Wound healing in the alpha2beta1 integrin-deficient mouse: altered keratinocyte biology and dysregulated matrix metalloproteinase expression

The alpha2beta1 integrin, a collagen/laminin receptor, is expressed at high level in the basal cell layer of the epidermis. To define the role of the alpha2beta1 integrin in wound healing, wound repair was extensively evaluated in wild-type and alpha2-null mice in vivo. In addition, the impact of alpha2beta1 integrin-deficiency on the function of primary murine keratinocytes in vitro was analyzed. Our in vivo findings demonstrate that genetic deletion of the alpha2beta1 integrin does not significantly alter the rate of re-epithelialization, collagen deposition, or tensile strength during wound closure in mice. In marked contrast to the observed similarities in wound healing, deletion of the alpha2beta1 integrin resulted in a dramatic increase in neoangiogenesis in the wound microenvironment. In contrast to in vivo studies, primary keratinocytes from alpha2-null mice adhered poorly and displayed impaired migration on type I collagen in vitro. We demonstrate that alpha2beta1 integrin-ligation negatively regulates expression of genes including matrix metalloproteinases both in vivo and in vitro. Furthermore, the changes in gene expression could potentially account for relatively normal wound healing in the alpha2-deficient mouse and our recent observation that suggests an antiangiogenic role for the alpha2beta1 integrin in vivo.     

Role of p38 MAPK in transforming growth factor beta stimulation of collagen production by scleroderma and healthy dermal fibroblasts

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.     

Integrin alpha2beta1 is required for regulation of murine wound angiogenesis but is dispensable for reepithelialization

The alpha2beta1 integrin functions as the major receptor for collagen type I on a large number of different cell types, including keratinocytes, fibroblasts, endothelial cells, and a variety of inflammatory cells. Recently, we demonstrated that adhesion of keratinocytes to collagen critically depends on alpha2beta1, whereas fibroblasts can partly compensate for loss of alpha2beta1 in simple adhesion to collagen. However, in three-dimensional collagen matrices, alpha2beta1-null fibroblasts are hampered in generating mechanical forces. These data suggested a pivotal role for alpha2beta1 during wound healing in vivo. Unexpectedly, reepithelialization of excisional wounds of alpha2beta1-null mice was not impaired, indicating that keratinocytes do not require adhesion to or migration on collagen for wound closure. Whereas wound contraction and myofibroblast differentiation were similar, wound tensile strain was reduced in alpha2beta1-null mice, suggesting subtle changes in organization of the extracellular matrix. In addition, we observed reduced influx of mast cells into the granulation tissue, whereas infiltration of other inflammatory cells was not impaired. Interestingly, ablation of alpha2beta1 resulted in strong enhancement of neovascularization of granulation tissue and sponge implants. Both ultrastructurally and functionally, these new blood vessels appeared intact. In conclusion, our data show unique and overlapping functions of alpha2beta1 integrin during murine cutaneous wound healing.     

