Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression

Aims:  Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids.
Methods and results:  Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions.
Conclusions:  These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.     

Increased plasminogen activator inhibitor-1 in keloid fibroblasts may account for their elevated collagen accumulation in fibrin gel cultures

Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts.     

Effects of hepatocyte growth factor on collagen synthesis and matrix metalloproteinase production in keloids

Keloids are pathologic proliferations of the dermal layer of the skin resulting from excessive collagen production and deposition. Hepatocyte growth factor (HGF) increases the expression of matrix metalloproteinase (MMP)-1 and suppresses collagen synthesis to modulate extracellular matrix turnover. To investigate the anti-fibrotic effects of HGF, we examine the mRNA expression of collagen types I and III and matrix metalloproteinase (MMP-1, MMP-3) on human dermal fibroblast (HDF) cell lines and keloid fibroblasts (KFs, n = 5) after adding various amount of HGF protein. We also evaluated the enzymatic activity of MMP-2, MMP-9 by zymograghy. In HDFs treated with TGF-β1 and HGF protein simultaneously, both type I and III collagen mRNA expression significantly decreased (P < 0.05). Expression of MMP-1, MMP-3 mRNA also decreased. However, the mRNA expression of MMP-1, MMP-3 significantly increased in KFs with increasing amount of HGF in dose dependent manner (P < 0.05). The enzymatic activities of MMP-2 increased with increasing HGF protein in a dose-dependent manner. However, the enzymatic activity of MMP-9 did not change. These results suggest that the anti-fibrotic effects of HGF may have therapeutic effects on keloids by reversing pathologic fibrosis.     

Collagenase in keloid biopsies and fibroblasts

Trypsin-activated collagenase activity was analyzed in media from keloid biopsies as well as keloid fibroblasts using radiolabeled guinea pig skin collagen as a substrate for collagenase digestion. Collagenase activity per tissue dry weight and DNA was similar in keloid and normal dermal biopsies; however, it was significantly elevated in keloid compared to normal dermal fibroblast media. In two paired cell lines where keloid and normal dermal fibroblasts were isolated from the same patient, relative collagen synthesis and trypsin-activated collagenase were increased to a similar extent in the keloid cell lines compared to normals. The biopsy data are in agreement with other findings, and further confirm the hypothesis that increased collagen deposition in keloids does not result from decreased collagen degradation. In vitro, however, the data presented here indicate that in some cell lines increased collagen synthesis may be associated with increased trypsin-activated collagenase.     

Mast cell chymase in keloid induces profibrotic response via transforming growth factor-β1/Smad activation in keloid fibroblasts

This study was to examine whether mast cell chymase exists in human keloids and exerts its profibrotic effect via transforming growth factor-β₁/Smad signaling pathway. The number of mast cells and the expression levels of chymase in keloids and normal skin were examined by immunohistochemistry assays. The mRNA expression and activity changes of chymase in keloids and normal skin were determined by real-time quantitative PCR and radioimmunoassay. After keloid fibroblasts were treated with different concentrations of chymase (0, 15, 30, 60, and 120 ng/mL) for various time periods, the proliferation of keloid fibroblasts, collagen synthesis, mRNA and protein expression of TGF-β₁, and the protein expression of phosphorylated Smad2/3, Smad2/3 and Smad7 were investigated using MTT assay, ELISA and Western blotting. Mast cells and chymase exist in keloid. Gene expression and activity of mast cell chymase in keloid are significantly higher than those in normal skin. Chymase promotes keloid fibroblast proliferation and collagen synthesis by activating TGF-β₁. The activation of Smad protein signaling pathway by chymase is related to the elevated P-Smad protein expression in keloid fibroblasts. Our data demonstrated that mast cell chymase plays an important role in keloid formation through TGF-β₁/Smad signaling pathway.     

