Differential effects of hexoses and sucrose, and platelet-derived growth factor isoforms on cyclooxygenase-1 and -2 mRNA expression in keloid, hypertrophic scar and granulation tissue fibroblasts

Abstract Cyclooxygenase (COX) is the key enzyme in the formation of prostaglandins in inflammation. In the present study the effects of biomedically relevant hexose sugars (glucose, fructose, galactose, mannose) and sucrose disaccharide on the expression of COX-1 and COX-2 genes were evaluated in granulation tissue fibroblasts, hypertrophic scar fibroblasts and keloid fibroblasts. The effects of three isoforms (AA, AB and BB) of PDGF on COX gene expression in granulation tissue fibroblasts were also examined. All cell lines expressed COX-1 mRNA, whilst fibroblasts derived from abnormal scars did not express COX-2 mRNA. COX-1 mRNA expression was decreased by sugars in granulation tissue fibroblasts and increased in hypertrophic scar fibroblasts. No major changes were seen in keloid fibroblasts. On the other hand, COX-2 mRNA expression in granulation tissue fibroblasts was decreased dramatically in the presence of fructose, mannose and sucrose and moderately in the presence of galactose. All isoforms of PDGF increased COX-1 and COX-2 mRNA expression in granulation tissue fibroblasts, the most marked increases being elicited by PDGF-BB. All fibroblast cell lines studied expressed the COX-1 gene while the COX-2 gene was not expressed by abnormal scar-derived fibroblasts. Further, granulation tissue fibroblasts seemed to behave differently under the influence of sugars compared to hypertrophic scar fibroblasts whilst keloid fibroblasts seemed to be relatively unaffected by sugars. In addition, the PDGF-BB isoform is a potent inducer of COX-2 gene expression in wound fibroblasts. These findings may be relevant to the development of abnormal scars and indicate the need for further studies. Received: 9 March 2000 / Revised: 28 July 2000 / Accepted: 23 November 2000     

Elevated prolidase activity in keloids: correlation with type I collagen turnover

BACKGROUND: Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide-cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover. OBJECTIVES: To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts. METHODS: Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis. RESULTS: Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids. CONCLUSIONS: The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.     

Reduced collagenase gene expression in fibroblasts from hypertrophic scar tissue

Summary The major histopathological feature of hypertrophic scar lesions is fibrosis. characterized by excessive accumulation of collagen. The purpose of this study was to determine if there is not only increased expression of collagen but also decreased expression of collagenase in hypertrophic scar fibroblasts. We compared the expression of mRNA for of α₁ (I) and α₁ (III) collagen, and collagenase in cultured fibroblasis from different portions of hypertrophic scars and normal dermis. the hypertrophic scar fibroblasts. increased levels of α₁ (I) and α₁ (III) collagen mRNAs were observed in fibroblasts from the edge and outside of scar tissue, while normal levels were noted in fibroblasts from the centre of this tissue. In contrast. decreased levels of collagenase mRNA were found in the hypertrophic scar fibroblasts. The reductions were centre (25% of the control) greater than the edge (43% of the control) greater than the outside (84% of the control). The changes in the collagenase mRNA levels of the hypertrophic scar fibroblasts correlated well with decreased collagenolytic activity as determined by the degradation rate of fluorescent isothiocyanate-labelled type I collagen in fibroblast culture supernatant. These results suggest that decreased expression of collagenase in hypertrophic scar fibroblasts may be one possible cause for the excessive accumulation of collagen in the skin lesions of hypertrophic scars.     

Increased expression of collagen types I and III in human skin as a consequence of radiotherapy

To study the mechanisms of irradiation-induced fibrosis, the expression of types I and III collagen was analysed in radiotherapy-treated human skin. The subjects were ten randomly chosen women who had been treated for breast cancer with surgery and radiotherapy. The subjects ranged in age from 42 to 68 years (mean 53 years) and the time from treatment ranged from 7 to 94 months. The irradiated skin was compared with a corresponding healthy skin area in the same subject. Suction blisters were induced on both skin areas. The aminoterminal propeptides of types I and III collagen (PINP and PIIINP), which reflect actual in vivo skin collagen synthesis, were determined in the suction blister fluid using radioimmunoassays. mRNA of types I and III collagen were determined in skin specimens using a nonradioactive in situ hybridization (ISH) technique. Immunohistochemical staining for PINP was also performed. The level of PINP in suction blister fluid was increased more than threefold and the level of PIIINP more than twofold in irradiated skin compared to control skin. The number of cells containing type I and type III collagen mRNA was increased in the upper dermis of irradiated skin. Immunohistochemical staining showed the amount of PINP-positive fibroblasts to be increased in irradiated skin. We conclude that skin collagen gene expression is increased as a result of irradiation and this leads to fibrosis and thickening of the dermis.     

