Plastic surgical perspectives on vascular endothelial growth factor as gene therapy for angiogenesis

The practice of plastic surgery has always remained at the frontier of medical science. Over the past few decades, this frontier has been marked by significant developments in the field of gene therapy. Gene therapy serves to replace, supplement, or manipulate a patient's genetic makeup to restore function that has been lost or to correct function that is aberrant. Recent technology may allow surgeons to augment the processes of wound healing and angiogenesis by transfecting genes encoding desirable proteins, such as vascular endothelial factor (VEGF), into ischemic tissues. VEGF is a vital growth factor in the development of blood vessels. Although its mechanisms of action are numerous, its sole function seems to be the augmentation of angiogenesis. VEGF is active in growth and development, in wound healing, and in various pathologic conditions, such as psoriasis and rheumatoid arthritis. The role of VEGF in the field of plastic surgery is just beginning to be explored; it may someday prove to be very rewarding.     

Vascular endothelial growth factor (VEGF) - part 1: in physiology and pathophysiology

Angiogenesis is an important component of many physiological processes, such as the female sexual cycle, placenta formation, the processes of growth and differentiation of tissues, and reparative processes including wound healing, fracture repair, and liver regeneration. The formation of new blood vessels during angiogenesis and vasculogenesis allows the growth and functioning of multicellular organisms. Pathological angiogenesis most commonly occurs in ischaemic, inflammatory and neoplastic diseases. Conditions in the pathogenesis of which angiogenesis plays an important role are sometimes labelled angiogenic diseases. To date, a number of pro-and anti-angiogenic factors have been defined. VEGF is the only specific mitogen for endothelial cells. It stimulates their growth and inhibits apoptosis, increases vascular permeability in many tissues, promotes vasculogenesis and angiogenesis. VEGF signalling activity in relation to the cell is dependent on having its specific membrane receptors (Flt-1, KDR, Flt-4). Angiogenesis plays a protective role in ischaemic heart disease and myocardial infarction. Angiogenesis extends life for patients after a stroke. Most of the facts about physiological angiogenesis are derived from studies into liver regeneration as a result of an acute injury or partial hepatectomy. Pathological hepatic angiogenesis occurs in the course of inflammation, fibrosis, hypoxia, and during tumourogenesis. There is interesting data relating to liver steatosis and obesity.     

Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non-malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro-survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy.     

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.     

Effect of ultrasound on the production of IL-8, basic FGF and VEGF.

Therapeutic angiogenesis is the controlled induction or stimulation of new blood vessel formation to reduce unfavourable tissue effects caused by local hypoxia and to enhance tissue repair. The effects of ultrasound on wound healing, chronic ulcers, fracture healing and osteoradionecrosis may be explained by the enhancement of angiogenesis. The aim of this study was to identify which cytokines and angiogenesis factors are induced by ultrasound in vitro.Two ultrasound machines were evaluated, a "traditional" (1 MHz, pulsed 1:4, tested at four intensities), and a "long wave" machine (45 kHz, continuous, also tested at four intensities). The ultrasound was applied to human mandibular osteoblasts, gingival fibroblasts and peripheral blood mononuclear cells (monocytes). The following cytokines and angiogenesis factors were assayed by ELISA techniques: interleukin-1beta(IL-1beta), IL-6, tumour necrosis factor alpha (TNF-alpha), IL-8, fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).A slight stimulation of IL-1beta was noted in all cell types. There was no difference in the IL-6 and TNF-alpha levels. The angiogenesis-related cytokines, IL-8 and bFGF, were significantly stimulated in osteoblasts, and VEGF was significantly stimulated in all cell types. Both ultrasound machines produced similar results, and the optimum intensities were 0.1 and 0. 4 W/cm2 (SATA) with 1 MHz ultrasound, and 15 and 30 mW/cm2 (SATA) with 45 kHz ultrasound.The results show that therapeutic ultrasound stimulates the production of angiogenic factors such as IL-8, bFGF and VEGF. This may be one of the mechanisms through which therapeutic ultrasound induces angiogenesis and healing.     

Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays

Angiogenesis plays a central role in a variety of important biological processes such as reproduction, tissue development, and wound healing, as well as being critical to tumor formation in cancer. The development of chromosomal substitution (consomic) rat strains has permitted the chromosomal localization of genetic factors critical to angiogenesis, but many questions remain as to the mechanisms involved. Here we utilize a novel cell capture assay to assess changes in the functional expression of vascular endothelial growth factor (VEGF) receptors on the surface of vascular endothelial cells isolated from rat strains that are normal or impaired in angiogenesis. We show that functional VEGF receptor expression is increased under hypoxic conditions in rat strains that exhibit normal angiogenesis but not in a strain impaired in angiogenesis. This result implicates the dysregulation of VEGF receptor expression levels on the endothelial cell surface as a key factor in impaired angiogenesis.     

Differential roles of VEGF: Relevance to tissue fibrosis

Excessive extracellular matrix deposition and pathological vascularization are characteristics of fibrosis, which compromises the normal functioning of organs. Although whether angiogenesis can be induced and can occur in parallel with the progression of fibrosis has not been definitely determined, angiogenesis undoubtedly plays a vital role in fibrosis. Since vascular endothelial growth factor (VEGF) is one of the most effective proangiogenic factors, VEGF-targeting interventions have been a focus for the development of therapeutic strategies against fibrosis. In this review, we will summarize the current knowledge of the role of VEGF and its relevant mechanisms in fibrotic biology. We especially expect to provide a comprehensive overview of the therapeutic potential of VEGF-targeted therapy strategies to restore vascular function in the organs affected by fibrosis.     