Premature Collagen Fibril Formation,Fibroblast-Mast Cell Interactions and Mast Cell-Mediated Phagocytosis of Collagen in Keloids

Keloids are benign hyper-proliferative growths of fibrous tissue where increased fibroblast activity results in abnormal collagen deposition. Excessive inflammation is a characteristic feature of keloids, but little is known about the underlying ultrastructural features of keloids related to collagen processing, fibril and fiber formation, the interaction between fibroblasts and associated collagen fibers and mast cells. In this study, the ultrastructure of the dermis of keloid patients was evaluated using light and transmission electron microscopy techniques. Abnormal intracellular premature collagen fibril formation was observed. Phagocytosis of collagen fibrils by mast cells was a common ultrastructural feature of keloid tissue as was a close or direct association between fibroblasts and mast cells. Based on these findings and recent advances in knowledge related to collagen synthesis, fibril formation and processing, we hypothesize that keloid formation is primarily due to abnormal collagen synthesis where the consequent accumulation of collagen fibers causes increased mast cell recruitment and collagen phagocytosis. Subsequent release of mast cell-derived mediators then promotes further collagen synthesis. The observation of early formation in keloid tissue of premature insoluble collagen fibrils supports previous studies that enzymes such as procollagen C-proteinase are important early therapeutic targets.     

Hsp70 Knockdown by siRNA Decreased Collagen Production in Keloid Fibroblasts

Purpose

There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts.

Materials and Methods

We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay.

Results

The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts.

Conclusion

Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.     

A novel truncated TGF-beta receptor II downregulates collagen synthesis and TGF-beta I secretion of keloid fibroblasts

Hypertrophic scars and keloid are dermal proliferative disorders in wound healing. Transforming growth factor beta (TGF-beta) has been implicated in scar formation through the activation of fibroblasts and the acceleration of collagen deposition. Our study aimed to design a novel truncated (27-123 residues) type II TGF-beta receptor (tTGFbetaRII) and to determine its effects on the proliferation of keloid fibroblasts and the collagen synthesis as well as TGF-beta I expression of the cells. The coding sequences of TGF-beta I and tTGFbetaRII were amplified using RT-PCR and then cloned into pGBKT7 and pGADT7 vectors. A yeast two-hybrid experiment and a glutathione S-transferase (GST)-pull down assay were performed to verify the affinity of tTGFbetaRII to TGF-beta I. Our results indicated that treatment with tTGFbetaRII inhibited the growth of keloid fibroblasts and suppressed the synthesis of type I collagen in keloid fibroblasts in a concentration-dependent manner. Moreover, northern and western blot analysis revealed a decline of the TGF-beta I expression at both mRNA and protein levels after exposure to 5, 10 or 20 mug/ml of tTGFbetaRII. Together, our data suggested that the exogenous tTGFbetaRII can efficiently trap TGF-beta I from access to wild-type receptors and can suppress TGF-beta I triggered signals. Thus it may potentially be clinically applied to scar therapy.     

Differential immunohistochemical localization of cytokeratins and collagen types I and III in experimentally-induced cirrhosis

Liver cirrhosis was induced in male Wistar rats by subcutaneous injection (1 ml of 30 g/l) of an aqueous solution of thioacetamide. Using the indirect immunoperoxidase technique, high molecular weight keratins were localized in bile ducts and ductules. Low molecular weight cytokeratins were present in regenerating hepatocytes in active cirrhosis; bile ducts were unstained. These results suggest that cytokeratin staining may be useful in distinguishing bile duct epithelium and hepatocytes in hepatobiliary diseases. Anticollagen type III antibody stained hepatocytes and thin connective tissue fibres, while anticollagen type I antibody stained thicker fibres and some sinusoidal cells but not hepatocytes. Collagens were usually undetectable in normal liver cells. It is suggested, therefore, that hepatocytes may play a major role in collagen type III production which precedes the deposition of collagen type I. By contrast, collagen type I may be produced by fibroblasts and some cells along sinusoids (e.g. perisinusoidal fat-storing cells) after liver injury.     

The effect of carboxymethyl-chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts

In this study, different molecular weight CM-chitosans were prepared and the effects on the growth and collagen secretion of normal skin fibroblasts and keloid fibroblasts were investigated in vitro. CM-chitosan promoted the proliferation of the normal skin fibroblast significantly but inhibited the proliferation of keloid fibroblast. The higher CM-chitosan concentration had a higher initial effect and the lower CM-chitosan concentration had a longer affecting time to the normal skin fibroblast. The lower molecular weight CM-chitosan had significant twofold activities. The CM-chitosan could reduce the ratio of type I/III collagen in keloid fibroblast by inhibiting the secretion of collagen type I; and had no effect on the secretion of types I and III collagen in the normal skin fibroblast.     

