Hydrogen sulfide accelerates wound healing in diabetic rats

Aim: The aim of this study was to explore the role of hydrogen sulfide on wound healing in diabetic rats. Methods: Experimental diabetes in rats was induced by intraperitoneal injection of streptozotocin (STZ) (in 0.1 mol/L citrate buffer, Ph 4.5) at dose of 70 mg/kg. Diabetic and age-matched non-diabetic rats were randomly assigned to three groups: untreated diabetic controls (UDC), treated diabetic administrations (TDA), and non-diabetic controls (NDC). Wound Healing Model was prepared by making a round incision (2.0 cm in diameter) in full thickness. Rats from TDA receive 2% sodium bisulfide ointment on wound, and animals from UDC and NDC receive control cream. After treatment of 21 days with sodium bisulfide, blood samples were collected for determination of vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), antioxidant effects. Granulation tissues from the wound were processed for histological examination and analysis of western blot. Results: The study indicated a significant increase in levels of VEGF and ICAM-1 and a decline in activity of coagulation in diabetic rats treated with sodium bisulfide. Sodium bisulfide treatment raised the activity of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) protein expression, and decreased tumor necrosis factor α (TNF-α) protein expression in diabetic rats. Conclusions: The findings in present study suggested that hydrogen sulfide accelerates the wound healing in rats with diabetes. The beneficial effect of H2S may be associated with formation of granulation, anti-inflammation, antioxidant, and the increased level of vascular endothelial growth factor (VEGF).     

Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing

Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium in chronic inflammatory wounds.     

Recombinant hepatocyte growth factor accelerates cutaneous wound healing in a diabetic mouse model

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.     

Nicotine accelerates angiogenesis and wound healing in genetically diabetic mice

Recently, we have discovered an endogenous cholinergic pathway for angiogenesis mediated by endothelial nicotinic acetylcholine receptors (nAChRs). Since angiogenesis plays a major role in wound repair, we hypothesized that activation of nAChRs with nicotine would accelerate wound healing in a murine excisional wound model. In genetically diabetic and control mice full-thickness skin wounds (0.8 cm) were created on the dorsum and topically treated over 7 days with either vehicle (phosphate-buffered saline, PBS) or nicotine (10(-8) mol/L, 10(-9) mol/L; each, n = 5). Wound size was measured over 14 days followed by resection, histological analysis, and quantitation of vascularity. In diabetic animals an agonist (epibatidine, 10(-10) mol/L) or antagonist (hexamethonium, 10(-4) mol/L) of nAChRs as well as the positive control basic fibroblast growth factor (bFGF, 25 microg/kg) were also tested. To further study the role of endothelial nAChRs in angiogenesis, we used an ex vivo vascular explant model. In diabetic mice wound healing was markedly impaired. Nicotine significantly accelerated wound healing as assessed by closure rate and histological score. The effects of nicotine were equal to bFGF and were mimicked by epibatidine and blocked by hexamethonium. Histomorphometry revealed increased neovascularization in animals treated with nicotine. Furthermore, capillary-like sprouting from vascular explants was significantly enhanced by nicotine. In conclusion, agonist-induced stimulation of nAChRs accelerates wound healing in diabetic mice by promoting angiogenesis. We have discovered a cholinergic pathway for angiogenesis that is involved in wound healing, and which is a potential target for therapeutic angiogenesis.     

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.     

