Expression of matrix metalloproteinase (MMP)-7 and MMP-13 and loss of MMP-19 and p16 are associated with malignant progression in chronic wounds

BACKGROUND: The risk of squamous cell carcinoma (SCC) is significantly increased in chronic leg ulcers. Very little is known about the molecular pathogenesis of these tumours, which are often undiagnosed for a long time. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated whether the pattern of epithelial MMP expression can predict development of SCC from pseudoepitheliomatous hyperplasia of chronic wounds. METHODS: Samples from nine patients with SCCs that had arisen in chronic wounds and 31 with venous leg ulcers were studied using immunohistochemistry for MMP-7, MMP-8, MMP-9, MMP-13, MMP-19 and the tumour suppressor p16. In situ hybridization was performed for MMP-1, MMP-3, MMP-7, MMP-12 and MMP-13. RESULTS: MMP-7 was expressed by malignantly transformed epithelium, while it was absent from chronic wounds. MMP-9 was detected in the epithelium in both SCCs and chronic wounds. Epithelial MMP-13 expression was strong in SCC, but was absent in chronic wounds. MMP-12 was expressed in the epithelium in two SCCs, while macrophages were positive in chronic wounds. MMP-19 was induced in proliferating epithelium of wounds, but was absent from invasive areas of SCC. p16 was expressed by keratinocytes in half of the chronic wounds and at superficial margins of SCCs, while invasive areas were negative. CONCLUSIONS: Our results suggest that epithelial expression of MMP-7, MMP-12 and MMP-13, but not that of MMP-1, MMP-3, MMP-8, MMP-9 and MMP-10, in chronic wounds provides a diagnostic clue for distinguishing SCCs from nonmalignant wounds. The loss of MMP-19 and p16 from the epithelium could aid in making the differential diagnosis between well-differentiated SCCs and nonmalignant chronic wounds.     

Transformation-specific matrix metalloproteinases (MMP)-7 and MMP-13 are expressed by tumour cells in epidermolysis bullosa-associated squamous cell carcinomas

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have an increased risk of developing rapidly progressive and metastatic cutaneous squamous cell carcinomas (SCC). It is unclear why these SCC behave more aggressively than sporadic SCC. Matrix metalloproteinases (MMP) are a family of endopeptidases that contribute to growth, invasion and metastasis of SCC. The role of MMP in RDEB-associated SCC is not known. OBJECTIVES: To investigate the expression of MMP-7, MMP-13 and MMP-9 in RDEB-associated SCC in comparison with sporadic SCC and Bowen's disease. METHODS: Immunohistochemical analysis of 25 RDEB-associated SCC, 61 sporadic SCC and 28 sporadic lesions of Bowen's disease was carried out using monoclonal antibodies for MMP-7, MMP-9, MMP-13 and E-cadherin and syndecan-1. RESULTS: MMP-7 was detected in all RDEB-associated SCC, in tumour cells within the invasive edge, where E-cadherin and syndecan-1 were markedly diminished or absent. MMP-7 expression was also observed in 98% of sporadic SCC and in 68% of Bowen's diseases. MMP-7 staining was significantly stronger in RDEB-associated SCC than in sporadic SCC, and was most abundant in poorly differentiated tumours. MMP-13 was detected in tumour cells in 96% of RDEB-associated SCC and in all sporadic cutaneous SCC. MMP-9 was detected in the inflammatory cells in all SCC examined. CONCLUSIONS: These results identify MMP-7 and MMP-13 as tumour cell-specific markers for SCC progression and as potential therapeutic targets in RDEB-associated SCC. The pattern of immunolabelling suggests that MMP-7 may shed E-cadherin and syndecan-1 from the SCC cell surface.     

Immunolocalizations of human gelatinase (type IV collagenase,MMP-9) and TIMP (tissue inhibitor of metalloproteinases) in normal epidermis and some epidermal tumors

Received: 15 March 1995     

MMP-2, TIMP-2 and MT1-MMP are differentially expressed in lesional skin of melanocytic nevi and their expression is modulated by UVB-light

