VEGF-binding aptides and the inhibition of choroidal and retinal neovascularization

Age-related macular degeneration and diabetic retinopathy are leading causes of blindness. Vascular endothelial growth factor (VEGF) is known to be the main factor that induces pathological angiogenesis in these diseases. In this study, we investigate the therapeutic potential and safety profiles of high-affinity peptides targeting VEGF which are identified using an ‘aptide’ technology. We show that two VEGF-binding aptides, APT_VEGF1 and APT_VEGF2, demonstrate high binding affinity and specificity to VEGF. Furthermore, they suppress VEGF-induced activation of VEGF receptor-2, in vitro angiogenesis, and in vivo pathological choroidal and retinal neovascularization. Despite potent anti-angiogenic effects, both VEGF-binding aptides do not induce any definite toxicity at the level of cellular viability, histological integrity, and gene expression. Our data show the therapeutic potential of VEGF-binding peptides for the treatment of choroidal and retinal neovascularization.     

The role of VEGF in normal and neoplastic hematopoiesis

VEGF is a secreted growth factor that mediates its biological effects by binding to two transmembrane tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The VEGF/receptor signaling system is involved in the regulation of two fundamental processes in vertebrates: the formation of blood vessels (angiogenesis) and of blood cells (hematopoiesis). Hematopoietic stem cells, capable of giving rise to all blood cell lineages, are often found in clusters with endothelial cells, the key cell type involved in the formation of blood vessels. Despite such proximity of VEGF-responsive cells, hematopoiesis occurs independently of neoangiogenesis in the adult bone marrow, suggesting that VEGF regulates the two processes by different mechanisms. In support of this hypothesis, the recently identified autocrine loop by which VEGF may control hematopoietic stem cell survival and repopulation, is fundamentally different from its paracrine effects regulating angiogenesis. Furthermore, coexpression of VEGF and its receptors, the prerequisite for autocrine loops, is frequently found in lymphomas and myelomas, suggesting that autocrine loops also play a role in hematological malignancies. Several therapeutic strategies blocking VEGF or VEGF-induced signaling are currently being investigated for the treatment of neoplastic diseases. They differ in their potential to interfere with the autocrine or paracrine effector functions of VEGF during angiogenesis, hematopoiesis, and tumor cell proliferation, properties which may ultimately determine their therapeutic potential.     

Vascular Endothelial Growth Factor (VEGF) in Autoimmune Diseases

Vascular endothelial growth factor (VEGF) is a potent stimulating factor for angiogenesis and vascular permeability. There are eight isoforms with different and sometimes overlapping functions. The mechanisms of action are under investigation with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins, which were not previously associated to angiogenesis. VEGF has important physiological actions on embryonic development, healing, and menstrual cycle. It also has a great role in pathological conditions that are associated to autoimmune diseases. There is considerable evidence in various autoimmune diseases such as in systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis of an interrelationship between the VEGF system and theses disorders. Serum levels of VEGF correlate with disease activity in a large number of autoimmune diseases and fall with the use of standard therapy. We raised the possible future therapeutic strategies in autoimmune diseases with the anti-VEGF or anti-VEGFR (receptor). So far, this therapy has been used in cancer and macular ocular degeneration in diabetes. This review outlines the evidence for VEGF participation in various autoimmune diseases and proposes lines for future research in this field.     

血管内皮生长因子的生物学及其在临床的初步应用

血管内皮生长因子(VEGF)是内皮细胞特异性的有丝分裂原。它诱导内皮细胞增殖、促进内皮细胞迁移，并抑制内皮细胞凋亡，在调节血管和淋巴管新生中起重要作用。VEGF也是胚胎发育、软骨内骨形成、女性生殖系统、以及肿瘤和眼球内血管新生所必需的。另外，VEGF也可诱导血小板粘附于血管内皮细胞而出现高凝状态。目前，有许多临床实验正在评价VEGF在血管新生依赖性疾病的促血管新生作用和抗血管新生药物用于治疗的效果。     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

VEGF and vascular changes in chronic neuroinflammation

A vascular component has long been associated with the pathological changes in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). Despite the codependence of angiogenesis and many chronic inflammatory disorders, only circumstantial evidence is available to support the existence of angiogenesis in MS or EAE. To determine if angiogenesis occurs in conjunction with clinical and pathological signs of CNS inflammatory disease we evaluated temporal and spatial blood vessel counts, VEGF immunoreactivity, and histopathological changes in the spinal cord of guinea pigs with chronic-progressive (CP)-EAE (day 0-90 post-immunization, n=64) and controls (n=17). The number of vessels per section increased in infiltrated and demyelinated lesions by day 15 post-immunization and remained significantly higher than controls throughout the course of the disease. The number of vessels correlated with both clinical and pathological scores for inflammation, infiltration and demyelination. Vascular endothelial growth factor (VEGF) expression increased during acute disease peaking at day 26, which was the transition from the acute-inflammatory to chronic-demyelinating phase, before gradually returning to baseline levels. These observations implicate angiogenesis as a component of chronic neuroinflammation and demyelination and may suggest alternative therapeutic strategies for multiple sclerosis.     