丹参对系统性硬化病高胶原合成成纤维细胞克隆Ⅰ型前胶原基因转录的调控

目的 探讨丹参对系统性硬化病患者皮损高胶原合成成纤维细胞克隆Ⅰ型前胶原基因转录的调控。方法 采用系统性硬化病患者皮损和正常人皮肤胶原合成异质性成纤维细胞克隆,通过四甲基偶氮唑盐比色法,检测1 g/L丹参注射液及其主要水溶性单体（20 mg/L丹参素钠、5 mg/L丹酚酸B、5 mg/L原儿茶醛）和脂溶性单体（5 mg/L丹参酮ⅡA）对系统性硬化病和正常人高、低胶原合成克隆增殖活性（A490）的影响。利用瞬时转染、双荧光素酶报告基因技术,检测上述药物对克隆Ⅰ型前胶原α1链（COL1A1）基因近端启动区活性的调控。结果 在早期（≤3 d）,丹参注射液及单体对成纤维细胞克隆增殖的抑制不显著（P > 0.05）。但随作用时间延长,它们对克隆增殖的抑制作用多逐渐增强,第5天,丹参注射液组与水溶性阴性对照组比较,差异有统计学意义（q′ ＝ 3.22,P < 0.01）;第7天,丹参注射液组、丹酚酸B组和原儿茶醛组与水溶性阴性对照组比较,差异均有统计学意义（q′ 分别为4.74、3.03、2.56,P值均 < 0.05）。第5天和第7天,丹参酮ⅡA组与脂溶性阴性对照组比较,差异均有统计学意义（t值分别为2.22和2.15,P值均 < 0.05）。丹参注射液、丹参酮ⅡA和原儿茶醛抑制系统性硬化病和正常人成纤维细胞克隆中COL1A1近端启动区的活性（P < 0.01）,前两种药优先下调系统性硬化病高胶原合成克隆中COL1A1近端启动区的活性,COL1A1近端启动区在系统性硬化病高、低胶原合成克隆中的活性水溶性阴性对照组为12.019 ± 0.830和5.388 ± 0.480,丹参注射液组为4.445 ± 1.061和2.856 ± 0.597,F ＝ 31.78,P < 0.01;脂溶性阴性对照组为14.155 ± 0.672和4.299 ± 0.252,丹参酮ⅡA组为9.638 ± 0.854和3.192 ± 0.450,F ＝ 24.10,P < 0.01。结论 丹参可抑制系统性硬化病高胶原合成成纤维细胞克隆Ⅰ型前胶原基因的转录,其单体丹参酮ⅡA和原儿茶醛可能发挥主要作用。     

Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin: incisional wound model

Enhanced wound healing is elicited by exogenous administration of transforming growth factor- beta 1 (TGF- beta 1) in split-thickness, excisional wounds in the pig (Quaglino, Lab Invest 63:307-319, 1990). A study was designed to investigate if the selective and localized effects of TGF-beta 1 found in the previous model were dependent upon the type of wound or could be considered a more general effect of the cytokine. Transdermal, sutured incisions in the pig were evaluated by conventional histology and by in situ hybridization to reveal locally affected gene expression of collagen, elastin, fibronectin, stromelysin, TGF- beta 1, and basic fibroblast growth factor. Granulation tissue formation was markedly enhanced at 6 d by a single injection of recombinant human TGF beta 1 at the time of wound closure. Although granulation tissue was confined within the margins of the incisional wound, prominent differences in hybridization signals were observed between control and treated wounds. The stimulatory effect of TGF- beta 1 on granulation tissue formation was accompanied by a distinct enhancement in cells expressing mRNA for several different extracellular matrix proteins including collagens type I and III and elastin, whereas a single injection of human recombinant TGF beta 1 (4 micrograms) at the wound site diminished the expression of the neutral metalloprotease, stromelysin, and enhanced the frequency and intensity of cells expressing TGF- beta 1. The data reinforce the concept that TGF- beta 1 can act as a potent, auto-inductive modulator of connective tissue remodeling during the repair process.     

Cell-Based Therapy for RDEB: How Does It Work?

No effective or specific treatment is currently available for recessive dystrophic epidermolysis bullosa (RDEB), a severe heritable blistering disorder caused by mutations in the type VII collagen gene (COL7A1). Recent studies have suggested that delivery of allogeneic fibroblasts to the skin of patients with RDEB may be beneficial in improving skin adhesion and increasing type VII collagen deposition at the dermal-epidermal junction. In this issue, Nagy et al. explore mechanisms of fibroblast therapy in a patient with RDEB displaying reduced type VII collagen protein expression at the dermal-epidermal junction. The results suggest that allogeneic fibroblast injection elicits expression of cytokines, including heparin binding-EGF-like growth factor (HB-EGF), that upregulate the expression of a patient-specific COL7A1 allele. Thus, fibroblast therapy may provide a novel way to improve skin therapy in a select subgroup of patients with this currently intractable disease.     

Angiogenesis in wound healing

During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-beta, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, alpha(v)beta3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: alpha(v)beta3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of alpha(v)beta3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of alpha(v)beta3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds.     