增生性瘢痕发生和演变过程中成纤维细胞生物学功能变化及其意义

目的 探索增生性瘢痕在发生和演变过程中,成纤维细胞生物学功能变化的规律及其意义.方法 选取人不同时期增生性瘢痕组织和正常皮肤组织,进行HE染色观察.另外,分离和培养不同时期瘢痕和正常皮肤中成纤维细胞,RT-PCR分别检测成纤维细胞在转移生长因子（TGF-β1）、血管内皮细胞生长因子（VEGF）、和Ⅰ、Ⅲ胶原mRNA表达水平的变化.结果 HE染色可见正常皮肤细胞和微血管数目较少,早期瘢痕增多,炎症细胞浸润明显.增生期瘢痕成纤维细胞和微血管增多.随病情进展,微血管呈缩窄倾向.消退期瘢痕成纤维细胞减少,微血管狭窄或闭塞.成熟期瘢痕见微血管、成纤维细胞数目进一步减少,微血管管腔小,大部分开放.RT-PCR检测结果发现,正常皮肤来源的成纤维细胞TGF-β1、VEGF和Ⅰ、Ⅲ胶原mRNA有较低表达,早期瘢痕成纤维细胞TGF-β1、VEGF和Ⅰ、Ⅲ胶原mRNA表达水平开始升高,在增生期表达到达高峰,消退期瘢痕的表达开始下降,到成熟期表达最低.结论 增生性瘢痕发生和演变过程中,成纤维细胞功能存在动态改变,这种动态改变与瘢痕的发生和消退成熟的病理变化相关.     

Distinct patterns of collagen gene expression are seen in normal and keloid fibroblasts grown in three-dimensional culture

The purpose of this study was to examine collagen gene expression in various types of scar fibroblasts as well as normal fibroblasts in a novel three-dimensional culture system and to compare them with those in a monolayer culture system. Cells in three-dimensional culture formed multiple layers within the self-produced dense extracellular matrix and formed a dermis-like structure. In monolayer culture, both normal and scar fibroblasts continued to express high levels of mRNA for proα1(I) and proα1(III) collagens. However, in three-dimensional culture, the mRNA levels gradually declined in normal fibroblasts. In contrast, mRNA levels remained high in keloid and hypertrophic scar fibroblasts. Atrophic scar fibroblasts demonstrated similar changes to normal fibroblasts in three-dimensional culture. When we compared mRNA expression in fibroblasts from the centre and the edge of hypertrophic scar, cells from the centre showed a persistently decreased level of collagenase mRNA expression. These results suggest that the mRNA expression pattern of proα1(I) and proα1(III) collagens varies depending on the culture system. Fibroblasts from keloids and hypertrophic scar may have a defective system of down-regulation in extracellular matrix metabolism.     

Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin

Radiation-induced fibrosis is a common side-effect of cancer treatment. The pathophysiological events leading to fibrosis are not known in detail. We analysed the effect of therapeutic irradiation on human skin collagen synthesis, skin thickness, gelatinases and their inhibitors. Twenty randomly chosen women who had been treated for breast cancer with surgery and radiation therapy participated in the study. In each patient, the irradiated skin area was compared with a corresponding non-treated skin area. Suction blister fluid (SBF) and serum samples were analysed for the aminoterminal propeptides of type I and type III procollagens (PINP and PIIINP), tissue inhibitors of matrix metalloproteinases (MMPs) 1 and 2 (TIMP-1 and TIMP-2) and MMP-9 and MMP-2/TIMP-2 complex. Skin biopsies were analysed for PINP and immunohistochemical staining was used for PIIINP. In irradiated skin, PINP, PIIINP, TIMP-1 and MMP-2/TIMP-2 complex levels in SBF and the number of PINP-positive fibroblasts in tissue sections were significantly higher in comparison with non-treated skin. The levels of TIMP-2 in irradiated and non-irradiated skin were similar. MMP-9 could not be detected in SBF with the assay used. The serum levels of MMP-9 were higher in the treated subjects than the reference values. The serum values of PINP, PIIINP, TIMP-1, TIMP-2 and MMP-2/TIMP-2 complex were not significantly affected. These results indicate increased local collagen synthesis and accumulation of connective tissue in irradiated skin. The marked upregulation of collagen synthesis as a result of irradiation offers a possibility to treat this complication with compounds such as topical steroids which downregulate collagen synthesis.     