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.     

Design of a variant of vascular endothelial growth factor-A (VEGF-A) antagonizing KDR/Flk-1 and Flt-1

Because of its central role in pathological angiogenesis, vascular endothelial growth factor (VEGF) has become a major target for anti-angiogenic therapies. We report here the construction of a heterodimeric antagonistic VEGF variant (HD-VEGF). In this antagonist, binding domains for the VEGF-receptors KDR/Flk-1 and Flt-1 are present at one pole of the dimer, whereas the other pole carries domain swap mutations, which prevent binding to either receptor. As HD-VEGF can only bind to monomeric receptors, it does not lead to signal transduction. Moreover, it antagonizes VEGF and possibly other members of the VEGF family, which are KDR/Flk-1 and Flt-1 ligands. We show here that HD-VEGF is a potent inhibitor of VEGF-mediated proliferation and tissue factor induction in endothelial cell cultures, requiring only a 20-fold and a 4-fold excess, respectively, to block the activity of wtVEGF completely. A 4-fold excess of HD-VEGF over wtVEGF was also sufficient to abrogate vascular permeability as determined in the Miles assay in vivo. Furthermore, HD-VEGF inhibited fetal bone angiogenesis in an ex vivo assay. Thus, HD-VEGF blocks KDR- and Flt-1-mediated VEGF activities that are crucial in the angiogenic process and is therefore a promising, multipotent compound in the treatment of angiogenesis-related diseases.     

The role of nuclear factor kappaB on angiogenesis regulation through monocyte chemotactic protein-1 in myeloma

Multiple myeloma is malignant proliferation of plasma cells and plasmacytoid cells. Vascular endothelial growth factor (VEGF) is known to be one of the most important if not the main regulator of physiologic and pathologic angiogenesis which triggers growth, survival and migration of myeloma cells. It has been shown that circulating mature or bone marrow driven endothelial precursor cells play an important role in neovascularisation. In accordance with these observations, current therapeutic approaches to myeloma include VEGF inhibitors. Since angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes many products with a different function it would be of benefit to determine the key molecule produced by transformed plasma cells which stimulates bone marrow environment to produce their homing "milieu" secreting different cytokines such as VEGF, IL-6, and monocyte chemotactic protein-1 (MCP-1). This molecule could be nuclear factor kappa B (NF-kappaB). It has been confirmed that myeloma cells express and produce NF-kappaB. It has been established recently that by blocking NF-kappaB production MCP-1 secretion is reduced up to 60%. If so, this would also reduce production of IL-6 and VEGF, since MCP-1 upregulates VEGF and IL-6 production. This way one could make bone marrow bad environment for myeloma cells to settle, followed with no disease progression. Targeting to NF-kappaB intended to inhibits its activation with receptor antagonist would possibly significantly inhibit lipopolysaccharide-induced IL-6, MCP-1 and TNF-alpha. All of them being stimulators for VEGF secretion and indirectly activation of angiogenesis. To conclude, angiogenesis could be induced by myeloma cells themselves through NF-kappaB activation pathway and by inhibiting its activation we might prevent myeloma expansion in bone marrow and progression of the disease by decreased MCP-1 secretion.     

Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.     

Vascular endothelial growth factor and endometriotic angiogenesis

Peritoneal endometriosis is a significant debilitating gynaecological problem of widespread prevalence. It is now generally accepted that the pathogenesis of peritoneal endometriosis involves the implantation of exfoliated endometrium. Essential for its survival is the generation and maintenance of an extensive blood supply both within and surrounding the ectopic tissue. The vascular endothelial growth factor (VEGF) family of angiogenic molecules is involved in both physiological angiogenesis, and a number of pathological conditions that are characterized by excessive angiogenesis. Increasing evidence suggests that the VEGF family may also be involved with both the aetiology and maintenance of peritoneal endometriosis. Sources of this factor include the eutopic endometrium, ectopic endometriotic tissue and peritoneal fluid macrophages. Important to its aetiology is the correct peritoneal environment in which the exfoliated endometrium is seeded and implants. Established ectopic tissue is then dependent on the peritoneal environment for its survival, an environment that supports angiogenesis. Our increasing knowledge of the involvement of the VEGF family in endometriotic angiogenesis raises the possibility of novel approaches to its medical management, with particular focus on the anti-angiogenic control of the action of VEGF.     