Genetic linkage to the collagen α1 (V) gene (COL5A1) in two British Ehlers–Danlos syndrome families with variable type I and II phenotypes

To investigate the role of COL5A1 as a candidate gene for Ehlers–Danlos syndrome (EDS), we have carried out linkage studies in two large British families with EDS type I/II and type II, respectively. Fourteen living, affected individuals were identified by family history, clinical examination and ultrastructural analysis. A polymorphic intragenic simple sequence repeat at the COL5A1 locus showed linkage to EDS without recombination to give a combined lod score of 5.7. We have previously reported linkage to COL5A1 in an EDS type I/II family which brings the total loci score to 9.8 at zero recombination. Taken together, these data implicate COL5A1 as an important cause of EDS and confirm that types I and II are allelic.     

A novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion of 30 nucleotides into the mature mRNA in a case of vascular type of Ehlers–Danlos syndrome

Vascular type of Ehlers–Danlos syndrome (EDS) is the most severe type of EDS. It is an autosomal dominantly inherited disorder that results from mutations within the α1 type III collagen gene (COL3A1). We report a novel point mutation at donor splice-site in intron 42 of type III collagen gene resulting in the inclusion of 30 nucleotides into the mature mRNA in a case of vascular type of EDS. Since the age of approximately 8 months, the patient had had repeated episodes of purpura and gradually developed thin, translucent skin. She had a past history of pneumothorax. At the initial examination, she was found to have the characteristic facies, i.e., bird-like face, of the vascular type of EDS, thinning of skin over the limbs and trunk, and scattered purpura. The blood vessels under the skin could be clearly visualized. She showed hypermobility of the small joints of all the four limbs and acrogeric changes of the hands and feet. Analysis of the amount of collagen synthesized from cultured dermal fibroblasts by SDS-polyacrylamide gel electrophoresis and fluorography was conducted based on the clinical suspicion of the vascular type of EDS, and a marked reduction in the synthesis of type III collagen was observed. Genetic analysis of the COL3A1 revealed a novel point mutation at the donor splice-site of intron 42, which resulted in the inclusion of 30 nucleotides into the mature mRNA of one allele.     

Differential contribution of dermal resident and bone marrow-derived cells to collagen production during wound healing and fibrogenesis in mice

Recent studies show that bone marrow (BM)-derived cells migrating into a dermal wound promote healing by producing collagen type I. However, their contribution to the repair process has not been fully verified yet. It is also unclear whether BM-derived cells participate in dermal fibrogenesis. We have addressed these issues using transgenic mice that harbor tissue-specific enhancer/promoter sequences of α2(I) collagen gene linked to either enhanced green fluorescent protein (COL/EGFP) or the luciferase (COL/LUC) reporter gene. Following dermal excision or subcutaneous bleomycin administration, a large number of EGFP-positive collagen-producing cells appeared in the dermis of COL/EGFP reporter mice. When wild-type mice were transplanted with BM cells from transgenic COL/EGFP animals and subjected to dermal excision, no EGFP-positive BM-derived collagen-producing cells were detected throughout the repair process. Luciferase assays of dermal tissues from COL/LUC recipient mice also excluded collagen production by BM-derived cells during dermal excision healing. In contrast, a limited but significant number of CD45-positive collagen-producing cells migrated from BM following bleomycin injection. These results indicate that resident cells in the skin are the major source of de novo collagen deposition in both physiological and pathological conditions, whereas BM-derived cells participate, in part, in collagen production during dermal fibrogenesis.     

Poly-L-Lactic acid increases collagen gene expression and synthesis in cultured dermal fibroblast (Hs68) through the TGF-β/Smad pathway

Objective

Poly-L-Lactic Acid (PLLA) is a synthetic polymer which possesses biocompatible and biodegradable properties, and is widely used in the clinical filler material. This study focuses on the potential role of PLLA on the collagen production of dermal fibroblasts and its mechanism.

