Matrix metalloproteinases and their tissue inhibitors in the lesions of cardiac and pulmonary sarcoidosis: an immunohistochemical study

The pathogenesis of the tissue damage and fibrosis in sarcoidosis is poorly understood. The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) must be considered in this regard, because they control the lysis of connective tissue components. Immunohistochemical studies (peroxidase and dual labeling for confocal microscopy) of reactivity for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and the 4 membrane-type-MMPs were made on tissues from patients with cardiac (n = 4) and pulmonary (n = 5) sarcoidosis. The granulomas were histochemically similar in both organs. The multinucleated giant cells (MGCs) showed moderate reactivity for MMP-1 and MMP-9 and variable reactivity for MMP-2 and MMP-3; in addition, they showed colocalization of MT-1-MMP, which activates MMP-2. The reactivity of epithelioid cells (ECs) was moderate for MMP-2 and mild for other MMPs. Macrophages showed weaker reactivity for MMPs than did MGCs and ECs. All 3 types of cells showed very low reactivity for TIMPs. Staining for type IV collagen showed focal damage to the basement membranes of cardiac myocytes and pulmonary alveoli near the granulomas. The cells in sarcoid granulomas contain an abundance of MMPs and a paucity of TIMPs. The MGCs also contain MT-1-MMP and thus can activate MMP-2 in the granulomas. The MMPs can cause damage to adjacent cardiac myocytes and pulmonary alveoli, leading to the interstitial fibrosis produced by sarcoidosis.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions

Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP-1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions.     

Expression of Matrix Metalloproteinases in Invasive Pulmonary Adenocarcinoma with Bronchioloalveolar Component and Atypical Adenomatous Hyperplasia

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.     

Immunohistochemical study of endothelin-1 and matrix metalloproteinases in plexogenic pulmonary arteriopathy

The matrix metalloproteinases (MMPs) and endothelin-1, a potent vasoconstrictor and mitogen for smooth muscle cells, have been shown to be involved in the pathogenesis of various vascular disorders. However, the expression of endothelin-1 and the activation of MMPs have not been fully evaluated in plexogenic pulmonary arteriopathy (PPA). Immunohistochemical and confocal microscopic studies were conducted to evaluate the reactivity of lung tissue from six patients with pulmonary hypertension for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, factor VIII, endothelin-1, various types of MMPs (MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9), membrane type-MMPs (MT-MMPs), tissue inhibitors of MMPs (TIMPs), and type IV collagen. Four major arterial morphological abnormalities were recognized in PPA: muscularization of pulmonary arterioles, onion-skin lesions, cellular and mature plexiform lesions, and atheromas in elastic pulmonary arteries. Reactivity for MMP-2 and MT-1-MMP was found in endothelial cells and, to a lesser extent, in myofibroblasts proliferating in various lesions of PPA. Increased expression of endothelin-1 was observed in the latter cells and in endothelial cells. Some myofibroblasts were positive for MMP-3 and MMP-7 in the vascular lesions except for mature plexiform lesions. MMP-1, MMP-9 and TIMP-2 tended to be positive only in the atheromatous lesions. Staining for type IV collagen showed focal thinning and discontinuities of the endothelial basement membrane in plexiform lesions. This study demonstrates colocalization of MMP-2 with MT-1-MMP and increased expression of endothelin-1 in various arterial lesions of PPA. These changes may play important roles in the remodeling of arterial structures, particularly of basement membranes, in this disorder.     

