Increased expression of matrix metalloproteinases in the uterine cervix of postmenopausal women

OBJECTIVE.: To determine whether atrophy of the uterine cervix in menopausal women is associated with an increased expression of matrix metalloproteinases (MMP), a decrease in their counter regulatory proteins (tissue inhibitors of matrix metalloproteinase [TIMP]), and a decrease in type I collagen. MATERIALS AND METHODS.: A pilot study was performed on cervical stroma harvested from 10 premenopausal and 9 postmenopausal women undergoing a hysterectomy. The amount of pro-MMP-2 and pro-MMP-9 in protein extracts from the two groups was compared by gelatin zymography. The membrane-type (MT)1-MMP, TIMP-1, and TIMP-2 were quantitated by Western immunoblotting. Total collagen was estimated by measuring hydroxyproline content. A primary fibroblast culture was developed to study estrogen regulation of MMP expression in vitro. RESULTS.: Pro-MMP-2 and pro-MMP-9 were increased in postmenopausal extracts. No difference in the amount of MT1-MMP, TIMP-1, TIMP-2, or total collagen was detected. In primary cervical fibroblast cultures, only active MMP-2 was suppressed by estrogen. CONCLUSIONS.: The protein expression of pro-MMP-2 and pro-MMP-9 is increased in cervical stroma of postmenopausal women relative to premenopausal women.     

基质金属蛋白酶-13在骨性关节炎发病中的活性调控研究

目的 研究一氧化氮(NO)是否通过膜型基质金属蛋白酶-1(MT1-MMP)间接激活基质金属蛋白酶-13酶原(pro-MMP-13).方法 购买并传代人软骨肉瘤细胞(SW1353),用NO供体S-亚硝基-N-乙酰基青霉胺(SNAP),SNAP+NO清除荆氧合血红蛋白(OxyHb)和SNAP+组织金属蛋白酶抑制物-2(TI...     

ONO-4817, a novel matrix metalloproteinase inhibitor, attenuates allograft vasculopathy in a rat cardiac transplant.

BACKGROUND: Cardiac allograft vasculopathy (CAV), a disorder characterized by rapid development and progression of obliterative vasculopathy in the transplanted heart, continues to be a major cause of graft failure in long-surviving human transplants. The mechanisms and histopathologic processes of CAV remain unknown. Previous animal studies have shown that inhibition of matrix metalloproteinase (MMP) prevents migration and proliferation of smooth muscle cells in CAV. In this study, we hypothesized that MMPs may be expressed in and may play an important role in CAV. METHODS: An F344-to-WKAH rat heterotopic heart transplantation model was used. Tacrolimus was administered intramuscularly 14 days after transplantation to prevent acute rejection and to allow the development of CAV. We divided the animals into 2 groups according to post-operative treatment: an ONO group received an MMP inhibitor (ONO-4817) daily by oral gavage for 14 days after transplantation (n = 6), and a control (n = 6) group received no treatment. Grafts were harvested 60 days after treatment. RESULTS: Immunohistochemical staining revealed that MMP-2 and tissue inhibitors of metalloproteinase-2 (TIMP-2) were expressed more strongly in the neointima and media of the control CAV animals than in the ONO-CAV animals. The animals given ONO-4817 exhibited a significant decrease in the percentage of affected vessels, in the percentage of intimal proliferation, in the intima-to-media ratio, and in the expression of MMP-2 and TIMP-2. CONCLUSION: These results suggest that MMP-2 and TIMP-2 play an important role in the development of CAV and that the use of an MMP inhibitor (ONO-4817) may prevent neointimal proliferation in patients with CAV.     

