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.     

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.     

Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-β-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.     

PDGF and FGF stimulate wound healing in the genetically diabetic mouse.

To examine the effects of recombinant growth factors in vivo, impaired wound healing was studied in genetically diabetic C57BL/KsJ-db/db mice. Large full-thickness skin wounds made on the backs of these mice exhibited significant delays in the entry of inflammatory cells into the wound, the formation of granulation tissue, and in wound closure when compared to their nondiabetic littermates. Recombinant human platelet-derived growth factor (rPDGF-BB, 1 or 10 micrograms), recombinant human basic fibroblast growth factor (rbFGF, 1 micrograms), or combinations of both were applied topically to the wounds for 5 to 14 days after wounding. Diabetic mouse wounds treated with rPDGF-BB or rbFGF had many more fibroblasts and capillaries in the wound bed at 10 and 21 days than did wounds treated with the vehicle alone. The animals treated with growth factors also had significantly greater wound closure at 21 days than those treated with the vehicle. Combinations of rPDGF-BB and rbFGF improved all parameters of healing but not to a greater extent than either growth factor alone. The effectiveness of rPDGF-BB and rbFGF suggest that recombinant growth factors may be useful in the treatment of patients with deficient wound repair.     

Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice

Diabetic skin ulcers are difficult to heal spontaneously due to the reduced levels and activity of endogenous growth factors. Recombinant human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are known to stimulate cell proliferation and accelerate wound healing. Direct delivery of VEGF and bFGF at the wound site in a sustained and controllable way without loss of bioactivity would enhance their biological effects. The aim of this study was to develop a poly(ether)urethane–polydimethylsiloxane/fibrin-based scaffold containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with VEGF and bFGF (scaffold/GF-loaded NPs) and to evaluate its wound healing properties in genetically diabetic mice (db/db). The scaffold application on full-thickness dorsal skin wounds significantly accelerated wound closure at day 15 compared to scaffolds without growth factors (control scaffold) or containing unloaded PLGA nanoparticles (scaffold/unloaded NPs). However, the closure rate was similar to that observed in mice treated with scaffolds containing free VEGF and bFGF (scaffold/GFs). Both scaffolds containing growth factors induced complete re-epithelialization, with enhanced granulation tissue formation/maturity and collagen deposition compared to the other groups, as revealed by histological analysis. The ability of the scaffold/GF-loaded NPs to promote wound healing in a diabetic mouse model suggests its potential use as a dressing in patients with diabetic foot ulcers.     

Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice

Receptor for advanced glycation end-products (RAGE), and two of its ligands, AGE and EN-RAGEs (members of the S100/calgranulin family of pro-inflammatory cytokines), display enhanced expression in slowly resolving full-thickness excisional wounds developed in genetically diabetic db+/db+ mice. We tested the concept that blockade of RAGE, using soluble(s) RAGE, the extracellular ligand-binding domain of the receptor, would enhance wound closure in these animals. Administration of sRAGE accelerated the development of appropriately limited inflammatory cell infiltration and activation in wound foci. In parallel with accelerated wound closure at later times, blockade of RAGE suppressed levels of cytokines; tumor necrosis factor-alpha; interleukin-6; and matrix metalloproteinases-2, -3, and -9. In addition, generation of thick, well-vascularized granulation tissue was enhanced, in parallel with increased levels of platelet-derived growth factor-B and vascular endothelial growth factor. These findings identify a central role for RAGE in disordered wound healing associated with diabetes, and suggest that blockade of this receptor might represent a targeted strategy to restore effective wound repair in this disorder.     

Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db mice

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) resulted within 30s in an insoluble, flexible hydrogel. About 20% of the FGF-2molecules were released from the FGF-2-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which no further significant release occurred under in vitro non-degradation conditions of the hydrogel. The FGF-2molecules retained in the chitosan hydrogel remained biologically active, and were released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In order to evaluate its accelerating effect on wound healing, full thickness skin incisions were made on the back of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure in both db/db and db/+ mice. However, the addition of FGF-2 in the chitosan hydrogel further accelerated wound closure in db/db mice, although not in db/+ mice. Histological examination also has demonstrated an advanced granulation tissue formation, capillary formation and epithelialization in wounds treated with FGF-2-incorporated chitosan hydrogels in db/db mice.     

