Regulation of scar formation by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF-A) is known for its effects on endothelial cells and as a positive mediator of angiogenesis. VEGF is thought to promote repair of cutaneous wounds due to its proangiogenic properties, but its ability to regulate other aspects of wound repair, such as the generation of scar tissue, has not been studied well. We examined the role of VEGF in scar tissue production using models of scarless and fibrotic repair. Scarless fetal wounds had lower levels of VEGF and were less vascular than fibrotic fetal wounds, and the scarless phenotype could be converted to a scar-forming phenotype by adding exogenous VEGF. Similarly, neutralization of VEGF reduced vascularity and decreased scar formation in adult wounds. These results show that VEGF levels have a strong influence on scar tissue formation. Our data suggest that VEGF may not simply function as a mediator of wound angiogenesis, but instead may play a more diverse role in the wound repair process.     

Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair

It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).     

Vascular endothelial growth factor-C accelerates diabetic wound healing

Diabetes impairs numerous aspects of tissue repair. Failure of wound angiogenesis is known to delay diabetic wound healing, whereas the importance of lymphangiogenesis for wound healing is unclear. We have examined whether overexpression of vascular endothelial growth factor (VEGF)-C via an adenoviral vector could improve the healing of full-thickness punch biopsy wounds in genetically diabetic (db/db) mice. We found that VEGF-C enhanced angiogenesis and lymphangiogenesis in the wound and significantly accelerated wound healing in comparison to the control wounds. VEGF-C also recruited inflammatory cells, some of which expressed VEGFR-3. On the other hand, when the function of endogenous VEGF-C/VEGF-D was blocked with a specific inhibitor, wound closure was delayed even further. These results suggest a function for VEGF-C in wound healing and demonstrate the therapeutic potential of VEGF-C in the treatment of diabetic wounds.     

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.     

Abrogation of TGF-beta by antisense oligonucleotides modulates expression of VEGF and increases angiogenic potential in isolated fibroblasts from radiated skin

The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer good prospects for the modulation of wound healing, specifically those targeting TGF-beta. The aim of this study was to analyze the effect of TGF-beta targeting on the expression of angiogenic vascular endothelial growth factor (VEGF), a key regulator of angiogenesis and in vitro angiogenic activity in fibroblasts isolated from radiation-induced chronic dermal wounds. The expression of angiogenic VEGF in tissue samples from radiation-induced chronic dermal wounds was investigated by immunohistochemistry and microarray technique. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of VEGF in isolated fibroblasts was analyzed by ELISA and multiplex RT-PCR. Human endothelial cells (ECs) were grown in conditioned medium produced from the treated fibroblasts. EC migration was measured using a modified Boyden chamber; EC tube formation was analyzed under a light microscope. Immunohistochemical investigation and microarray analysis demonstrated a decreased expression of VEGF protein and mRNA in tissue samples from radiation-induced chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment significantly up-regulated VEGF secretion in vitro. Addition of conditioned medium from TGF-beta antisense-treated fibroblasts resulted in an increase in EC cell migration and tube formation. In conclusion, our results demonstrate that TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for stimulation of angiogenesis in radiation-induced dermal wounds.     

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.     

VEGF signaling through NADPH oxidase-derived ROS

Angiogenesis is a key process involved in normal development and wound repair, as well as ischemic heart and limb diseases, and atherosclerosis. Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, stimulates proliferation, migration, and tube formation of endothelial cells (ECs), primarily through the VEGF receptor type2 (VEGFR2). Reactive oxygen species (ROS) function as signaling molecules to mediate biological responses. In ECs, NADPH oxidase is one of the major sources of ROS and consists of catalytic subunits (Nox1, Nox2, and Nox4), p22phox, p47phox, p67phox, and the small GTPase Rac1. VEGF stimulates ROS production via activation of gp91phox (Nox2)-based NADPH oxidase, and ROS are involved in VEGFR2-mediated signaling linked to EC migration and proliferation. Moreover, ROS derived from NADPH oxidase are involved in postnatal angiogenesis. Localizing NADPH oxidase and its regulators at the specific subcellular compartment is an important mechanism for activating specific redox signaling events. This review focuses on a role of NADPH oxidase-derived ROS in angiogenesis and critical regulators involved in generation of spatially and temporally restricted ROS-dependent VEGF signaling at leading edge, focal adhesions/complexes, caveolae/lipid rafts, and cell-cell junctions in ECs. Understanding these mechanisms should facilitate the development of new therapeutic strategies to modulate new blood vessel formation.     

