Increased Expression And Signification Of HIF-1α In Hypertrophic Scar And Keloid

BACKGROUND:Hypertrophic scar and keloid scar are general abnormal scars seen in clinic, which are result of over wound-healing. Keloids are locally aggressive and, in contrast to hypertrophic scar, they proliferate beyond the boundaries of the initial wound. They often recur after various types of therapy and persist for many years. Though a lot of researches have been performed on abnormal scars, pathogenesis of abnormal scars were still unclear. It is still one of key points of plastic surgery to approach the pathogenesis of abnormal scars.In prophase of our study, we identified differentially expressed genes between pathological scar tissues and normal skin tissues using microarray-based detection that composed of 8,064 clones. The results indicated that 171 genes were different expression genes between hypertrophic scar with normal skin, where 99 genes were up-regulated and 72 genes were down-regulated; 277 genes were different expression genes between keloid scar with normal skin, where 163 genes were up-regulated and 114 genes were down-regulated. These datas indicated that the formation of pathological scar was involved of differentially expression of mutiple genes. These genes were relative to immune, apoptosis, proto oncogene, anti oncogene and cytoskeleton movement and so forth. How to analyse these dates, extract useful informations and guide further study are vital.Therefore, a mining technique based on the analysis of literature profiles generated by extracting the frequencies of certain terms from thousands of abstracts about these different expression genes stored in the Medline literature database was used to annotate these genes. Terms are then filtered on the basis of both repetitive occurrence and co-occurrence among multiple gene entries and suppositional pathogenesis theory of abnormal scars to get terms at last. Finally, clustering analysis with Cluster and Treeview program is performed on the retained frequency values, shaping a coherent picture of the functional relationship among these large and heterogeneous lists of genes. Such data treatment also provides information on the nature and pertinence of the associations that were formed.197 different expression genes with known names in abnormal scars were further analyzed with literature mining methods.87,282 records about these genes were extracted from PubMed. After text analysis and data filtering,23 terms were obtained to be clustered analysis. Results indicated that HIF1A was related with terms "hypoxia", "proliferation", "tumor", "apoptosis", "fibroblast", and "scar". So we presume that HIF-la could be a significantly new gene that play a important role in abnormal scarring.As early as 1970s, Kischer presumed that hypoxia played an important role in pathological scar formation. Hypoxia can affect the activity of fibroblasts and collagen synthesis and degradation and growth factor secretion that were three main factors of pathological scar formation. Literatures suggested that in the early pathological scar, due to high cell density and high cell activity may lead to a hypoxia state of scars. Vessels in keloid are significantly lower than that in hypertrophic scar. Scanning electron microscopy and transmission electron microscopy showed that vessel blocking by microvascular endothelial cell proliferation in keloid tissue lead to tissue hypoxia. Therefore, it is presumed that less blood vessels and hypoxia are one of pathophysiology of abnormal scars.Hyman body can feel O2 concentration, and make adaptive responses to hypoxia. Regulation of gene expression is such a major way of adaptive responses. Hypoxia can activate the transcription of many genes for maintenance of tissue oxygen supply and enhance of cell viability under hypoxia. With the deepening of research about hypoxia regulating gene expression, we found that HIF-1αplayed not only a centrum role in physiologic reaction at hypoxic condition, but also an important role in normal development of embryo, and in metabolism, angiogenesis and metastasis of tumor.HIF-1 gene located in human 14q21-24, is a heterodimers (or tetramers) composed by HIF-1αand HIF-1βsubunits. The biological activity of HIF-1 is determined by HIF-1αsubunit that is a direct receptor of hypoxia, its expression is strictly regulated by O2 concentration. In normoxic conditions, HIF-1αwas controlled by the structure of the oxygen-dependent degradation domain (ODD), rapidly degraded through the ubiquitin-proteasome pathway. So HIF-1αprotein is almost undetectable in normoxic conditions. Contrastively