Methods

The dermal fibroblast Hs60 was treated with different concentration of PLLA. RT-qPCR was conducted for the determination of mRNA levels of collagen type I (COL1) alpha 1 (COL1A1), COL1 alpha 2 (COL1A2), elastin, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. Procollagen Type I C-peptide (PIP) enzyme immunoassay (EIA) Kit assay was carried out to analyze procollagen production. Western Blot was employed to examine the effect of PLLA and transforming frown factor (TGF-β) receptor-specific inhibitor (SB431542) on protein levels of COL1A1 and TGF-β/Smad signaling pathway related proteins.

Results

With the increase of PLLA concentration, the production of procollagen gradually increased, and both protein and mRNA levels of COL1A1 and COL1A2 gradually increased (p < 0.001). Elevated PLLA concentrations increased elastin, TIMP-1, and TIMP-2 levels and attenuated MMP-1 expression. PLLA increased TGF-β levels in a dose-dependently manner. p-Smad2 and p-Smad3 protein levels were also increased by PLLA, but the influences were reversed by SB431542 (p < 0.001). Similarly, increased levels of COL1A1, COL1A2, TIMP-1, and TIMP-2 caused by PLLA were significantly inhibited by SB431542, whereas MMP-1 was typically elevated (p < 0.001).

Conclusion

Poly-L-Lactic Acid promotes the collagen production of dermal fibroblasts by activating the TGF-β/Smad signaling pathway. The findings may lay a foundation for clinical material applications of PLLA.     

Isolation and characterization of type V collagen from human post-burn granulation tissues

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of pepsin-solubilized collagens from post-burn granulation tissues revealed that type V collagen consisted of 3 alpha chains: alpha 1(V), alpha 2(V), and alpha 3(V). The mean value (0.12 +/- 0.01 SD) of the type V/type I ratio in the granulation tissues was significantly higher (p less than 0.001) than that (0.03 +/- 0.01 SD) of the ratio in normal skin. The average ratio of alpha 1(V):alpha 2(V):alpha 3(V) of type V collagen purified from the granulation tissues was determined to be about 5:3:1. SDS-polyacrylamide gel electrophoresis patterns of 3 alpha chains were not affected in the presence or absence of 2-mercaptoethanol. Purified type V collagen was degraded by bacterial collagenase, but remained intact after tadpole collagenase digestion, in contrast to type I and type III collagens. Amino acid analyses of each alpha chain separated on SDS-gel electrophoresis of type V collagen revealed that all 3 alpha chains of type V collagen were poor in alanine, rich in hydroxylysine, and had high ratios of hydroxylysine/lysine, which are typical features of type V collagen. The purified type V collagen was further fractionated by ammonium sulfate into 2 molecular species, [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V). Our data demonstrate that type V collagen in preparations from human post-burn granulation tissues consists of 3 alpha chains and can be resolved into 2 distinct heterotrimers.     

Construction of a full-length murine pro alpha 2(I) collagen cDNA by the polymerase chain reaction.

Construction of large collagen cDNA has been hindered by the relatively large size and high G-C content of processed mRNA. We describe here the development of a rapid and efficient method for obtaining large full-length collagen cDNA. A full-length (4.3 kb) murine pro alpha 2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT17-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Pro alpha 2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a pro alpha 2(I) specific primer as the 5' primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine pro alpha 2(I) collagen gene, COL1A2), and with the adapter sequence 5' to the dT17 as the 3' primer. The PCR conditions were optimized to allow amplification of the expected 4.0-5.0-kb product; a major 4.3-kb product was visualized by ethidium bromide, identified by in situ gel hybridization, and cloned. DNA sequencing determined that it contained the correct 5' sequence and the 3' end had a 68 basepair (bp) 3' untranslated region. The entire sequence that codes the amino-terminal propeptide domain has been determined and compared to the human sequence. The homology between human and mouse is less in the amino terminal propeptide than in the triple helical domain; exon 5 of murine COL1A2 codes for an additional six amino acids not found in human COL1A2.