Implication of extracellular matrix metalloproteinases in the course of chronic inflammatory airway diseases

Matrix metalloproteinases (MMPs) are major proteolytic enzymes that are involved in extracellular matrix (ECM) turn over. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) cleave type IV collagen, which is an important constituent of basement membrane. These enzymes play an important role in normal tissue homeostasis, but imbalance between MMPs and their tissue inhibitors (TIMPs) is thought to be a critical factor in regulating tissue remodeling. MMP-2 is produced by fibroblasts, endothelial, and epithelial cells, while MMP-9 is mainly produced by inflammatory cells. The role of MMPs was investigated through biochemical analysis or in situ expression, in the pathogenesis of two chronic inflammatory airway diseases, asthma and nasal polyposis. Both are characterized with the accumulation of active inflammatory cells, matrix remodeling and epithelial changes. Increased levels of MMP-9 and TIMP-1 were found in asthmatic subjects and NP. In NP, MMP-9 expression was detected in epithelial, endothelial and inflammatory cells. In this setting, MMP-9 could play a crucial role in the transmigration of basement membrane components by inflammatory cells leading to inflammatory cell accumulation and maintenance of inflammation in airway. Moreover, MMP-9 may contribute to cell migration, an important mechanism involved in the repair of the respiratory epithelium.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development

AIMS : Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in lung development because they play an important role in the turnover of the extracellular matrix. Although limited data on MMP and TIMP expression are available from animal studies during prenatal pulmonary development, little is known about their expression during human fetal lung development. The aim of this study was to investigate the expression of MMP-1, -2, -9, TIMP-1, -2 and -3 in human fetal lungs from 9 to 42 weeks of gestation. METHODS AND RESULTS : Forty-five normal human fetal lung samples were analysed by immunohistochemistry. MMP-1, -9, TIMP-1, -2 and -3, but not MMP-2, were expressed in the epithelium at all gestational ages. The endothelium of all vessels and the arterial smooth muscle cells expressed MMP-1, -2, -9, TIMP-2 and -3, but not TIMP-1, at all developmental stages. CONCLUSION : The extensive distribution of MMPs and TIMPs throughout all stages of human lung development suggests that they play a significant role in the remodelling that occurs in the interstitium and epithelial basement membrane during lung development and in pulmonary vascular development. These data will serve as a base line for comparison with neonatal lung pathology, including pulmonary hypertension.     

Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinase, collagens, and Ki67 antigen in pleural malignant mesothelioma: an immunohistochemical and electron microscopic study

Because matrix metalloproteinases (MMPs) degrade extracellular matrix, including basement membrane, and because tissue inhibitors of MMP (TIMPs) suppress MMP activities, MMPs and TIMPs are considered to play important roles in invasion and metastasis in many malignancies. We examined immunohistochemically the expression of MMPs (MMP-1, -2, -3, -7, and -9), TIMPs (TIMP-1 and -2), and collagens (types I, III, and IV) in 16 patients with pleural malignant mesothelioma (PMM; 8 with the epithelial, 4 with the sarcomatous, and 4 with the biphasic type). Electron microscopy revealed that the tumor cells in all types possessed the characteristics of malignant mesotheliomas, including numerous microvilli and moderate amounts of intermediate filaments. Basement lamina was present only focally. The proliferative Ki67 index was at a high level, compared with values reported in various other malignancies. Positive staining for MMP-1 was observed in most tumor cells in all 16 patients (100%). MMP-2 was expressed in most tumor cells in 2 patients (13%). In contrast, MMP-3, -7, and -9 were not detected in any PMM. TIMP-1 and TIMP-2 were expressed in 3 patients (19%) and 2 patients (13%), respectively. The stromal cells were simultaneously positive for MMPs or TIMPs in the patients whose tumor parenchymal cells were positive for each enzyme. These results indicate that the expression of MMP-1 and MMP-2 may be related to PMM invasion and spread. In particular, as MMP-1 was overexpressed in contrast to the lower expression of TIMP-1, MMP-1 is strongly suggested to play an important role in PMM invasion by degrading the tumor stroma. In spite of general agreement that epithelial-type PMM has a better prognosis than other types, there was no significant difference in the Ki67 index among the histological types of PMM.     

Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis

The role of various matrix metalloproteinases (MMP) and tissue Inhibitor of metalloprotelnases-2 (TIMP-2), and the gelatholytic activities of MMP involved in the process of bleomycin-induced pulmonary fibrosis in rabbits were Investigated. Male Japanese white rabbits were intubated with tracheal tubes under anesthesia, and bleomycin hydrochloride in sterile saline or only sterile saline was administered through the tracheal tubes. The animals were killed 1, 3, 7, 14 and 28 days after the administration of bleomycln ( n = 3) or saline ( n = 2). Light microscopic lmmunohistochemlstry for MMP-1 (interstitial collagenase), MMPP (gelatinase A), MMP-9 (gelatinase B) and TIMP-2 was performed. The gelatinolytic activities of lung tissue homogenates were studied by gelatin zymography. In the early stages, the gelatholytic activity of MMP-9 was predominant. MYP-9 localized in the infiltrating neutrophils, macrophages, bronchial and bronchiolar epithelial cells. The alveolar epithelial basement membrane was frequently disrupted in the early stages, where MMP-9 possibly contributed to the disruption. In the late stages, the gelatinolytic activities of the latent and active forms of MMP-2 were predominant, and MMPP localized in the regenerated alveolar epithelial cells in addition to the bronchial epithelial cells. MMP-2, especially its active form, possibly plays a role in alveolar epithelial cell regeneration. The localization of MMP-1 was similar to that of MMP-9. TIMP-2 localized in the epithelial cells and in some fibroblasts in fibro tic lesions. TIMP-2 possibly plays a role in extracellular matrix deposition in balance with MMP.     

Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid

BACKGROUND: Matrix metalloproteinase (MMP)-2, MMP-9 and MMP-13 can degrade type IV collagen which is the major component of the basement membrane zone (BMZ). In bullous pemphigoid (BP), the separation occurs within the BMZ. OBJECTIVE: To evaluate the involvement of MMPs in the pathogenesis of BP, we examined the expression of MMP-2, MMP-9 and MMP-13 in the lesional skin of BP patients. METHODS: The expression of MMP-2, MMP-9, MMP-13, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 was analyzed by immunohistochemistry in the lesional skin of BP patients in comparison with that in normal human skin. Next, the cellular sources of MMP-2, MMP-9 and MMP-13 were analyzed by double immunohistochemistry. Finally, the levels of these MMPs in the serum and blister fluid of BP patients were measured by ELISA. RESULTS: The number of cells expressing MMP-2, MMP-9 and MMP-13 were significantly increased in the lesional skin of BP patients as compared to that in normal skin. Although the number of cells expressing TIMP-1 and TIMP-2 were also increased in the lesional skin of BP patients as compared to that in normal skin, the ratio of MMPs to TIMPs in the lesional skin of BP patients was high (2.4:1). T cells comprised the major source of MMP-2, MMP-9 and MMP-13, while a proportion of mast cells and eosinophils also expressed these MMPs. Furthermore, marked expression of MMP-2 was detected in the epidermal keratinocytes. The levels of these MMPs in the blister fluid were significantly greater than those in the serum. CONCLUSION: These results suggest that MMP-2, MMP-9 and MMP-13 may be involved in the mechanism of blister formation in BP and that besides infiltrating inflammatory cells, structural cells like epidermal keratinocytes may also participate in the induction of blister formation in BP.     

Role of MMP-2 in alveolar epithelial cell repair after bleomycin administration in rabbits