Structural Insights into the Binding of Vascular Endothelial Growth Factor-B by VEGFR-1D2: RECOGNITION AND SPECIFICITY*

The formation of blood vessels (angiogenesis) is a highly orchestrated sequence of events involving crucial receptor-ligand interactions. Angiogenesis is critical for physiological processes such as development, wound healing, reproduction, tissue regeneration, and remodeling. It also plays a major role in sustaining tumor progression and chronic inflammation. Vascular endothelial growth factor (VEGF)-B, a member of the VEGF family of angiogenic growth factors, effects blood vessel formation by binding to a tyrosine kinase receptor, VEGFR-1. There is growing evidence of the important role played by VEGF-B in physiological and pathological vasculogenesis. Development of VEGF-B antagonists, which inhibit the interaction of this molecule with its cognate receptor, would be important for the treatment of pathologies associated specifically with this growth factor. In this study, we present the crystal structure of the complex of VEGF-B with domain 2 of VEGFR-1 at 2.7 Å resolution. Our analysis reveals that each molecule of the ligand engages two receptor molecules using two symmetrical binding sites. Based on these interactions, we identify the receptor-binding determinants on VEGF-B and shed light on the differences in specificity towards VEGFR-1 among the different VEGF homologs.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.     

Copper-induced vascular endothelial growth factor expression and wound healing

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.     

Angiogenic factors in bone local environment

Angiogenesis plays an important role in physiological bone growth and remodeling, as well as in pathological bone disorders such as fracture repair, osteonecrosis, and tumor metastasis to bone. Vascularization is required for bone remodeling along the endosteal surface of trabecular bone or Haversian canals within the cortical bone, as well as the homeostasis of the cartilage-subchondral bone interface. Angiogenic factors, produced by cells from a basic multicellular unit (BMU) within the bone remodeling compartment (BRC) regulate local endothelial cells and pericytes. In this review, we discuss the expression and function of angiogenic factors produced by osteoclasts, osteoblasts and osteocytes in the BMU and in the cartilage-subchondral bone interface. These include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), BMP7, receptor activator of NF-κB ligand (RANKL) and epidermal growth factor (EGF)-like family members. In addition, the expression of EGFL2, EGFL3, EGFL5, EGFL6, EGFL7, EGFL8 and EGFL9 has been recently identified in the bone local environment, giving important clues to their possible roles in angiogenesis. Understanding the role of angiogenic factors in the bone microenvironment may help to develop novel therapeutic targets and diagnostic biomarkers for bone and joint diseases, such as osteoporosis, osteonecrosis, osteoarthritis, and delayed fracture healing.     

Vascular endothelial growth factor--structure and functions

Vascular endothelial cell growth factor (VEGF), originally described as a vascular permeability factor, is currently known as one of the main factors which regulate angiogenesis. It plays an important role in the regulation of normal as well as pathological angiogenesis. In this paper we try to shortly review the actual knowledge on VEGF protein family, its expression, VEGF receptors and role of VEGF in signal transduction. The aim of this review is also to summarize recent achievements in research on biological functions of vascular endothelial growth factor and their clinical applications.     

Extracellular regulation of VEGF: Isoforms,proteolysis, and vascular patterning

The regulation of vascular endothelial growth factor A (VEGF) is critical to neovascularization in numerous tissues under physiological and pathological conditions. VEGF has multiple isoforms, created by alternative splicing or proteolytic cleavage, and characterized by different receptor-binding and matrix-binding properties. These isoforms are known to give rise to a spectrum of angiogenesis patterns marked by differences in branching, which has functional implications for tissues. In this review, we detail the extensive extracellular regulation of VEGF and the ability of VEGF to dictate the vascular phenotype. We explore the role of VEGF-releasing proteases and soluble carrier molecules on VEGF activity. While proteases such as MMP9 can ‘release’ matrix-bound VEGF and promote angiogenesis, for example as a key step in carcinogenesis, proteases can also suppress VEGF's angiogenic effects. We explore what dictates pro- or anti-angiogenic behavior. We also seek to understand the phenomenon of VEGF gradient formation. Strong VEGF gradients are thought to be due to decreased rates of diffusion from reversible matrix binding, however theoretical studies show that this scenario cannot give rise to lasting VEGF gradients in vivo. We propose that gradients are formed through degradation of sequestered VEGF. Finally, we review how different aspects of the VEGF signal, such as its concentration, gradient, matrix-binding, and NRP1-binding can differentially affect angiogenesis. We explore how this allows VEGF to regulate the formation of vascular networks across a spectrum of high to low branching densities, and from normal to pathological angiogenesis. A better understanding of the control of angiogenesis is necessary to improve upon limitations of current angiogenic therapies.     

Binding to the Extracellular Matrix and Proteolytic Processing: Two Key Mechanisms Regulating Vascular Endothelial Growth Factor Action

Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. One feature of VEGF is the existence of multiple isoforms arising from alternative exon splicing. Our initial biochemical and biological studies indicated that such isoforms are uniquely suited to generate angiogenic gradients by virtue of their differential ability to interact with the extracellular matrix (ECM). Although ECM-bound VEGF was bioactive, processing by physiologically relevant proteases such as plasmin was identified as a key mechanism to convert ECM-bound VEGF into freely diffusible forms. This retrospective article examines the early studies and also emphasizes the subsequent progress in our understanding of these processes in health and disease.     

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.