Gingival Fibroblasts Inhibit MMP-1 and MMP-3 Activities in an Ex-Vivo Artery Model

The main arterial pathologies can be associated with a deregulation of remodeling involving matrix metalloproteinases (MMPs), whereas gingival healing is characterized by an absence of fibrosis or irreversible elastin/collagen degradation. The aim of our study was to evaluate the effect of gingival fibroblasts on MMP-1 and MMP-3 secretion in an organotypic artery culture. MMP-1 and MMP-3 secretions and activities (dot blots, zymography, ELISA) were evaluated in coculture of rabbit artery in the presence or not of gingival fibroblasts. MMP-1/TIMP-1 and MMP-3/TIMP-1 complexes forms were measured by ELISA. Complementary studies were performed using human aortic smooth muscle cells cocultured with adventitial, dermal, or gingival fibroblasts. Our results indicated that MMP-1 and MMP-3 free-forms activities were significantly reduced in coculture. This inhibition was linked to a significant increase of TIMP-1 leading to formation of TIMP-1/MMPs complexes. Due to the presence of gingival fibroblasts, the decrease in MMP-1 and MMP-3 efficiency thus contributes to diminish the degradation of artery. This cellular therapy strategy could be promising in artery pathologies treatment.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

缺氧对肺动脉成纤维细胞MMP-2、TIMP-1表达的影响

目的：探讨缺氧对肺动脉成纤维细胞（Fpa）分泌基质金属蛋白酶（MMPs）、金属蛋白酶组织抑制剂（TIMPs）的影响。 方法： 采用酶谱法测定Fpa培养基中MMP-2的酶活性，免疫印迹法检测培养基中MMP-2、TIMP-1 的蛋白水平，免疫组化法测定细胞原位的蛋白表达， RT-PCR法检测mRNA表达量。 结果： 缺氧后Fpa分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；而TIMP-1的表达则呈相反变化。 结论： 缺氧可使肺动脉成纤维细胞MMP-2/TIMP-1的表达失衡，可能参与缺氧性肺血管重建。     

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.     

内皮素-1对人系膜细胞表达MMP-3及TIMP-1的影响

目的研究内皮素-1(ET-1)对人系膜细胞(MC)基质金属蛋白酶-3(MMP-3)及其组织抑制剂-1(TIMP-1)表达的影响。方法采用体外MC培养,应用细胞ELISA法测定MC内MMP-3、TIMP-1蛋白的表达。结果ET-1促进MC内MMP-3、TIMP-1蛋白表达,但MMP-3/TIMP-1比值呈剂量及时间依赖性下降。结论ET-1可能是人系膜细胞内MMP-3/TIMP-1比值下降的原因之一,这与肾小球细胞外基质降解受抑密切相关。     

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.     

Differential expression of stromal MMP-1, MMP-9 and TIMP-1 in basal cell carcinomas of immunosuppressed patients and controls

Matrix metalloproteinases (MMPs) have an important role in the initiation, growth, and invasion of malignant tumors. Basal cell cancer (BCC) is the most common human malignancy. The risk of BCC is 10–16 times higher among organ transplant recipients compared with the nontransplanted population. The aim of this study was to compare the expression of several MMPs and their tissue inhibitors (TIMPs) in BCCs from kidney transplant recipients and controls. Expression of MMPs-1, -7, -8, -9, -10, -13, -26, and TIMPs-1 and -3 was evaluated by immunohistochemistry in 25 samples of BCC of kidney transplant recipients and 25 matched controls representing superficial and nodular subtypes. No significant differences were detected in MMP expression of BCC tumor cells between immunocompetent and immunodeficient patients. However, MMPs-1 and -9 and TIMP-1 were expressed more frequently in stromal macrophages in the BCCs of immunocompetent patients. When tumor subtypes were compared irrespective of the patient group, more MMP-1-positive fibroblasts and MMP-9-positive neutrophils were detected in the superficial subtype, while stromal MMP-10 expression was more abundant in nodular tumors. Our results suggest that abundant peritumoral expression of TIMP-1 in non-immunocompromised patients limits ECM degradation permissive for cancer cell migration.     