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.     

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.     

Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors

Recent evidence indicates that topical application of adenosine A(2A) receptor agonists, unlike growth factors, increases the rate at which wounds close in normal animals and promotes wound healing in diabetic animals as well as growth factors, yet neither the specific adenosine receptor involved nor the mechanism(s) by which adenosine receptor occupancy promotes wound healing have been fully established. To determine which adenosine receptor is involved and whether adenosine receptor-mediated stimulation of angiogenesis plays a role in promotion of wound closure we compared the effect of topical application of the adenosine receptor agonist CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosine) on wound closure and angiogenesis in adenosine A(2A) receptor knockout mice and their wild-type littermates. There was no change in the rate of wound closure in the A(2A) receptor knockout mice compared to their wild-type littermates although granulation tissue formation was nonhomogeneous and there seemed to be greater inflammation at the base of the wound. Topical application of CGS-21680 increased the rate of wound closure and increased the number of microvessels in the wounds of wild-type mice but did not affect the rate of wound closure in A(2A) receptor knockout mice. Similarly, in a model of internal trauma and repair (murine air pouch model), endogenously produced adenosine released into areas of internal tissue injury stimulates angiogenesis because there was a marked reduction in blood vessels in the walls of healing air pouches of A(2A) receptor knockout mice compared to their wild-type controls. Inflammatory vascular leakage and leukocyte accumulation in the inflamed air pouch were similarly reduced in the A(2A) receptor knockout mice reflecting the reduced vascularity. Thus, targeting the adenosine A(2A) receptor is a novel approach to promoting wound healing and angiogenesis in normal individuals and those suffering from chronic wounds.     

血管紧张素Ⅱ2型受体阻滞剂对小鼠全层皮肤缺损创面愈合的影响

 目的： 观察阻断血管紧张素II （Ang II）及其2型受体（AT2R）对创面愈合过程的创面愈合率、上皮爬行、肉芽组织形成以及创伤局部生长因子表达的影响,探讨Ang II及AT2R影响创伤愈合的机制。方法：建立小鼠背部全层皮肤缺损创面模型,直径6 mm,在创面模型建立同时腹腔注射给予特异性AT2R阻断剂PD123319（每天10 mg/kg）,于创面形成后第3、5、7、9、11、13和15天切取创面组织标本,采用HE染色观察PD123319对创面愈合过程中创面愈合率、上皮爬行和肉芽组织生长的影响;采用ELISA法检测PD123319对创面内与创伤愈合密切相关的表皮生长因子（EGF）、血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）表达的影响。结果：对照组在创面形成后第5天和第7天的愈合率分别为(63.55±2.57)%和(80.78±4.65)%。PD123319处理组在创面形成后第5天和第7天分别为(79.89±4.56)%和(88.98±3.83)%,两组差异有统计学意义（P＜0.05）。在伤后第5天和第7天, 对照组创面上皮爬行距离分别为(1.22±0.15)mm和(1.93±0.17)mm,PD123319处理组创面上皮爬行距离分别为(1.65±0.12)mm和(2.36±0.18) mm,两组差异有统计学意义（P＜0.05）。在伤后第5天和第7天对照组创面肉芽组织的面积分别为(9.37±0.53)mm2和(7.15±0.42)mm2,PD123319处理组创面肉芽组织面积分别为(11.51±0.98) mm2和(9.32±0.67) mm2,两组差异有统计学意义（P＜0.05）。在伤后第5天和第7天,PD123319处理组创面局部EGF、 VEGF和bFGF的含量明显高于对照组,差异有统计学意义（P＜0.05）。结论：AT2R阻滞剂PD123319能够促进创面愈合。PD123319促进创面愈合可能与其促进创面内上皮爬行、肉芽组织形成及EGF、VEGF、bFGF等生长因子的表达有关。     

The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation

Background

Continuous diabetes-associated complications are a major source of immune system exhaustion and an increased incidence of infection. Diabetes can cause poor circulation in the feet, increasing the likelihood of ulcers forming when the skin is damaged and slowing the healing of the ulcers. Whey proteins (WPs) enhance immunity during childhood and have a protective effect on some immune disorders. Therefore, in this study, we investigated the effects of camel WP on the healing and closure of diabetic wounds in a streptozotocin (STZ)-induced type I diabetic mouse model.