Decreased macrophage number and activation lead to reduced lymphatic vessel formation and contribute to impaired diabetic wound healing

Impaired wound healing is a common complication of diabetes. Although it is well known that both macrophages and blood vessels are critical to wound repair, the role of wound-associated lymphatic vessels has not been well investigated. We report that both the presence of activated macrophages and the formation of lymphatic vessels are rate-limiting to the healing of diabetic wounds. We have previously shown that macrophages contribute to the lymphatic vessels that form during the acute phase of corneal wound healing. We now demonstrate that this is a general phenomenon; cells that co-stain for the macrophage marker F4/80 and the lymphatic markers LYVE-1 (lymphatic vascular endothelium hyaluronate receptor) and podoplanin contribute to lymphatic vessels in full-thickness wounds. LYVE-1-positive lymphatic vessels and CD31-positive blood vessels were significantly reduced in corneal wound healing in diabetic mice (db/db) (P < 0.02) compared with control (db/+) mice. Glucose treatment of control macrophages led to the down-regulation of the lymphatic-specific receptor VEGFR3 and its ligands, vascular endothelial growth factor-C and -D (VEGF-C, -D). Interleukin-1beta stimulation rescued diabetic macrophage function; application of interleukin-1beta-treated db/db-derived macrophages to wounds in db/db mice induced lymphatic vessel formation and accelerated wound healing. These observations suggest a potential therapeutic approach for healing wounds in diabetic patients.     

Placenta growth factor in diabetic wound healing: altered expression and therapeutic potential

Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.     

HOXA9 regulates angiogenesis in human hypertrophic scars: induction of VEGF secretion by epidermal stem cells

Hypertrophic scars are fibroproliferative disorders of excessive wound healing after skin injury. Vascular endothelial growth factor (VEGF)-induced angiogenesis plays a major role in fibrogenesis and hypertrophic scar formation. Over recent years, there has been a major interest in homeobox gene regulation of VEGF-VEGFR mediated angiogenesis in dermal tissue. In the current study, we investigated the role of homeobox genes in the epidermis, for their role in angiogenesis, with a focus on epidermal-mesenchymal interactions. As epidermal stem cells (ESCs) have a central role in epidermal homeostasis, we tested the hypothesis that these cells play a key role in the pathogenesis of hypertrophic scars through the HOXA9-VEGF/VEGFR signaling pathways. We found significant differences in the expression of homeobox A9 in hyperplastic scar tissue during different phases of development. These differences coincided with similar regulations in VEGF expression and with the distribution of ESCs. HOXA9 is expressed in cultured human ESCs in vitro. Antisense suppression of HOXA9 expression was found to suppress VEGF levels in ESCs. Together these findings indicate that homeobox A9 regulates the expression of VEGF in ESCs.     

Could chronic wounds not heal due to too low local copper levels?

Copper is an essential trace element involved in numerous human physiological and metabolic processes. It plays a key role in many of the processes that together comprise wound healing, including induction of endothelial growth factor, angiogenesis and expression and stabilization of extracellular skin proteins. We hypothesize that in individuals with diabetic ulcers, decubitus, peripheral vascular, or other wounds which might have compromised circulation to the wound site, that part of the incapacity of the wounds to heal is due to low local copper levels. Contamination of wounds is also an important factor causing impaired wound healing. Importantly, copper has potent broad biocidal properties. In contrast, the risk of adverse skin reactions due to exposure to copper is extremely low. We thus hypothesize that introducing copper into wound dressings would not only reduce the risk of wound and dressing contamination, as silver does but, more importantly, would stimulate faster wound repair directly. This would be done by the release of copper from the wound dressings directly into the wound site inducing angiogenesis and skin regeneration.     

Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing.

Angiogenesis is an essential component of normal wound repair, yet the primary mediators of wound angiogenesis have not been well described. The current study characterizes the contribution of vascular endothelial cell growth factor (VEGF) to the angiogenic environment of human surgical wounds. Surgical wound fluid samples (n = 70) were collected daily for up to 7 postoperative days (POD) from 14 patients undergoing mastectomy or neck dissection. VEGF levels in surgical wound fluid were lowest on POD 0, approximating values of serum, but increased steadily through POD 7. An opposite pattern was noted for basic fibroblast growth factor-2. Fibroblast growth factor-2, which has been previously described as a wound angiogenic factor, exhibited highest levels at POD 0, declining to near serum levels by POD 3. Surgical wound fluid form all time points stimulated marked endothelial cell chemotaxis and induced a brisk neovascular response in the rat corneal micropocket angiogenesis assay. Antibody neutralization of VEGF did not affect the in vitro chemotactic or the in vivo angiogenic activity early wound samples (POD 0). In contrast, VEGF neutralization significantly attenuated both chemotactic activity (mean decrease 76 +/- 13%, P < 0.01) and angiogenic activity (5 of 5 samples affected) of later wound samples (POD 3 and 6). The results suggest a model of wound angiogenesis in which an initial angiogenic stimulus is supplied by fibroblast growth factor-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.     

Impaired wound repair and delayed angiogenesis in aged mice

Wound repair is a multistep process consisting of hemostasis, inflammatory cell infiltration, tissue regrowth, and remodeling. In aged individuals, this progression of events is altered, resulting in wounds that heal more slowly than wounds in the young. These studies were designed to examine the proliferative phase of repair in young and aged mice, with attention to the angiogenic process. Using a standardized excisional injury model, wound re-epithelialization, collagen accumulation, and angiogenesis were examined. Re-epithelialization and collagen synthesis were substantially delayed in aged mice as compared with young mice. Angiogenesis in wounds from aged mice was also delayed, with significantly more capillary growth in wounds from young mice than aged mice. In addition, wounds from aged mice contained significantly less of the angiogenic mediators fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) than wounds from young animals (p < 0.05). Because macrophages are a rich source of angiogenic factors in wounds, macrophage production of VEGF was examined. Macrophages from aged mice produced significantly less VEGF than cells from young mice. To examine the in vivo endothelial cell responsiveness, a defined amount of rFGF-2 was suspended in Matrigel and placed subcutaneously in either young or aged mice. In response to FGF-2, capillary growth into Matrigel was significantly less in aged than young mice. The results suggest that a decline in angiogenic growth factor production, as well as a decline in endothelial responsiveness to specific factors, may account for the delayed wound angiogenesis in aged mice. These results also indicate that age-related alterations in macrophage function might partially account for the overall delay in the wound repair process.     

Vascular endothelial growth factor improves functional outcome and decreases secondary degeneration in experimental spinal cord contusion injury

Spinal cord injury leads to acute local ischemia, which may contribute to secondary degeneration. Hypoxia stimulates angiogenesis through a cascade of events, involving angiogenesis stimulatory substances, such as vascular endothelial growth factor (VEGF). To test the importance of angiogenesis for functional outcome and wound healing in spinal cord injury VEGF165 (proangiogenic), Ringer's (control) or angiostatin (antiangiogenic) were delivered locally immediately after a contusion injury produced using the NYU impactor and a 25 mm weight-drop. Rats treated with VEGF showed significantly improved behavior up to 6 weeks after injury compared with control animals, while angiostatin treatment lead to no statistically significant changes in behavior outcome. Furthermore, VEGF-treated animals had an increased amount of spared tissue in the lesion center and a higher blood vessel density in parts of the wound area compared with controls. These effects were unlikely to be due to increased cell proliferation as determined by bromo-deoxy-uridine-labeling. Moreover, VEGF treatment led to decreased levels of apoptosis, as revealed by TUNEL assays. In situ hybridization demonstrated presence of mRNA for VEGF receptors Flt-1, fetal liver kinase-1, neuropilin-1 and -2 in several important cellular compartments of the spinal cord. The different experiments indicate that beneficial effects seen by acute VEGF delivery was attributable to protection/repair of blood vessels, decreased apoptosis and possibly also by other additional effects on glial cells or certain neuron populations.     