MMP-7、MMP-10和TIMP-4在心力衰竭心室重构中的表达

目的:应用细胞因子抗体芯片技术筛选与心力衰竭心室重构关系密切的基质蛋白酶。方法:从本院的心脏病组织库中挑选6例病理诊断明确和各方面资料比较齐全的致心律失常性右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行细胞因子抗体芯片分析,筛选在致心律失常性右室心肌病引起的心力衰竭中差异表达的基质蛋白酶,并应用酶联免疫分析和免疫组织化学的方法加以验证。结果:在所筛选的17种基质金属蛋白酶中,只有MMP-7和MMP-10在致心律失常性右室心肌病引起心力衰竭中高表达,而在4种基质金属蛋白酶内源性组织抑制剂中,只有TIMP-4 低表达。经酶联免疫分析和免疫组织化学的方法证实,不仅在致心律失常性右室心肌病引起心力衰竭的心肌,在缺血性心肌病和扩张性心肌病引起的心力衰竭心肌中也发现MMP-7和MMP-10 的高表达及TIMP-4的低表达。结论:心肌组织中MMP-7和MMP-10的高表达及TIMP-4的低表达在不同心肌病引起的心力衰竭心室重构分子机制中可能发挥重要的作用。     

Expression and activities of matrix metalloproteinases under oscillatory shear in IL-1-stimulated synovial cells

Matrix metalloproteinases (MMPs) are known to play a critical role in tissue disintegration, and an elevated level of MMPs is observed in synovium and synovial fluid of joints with rheumatoid arthritis. During joint movement, synovial tissue receives various mechanical stimuli, but effects of mechanical challenges on regulation of MMPs in rheumatic synovium are poorly understood. Focusing on cellular responses to oscillatory fluid shear in human synovial cells, we determined the expression of MMP-1 and MMP-13 by polymerase chain reaction and immunoblotting as well as proteolytic activities of total MMPs by a fibril degradation assay and zymography. The results revealed that approximately 0.5 dyn/cm2 oscillatory shear at 1 Hz not only reduced an mRNA level and a protein level of MMP-1 and MMP-13, but it also decreased collagenase and gelatinase activities of total MMPs. Furthermore, the induction of the MMP expression and activities by interleukin-1 was suppressed by the oscillatory shear. Interestingly, the oscillatory shear upregulated the mRNA expression of TIMP-1 and TIMP-2. Our results support a potential role of oscillatory shear in regulating expression and activities of MMPs in the presence and the absence of proinflammatory cytokine.     

Expression and Activities of Matrix Metalloproteinases under Oscillatory Shear in IL-1-Stimulated Synovial Cells

Matrix metalloproteinases (MMPs) are known to play a critical role in tissue disintegration, and an elevated level of MMPs is observed in synovium and synovial fluid of joints with rheumatoid arthritis. During joint movement, synovial tissue receives various mechanical stimuli, but effects of mechanical challenges on regulation of MMPs in rheumatic synovium are poorly understood. Focusing on cellular responses to oscillatory fluid shear in human synovial cells, we determined the expression of MMP-1 and MMP-13 by polymerase chain reaction and immunoblotting as well as proteolytic activities of total MMPs by a fibril degradation assay and zymography. The results revealed that ~0.5 dyn/cm 2 oscillatory shear at 1 Hz not only reduced an mRNA level and a protein level of MMP-1 and MMP-13, but it also decreased collagenase and gelatinase activities of total MMPs. Furthermore, the induction of the MMP expression and activities by interleukin-1 was suppressed by the oscillatory shear. Interestingly, the oscillatory shear upregulated the mRNA expression of TIMP-1 and TIMP-2. Our results support a potential role of oscillatory shear in regulating expression and activities of MMPs in the presence and the absence of proinflammatory cytokine.     

两种冠边缘设计对龈沟液中MMP-8及其组织抑制剂-1水平的影响

目的:探讨两种冠边缘设计对龈沟液(GCF)中基质金属蛋白酶-8(MMP-8)及其组织抑制剂-1(TIMP-1)水平的影响.方法:收集行烤瓷熔附金属全冠修复的患者16例(26颗牙),分为龈下组(8例,14颗牙)和平龈组(8例,12颗牙).分别于修复前、修复后1个月、3个月、6个月时收集两组受试牙的GCF,应用ELISA法测定GCF中MMP-8及TIMP-1的含量.结果:以修复前GCF中MMP-8和TIMP-1的含量为基线,龈下组GCF中MMP-8的含量在修复后1个月时明显高于基线水平(P＜0.05),修复后3个月时持续升高,在修复后6个月时开始下降,但仍高于基线水平(P＜0.05).而平龈组GCF中MMP-8的含量在修复后1个月、3个月时有所升高,但与基线水平比较差异无显著性,在修复后6个月时下降,恢复至基线水平.龈下组和平龈组GCF中TIMP-1的含量在修复后1个月、3个月、6个月时,均呈逐渐升高的趋势,两组在修复后各时间点TIMP-1的含量均明显高于基线水平(P＜0.05).组间比较,平龈组修复后各时间点GCF中MMP-8的含量均明显低于龈下组(P＜0.05);而TIMP-1的含量则均明显高于龈下组(P＜0.05).结论:冠边缘平龈设计可能更有利于MMP-8/TIMP-1系统在牙周组织中更新、代谢和改建过程中的表达作用,以及牙龈组织的健康.     