血管内皮生长因子与糖尿病性视网膜病变

随着糖尿病发病率的增高 ,其并发症——糖尿病性视网膜病变 (DR)已成为致盲的主要原因之一 ,并日益受到人们的重视。目前发现一种细胞生长因子——血管内皮生长因子 (VEGF)与糖尿病性视网膜病变的发生发展有着极为密切的关系。本文就 VEGF的生物学特点及其在 DR中的作用简要做一综述     

Nerve growth factor accelerates the early cellular events associated with wound healing

The effects of injecting nerve growth factor (NGF) into subdermal air sacs on the backs of mice was studied. Sequential infiltration of cells into the lining of the sacs was observed. The initial cell type to infiltrate was the polymorphonuclear leukocyte, followed by highly vacuolated mononuclear cells, and then by fibroblast-like cells. This resembles the classical pattern of cellular responses during the normal process of wound healing. The kinetics for the peak accumulation of each cell type were dependent upon the concentration of NGF injected, and significant acceleration of infiltration of each cell type was observed with as little as 1 nM NGF. A similar acceleration of cellular infiltration was observed when mice were injected with the synthetic chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine. This finding suggests that application of any chemotactic factor to wound sites may lead to accelerated tissue repair and an enhanced rate of wound contraction. The observation that NGF can act as a chemotactic factor in vivo may explain its ability to accelerate the healing of experimentally induced wounds in mice.     

Neuregulin-1 accelerates corneal epithelial wound healing

Corneal epithelial cells (CECs) play an important role in the function of the cornea, and are maintained by corneal epithelial stem cells (CESCs). Recent studies have shown that neuronal growth factors affect the proliferation and migration of CESCs. Neuregulin-1 (NR-1) is a neuronal growth factor that is expressed in the early stages of brain development. The aim of this study was to determine whether NR-1 activates corneal wound healing. We observed that NR-1 activated both proliferation and migration of CECs. In addition, the colony-forming efficacy of CESCs was enhanced. In mice, NR-1 treatment improved corneal wound healing. Furthermore, the expression of markers of corneal epithelium maintenance (ΔNp63) and CESC proliferation (Bmi-1 and Abcg2) was increased. These effects were mediated by intracellular signalling pathways (Stat3, Erk1/2 and p38). Taken together, our results suggest that NR-1 accelerates the recovery of corneal wounds, and may represent a novel treatment for corneal damage.     

Hyperoxia, endothelial progenitor cell mobilization, and diabetic wound healing

Diabetic foot disease is a major health problem, which affects 15% of the 200 million patients with diabetes worldwide. Diminished peripheral blood flow and decreased local neovascularization are critical factors that contribute to the delayed or nonhealing wounds in these patients. The correction of impaired local angiogenesis may be a key component in developing therapeutic protocols for treating chronic wounds of the lower extremity and diabetic foot ulcers. Endothelial progenitor cells (EPCs) are the key cellular effectors of postnatal neovascularization and play a central role in wound healing, but their circulating and wound-level numbers are decreased in diabetes, implicating an abnormality in EPC mobilization and homing mechanisms. The deficiency in EPC mobilization is presumably due to impairment of eNOS-NO cascade in bone marrow (BM). Hyperoxia, induced by a clinically relevant hyperbaric oxygen therapy (HBO) protocol, can significantly enhance the mobilization of EPCs from the BM into peripheral blood. However, increased circulating EPCs failed to reach to wound tissues. This is partly a result of downregulated production of SDF-1alpha in local wound lesions with diabetes. Administration of exogenous SDF-1alpha into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, neovascularization, and wound healing.     

Vascular endothelial growth factor expression in rat skin incision wound

Vascular endothelial growth factor (VEGF) is a glycoprotein that enhances vascular permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes growth of vascular endothelial cells. We report that infiltrating polymorphonuclear leukocytes in an incision wound in rat skin express VEGF from 1 day after the injury, as shown by immunohistochemistry. VEGF is also present in macrophages, fibroblast-like cells, and endothelial cells 3 and 7 days after the injury. mRNA for VEGF is statistically significantly increased in the wound area in the tissue 1 day after the skin incision compared with 3 and 7 days after the incision. The VEGF protein content in the wound tissue is statistically significantly higher in the wound than in control skin at 1 and 3 days after skin incision. Our results indicate that VEGF is produced by inflammatory cells to induce vascularization in the early stage of the wound healing process.     