Matrix metalloproteinases (MMPs) have been implicated in the pathological processes of interstitial lung diseases. However, underlying mechanisms, particularly for activity levels and distribution of activated MMP-2 in the disease process, are yet to be elucidated. The present study investigated the immunolocalization of MMP-2, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-2, p53, and Ki-67 in a rabbit model of bleomycin-induced pulmonary fibrosis. Gelatin zymography and in situ zymography were used to examine the activity and the localization of MMP-2. Furthermore, we performed Western blot and in situ hybridization for MT1-MMP, an activator for MMP-2. The total MMP-2 level estimated by gelatin zymography increased significantly at 3, 7, and 14 days after bleomycin administration, compared with controls. In the immunohistochemical study, immunoreaction for MMP-2 was strongest in alveolar epithelial cells among the cell populations. Swollen and/or elongated type II alveolar epithelial cells showed strong immunoreactions for MMP-2, MT1-MMP, and TIMP-2. After bleomycin administration, immunoreaction for p53 was observed in bronchiolar and alveolar epithelial cells. The proportion of p53-positive cells was high in epithelial cells from 1 to 14 days as MMP-2 levels were increased, suggesting that p53 may be responsible, at least in part, for the increase of MMP-2. The ratio of activated MMP-2 to total MMP-2 estimated by gelatin zymography increased significantly at 3, 7, 14, and 28 days after bleomycin treatment. In situ zymography revealed that type II alveolar epithelial cells degraded gelatin. An increased expression of MT1-MMP protein was observed by Western blot following administration of bleomycin. In situ hybridization demonstrated that type II alveolar epithelial cells gave intense signal for MT1-MMP mRNA. These results suggest that type II alveolar epithelial cells express MT1-MMP and activate MMP-2 on their cell surfaces, which may lead to the elongation and migration of alveolar epithelial cells in the repair process of bleomycin-induced pulmonary fibrosis.     

Tissue inhibitor of metalloproteinase-1 deficiency abrogates obliterative airway disease after heterotopic tracheal transplantation

Obliterative bronchiolitis (OB) is a major cause of allograft dysfunction after lung transplantation and is thought to result from immunologically mediated airway epithelial destruction and luminal fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the regulation of lung inflammation, airway epithelial repair, and extracellular matrix remodeling and therefore may participate in the pathogenesis of OB. The goals of this study were to determine the expression profiles of MMPs and TIMPs and the role of TIMP-1 in the development of airway obliteration using the murine heterotopic tracheal transplant model of OB. We demonstrate the selective induction of MMP-3, MMP-9, MMP-12, and TIMP-1 in a temporally restricted manner in tracheal allografts compared with isografts. In contrast, the expression of MMP-7, TIMP-2, and TIMP-3 was decreased in allografts relative to isografts during the period of graft rejection. TIMP-1 protein localized to epithelial, mesenchymal, and inflammatory cells in the tracheal grafts in a temporally and spatially restricted manner. Using TIMP-1-deficient mice, we demonstrate that the absence of TIMP-1 in the donor trachea or the allograft recipient reduced luminal obliteration and increased re-epithelialization in the allograft compared with wild-type control at 28 d after transplantation. Our findings provide direct evidence that TIMP-1 contributes to the development of airway fibrosis in the heterotopic tracheal transplant model, and suggest a potential role for this proteinase inhibitor in the pathogenesis of OB in patients with lung transplant.     

Temporospatial distribution of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinases during murine secondary palate morphogenesis

Extracellular matrix (ECM) molecules are known to play a pivotal role in the morphogenesis of the secondary palate. The maintenance and degradation of the ECM is mediated in part by the matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs. MMPs and TIMPs have previously been shown to be developmentally regulated within the palatal shelf during secondary palate morphogenesis. This study was conducted to examine the temporospatial distribution of these enzymes and their inhibitors within the palatal shelves using immunofluorescent localization to determine if specific changes occur in their distribution concomitant with events in palatal shelf formation and reorientation. Frontal sections through the posterior palatal shelves at gestational day (gd) 12, 13 and 14 were immunofluorescently stained for MMPs 2, 3, 9, and 13 and TIMPs 1, 2, and 3 using standard protocols and commercially available antibodies. The results demonstrated that MMPs and TIMPs were already present within the palatal shelf mesenchyme 30 h prior to reorientation and closure and that their expression within the shelf mesenchyme increased as the shelves remodeled, then decreased with closure and fusion. Increased distribution of MMPs and TIMPs within specific regions of the palatal mesenchyme and palatal epithelial basement membrane preceded decreases previously observed within these areas for their substrates, fibronectin, collagen III and collagen I. In addition, MMP-3 and TIMP-3 were immunolocalized to regions of the palatal epithelium that undergo reorganization concomitant with reorientation. The results of this study indicate that MMPs and TIMPs are developmentally regulated during palatal shelf morphogenesis and that their distribution correlates with the distribution of the ECM components of the palatal shelf they regulate. These results provide support for the idea that temporospatially controlled interactions between MMPs and their substrates may be pivotal in modulating events in palatal morphogenesis. Accepted: 3 March 2000     