Differential regulation of matrix metalloproteinase activities in abdominal aortic aneurysms

BACKGROUND: The increased synthesis of matrix metalloproteinases (MMPs) by aortic smooth muscle cells (SMCs) is thought to be involved in the etiopathogenesis of abdominal aortic aneurysms (AAAs), but the functional regulation and the activation states of these MMPs remain unclear. In this study, we assessed the expression levels and the functional regulation of several MMPs in the pathogenesis of AAAs. METHODS: Human healthy aorta and AAA specimens were homogenized, and the proteolytic activities of MMP-2 and MMP-9 and of the macrophage metalloelastase (MMP-12) were assessed with zymography. Protein expression of MMP-1, MMP-12, membrane-type 1 MMP (MT1-MMP), tissue inhibitor of MMP 1 (TIMP-1), TIMP-2, TIMP-3, alpha-actin, and beta-actin was analyzed with electrophoresis on sodium dodecyl sulfate gels and immunoblotting. RESULTS: MMP-1, MMP-9, and MMP-12 zymogen levels and proteolytic activities were increased in AAAs when compared with healthy aorta. A severe reduction in alpha-actin--positive vascular SMCs was observed in all the AAA specimens and was correlated with an increase in TIMP-3 but not TIMP-1 or TIMP-2 potential activities. Although pro--MMP-2 activity was decreased, the extent of activated MMP-2 remained unaffected in the AAAs. In accordance with this result, a highly activated MT1-MMP form was also observed in AAAs. CONCLUSION: These data suggest that chronic aortic wall inflammation is mediated by macrophage infiltration, which may account for the destruction of medial elastin, as reflected by SMC down regulation, through increased levels of active MMP-1 and MMP-12. Moreover, altered MT1-MMP proteolytic turnover and differential regulation of TIMP expression in AAAs suggest that tight regulatory mechanisms are involved in the molecular regulation of MMP activation processes in the pathogenesis of AAAs.     

Significance of matrix metalloproteinases and tissue inhibitors of metalloproteinase expression in the recurrence of superficial transitional cell carcinoma of the bladder

PURPOSE: We determined whether expression levels of matrix metalloproteinase (MMP)-2, MMP-9, membrane-type MMP-1 (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 messenger (m) RNA in superficial transitional cell carcinoma of the bladder may be used as predictors of tumor recurrence. MATERIALS AND METHODS: Total RNA was extracted from 51 superficial transitional cell carcinomas of the bladder, and expression levels of MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 messenger mRNA in these specimens were measured by Northern blot analysis. Results were evaluated in regard to tumor recurrence. RESULTS: Mean MMP-9 and TIMP-2 mRNA expression in the tumors of patients with recurrence were 2.5 and 3-fold higher, respectively, than in those of patients without recurrence despite no significant differences in MMP-2, MT1-MMP or TIMP-1 expression. The recurrence-free survival rate of patients with elevated MMP-9 and TIMP-2 mRNA expression was significantly lower than that of patients with normal MMP-9 and TIMP-2 expression, respectively. In addition, Cox's multivariate analysis revealed that elevated MMP-9 and TIMP-2 were strongly associated with a high incidence of intravesical recurrence of superficial bladder cancer. CONCLUSIONS: These results indicate that MMP-9 and TIMP-2 are strongly expressed in the tumors of patients with recurrence compared with those without recurrence and elevated MMP-9 and TIMP-2 may be used as predictors of recurrence in patients with superficial transitional cell carcinoma.     

