in hypoxia, with concomitant augmentation of VEGF production, which was further potentiated by hemin stimulation. Accordingly, conditioned media from keratinocytes overexpressing HO-1 enhanced endothelial cell proliferation and augmented formation of capillaries in angiogenic assay in vitro. CONCLUSIONS: HO-1 is involved in hemin-induced VEGF expression in HaCaT and may play a role in hypoxic regulation of this protein. HO-1 overexpression may be beneficial in restoring the proper synthesis of VEGF disturbed in diabetic conditions.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.