The distribution of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the lungs of congenital diaphragmatic hernia patients and age-matched controls

AIMS: In congenital diaphragmatic hernia (CDH), the pathogenesis of abnormal pulmonary morphology is still incompletely understood. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are known to play an important role in the turnover of the extracellular matrix (ECM) during development and in remodelling of tissue. The aim of this study was to investigate differences in the expression of MMPs and TIMPs between CDH lungs and controls, against the background of the abnormal pulmonary vasculature in CDH. METHODS: We studied 12 lungs of term CDH patients who died < 24 h after birth and 11 normal age-matched control lungs, by immunohistochemistry with antibodies against human MMP-1, -2, -9, TIMP-1 and -2. RESULTS: There was a clear increase in the number of MMP-1-reactive capillaries and fibroblasts in CDH lungs compared with controls. In contrast, TIMP-2 reactivity in these structures was decreased in CDH lungs. The arterial endothelium and medial smooth muscle expressed MMP-2, -9 and TIMP-2 in both CDH and control lungs. In small arteries (< 100 microm in diameter), the positive surface area of MMP-2, -9 and TIMP-2 was significantly larger in CDH lungs than in controls. There was no difference in the distribution and expression of TIMP-1 between CDH lungs and normal controls. CONCLUSION: The differences in staining pattern of MMPs and TIMPs between normal and CDH lungs suggest that these enzymes might play a role in the abnormal remodelling of the interstitium and the pulmonary arteries in CDH lungs. This could contribute to our understanding of the abnormal lung morphology and the occurrence of pulmonary hypertension, which forms one of the major obstacles to the successful treatment of these patients.     

Matrix metalloproteinase 9 (gelatinase B) is expressed in multinucleated giant cells of human giant cell tumor of bone and is associated with vascular invasion.

Human giant cell tumor (GCT) consists of multinucleated giant cells and mononuclear stromal cells, and is characterized by frequent vascular invasion without distant metastases. To study the role of matrix metalloproteinases (MMPs) in the vascular invasion, we examined production of MMP-1 (tissue collagenase), -2 (gelatinase A), -3 (stromelysin-1), -9 (gelatinase B), and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in GCT. MMP-9 was highly and predominantly expressed in giant cells by both immunohistochemistry and in situ hybridization. Expression of other MMPs was also observed in some cases but was inconstant. Sandwich enzyme immunoassays demonstrated that MMP-9 is the predominant MMP secreted by GCT. There was a definite imbalance between the amounts of MMP-9 and those of TIMPs in the culture media of GCT, leading to detectable gelatinolytic activity in an assay using 14C-gelatin. Gelatin zymography demonstrated the main activity at about 90 kd, which was identified as the zymogen of MMP-9 by immunoblotting. Immunohistochemistry for type IV collagen and laminin, major basement membrane components, showed that disappearance of the proteins is closely associated with MMP-9-positive giant cells. These results indicate the production of MMP-9 by multinucleated giant cells and suggest that the metalloproteinase may contribute to proteolysis associated with vascular invasion and local bone resorption in human GCT.     

Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

Purpose

Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.

Methods

Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.

Results

At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.

Conclusions

We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.