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。     

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.     

Dynamics of the healing of skin wounds in the horse as compared with the rat.

The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of prolyl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.     

Growth factors bFGF and TGB beta accelerate the rate of wound repair in normal and in diabetic rats

The effects of basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) on the rate of wound repair in both normal and streptozotocin-induced diabetic rats were investigated using two model systems of wound repair, namely incisional wounding and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. Both models showed the expected wound-healing defects of the diabetic rats. Granulation tissue collected from the implanted PVA sponges showed that the diabetic rats had reduced amounts of collagen, DNA and protein present at the wound site at two time points tested (7 and 9 days post-implantation). Fresh tensile strength of the incisional wounds, a measure of the collagen organization in a wound, was reduced to 53% of normal in diabetic rats on day 7 post-wounding, and was only 29% of normal by day 21. Formalin-fixed tensile strength, a measure of collagen content of the wound, was 41% of normal on day 7, and 78% of normal by day 21, giving evidence that while the collagen concentration of the diabetic wounds approached that of normal wounds, it did not undergo the normal maturation process. A single injection of 2 micrograms of TGF beta directly into the incision three days after wounding resulted in little difference in the fresh and fixed tensile strength of diabetic wounds when tested at 7, 14 and 21 days post-wounding. Normal rats, however, responded well, resulting in a greater than 30% increase in both fresh and fixed tensile strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and activity of matrix metalloproteinase-2 and -9 in experimental granulation tissue

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix-modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP-2 and MMP-9 and to study the regulation of MMP-2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP-2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP-9 gene expression was low between days 8-21 and increased after 4 weeks of granulation tissue formation. Membrane-type 1 MMP (MT1-MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP-2) mRNA later during wound healing. In in situ hybridization the expression of MMP-2 mRNA was seen mostly in fibroblast-like cells and MMP-9 mRNA in macrophage-like cells. MMP-9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage-like cells on days 3-8. MMP-9 proteolytic activity was observed only on days 3-8. The active form of the MMP-2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP-2 did not show any apparent changes during the experimental period. We conclude that MMP-2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP-9 was demonstrated only in early wound healing, and the MMP-9 gene is upregulated when the granulation tissue matures.     

Type V collagen in experimental granulation tissue

To evaluate the spatial and temporal expression of type V collagen in a wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 3, 5, 8, 14, 21, 30, 59 and 84. Acid soluble collagens were extracted and the relative amount of type V collagen was quantified by SDS-PAGE. Specific antibodies to type I, III and V collagens were used in immunohistochemistry and specific RNA probes to proalpha1(I), proalpha1(III) and proalpha1(V) collagen in in situ hybridization. Type V collagen content increased relative to type I and III collagens up to day 8 and remained at the same level for up to the three months. Type V collagen was expressed strongly in blood vessel walls as seen in immunohistochemistry. In situ hybridization showed that all of the three types of collagen were expressed mostly in fibroblast-like cells and also in rounded cells, especially type V collagen. In conclusion, type V collagen was seen in the wound healing model in increasing amounts from day 3 onwards, its localization being highly associated with blood vessels in granulation tissue and it was synthesized by fibroblast-like and rounded cells.     

Role of elevated plasma transforming growth factor-beta1 levels in wound healing

Transforming growth factor (TGF)-beta1 plays a central role in wound healing. Wounds treated with neutralizing antibody to TGF-beta1 have a lower inflammatory response, reduced early extracellular matrix deposition, and reduced later cutaneous scarring, indicating the importance of local tissue TGF-beta1. By contrast, increasing the local, tissue levels of TGF-beta1 increases the early extracellular matrix deposition but does not alter scar formation. Increased levels of plasma TGF-beta1 correlate with increased fibrogenesis in the lung, kidneys, and liver. The aim of the present study was to investigate the role of elevated systemic levels of TGF-beta1 on wound healing. We used transgenic mice that express high levels of active TGF-beta1 and have elevated plasma levels of TGF-beta1 and wild-type mice of the same strain as controls. Incisional wounds and subcutaneously implanted polyvinyl alcohol (PVA) sponges were analyzed. Surprisingly, cutaneous wounds in transgenic, TGF-beta1-overexpressing mice healed with reduced scarring accompanied by an increase in the immunostaining for TGF-beta3 and TGF-beta-receptor RII and a decrease in immunostaining for TGF-beta1 compared with wounds in control mice. By contrast, the PVA sponges showed the opposite response, with PVA sponges from transgenic mice demonstrating an enhanced rate of cellular influx and matrix deposition into the sponges accompanied by an increase in the immunostaining for all three TGF-beta isoforms and their receptors compared with PVA sponges from control mice. Together, the data demonstrate that increased circulating levels of TGF-beta1 do not always result in increased expression or activity in selected target tissues such as the skin. The two wound models, subcutaneously implanted PVA sponges and cutaneous incisional wounds, differ significantly in terms of host response patterns. Finally, the data reinforce our previous observations that the relative ratios of the three TGF-beta isoforms is critical for control of scarring.     