Results

Diabetic mice exhibited delayed wound closure characterized by a significant decrease in an anti-inflammatory cytokine (namely, IL-10) and a prolonged elevation of the levels of inflammatory cytokines (TNF-??, IL-1?? and IL-6) in wound tissue. Moreover, aberrant expression of chemokines that regulate wound healing (MIP-1??, MIP-2, KC and CX3CL1) and growth factors (TGF-??) were observed in the wound tissue of diabetic mice compared with control nondiabetic mice. Interestingly, compared with untreated diabetic mice, supplementation with WP significantly accelerated the closure of diabetic wounds by limiting inflammatory stimuli via the restoration of normal IL-10, TNF-??, IL-1?? and IL-6 levels. Most importantly, the supplementation of diabetic mice with WP significantly modulated the expression of MIP-1??, MIP-2, KC, CX3CL1 and TGF-?? in wound tissue compared with untreated diabetic mice.

Conclusion

Our data demonstrate the benefits of WP supplementation for improving the healing and closure of diabetic wounds and restoring the immune response in diabetic mice.     

Platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair.

Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growth factor beta 1 (rTGF-beta 1), and basic fibroblast growth factor (rbFGF) can accelerate healing of soft tissues. However, little information is available characterizing the components of wound matrix induced by these growth factors and the molecular mechanisms underlying accelerated repair and wound maturation. In this study, the composition, quantity, and rate of extracellular matrix deposition within growth factor-treated lapine ear excisional wounds were analyzed at different stages of healing using specific histochemical and immunohistochemical stains, coupled with image analysis techniques. Single application of optimal concentrations of each growth factor accelerated normal healing by 30% (P less than 0.0003); rPDGF-BB markedly augmented early glycosaminoglycan (GAG) and fibronectin deposition, but induced significantly greater levels of collagen later in the repair process, compared with untreated wounds rTGF-beta 1 treatment led to rapidly enhanced collagen synthesis and maturation, without increased GAG deposition. In contrast, rbFGF treatment induced a predominantly angiogenic response in wounds, with a marked increase in endothelia and neovessels (P less than 0.0001), and increased wound collagenolytic activity (P less than 0.03). rbFGF-treated wounds did not evolve into collagen-containing scars and continued to accumulate only provisional matrix well past wound closure. These results provide new evidence that growth factors influence wound repair via different mechanisms: 1) rPDGF-BB accelerates deposition of provisional wound matrix; 2) rTGF-beta 1 accelerates deposition and maturation of collagen; and 3) rbFGF induces a profound monocellular angiogenic response which may lead to a marked delay in wound maturation, and the possible loss of the normal signal(s) required to stop repair. These results suggest that specific growth factors may selectively regulate components of the repair response by differing mechanisms, offering the potential for targeted therapeutic intervention.     

Transgenic overexpression of keratinocyte-specific VEGF and Ang1 in combination promotes wound healing under nondiabetic but not diabetic conditions