基质金属蛋白酶和组织金属蛋白酶抑制剂在大肠癌中的表达

目的　研究基质金属蛋白酶 (MMPs)及组织金属蛋白酶抑制剂 (TIMPs)在大肠癌中的表达特点 ,与肿瘤发生发展的关系 ,以及在肿瘤治疗中的应用前景。　方法　采用RT PCR方法测定 2 8例大肠癌患者肿瘤组织和周围正常粘膜的基质金属蛋白酶 2 (MMP 2 )、膜型 1 基质金属蛋白酶 (MT1 MMP)、基质溶素 (MMP 7)、组织金属蛋白酶抑制剂 2 (TIMP 2 )、组织金属蛋白酶抑制剂 3(TIMP 3)的mRNA表达状况 ,并将其结果与临床及病理学资料进行统计学分析。　结果　 (1) 2 7例患者肿瘤组织中MMP 7mRNA表达阳性 ,MMP 2、MT1 MMP、TIMP 2和TIMP 3在肿瘤组织和正常粘膜中均有高表达 ;(2 )肿瘤组织中MMP 7mRNA的表达水平与大肠癌患者的Dukes′分期相关 (P <0 0 1) ;(3)淋巴结阳性患者的肿瘤组织TIMP 2表达水平为 (1 2 5± 0 46 )明显高于淋巴结阴性患者的 (0 75± 0 41) ,差异有显著性意义 (P <0 0 1) ;(4)大肠癌患者癌周正常粘膜TIMP 3mRNA表达随患者Duke′s分期的进展和肿瘤浸润深度的增加而降低 (P <0 0 1) ;(5 )TIMPs与MMPs之间无明显相关关系 (P >0 1)。　结论　MMP 7可望成为诊断大肠癌的敏感指标 ;人工诱导TIMP 2、TIMP 3或阻断MMP 7、MMP 2、MT1 MMP的表达可能抑制肿瘤的浸润和转移 ,成为肿瘤治疗的新途径。     

Modulation of matrix gelatinases and metalloproteinase-activating process in acute kidney rejection

Changes in matrix metalloproteinase (MMP) activities would contribute to the accumulation of extracellular matrix during acute kidney allograft rejection. MMP-2 and MMP-9 and other gelatinolytic activities were examined in the rejected graft and the urine of a rat model of acute kidney rejection (orthotopic allotransplantation from a Buffalo donor to a Wistar-Furth recipient) by either zymography or fluorescence assay. MMP-2, membrane type 1 (MT1)-MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 were also examined by immunodetection. The proMMP-2 activity and protein level increased in the graft during rejection when compared with normal Buffalo kidney, whereas activated MMP-2 decreased. TIMP-2 protein levels were markedly decreased and MT1-MMP proteolytic fragments (44-40 kDa) were undetectable. This suggests an altered MT1-MMP-dependent processing of proMMP-2 into active MMP-2 due to a diminished TIMP-2 level in acute kidney rejection. In the urine the overall gelatinolytic activity decreased considerably, although activity associated with an as yet unidentified 78-kDa protein appeared 6 days after transplantation.     

Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines

Stromal expression of some matrix metalloproteinases (MMPs) has been associated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaP-derived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer.     

MMP-2、MMP-9、TIMP-1在血管成形术后再狭窄中的表达及其意义的实验研究

目的观察PTA后MMP-2、MMP-9和TIMP-1在血管壁各层随时间的演变规律,探讨其在血管再狭窄过程中的表达及意义。方法48只大鼠制作成颈动脉再狭窄动物模型,选取30只内膜增生满意的标本,分别代表动脉损伤后1、3、7、14、28、42天6个观察时间点,对胶原纤维进行Masson染色。MMP-2、TIMP-1、PCNA进行免疫组织化学染色,MMP-9进行免疫组织化学和原位杂交两种染色。分析它们的表达与内膜增生和血管重塑的关系。结果正常血管不表达MMP-9mRNA及蛋白,损伤后第3天中膜、外膜mRNA表达达高峰,第7天内膜表达达高峰。MMP-9蛋白第7天内膜表达达高峰,且阳性细胞率高于中膜和外膜。第14、28天,血管壁各层表达逐渐下降至基线水平,内膜的阳性细胞主要集中在新生内膜靠近管腔的一侧。内膜MMP-2表达高峰出现晚至第14天,且近内弹力板处MMP-2也有明显的表达。正常血管壁不表达TIMP-1,损伤后第3天,中膜和外膜表达达高峰。第7天,新生内膜表达达高峰,中膜和外膜仍有较高水平表达。结论MMP-9、MMP-2、TIMP-1参与再狭窄,内膜MMP-9表达与早期增殖的细胞向内膜迁移形成新生内膜有关。MMP-2表达与晚期内膜形成、内膜重塑有关。TIMP-1的表达对内膜增生和重塑没有直接作用,其表达升高是机体为适应MMPs升高做出的代偿反应,在维持自身平衡中起作用。     