in hypoxic conditions, HIF-1αubiquitination and degradation process was inhibited, resulting in HIF-1αprotein in intracellular accumulation, polymerizing with HIF-1β, and forming active HIF-1 protein that regulated many genes such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-2), epidermal growth factor receptor (EGFR), Matrix Metalloproteinase (MMP), erythropoietin (EPO) and more than 60 genes transcription and expression, causing cells to generate a series of adaptive responses to hypoxia. It has been proven that parts of these genes have close relative with the formation of pathological scar. Foreign scholars have also found that HIF-1αexists up-regulated expression in keloid. Li Gaofeng has found that HIF-1αmay play an important role in the process of hypoxia reduction in the burn scars. But still no one has explored a HIF-1 induced hypoxia pathway. Thus, to identify the mechanism of hypoxia pathway of keloid has important theoretical and practical significance in improving the level of clinical prevention and treatment of keloidOBJECTIVES:1. To compare the expression of HIF-1 A among normal skin tissues, hypertrophic scar tissues and keloid tissues, and among fibroblasts of normal skins, hypertrophic scars and keloids under a hypoxia condition in order to analyse the effect of HIF-1 in pathological scarring.2. To investigate the correlation of HIF-1αand hypoxia in keloids fibroblasts. To investigate the effects of normoxia and different hypoxia on proliferation, cell cycle and collagen synthesis of keloid fibroblasts. Sequentially to investigate the mechanism that hypoxia advance abnormal scarring by HIF-1αpathway.PATIENTS AND METHODS:1. Surgical ExcisionThe study was approved by the medical and ethical committees of the participating hospitals, and patients signed an informed consent before the study. Four patients with keloid and hypertrophic scar were prepared for investigation. Four keloid samples were resected from ear lobe keloid patients. None of the patients had received previous treatment for their keloids. Hypertrophic scars were on the extremities in the four patients. All lesions were resected for aesthetic and functional improvement. Four normal skin tissue were from the patients who was circumcised. The patients'ages ranged from 12 to 40 years.2. Methodsa. RT-PCR was performed to detect the expressions of HIF-1αprotein.b. Western Blotting was performed to detect the expression of HIF-1αmRNA.c. MTT assay were performed to evaluate the cell proliferation of the keloid fibroblasts.d. Flow cytometer was performed to evaluate the cell cycle of the keloid fibroblasts.e. Hydroxyproline chromatometry was performed to detect collagen synthesis of the keloid fibroblasts.RESULTS:1. The expression of HIF-1A in pathological scar biopsiesThe results of RT-PCR and Western Blotting demonstrated that HIF-la was higher expressed in pathologic scar biopsies than in normal skin in both mRNA and protein levels.2. The expression of HIF-1A in pathological scar fibroblastsThe results of RT-PCR and Western Blotting demonstrated that HIF-1αwas higher expressed in pathologic scar fibroblasts cultured under hypoxia in vitro than in normal skin fibroblasts in both mRNA and protein levels.3. The expression of HIF-1 A in keloid fibroblasts under different hypoxiaRelative amounts of HIF-1αin keloid fibroblasts cultured under O2 concentrations at 20%,10%,5%,1%were 0.007±0.006,0.133±0.006,0.537±0.015, 0.903±0.021. It indicated that hypoxia can increase the expression of HIF-1αin keloid fibroblasts.4. Cell proliferation, S phase and collagen synthesis of keloid firoblastsWith the O2 concentrations decreasing, the cell proliferation and the proportion of the keloid fibroblasts in the S phase of cell cycle also became more and more higher. When compared with the control groups, hypoxia can markedly increase collagen synthesis of the cultured keloid fibroblasts.CONCLUSIONS:1. The up-regulated expressed HIF-1αhinted that the up-regulated expressions of HIF-1αand the hypoxia pathway induced by it subsequently may correlated with the tumor-like characteristics of hypertrophic scars and keloids.2. Hypoxia can induce the expression of HIF-1αin fibroblasts of keloids. Moreover, there still is a positive relation between hypoxia and the expression of HIF-1α.3. Hypoxia can induce keloid fibroblast proliferation and increase collagen synthesis. The mechanism may be by the up-regulated expressions of HIF-1αand the hypoxia pathway induced by it subsequently. Decreasing the pathway may be advantageous to prevent scar proliferation and reduce the collagen deposition in abnormal scarring.