Overexpression of miR‐200b inhibits the cell proliferation and promotes apoptosis of human hypertrophic scar fibroblasts in vitro

Hypertrophic scarring leads to a deformed appearance and contracted neogenetic tissue, resulting in physiological and psychological problems for patients. Millions of people suffer these discomforts each year. Emerging evidence has reported that miRNA contributed to hypertrophic scarring or keloid formation. In this study, nine hypertrophic scar samples and the matched normal skin tissues were used to perform a miRNA microarray. The results of miRNA array showed that miR‐200b was downregulated by more than 2‐fold, validated by qPCR in hypertrophic scar tissues and human hypertrophic scar fibroblasts, suggesting that there was an important correlation between miR‐200b and hypertrophic scarring. We also found that miR‐200b affected hypertrophic scarring through regulating the cell proliferation and apoptosis of human hypertrophic scar fibroblasts by affecting the collagen I and III synthesis, fibronectin expression and TGF‐β1/α‐SMA signaling. Thus, our study provides evidence to support that miR‐200b may be a useful target for hypertrophic scarring management.     

A fibroblast cell line cultured from a hypertrophic scar displays selective downregulation of collagen gene expression: barely detectable messenger RNA levels of the proα1(III) chain of type III collagen

The present study was designed to investigate the expression of type I, III and VI collagens by a fibroblast cell line initiated from a hypertrophic scar. The same tissue has previously been demonstrated to display markedly elevated expression of type I and III collagen mRNAs in vivo. Unexpectedly, slot-blot and Northern hybridizations revealed a barely detectable steady-state level of pro1(III) collagen chain mRNA in cultured hypertrophic scar fibroblasts. The levels of pro1(I) and 2(VI) collagen chain mRNAs were essentially the same in fibroblasts cultured from hypertrophic scar and in fibroblasts cultured from normal skin. However, Northern blot analyses indicated that the ratio of 5.8 kb to 4.8 kb species of pro1(I) collagen mRNA was slightly reduced in fibroblasts originating from the hypertrophic scar compared to that in normal fibroblasts. When normal fibroblasts were incubated in conditioned medium from hypertrophic scar cultures, the expression of pro1(III) collagen chain mRNA decreased to a markedly lower level. Our studies suggest that collagen synthesis by fibroblasts in hypertrophic scars is stimulated by humoral factors which are active only in vivo. Furthermore, the results suggest that fibroblasts cultured from hypertrophic scar display a selective downregulation of different collagen genes and that this downregulation is exerted through an autocrine mechanism.     

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.     

Collagen mRNA expression detected by in situ hybridization in keloid tissue

The keloid fibroblasts exhibited increased extracellular matrix gene expression, and prominent elevated type I procollagen mRNA when compared to control fibroblasts cultured from the uninvolved skin of normal people. It also showed markedly elevated type I/III procollagen mRNA ratios, but no synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. By in situ hybridization in keloid tissue, high levels of type I and type III procollagen mRNAs were detected in most of the fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of type I and type III procollagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens. And concentration of type I procollagen mRNA was found more predominantly than was type III. These results suggest that deposition of collagen in keloid could result from activation of certain fibroblasts responsible for type I procollagen production.     

Studies of transforming growth factors beta 1-3 and their receptors I and II in fibroblast of keloids and hypertrophic scars