Vascular endothelial growth factor-C expression in human gallbladder cancer and its relationship to lymph node metastasis

Lymph node metastasis is a major prognostic factor in human cancer. Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic polypeptide that has been implicated in several human solid tumors. However, the clinical significance of VEGF-C has remained unknown in gallbladder carcinoma. Paraffin-embedded tumor specimens of 52 surgically resected gallbladder cancers were immunohistochemically stained for VEGF-C, VEGF, and CD34. The correlations among VEGF-C expression, VEGF expression, microvessel density (MVD), clinicopathologic features, and clinical outcomes were statistically analyzed. Thirty-three (63%) of the 52 gallbladder cancers were highly positive for VEGF-C protein by immunohistochemistry. VEGF-C expression was significantly correlated with lymphatic vessel involvement, lymph node metastasis, and worse outcomes after operation (p<0.001, p<0.001, p<0.001, respectively), but not with MVD. By the Cox regression model, lymphatic vessel involvement emerged as an independent prognostic parameter. These results suggest that VEGF-C may play a role in tumor progression via lymphangiogenesis and lymph node metastasis in human gallbladder cancer.     

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.     

低强度脉冲电磁场加速2型糖尿病表皮软组织创伤愈合实验

目的:研究低强度的脉冲电磁场（PEMF）对于2型糖尿病软组织创伤愈合的治疗效果。方法:32只3月龄雄性2型糖尿病db/db小鼠及16只同背景的野生对照小鼠用于本研究,分为Wild type野生型小鼠组（Wild type）、db/db小鼠组（db/db）及db/db施加以PEMF刺激组（db/db+PEMF）,每组16只。分别于创伤模型构建的第5、12、19天处死每组的4只小鼠。全部小鼠均构建背部表皮创伤模型,db/db+PEMF组小鼠施以全身PEMF刺激,每天刺激2 h。分别通过伤口闭合率、伤口组织抗张强度、伤口组织病理和ELISA检测分析评估其作用效果。结果:PEMF刺激在术后的第5、12、19天均显著提升了db/db小鼠伤口的愈合速率（P<0.05）,降低了db/db小鼠的伤口总愈合时间（P<0.05）,在术后的第5、12、19天均显著提升了伤口组织的抗张强度（P<0.05）,降低了伤口组织的炎性细胞数量且显著降低了炎性因子IL-1β和TNF-α的表达（P<0.05）,并上调了VEGF和IGF-1的表达（P<0.05）。结论:低强度PEMF能够显著加速2型糖尿病db/db小鼠的软组织修复进程,这一效应可能与PEMF的抗炎性反应及促胶原沉积和血管再生相关。     

Inhibition of indoleamine 2,3-dioxygenase activity accelerates skin wound healing

Skin wound healing is a complex process involving several stages that include inflammation, proliferation, and remodeling. In the inflammatory phase, pro-inflammatory cytokines and chemokines are induced at the wound site and, they contribute to the development of wound healing. These cytokines also induce indoleamine 2,3-dioxygenase (IDO1) activity; this is the rate-limiting and first enzyme in the l-tryptophan (TRP)-l-kynurenine (KYN) pathway. This study examined the effect of IDO1 on the process of skin wound healing. The expression of the Ido1 mRNA was enhanced after creating a wound in wild-type (WT) mice. TRP concentration was simultaneously reduced at the wound site. The rate of wound healing in IDO1 knockout (IDO-KO) mice was significantly higher than that in WT mice. 1-Methyl-dl-tryptophan (1-MT), a potent inhibitor of IDO1, increased the rate of wound healing in WT mice. The administration of TRP accelerated wound healing in vivo and in an in vitro experimental model, whereas the rate of wound healing was not affected by the administration of KYN. The present study identifies the role of IDO1 in skin wound healing, and indicates that the local administration of 1-MT or TRP may provide an effective strategy for accelerating wound healing.     

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     

Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.