The role of matrilysin (MMP-7) in leukaemia cell invasion

The matrix metalloproteinases (MMPs) are important in tumour cell invasion and metastasis in many common cancers. However, relatively few studies have investigated the role of MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in leukaemia cell invasion. This study examined two leukaemia cell lines, K562 and HL-60 and showed that the K562 cell line was four times more invasive than the HL-60 cell line. The expression of MMP-2, matrilysin (MMP-7), MMP-9, TIMP-1, TIMP-2 and TIMP-3 was analysed. Both cell lines produced similar amounts of MMP-2, MMP-9 and TIMP-2. The K562 cells expressed more TIMP-1 than the HL-60 cells and neither cell line expressed TIMP-3. Interestingly, only the K562 cells expressed matrilysin suggesting a potential role for matrilysin in leukaemia cell invasion. In vitro invasion assays performed in the presence of a matrilysin blocking antibody showed a 40% reduction in invasive ability. This data suggests that matrilysin plays an important role in leukaemia cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Suppressive activity of fexofenadine hydrochloride on metalloproteinase production from nasal fibroblasts in vitro

BACKGROUND: Allergic rhinitis (AR) is an inflammatory disease characterized by nasal wall remodelling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are the major proteolytic enzymes that induce airway remodelling. These enzymes are also important in the migration of inflammatory cells through basement membrane components. OBJECTIVE: We evaluated whether fexofenadine hydrochloride (FEX), the carboxylic acid metabolite of terfenadine with selective H(1)-receptor antagonist activity, could inhibit MMP production from nasal fibroblasts (NFs) in response to TNF-alpha stimulation in vitro. METHODS: NFs were established from nasal polyp-derived fibroblasts (PFs) taken from patients with AR. Nasal mucosal fibroblasts (MFs) were also induced from nasal mucosal tissues from septal deformity patients without allergy. PF and MF (2 x 10(5) cells/mL, each) were stimulated with TNF-alpha in the presence of various concentrations of FEX. After 24 h, culture supernatants were obtained and assayed for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 levels by ELISA. The influence of FEX on mRNA expression of MMPs and TIMPs in 4 h-cultured cells was also evaluated by real-time RT-PCR. Furthermore, nuclear factor-kappa B (NF-kappa B) activation in fibroblasts treated with FEX for 4 h was examined by ELISA. RESULTS: FEX at more than 350 ng/mL inhibited the production of MMP-2 and MMP-9 from both PF and MF in response to TNF-alpha stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected by FEX. FEX also inhibited MMP mRNA expression and NF-kappa B activation in PF and MF after TNF-alpha stimulation. CONCLUSION: The present data suggest that the attenuating effect of FEX on MMP-2 and -9 production from NFs induced by inflammatory stimulation may underlie the therapeutic mode of action of the agent on allergic diseases, including AR.     

Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro

Recent studies have demonstrated important pro-inflammatory roles for two matrix metalloproteinases (MMPs)-MMP-3 (stromelysin-1) and MMP-9 (gelatinase B)-in acute lung injury [Am. J. Respir. Cell Mol. Biol. 24 (2001) 1]. A role for MMP-3 in skin inflammation has also been demonstrated [Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6885]. While leukocytes (neutrophils and macrophages) are known to elaborate these tissue-destructive enzymes, parenchymal cells are also capable of synthesizing MMPs. In the present study, we examined the production of MMP-3 and MMP-9 by rodent lung fibroblasts, type II epithelial cells, and vascular endothelial cells. Dermal fibroblasts were also examined. Cells were examined under control conditions and in response to agonists that induce acute inflammatory tissue injury (IgG-containing immune complexes and lipopolysaccharide [LPS]). In the absence of stimulation, MMP-3 and MMP-9 were not detected or were present at low level. However, upon stimulation with either of the two pro-inflammatory agonists, production of both enzymes occurred in fibroblasts and epithelial cells (though not in endothelial cells). The observation that resident cells in the tissue parenchyma can elaborate MMPs in direct response to pro-inflammatory stimuli provides insight into possible mechanisms by which tissue damage occurs in acute inflammation.     