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterized by an abnormal deposition of extracellular matrix components, particularly collagen. There is uncertain evidence that transforming growth factor-beta (TGFss) is involved in keloid formation. Therefore we investigated the expression of TGFss1, 2 and 3 and their receptors in keloids, hypertrophic scars and normal skin. Dermal fibroblasts were obtained from punch biopsies of patients with keloids and hypertrophic scars and from normal skin of healthy individuals. Total RNA was isolated and the expression of TGFss1, 2 and 3 and of TGFss receptors I and II (TGFssRI and II) was analysed by real-time PCR using the Lightcycler technique. Our data demonstrate significantly lower TGFss2 mRNA expression in hypertrophic scar fibroblasts as compared with fibroblasts derived from keloids and normal skin (p<0.05). In contrast, TGFss3 mRNA expression was significantly lower in keloid fibroblasts in comparison with fibroblasts derived from hypertrophic scar and normal skin (p<0.01). TGFssRI mRNA expression was significantly decreased in hypertrophic scar fibroblasts (p<0.01) and TGFssRII mRNA expression was decreased in keloids compared with hypertrophic scar fibroblasts (p<0.001). The ratio of TGFssRI/TGFssRII expression was increased in keloids compared with hypertrophic scar and normal skin fibroblasts. As recently supposed, an increased TGFssRI/TGFssRII ratio could promote fibrosis. Therefore our data support a possible role of TGFssRI and TGFssRII in combination with a certain TGFss expression pattern as fibrosis-inducing factors in keloids.     

Decorin‐expressing adenovirus decreases collagen synthesis and upregulates MMP expression in keloid fibroblasts and keloid spheroids

Decorin is a natural transforming growth factor‐β1 (TGF‐β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid‐derived fibroblasts (KFs) were transduced with decorin‐expressing adenovirus (dE1‐RGD/GFP/DCN), and we examined the therapeutic potential of decorin‐expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1‐RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF‐β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase‐1 (MMP‐1) and matrix metalloproteinase‐3 (MMP‐3) mRNA levels were measured by real‐time RT‐PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1‐RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1‐RGD/GFP/DCN, secreted TGF‐β1 and EGFR protein expressions were decreased in TGF‐β1‐treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP‐1 and MMP‐3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1‐RGD/GFP/DCN‐transduced keloid spheroids. These results support the utility of decorin‐expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.     

Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring

Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix‐associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type‐I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three‐dimensional type‐I collagen lattices. Inhibition of Rho‐associated protein kinase activity significantly attenuated the effects of matrix‐associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix‐associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho‐associated protein kinase‐dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.     

Adenovirus-relaxin gene therapy for keloids: implication for reversing pathological fibrosis

Background Keloids or hypertrophic scars are pathological proliferations of the dermal skin layer resulting from excessive collagen deposition. Because the hormone relaxin (RLX) inhibits collagen synthesis and expression in stimulated fibroblasts, an adenovirus expressing RLX (dE1‐RGD/lacZ/RLX) was generated. Objectives To investigate the effect of RLX‐expressing adenovirus on expression of various extracellular matrix (ECM) components in primary keloid spheroids. Methods The expression levels of type I and III collagen, fibronectin and elastin were investigated by immunohistochemistry in primary keloid spheroids transduced with the RLX‐expressing adenovirus. Results Immunohistochemical analysis showed that expression of major ECM components (e.g. type I and III collagen, elastin and fibronectin) was markedly reduced in primary keloid spheroids transduced with dE1‐RGD/lacZ/RLX. Conclusions These results suggest that the antifibrotic effect of RLX‐expressing adenovirus may have therapeutic effects on keloids by reversing pathological fibrosis and preventing keloid recurrence after surgical excision.     

Inverse correlation between CD34 expression and proline-4-hydroxyase immunoreactivity on spindle cells noted in hypertrophic scars and keloids

The CD34 positive (CD34+) spindle cells constitute a special population of spindle cells which show a unique distribution in the skin. So far, however, the functional role of CD34+ spindle cells and the regulation of CD34 expression on dermal spindle cells are totally unknown. We examined immunohistologically the pattern of the expression of CD34 and proline-4-hydroxylase, a marker for the fibroblasts that participate in active collagen synthesis, on dermal spindle cells at various stages of scar and keloidal tissues.
Dermal spindle cells in the lesions of hypertrophic scar and those at inflammatory expanding borders of keloids totally lost CD34 expression, but they strongly expressed proline-4-hydroxylase. On the other hand, they expressed CD34, together with decreased immunoreactivity to anti-proline-4-hydroxylase antibody, in non-inflammatory scars or in a non-inflammatory central portion of keloid. In two cases of scars, in which inflammation began to subside, double immunofluorescence demonstrated that both CD 34 and proline-4-hydroxylase were expressed on the same spindle cells.
CD34 expression, once disappeared from the lesions of hypertrophic scar or keloid, seems to return on CD34–proline-4-hydroxylase+ cells, when the initial inflammatory changes begin to regress. There is a reverse correlation between CD34 expression on spindle cells and the synthesis of type I collagen in the skin.