Combined use of in situ hybridization and unlabeled antibody peroxidase anti-peroxidase methods: simultaneous detection of type I procollagen mRNAs and factor VIII-related antigen epitopes in keloid tissue.

In this study, we developed methodology that allows the combined use of in situ hybridization and peroxidase anti-peroxidase techniques on the same tissue section. A human pro alpha 1(I) collagen cDNA and antibodies to factor VIII-related antigen were used on keloid tissue sections as a model for a fibrotic reaction. The basic protocols of the techniques were modified to obtain optimal results. The feasibility of this new method was demonstrated by elucidation of type I procollagen gene expression in the cells of blood vessel wall and the adjacent fibroblasts. In the case of capillaries, pro alpha 1(I) collagen mRNAs were detected within endothelial cells identified by the presence of factor VIII-related antigen. Pro alpha 1(I) collagen mRNAs were also found in close proximity of medium-size blood vessels, but in this context clearly outside the vessel wall. These results may contribute to the understanding of pathogenetic aspects of keloids and other fibrotic conditions. Thus, the combination of in situ hybridization and peroxidase anti-peroxidase techniques provides a useful tool to examine gene expression simultaneously both at mRNA and protein levels in fibrotic tissues. This methodology is also applicable to a variety of other biologic and pathologic situations.     

瘢痕疙瘩组织中Livin、Smac、Caspase-3的表达及其相关性

BACKGROUND: Currently, there is no effective treatment for keloids that often recur. Its pathogenesis is still entirely unclear, and fibroblast proliferation and apoptosis have become a research hotspot. OBJECTIVE: To investigate the expression of Livin, Smac and Caspase-3 in keloids and to analyze their relationship so as to preliminarily explore the significance of Livin, Smac and Caspase-3 in the pathogenesis of keloids. METHODS: RT-PCR and immunohistochemical methods were used to detect the mRNA and protein expressions of Livin, Smac and Caspase-3 in keloids (n=20) and normal skin tissues (n=20). RESULTS AND CONCLUSION: Compared with the normal skin tissue, the mRNA and protein positive expressions of Livin were significantly higher in keloids (P < 0.05), while the mRNA and protein positive expressions of Smac and Caspase-3 were lower in keloids (P < 0.05). There was a negative association between Livin and Smac, Caspase-3 protein expression in keloids. These findings indicate that the high mRNA expression of Livin may cause the imbalance between proliferation and apoptosis of fibroblasts by inhibiting the mRNA expression of Smac and Caspase-3, and eventually lead to the formation of keloid.      

Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-beta1 and detection in human gingiva

Gingival overgrowth is characterized by excess extracellular matrix accumulation and elevated levels of cytokines, including transforming growth factor-beta1 (TGF-beta1). The functional relationships between altered cytokine levels and extracellular matrix accumulation have not been extensively investigated in gingival cells and tissues. Lysyl oxidase catalyzes the final known enzymatic step required for cross-linking collagen and elastin in the synthesis of a functional extracellular matrix. This study investigated the regulation by TGF-beta1 of lysyl oxidase and its collagen and elastin substrates in early passage human gingival fibroblasts. In addition, TGF-beta1 regulation of connective tissue growth factor (CTGF) was assessed in human gingival cells and tissues. The results show that TGF-beta1 increases lysyl oxidase enzyme activity and mRNA levels for lysyl oxidase and alpha-1-type I collagen, but not elastin, in a dose- and time-dependent manner. Maximal stimulation of lysyl oxidase activity and mRNA levels for both lysyl oxidase and collagen occurs after 48 hours of treatment of gingival fibroblastic cells with 400 pM of TGF-beta1. This study shows for the first time that CTGF mRNA and protein are strongly and rapidly induced by TGF-beta1 in human gingival fibroblasts. Exogenous addition of 1 to 50 ng/ml CTGF to gingival fibroblasts stimulates production of lysyl oxidase enzyme activity up to 1.5-fold after 48 hours, and 50 ng/ml CTGF stimulated insoluble collagen accumulation 1.5- to 2.0-fold after 4, 11, and 18 days of treatment. It is interesting to note that the addition of CTGF-blocking antibodies in the presence of TGF-beta did not block TGF-beta stimulation of collagen mRNA levels. Thus, although CTGF itself contributes to increased insoluble collagenous extracellular matrix accumulation, CTGF does not mediate all of the effects of TGF-beta1 on stimulation of collagen mRNA levels in human gingival fibroblasts. Immunohistochemistry studies of gingival overgrowth tissue samples indicate for the first time detectable levels of CTGF protein in Dilantin-induced hyperplasia tissues also positive for TGF-beta1. CTGF was not found in TGF-beta1-negative samples. In addition, extracellular lysyl oxidase protein was detected in vivo. Taken together, these studies support mostly independent roles for TGF-beta1 and CTGF in stimulating collagenous extracellular matrix accumulation in human gingival fibroblasts and tissues.     