Angiogenesis: now and then

Angiogenesis or new blood vessel formation plays an essential role during embryogenesis, adult vascular remodeling and in several pathological disorders, as in tumor development. Although sprouting of blood vessels is the principal angiogenic mechanism, additional ones, such as the recruitment of bone marrow-derived cells, have recently been described. These processes are controlled by several molecules, although members of the VEGF family of angiogenic factors and its receptors seem to be the main mediators. Initially, VEGF receptors were described as endothelial specific; however, further studies have reported their presence in several types of cells of non-endothelial origin, such as tumor cells. This VEGF receptor altered expression has suggested an angiogenesis-independent growth advantage mechanism on certain types of cancers by the generation of autocrine loops. A possible role in tumorigenesis and a potential novel target in cancer therapy have been hypothesized. Detection of other receptors and molecules considered to be angiogenic players has also been observed on tumor cells. Currently, their clinical significance as well as their potential as therapeutic targets for the treatment of certain cancers is being evaluated, having in mind the future development of promising mechanism-based therapies. The aspects mentioned above are the main focus of this review, which aims to throw light on recent findings respecting angiogenesis and novel therapeutic approaches.     

血管内皮生长因子复合人工骨材料修复骨缺损

背景：由创伤、感染、肿瘤切除以及某些先天性疾病等所导致的四肢骨缺损的治疗仍是骨科临床最常见、最棘手的一大难题。 目的：文章综述了血管内皮生长因子的生物学特性、及其复合人工骨的作用机制、局部基因治疗在治疗骨缺损中的应用。 方法：应用计算机检索CNKI和PubMed数据库中1999-01/2009-12关于血管内皮生长因子和骨缺损修复相关的内容的文章,在标题和摘要中以“血管内皮生长因子,骨缺损,人工骨,基因治疗”或“VEGF,Bone defect,Bone substitute,Gene therapy”为检索词进行检索。选择文章内容与血管内皮生长因子和骨缺损修复相关,同一领域文献则选择近期发表或发表在权威杂志文章。 结果与结论：初检得到285篇文献,根据纳入标准选择45篇文章进行综述。血管内皮生长因子是增加血管渗透性和血管发生的特殊生长因子,在骨的生长、发育和重建中密切协调血管发生和骨发生的关系,而目前的人工骨材料只具有传导成骨的特性,所以改善人工骨材料中的血管生成可促进血运丰富骨组织的形成。     

Tumour proliferation, angiogenesis, and ploidy status in human colon cancer

AIMS: Tumour angiogenesis is essential for carcinogenesis and facilitates the process of tumour development and metastasis. Vascular endothelial growth factor (VEGF) is a well characterised angiogenetic factor and is known to play a crucial role in new vessel development. To gain further insight into the effects of microvessel density and VEGF expression in colon cancer, their relation with tumour proliferation, ploidy status, and p53 expression was investigated in colon cancer. METHODS: Tissue samples of 50 archived colon cancers were analysed by immunohistochemistry for VEGF, p53, and the endothelial cell marker, von Willebrand factor (VWF), using specific antibodies. The same samples were re-cut for flow cytometric studies to obtain S phase fraction (SPF) and ploidy status. RESULTS: A positive significant correlation was found between SPF and angiogenesis. The median microvessel count in high SPF tumours was significantly higher than in low SPF ones. No association was found between VEGF expression and SPF. A positive correlation was found between ploidy status and p53 expression and microvessel count. Furthermore, a positive correlation was established between DNA ploidy, VEGF expression, and microvessel count. CONCLUSION: This study provides evidence that in colon cancer, tumour growth may be stimulated by vascular supply, and the lack of a correlation between tumour cell proliferation and VEGF expression indicates that these two parameters may be regulated by separate mechanisms. Furthermore, the positive correlation between microvessel density, VEGF expression, and ploidy status provides more evidence that genetic alterations are involved in tumour angiogenesis.     