VEGF and Angiopoietin (Ang)1 are growth factors that independently improve wound healing outcomes. Using a tet-repressible mouse model coupled with streptozotocin-induced diabetes, we examined wound healing in diabetic and nondiabetic mice engineered to overexpress keratinocyte-specific (K5) VEGF, Ang1 or Ang1-VEGF combined. All nondiabetic mice healed more rapidly than their diabetic counterparts; however overexpression of VEGF, Ang1 or the combination failed to improve wound closure under diabetic conditions. Conversely, under nondiabetic conditions, combining Ang1 and VEGF resulted in rapid wound closure. Molecular analyses of diabetic and nondiabetic K5-Ang1-VEGF skin revealed no differences in VEGF expression but an 80% decrease in Ang1 under diabetic conditions, suggesting an integral role for Ang1. Nondiabetic K5-Ang1 mice healed more quickly and had significant increases in granulation tissue and a 60% decrease in re-epithelialization 7 days after wounding. Furthermore, Ang1 stimulated primary mouse keratinocytes showed significantly less migration into a wound bed in an in vitro wound healing bioassay and had decreased pMAPK, pNFκB, pAkt, and pStat3 signaling. These data suggest that combined Ang1-VEGF overexpression cannot overcome diabetes-induced delays in wound healing but is efficacious under nondiabetic conditions possibly via Ang1-mediated delays in re-epithelialization and enhancement of granulation tissue formation, thereby allowing more rapid secondary intention healing.     

Accelerated wound closure in neutrophil-depleted mice

The infiltration of neutrophils into injured tissue is known to protect wounds from invading pathogens. However, more recent studies suggest that neutrophils might inhibit the wound repair process. To investigate the role of neutrophils in wounds, mice were neutrophil-depleted by injection with rabbit anti-mouse neutrophil serum. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in neutropenic than control mice (77.7+14.2% vs. 41.2+0.9%, P<0.02 at day 2). Dermal healing was not affected by neutrophil depletion, as neither collagen deposition nor wound-breaking strength was significantly different between neutropenic and control mice. As the delayed repair of diabetic individuals exhibits robust inflammation, the effect of neutrophil depletion on diabetic wound healing was investigated. Similar to the observations in wild-type mice, wound closure was accelerated by nearly 50% in neutropenic, diabetic mice. The results suggest that although neutrophils may provide protection against infection, they may retard wound closure.     

Diverse and potent activities of HGF/SF in skin wound repair

Genetic studies in the mouse have highlighted essential roles for several growth factors in skin repair and have offered a rationale for their use in therapy. The present study shows that the plasminogen-related growth factor HGF/SF (hepatocyte growth factor/scatter factor) promotes wound repair in homozygous diabetic db/db mice by recruiting neutrophils, monocytes, and mast cells to the wound; by promoting the migration of endothelial cells to the injured area; and by enhancing keratinocyte migration and proliferation. As a result, granulation tissue formation, wound angiogenesis, and re-epithelialization are all increased. The results demonstrate that HGF/SF affects and sustains all key cellular processes responsible for wound repair and point to a unique potential of this molecule for the therapy of chronic skin wounds.     

Effect of chitosan film containing basic fibroblast growth factor on wound healing in genetically diabetic mice

Basic fibroblast growth factor (bFGF) has been shown to stimulate wound healing. However, consistent delivery of bFGF has been problematic. We studied the stability of bFGF incorporated into a chitosan film as a delivery vehicle for providing sustained release of bFGF. The therapeutic effect of this system on wound healing in genetically diabetic mice was determined as a model for treating clinically impaired wound healing. A chitosan film was prepared by freeze-drying hydroxypropylchitosan (a water-soluble derivative of chitosan) acetate buffer solution. Growth factor was incorporated into films before drying by mixing bFGF solution with the hydroxypropylchitosan solution. bFGF activity remained stable for 21 days at 5 degrees C, and 86.2% of activity remained with storage at 25 degrees C. Full-thickness wounds were created on the backs of diabetic mice, and chitosan film or bFGF-chitosan film was applied to the wound. The wound was smaller in after 5 days in both groups, but the wound was smaller on day 20 only in the bFGF-chitosan group. Proliferation of fibroblasts and an increase in the number of capillaries were observed in both groups, but granulation tissue was more abundant in the bFGF-chitosan group. These results suggest that chitosan itself facilitates wound repair and that bFGF incorporated into chitosan film is a stabile delivery vehicle for accelerating wound healing.