Type I collagen synthesis parallels the conversion of keratinocytic intraepidermal neoplasia to cutaneous squamous cell carcinoma

Neoplastic progression of solid tumours is often characterized by a simultaneous increase in matrix protein (eg collagen) synthesis and degradation, and results in the formation of a tumour stroma. At the tumour periphery, this process is believed to facilitate angiogenesis and invasive growth of tumour cells. In various types of carcinoma, differentiation of fibroblasts towards myofibroblasts is thought to play an important role in extracellular matrix remodelling as their emergence coincides with architectural changes in the tumour stroma. Here, distinct architectural changes in collagen fibres are reported in cutaneous squamous cell carcinomas (cSCC) with respect to normal skin and precursor lesions, ie keratinocytic intraepidermal neoplasia (KIN). Simultaneously, type I collagen mRNA was observed in fibroblasts in close proximity to cSCC lesions (19/19) but only in 2 of 10 KIN lesions tested. Interestingly, whereas emerging of myofibroblasts correlated with reduced differentiation of cSCCs, it was not a prerequisite for type I collagen synthesis. These data indicate that type I collagen synthesis by fibroblasts parallels the malignant transformation of human KIN to cSCC.     

The role of nitric oxide in the formation of keloid and hypertrophic lesions

Keloid and hypertrophic scars are a type of scarring pathology which is characterised by excess collagen deposition produced during the wound healing process. The mechanism by which this occurs is not understood and although hypertrophic scars can regress spontaneously, keloids do not, and currently no effective treatment exists. In this paper we hypothesise that nitric oxide, a free radical molecule synthesised by numerous mammalian cells, is involved in the formation of these scars. We suggest that the excess collagen production in keloid lesions can be attributed to higher than normal levels of nitric oxide, as the free radical is a known stimulus for fibroblast collagen synthesis. Furthermore, we propose that the basal epidermis is a source of this additional nitric oxide and we discuss this in relation to known histological characteristics of keloid and hypertrophic lesions.     

Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease

Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.     

Is survivin a novel pathway for the treatment and pathogenesis of keloid?

Keloids behave like benign tumors as they grow beyond the boundaries of the original wound margin, do not regress spontaneously, and recur despite treatments. Recently, accumulating evidences showed that survivin played an important role in cell growth, apoptotic resistance, and cell cycle control. More than that, survivin was confirmed to be associated with tumor angiogenesis and chemoresistance. Survivin blocker therapy has been proved to be a novel treatment in some kinds of tumors. Our preliminary work showed that survivin expression was significantly higher in keloids than in normal skin. The mRNA and protein levels of survivin were downregulated in keloid fibroblasts by survivin-siRNA. Therefore, we hypothesize that survivin has a profound effect on keloid formation and progression. Therefore, survivin may be a potential therapeutic target for keloids. Our hypothesis sheds light for the first time on the role of survivin involves in keloid pathophysiology and provides with novel therapeutic implications for keloids that are associated with apoptosis.