BACKGROUND: In malignant melanoma, recent studies have demonstrated an important role of matrix-metalloproteinase 2 (MMP-2), its co-activating enzyme membrane-type matrix-metalloproteinase 1 (MT1-MMP), and the endogenous inhibitor of MMP-2, tissue-inhibitor of matrix metalloproteinase 2 (TIMP-2). Melanocytic nevi are benign neoplasms of the melanocytic lineage, but may exhibit dysplastic features that can be difficult to distinguish from early stage melanoma. As shown in earlier studies, nevi show important morphological and phenotypical changes in response to ultraviolet light (UVB) irradiation. OBJECTIVE: To clarify the role of MMP-2, TIMP-2 and MT1-MMP in UVB-irradiated vs. non-irradiated melanocytic nevi. METHODS: Immunohistochemical comparison of the MMP-2, TIMP-2 and MT1-MMP expression pattern. RESULTS: MMP-2 is expressed by lesional keratinocytes and its expression is up-regulated by UVB-irradiation. MMP-2 expression was not observed in melanocytic cells. TIMP-2, by contrast, is predominantly expressed by melanocytic nevus cells, and its expression is in part down-regulated by UVB-irradiation. MT1-MMP is expressed by basal keratinocytes and to a weaker extent by melanocytic nevus cells. CONCLUSIONS: MMP-2 expression by keratinocytes in nevi probably represents the result of activation of keratinocyte turnover in lesional epidermis. MMP-2 could play a role in the downward movement of junctional nevus cells into the dermis. The reduction of TIMP-2 expression in melanocytic cells by UV-light together with the enhanced expression of MMP-2 in the adjacent epidermis may promote basement membrane degradation. The expression pattern of MT1-MMP in close proximity to epithelial-mesenchymal interfaces underlines the synergistic role of MT1-MMP in this process.     

溶菌酶对体外培养的人成纤维细胞基质金属蛋白酶-1和12及赖氨酸氧化酶基因表达的影响

目的 探讨溶菌酶对体外培养的人成纤维细胞基质金属蛋白酶-1和12及赖氨酸氧化酶基因表达的影响。方法 采用酶消化法进行体外人皮肤成纤维细胞原代培养,然后将不同浓度的溶菌酶（0,1 × 10-8,1 × 10-7 mol/L）加入体外培养的人皮肤成纤维细胞中,待药物作用后,提取总RNA,通过逆转录反应获得cDNA并进行体外扩增,对扩增产物进行琼脂糖凝胶电泳,根据条带的平均光密度A值的比值来判断人成纤维细胞中基质金属蛋白酶（MMP）-1、MMP-12及赖氨酸氧化酶（LOX）mRNA的表达水平。结果 对体外培养的人成纤维细胞进行溶菌酶干预,经β肌动蛋白内参校正后,RT-PCR示对照组、低剂量组、高剂量组MMP-1、MMP-12 mRNA表达水平三组间差异具有统计学意义（F值分别为6.98和4.44,P值均 < 0.05）。SNK-q检验显示,低剂量组与对照组、高剂量组比较,差异均无统计学意义（P > 0.05）,高剂量组与对照组相比,差异有统计学意义（P < 0.05）。LOX mRNA表达水平三组间差异具有统计学意义（F = 5.24,P < 0.05）,SNK-q检验显示,低剂量组、高剂量组与对照组相比,差异具有统计学意义（P < 0.05）,低剂量组与高剂量组相比,差异无统计学意义（P > 0.05）。结论 溶菌酶可以下调MMP-1和MMP-12及上调LOX基因的转录水平。     

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Please cite this paper as: Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes. Experimental Dermatology 2010; 19 : e44–e49. Abstract: Toll‐like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP‐1 and MMP‐9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose‐dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IκB‐α and activated the nuclear translocation of NF‐κB. MMP‐1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11‐7082 (NF‐κB inhibitor) or SP600125 (JNK inhibitor), whereas MMP‐9 expression was inhibited by pretreatment with BAY11‐7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP‐1 and MMP‐9 in human epidermal keratinocytes. In addition, NF‐κB or JNK mediated the MMP‐1 expression induced by TLR2, TLR3 and TLR5, whereas NF‐κB, JNK or p38 MAPK mediated the MMP‐9 expression induced by TLR2, TLR3 and TLR5.     

Regulation of matrix metalloproteinase (MMP) expression in cutis laxa fibroblasts: upregulation of MMP-1, MMP-3 and MMP-9 genes but not of the MMP-2 gene

A major histopathological abnormality in cutis laxa (CL) is a paucity of elastic structures. The aim of this study was to investigate the gene expression levels of the major matrix degrading factors matrix metalloproteinase (MMP) 1, MMP-2, MMP-3 and MMP-9 in CL. The gene expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 in cultured CL fibroblasts were measured by northern blot, immunoblot and gelatin zymographic analysis. Markedly increased mRNA levels of MMP-1 (8.4-fold), MMP-3 (7.2-fold) and MMP-9 (more than 10-fold) were found in CL fibroblasts, whereas MMP-2 mRNA levels in these fibroblasts were unaltered. Increased protein production levels of MMP-1 (4.6-fold) and MMP-3 (5.1-fold) in CL fibroblasts were shown by immunoblot analysis. On gelatin zymographic analysis, the gelatinolytic activities of MMP-9 but not of MMP-2 were increased (2.2-fold). These results suggest that increased gene expression levels of MMP-1, MMP-3 and MMP-9 in CL fibroblasts may contribute to the histopathological abnormality in CL.     

Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.     

尖锐湿疣患者皮损中HPV的检测分型及HPV6b L1变异分析

目的 探讨温州地区尖锐湿疣（CA）患者人乳头瘤病毒（HPV）感染型别及HPV6基因多态性。 方法 采用基因芯片检测73例CA患者皮损组织HPV 21种基因型的感染率,对所获得的11例患者的HPV6阳性者行HPV6b L1基因测序分析。 结果 73例CA患者标本中检出HPV阳性61例,总的阳性检出率为83.56％。在61例HPV阳性患者,其中单一型别阳性检出率为93.44％,单一型别感染中以HPV11（29.51％）型为主,其次为HPV6 （18.03％）型;混合型感染阳性检出率为6.56％,以HPV6+11型感染为主。此外,CA患者HPV 6b L1基因测序结果表明,6个位点的核苷酸存在多态性,其中810位点A/T变异存在于所有标本,可能为温州地方株突变。结论 HPV11、6型感染为CA的主要致病型别,HPV 6b L1基因存在多处突变。     

Intra-individual comparison of the cutaneous safety and efficacy of calcitriol 3 microg g(-1) ointment and calcipotriol 50 microg g(-1) ointment on chronic plaque psoriasis localized in facial,hairline, retroauricular or flexural areas

BACKGROUND: Psoriasis involving sensitive skin areas remains difficult to treat because of the side-effects of topical corticosteroids and the irritancy potential of vitamin D3 derivatives. Several clinical trials have demonstrated that calcitriol, the naturally occurring and hormonally active form of vitamin D3, is effective and safe at the dose of 3 microg g(-1) for the treatment of psoriasis affecting the trunk and limbs. METHODS: We compared the safety and efficacy of calcitriol 3 microg g(-1) ointment and calcipotriol 50 microg g(-1) ointment in a multicentre, randomized, investigator-blinded, left-right comparison in mild to moderate chronic plaque psoriasis affecting sensitive areas, defined as being the face, hairline, retroauricular and flexural areas. One pair of symmetrical and bilateral target lesions was selected from each area and assessed for perilesional erythema, oedema, and stinging/burning. Global assessment of local tolerability and global improvement were rated by the investigator, and the subjects were asked to evaluate the tolerability and efficacy of each product and to express their global preference. RESULTS: In the 75 subjects, calcitriol and calcipotriol both led to clearing of at least one target lesion in 21 (28%) of the subjects each. Perilesional erythema (P < 0.001), perilesional oedema (P < 0.02) and stinging/burning (P < 0.001) were all significantly less severe with calcitriol than with calcipotriol. The subjects' evaluation of local tolerability was significantly (P < 0.0001) in favour of calcitriol. Ten treatment-related dermatological events occurred in eight subjects, including one subject who experienced skin discomfort on both sides. All other events occurred only on the calcipotriol-treated side (irritant dermatitis, six subjects; contact dermatitis, one subject). Global assessment of improvement from baseline by the investigators was significantly greater for the calcitriol-treated lesions (P < 0.02). The subjects' global preference was significantly in favour of calcitriol (P < 0.02). CONCLUSIONS: In the present study, calcitriol ointment was found to be better tolerated and would appear to be more effective than calcipotriol ointment in the treatment of psoriasis in sensitive areas.     

Comparison of cutaneous tolerance and efficacy of calcitriol 3 microg g(-1) ointment and tacrolimus 0.3 mg g(-1) ointment in chronic plaque psoriasis involving facial or genitofemoral areas: a double-blind, randomized controlled trial