Low molecular weight hyaluronic acid prevents oxygen free radical damage to granulation tissue during wound healing

Hyaluronic acid protects granulation tissue from oxygen free radical damage and stimulates wound healing, but its molecular weight prevents it from permeating the epidermal barrier A low molecular weight hyaluronic acid preparation is able to permeate the skin, but it is unknown whether or not it retains the scavenging effects of oxygen free radicals in granulation tissue. Our experiments were conducted in rats with excisional or incisional wounds. Wound contraction over 11 days and breaking strength on the fifth day were measured. Oxygen free radical production was induced by intraperitoneal administration of two different xenobiotics: phenazine methosulfate and zymosan. The wounds were treated topically with low molecular weight hyaluronic acid (0.2%) cream or placebo. In the incisional wound group, the effects of superoxide dismutase were also determined. Absolute controls received wounds and placebo but no xenobiotics. Wound healing was significantly slower in the xenobiotic group than in the control groups. These effects were strongly reduced by topical administration of low molecular weight hyaluronic acid (0.2%) cream and in incisional wounds by topically injected superoxide dismutase. Low molecular weight hyaluronic acid is effective as the native compound against oxygen free radicals. Its pharmacological effects through transdermal administration should be tested in appropriate models.     

Role of endothelial selectins in wound repair.

P- and E-selectins are adhesion molecules expressed on activated endothelium and platelets at sites of vascular injury and inflammation. The selectins are important for leukocyte recruitment. Because little is known about the role of selectins in wound healing, we studied cutaneous wound repair of full-thickness excisional skin wounds in mice lacking P-selectin, E-selectin, or both of these selectins. The absence of either selectin alone had no notable effect on healing, and the only deficit observed was a delay in early neutrophil extravasation in the P-selectin-deficient mice. Mice deficient in both P- and E-selectins had markedly reduced recruitment of inflammatory cells and impaired closure of the wounds. Wound sections, studied up to 3 days after wounding, showed significant impairment of neutrophil influx. Macrophage numbers were also reduced in the double mutants at 3 and 7 days after wounding as compared with wild-type mice. Additionally, a wider epithelial gap in the wounds of the P- and E-selectin-double-deficient mice 3 days after wounding indicated delayed keratinocyte migration. These results demonstrate an important combined role for P- and E-selectins in processes leading to wound healing.     

Macroscopic aspects of wound healing (contraction and epithelialisation) after topical administration of allicin in dogs

Macroscopical aspects of second-intention healing of full-thickness excisional wounds were studied in five normal male mixed-breed dogs. Test wounds were treated topically with allicin 0.5% in methyl cellulose gel, and control wounds were treated with methyl cellulose gel only. Wound treatment started 24 h after wounding. The wounds were evaluated over a 4-week period. At days 0, 3, 7, 10, 14, 17, 21, 24 and 28, digital photographs were taken of all wounds. Rulers were held vertically and horizontally close to the wound as a reference. The area of epithelialisation and granulation tissue were measured for each wound using Scion Image software. Percentage wound contraction, epithelialisation and healing were calculated for each wound. Initially, all wound areas increased in size. After the initial enlargement, wound areas decreased rapidly in size between days 7 and 17 in both the test and control groups. Epithelialisation was first noticed at day 3 in control and day 5 in the test wounds. No significant differences were observed in the percentage of wound contraction, epithelialisation and healing between the test and control wounds (P> 0.05).     

TGF-beta 1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits.

TGF-beta modulates events of normal wound healing through multiple pathways that influence cell infiltration, proliferation, angiogenesis, extracellular matrix synthesis and remodeling. The effects of topically applied TGF-beta 1 on wound healing in two models of healing were evaluated when the healing response was impaired by the administration of methylprednisolone to rats or rabbits. TGF-beta 1 increased the healing of linear incision wounds on rats, as measured by breaking strength, to that of normal rats. Full thickness open wounds were also created on the inner ears of rabbits to simulate a non-contracting wound with limited blood supply. Healing was further impaired by the administration of methylprednisolone. The single application of TGF-beta 1 improved the healing of open wounds. TGF-beta 1 stimulated increased granulation tissue formation, as well as reepithelialization. The amount of granulation tissue and epithelialization were similar to wounds from normal-healing control rabbits. The delayed healing caused by methylprednisolone permitted the evaluation of multiple applications of TGF-beta 1 to wounds. Two applications of TGF-beta 1 spaced 7 days apart further improved the healing response when compared to a single application. Thus, single or multiple topical applications of TGF-beta 1 reversed impaired healing conditions secondary to methylprednisolone when used on incisional or open wounds. These observations support the hypothesis that growth factors, such as TGF-beta 1, may be useful as accelerators of wound repair in patients with impaired healing conditions.     

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.     

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.     

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.     

Effects of cyclophosphamide on open, granulating skin wounds in rats.

The effect of cyclophosphamide on the healing of open cutaneous wounds was studied in rats. Following intraperitoneal injections of 25 mg/kg body weight every second day for 9 days, only about 7% of the wounds were completely covered by epithelium after 15 days, whereas in the control animals 60% of the wounds were completely epithelialized. Measurements of wound diameters in circular skin wounds revealed unhealed wound areas in the drug treated animals which were significantly larger than those of the control animals. Cyclophosphamide was found to reduce the occurrence of H3-labelled cells in the granulation tissue when evaluated after 11 days. At 15 days there was no difference in the labelling frequency between treated and control animals, indicating reversal of the drug effect.