低能量体外震波对糖尿病慢性创面模型血管新生和血管内皮细胞生长因子表达的影响**☆

背景：体外震波作为一种非侵入性的物理刺激近年来发现具有促进新生血管形成、促进组织修复的功能。 目的：观察体外震波对创面内血管内皮细胞生长因子表达和新血管形成的影响及促进创面愈合的作用。 方法：72只SD大鼠随机均分为治疗组、糖尿病组及对照组。治疗组和糖尿病组制作糖尿病慢性创面模型。建模后1 d治疗组创面用体外震波处理,糖尿病组和对照组仅涂抹耦合液。观察创面的肉芽组织和新血管形成情况,检测血管内皮细胞生长因子蛋白含量及mRNA的表达水平。 结果与结论：与糖尿病组比较,治疗组创面的闭合率增加。治疗后3 d开始,治疗组创面内毛细血管数量比糖尿病组增多,肉芽组织相应增加。与对照组比较,糖尿病组血管内皮细胞生长因子蛋白含量和mRNA表达水平在3 d和7 d均降低,在7 d出现下降。经体外震波治疗后,各时间段表达均增高,在14 d出现下降。说明糖尿病创面局部血管内皮细胞生长因子分泌量降低和高表达时段缩短是其难愈的重要因素之一,体外震波治疗可增加糖尿病创面组织内血管内皮细胞生长因子的表达强度,延长其高表达的时间,从而促进创面内新血管形成,最终加快肉芽组织生长和创面愈合。关键词：体外震波;糖尿病创面;血管内皮细胞生长因子;血管新生;愈合 缩略语注释：VEGF: vascular endothelial growth factor,血管内皮细胞生长因子 doi:10.3969/j.issn.1673-8225.2012.15.019     

Possible participation of pICln in the regulation of angiogenesis through alternative splicing of vascular endothelial growth factor receptor mRNAs.

In this study, the authors applied a modified Antisense Display method to human vascular endothelial cells (ECs) in culture to isolate new angiostatic genes. Screening of a 10mer antisense oligodeoxyribonucleotide (oligo) repertoire identified a subpool that consistently stimulated EC growth. Subsequent screening of oligos with increasing chain length led to the isolation of a unique growth-stimulatory 14mer, 5'-TTCCACATCATATT-3'. cDNA/EST data-base search and expression analyses in ECs indicated pICln as the corresponding gene. A longer unique antisense oligo against a different region of pICln mRNA was found to also enhance EC growth and tube formation and to decrease mRNAs for soluble Flt-1 and neuropilin-1 vascular endothelial growth factor (VEGF) receptors, the angiostatic factors that are generated by alternative RNA splicing. Conversely,pICln overexpression suppressed EC growth and increased the mRNAs for both soluble Flt-1 and soluble neuropilin-1. The present findings thus suggest that pICln plays a role in autocrine regulation of angiogenesis, probably through alternative splicing.     

Combined effects of artificial dermis and vascular endothelial growth factor concentration gradient on wound healing in diabetic porcine model

Wounds in patients with diabetes mellitus are one of the most prevalent impaired wounds in the world. Vascular endothelial growth factor (VEGF) is one of the most important proangiogenic mediators. Artificial dermal (AD) such as Pelnac^® has been shown, in humans and animal models, a great therapeutic potential in full-thickness skin wounds. We attempt to promote the wound healing in diabetic porcine models through combined use of AD and constant concentration of VEGF or VEGF concentration gradient. We created full-thickness excisional wounds in diabetic animal models. Analyzed the healing process through images, histology and immunohistochemistry. Results show that the combination of AD and concentration gradient of VEGF could provide an appropriate angiogenesis, improve granulation formation, increase epithelization and maintain the VEGF levels of the wound bed. Eventually accelerate the direct healing of diabetic wounds or make good preparation for secondary skin graft.