Matrix metalloproteinases in premature coronary atherosclerosis: influence of inhibitors, inflammation, and genetic polymorphisms

Matrix metalloproteinases (MMPs) are thought to participate in the pathogenesis of coronary artery disease (CAD), particularly in the occurrence of acute coronary syndrome (ACS). Little is known about human in vivo MMP regulation in CAD. The expression and regulation of MMPs and their tissue inhibitors (TIMPs) were evaluated in premature CAD. The distribution of MMP-3 5A/6A and MMP-9 C/T promoter polymorphisms and MMP-9 A/G exon-6 polymorphism were investigated in 200 consecutive male premature CAD patients (aged < or = 55 years) and 201 age-matched male blood donors. Plasma concentrations/activities of MMP-2 and MMP-9 were also measured, as were plasma concentrations of MMP-3, TIMP-1, and TIMP-2 in 80 patients (49 with ACSs and 31 with stable CAD) and 40 controls. Inflammation markers were also obtained. MMP genetic polymorphism distributions did not vary between patients and controls and did not seem to influence their respective MMP plasma levels. Patients showed increased MMP-9 and TIMP-1 concentrations and decreased TIMP-2 concentration and MMP-2 total activity (all P < or = 0.002). Overall, TIMP-1 correlated with C-reactive protein (CPR) (r = 0.594, P < 0.001) and haptoglobin (r = 0.276, P = 0.005), whereas MMP-2 activity correlated inversely with haptoglobin (r = -0.195, P = 0.032). Blood glucose correlated positively with TIMP-1 concentration (r = 0.711, P < 0.001) and negatively with MMP-2 activity (r = -0.250, P = 0.006). In conclusion, MMP and TIMP plasma levels in premature CAD are linked to clinical presentation and markers of inflammation and metabolic disorders rather than to genetic polymorphisms.     

咪达普利拉对人心脏成纤维细胞基质金属蛋白酶-2和抑制剂-2的作用及机制研究

目的：研究血管紧张素转化酶抑制剂（angiotensin converting enzyme inhibitor，ACEI）咪达普利活性代谢物咪达普利拉对白细胞介素－1β（interleukin -1β，IL-1β）诱导的心脏成纤维细胞基质金属蛋白酶（matrix metalloproteinases，MMPs）和基质金属蛋白酶2型抑制剂（type 2 tissue inhibitor of matrix metalloproteinase，TIMP-2）表达的影响及其可能机制。方法:原代人心脏成纤维细胞从美国细胞应用所购买。用RT-PCR检测心脏成纤维细胞MMP-2、MMP-9和TIMP-2 mRNA水平的变化；用凝胶酶谱法分析心脏成纤维细胞中MMP-2、MMP-9的活性；用Griess方法检测培养上清一氧化氮（nitric oxide，NO）水平。结果:通过凝胶酶谱分析，RT-PCR和Griess方法发现IL-1β能显著增加MMP-2基因转录以及活性(P<0.05)，同时也显著增加了细胞培养上清中NO的产生(P<0.05)。IL-1β的这些效应可以被咪达普利拉和外源性NO合酶抑制剂L-单甲基精氨酸（NG-methyl L-Arginine，L-NMMA）所抑制(P<0.05)，而外源性NO供体亚硝基铁氰化钠(sodium nitroprusside，SNP）可以取消咪达普利拉对于NO、MMP-2的有效作用(P<0.05)。实验中发现IL-1β以及咪达普利拉对TIMP-2基因转录无明显作用(P>0.05)。结论:咪达普利拉能通过NO途径抑制IL-1β诱导的心脏成纤维细胞MMP-2的合成和活性；提示ACEI抗心肌重塑以及心力衰竭的部分作用可能是通过MMPs途径实现的。     

Identification of a calcium-dependent matrix metalloproteinase complex in rat chorioallantoid membranes during labour

The induction of the expression of matrix metalloproteinases (MMPs) and their extracellular activation are key processes in connective tissue degradation in the chorioallantoid membrane during rat labour. However, the regulatory mechanisms remain largely unknown. Here, we report the identification of a calcium-dependent high molecular weight complex composed of MMP-9, MMP-3, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, identified by zymography and western blotting. Molecular sieve chromatography confirmed the presence of a complex of MMPs and TIMPs with an exclusion volume >670 kDa. Differential scanning calorimetry of the complex confirmed the existence of a macromolecular complex that unfolds with a broad transition; it is denatured over a wide range of temperatures and has a T(m) of 72 degrees C in the presence of Ca(2+). When denatured in the absence of Ca(2+), there were at least eight transitions with T(m)s that corresponded to pro-MMP-9, MMP-9, pro-MMP-3, MMP-3, pro-MMP-2, MMP-2, TIMP-1 and TIMP-2. Co-localization of the same molecular components was demonstrated by confocal microscopy using cell-depleted chorioallantoid membranes. The assembly and disassembly of the complex can be reproduced at physiological concentrations of Ca(2+). This complex provides a potential mechanism for the enzymatic regulation of MMPs, which may participate in connective tissue degradation leading to the rupture of the fetal membranes during labour.