BACKGROUND: Two nonsteroidal topical agents, calcitriol and tacrolimus, have been reported to be effective and safe for psoriatic lesions on sensitive areas. However, no comparative studies between calcitriol and tacrolimus have been reported. OBJECTIVES: To compare the tolerability and efficacy of calcitriol 3 microg g(-1) and tacrolimus 0.3 mg g(-1) ointment in chronic plaque psoriasis affecting facial and genitofemoral regions. METHODS: This is a double-blind, parallel, 6-week study of 50 patients who were randomized in a 1 : 1 ratio to apply calcitriol or tacrolimus twice daily. The primary efficacy variable was the mean reduction of the target area score (TAS), and the secondary efficacy variable was the percentage of patients with the Physician's Global Assessment (PGA) score of 5 (clear) and 4 (almost clear) at the end of the study. RESULTS: Both calcitriol and tacrolimus were well tolerated. Although calcitriol induced perilesional erythema in a statistically significant higher proportion of patients than tacrolimus (55% vs. 16% at week 6; P < 0.05), it did not necessitate treatment discontinuation. At the end of the study, tacrolimus was significantly more effective than calcitriol based on a significant reduction of mean TAS (67% vs. 51%; P < 0.05) as well as more patients achieving complete or almost complete clearance by PGA (60% vs. 33%; P < 0.05). CONCLUSIONS: Both calcitriol 3 microg g(-1) and tacrolimus 0.3 mg g(-1) are safe and well-tolerated therapeutic agents in the treatment of psoriasis in sensitive areas. Tacrolimus demonstrated a more effective clinical outcome compared with calcitriol.     

T-cell subsets and NCI (nuclear contour index) of their skin infiltrates in mycosis fungoides (MF)--a comparison of pre-and post-treatments using ACNU [1-(4-amino-2 methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl-3-nitrosourea)] and interferon alpha and the effectiveness of interferon alpha on Sézary cells in vitro

ACNU solution was applied to plaque lesions once daily for 4 weeks, while 3 million IU/day of Interferon α was injected locally into tumor lesions for 4 weeks. Clinically, ACNU and Interferon α were very effective. To determine the cytological effectiveness of both treatments, T-cell subsets and the NCI of their skin infiltrates in mycosis fungoides (MF) were compared pre- and post-treatment. Using either ACNU and Interferon α, in pre-treatment skin tissue, Leu3a positive cells were dominant, whereas in post-treatment skin tissue, Leu2a positive cells were dominant. As a result, the helper/suppressor T-cell (H/S) ratio in plaque lesion (3.1) was remarkably decreased (0.44) by ACNU treatment and the H/S ratio in tumor lesions was also reduced from 9.3 to 2.6 after injection of Interferon α. Moreover, the NCI of T-cells in plaque lesions decreased from 7.57 ± 1.71 to 4.30 ± 0.75 following treatment with ACNU, and that in tumor lesions was also reduced from 8.62 ± 1.89 to 4.35 ± 1.17 with Interferon α treatment. These immunohistos-chemical staining patterns and immunoelectron microscopic features in post-treatment periods are similar to those of control skin tissue which is not malignant. It was of interest that the antiproliferative effect of Interferon α on Hut 78 cells (Sézary cells) in vitro was demonstrated. Therefore, Interferon α might have direct cytotoxic effects on MF cells similar to that with Sézary cells. These results indicate that both treatments, ACNU and Interferon α, are effective not only clinically but also cytologically. The data also indicate that the NCI of T-cell and T-cell subsets might be used as an indicator of a treatment's effectiveness against MF.     

促血管生成素-1、2及其受体在寻常性银屑病中的表达

目的 探讨促血管生成素-1(Ang-1)、促血管生成素-2(Ang-2)及其受体Tie-2在寻常性银屑病皮损组织中的意义.方法 免疫组化SABC法检测30例寻常性银屑病患者皮损中的Ang-1、Ang-2及其受体Tie-2蛋白表达,以CD34标记新生血管内皮并计数微血管密度(MVD).另取15例正常人皮肤组织作为对照组.结果 寻常性银屑病患者皮损处Ang-2及其受体Tie-2表达明显强于正常人对照组(P<0.01),而Ang-1蛋白的表达与正常人对照组表达差异无统计学意义.进行期与静止期比较,Ang-2及Tie-2表达差异均有统计学意义(P<0.05),而Ang-1蛋白的表达差异无统计学意义(P>0.05).寻常性银屑病患者皮损中MVD值明显高于正常人对照组(P<0.01),高密度组(MVD≥15.46)与低密度组(MVD<15.46)的Ang-1表达差异无统计学意义(P>0.05),但Ang-2及其受体Tie-2表达差异有统计学意义(P<0.05).相关性分析表明,银屑病皮损Ang-1与Ang-2蛋白表达水平呈负相关(r=0.521,P<0.05);MVD值与Ang-2蛋白表达呈正相关关系(r=0.656,P<0.01),与Ang-1蛋白表达无相关性(r=0.237,P>0.05 o结论Ang-2及其受体Tie-2与银屑病的血管生成关系密切,促血管生成素及其受体系统在银屑病新生血管生成中的调节作用可能是以Ang-2的作用为主导的.