A differential effect of bone morphogenetic protein-2 and vascular endothelial growth factor release timing on osteogenesis at ectopic and orthotopic sites in a large-animal model

In bone tissue engineering, growth factors are widely used. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are the most well-known regulators of osteogenesis and angiogenesis. We investigated whether the timing of dual release of VEGF and BMP-2 influences the amount of bone formation in a large-animal model. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were loaded with BMP-2 or VEGF to create sustained-release profiles, and rapidly degrading gelatin was loaded with either growth factor for fast-release profiles. To study in vivo osteogenicity, the two delivery vehicles were combined with biphasic calcium phosphate (BCP) scaffolds and implanted in 10 Beagle dogs for 9 weeks, at both ectopic (paraspinal muscles) and orthotopic sites (critical-size ulnar defect). The 9 ectopic groups contained combined or single BMP/VEGF dosage, in sustained- or fast-release profiles. In the ulnae of 8 dogs, fast VEGF and sustained BMP-2 were applied to one leg, and the other received the opposite release profiles. The two remaining dogs received bilateral control scaffolds. Bone growth dynamics was analyzed by fluorochrome injection at weeks 3, 5, and 7. Postoperative and posteuthanization X-rays of the ulnar implants were taken. After 9 weeks of implantation, bone quantity and bone growth dynamics were studied by histology, histomorphometry, and fluorescence microscopy. The release of the growth factors resulted in both enhanced orthotopic and ectopic bone formation. Bone formation started before 3 weeks and continued beyond 7 weeks. The ectopic BMP-2 fast groups showed significantly more bone compared to sustained release, independent of the VEGF profile. The ulna implants revealed no significant differences in the amount of bone formed. This study shows that timing of BMP-2 release largely determines speed and amount of ectopic bone formation independent of VEGF release. Furthermore, at the orthotopic site, no significant effect on bone formation was found from a timed release of growth factors, implicating that timed-release effects are location dependent.     

血管内皮生长因子基因修饰骨髓间充质干细胞移植心肌梗死大鼠： 高压氧干预促进治疗性血管生成

背景：骨髓间充质干细胞移植入缺血心肌后梗死区周边心肌的缺血、缺氧、炎性反应、细胞凋亡等病理变化严重影响到移植骨髓间充质干细胞的存活,而高压氧能显著改善其缺氧状态。 目的：对心肌梗死模型大鼠缺血心肌局部移植血管内皮生长因子修饰的骨髓间充质干细胞,在移植后对大鼠进行高压氧干预,探讨其对骨髓间充质干细胞移植后所获得的治疗性血管生成效应的影响。 方法：将真核表达载体pcDNA3.1(-)/人血管内皮生长因子165转染大鼠骨髓间充质干细胞,再移植至心肌梗死模型大鼠的缺血区组织,移植前细胞行CM-DiI标记。移植后2周每日对大鼠行高压氧治疗干预。移植1个月后行心脏B超测量其左室射血分数,组织化学苏木精-伊红染色和Ⅷ因子染色并评价新生血管密度。 结果与结论：CM-DiI能有效标记骨髓间充质干细胞,标记率近100%。细胞移植1个月后高压氧干预的大鼠射血分数、再生血管密度较均明显增高(P < 0.05)。证实,高压氧干预能显著促进移植血管内皮生长因子基因修饰的骨髓间充质干细胞的心肌梗死大鼠所获得的治疗性血管生成作用,对心脏功能有明显改善作用。     

CAPE suppresses VEGFR-2 activation, and tumor neovascularization and growth

The growth and metastasis of human solid tumors and the development of conditions such as diabetic retinopathy, rheumatoid arthritis, inflammatory psoriasis, and others are regulated by the balance between angiogenic stimulators and inhibitors released in the angiogenic–pathological microenvironment. Vascular endothelial growth factor (VEGF), an angiogenic factor, is a potent endothelial-specific mitogen that activates endothelial cells in pathological angiogenesis. Recently, we demonstrated that caffeic acid phenethyl ester (CAPE) inhibits tumor growth, invasion, and metastasis. However, the precise molecular mechanism underlying the inhibitory effect of CAPE on VEGF-mediated angiogenesis remains unknown. Here, we show that CAPE suppressed VEGF-induced proliferation, tube formation, migration, the formation of actin stress fibers and loss of VE-cadherin at cell–cell contacts in endothelial cells, indicating the inhibition of VEGF-mediated VEGF receptor-2 (VEGFR-2) and its downstream signal activation in vitro. CAPE blocked VEGF-stimulated neovascularization in the Matrigel plugs assay, and reduced vascular permeability in mouse skin capillaries in vivo. CAPE inhibited the growth and neovascularization of primary tumor cells in C57BL/6 and BALB/c mice inoculated with Lewis lung carcinoma, colon carcinoma, and melanoma cells. These results suggest that CAPE negatively modulates VEGF-induced angiogenesis by suppressing VEGFR-2 activation, and might be a therapeutic avenue for anti-angiogenesis.     

Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.