Experimental hindlimb ischemia leads to neutrophil-mediated increases in gastrocnemius MMP-2 and -9 activity: a potential mechanism for ischemia induced MMP activation

INTRODUCTION: Matrix metalloproteinases (MMP)-2 and -9 are Type 4 collagenases instrumental in basement membrane degradation, a process necessary for angiogenesis to occur. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of both PMN-derived serine protease and membrane type 1 (MT1)-MMP, are able to activate pro-MMP-2 following hindlimb ischemia. We hypothesized that neutrophil depletion (ND) of animals prior to hindlimb ischemia (HI) would abrogate the activation of pro-MMP-2 and decrease the level of MMP-9 MATERIALS AND METHODS: 12 FVB/N Tie2/LacZ-182 SATO female mice were randomly divided into four blinded groups; HI + PBS, HI + anti-PMN antibody (GR-1), HI + isotype matched control antibody (IgG(2b,K)), and no HI + PBS. PMN depletion was achieved prior to the time of ischemia and maintained until sacrifice. HI was achieved by unilateral femoral artery ligation. Three days postligation the animals were sacrificed and the gastrocnemius muscle from each hindlimb was harvested. MMP-2 and -9 (gelatin zymography) and MT1-MMP (Western blot) expression and activation were quantified by densitometry and NIH Image Analysis software. MMP values were expressed as a ratio of ischemic-to-nonischemic hindlimbs and compared between groups. Statistical significance was determined with analysis of variance (ANOVA) RESULTS: Zymograms revealed a greater than 10-fold increase in active MMP-9 and greater than 4-fold increase in active MMP-2 from HI + PBS compared to no HI + PBS (P < 0.05). HI + anti-PMN antibody demonstrated reduction of both active MMP-2 and -9 levels to that of the nonischemic group. Pro-MMP-2 was constitutively expressed in all four groups with no significant differences between any group (P = NS). There was no difference between the HI + isotype-matched antibody group and the HI + PBS group throughout the experiments (P = NS). ND did not affect MT1-MMP activation or expression CONCLUSIONS: Limb ischemia causes activation of MMP-2 and -9, which is eliminated by ND. ND animals undergoing hindlimb ischemia exhibit identical levels of active MMP-2 and -9 as animals that did not have hindlimb ischemia. Neutrophils may be an important activator of MMP-2 and the suppliers of MMP-9 in the ischemic hindlimb and may be essential for tissue remodeling, basement membrane degradation, and angiogenesis in ischemic limbs     

Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP-2 activity in experimental rodent aortic aneurysms

OBJECTIVE: The objective of this study was to determine the significance of membrane type 1 matrix metalloproteinase (MT1-MMP) activation of MMP-2 in experimental abdominal aortic aneurysms. METHODS: Rat aortas were perfused with either saline as a control or elastase, and harvested on 2, 4, or 7 days after perfusion (n = 5 per treatment group/day). Aortic MT1-MMP and MMP-2 expression and protein were determined by real time polymerase chain reaction and Western blotting, respectively. Aortic explants were used to measure MMP-2 activity by zymography. Rat aortic smooth muscle cells in vitro were exposed to increasing doses of elastase and analyzed for MT-1 MMP expression. RESULTS: Aneurysms formed in 80% of the elastase-perfused aortas at 7 days, whereas none formed in the saline-perfused aortas. Significantly increased MT1-MMP expression was observed only on day 4, when levels were 6.5-fold higher in elastase-perfused aortas compared with saline-perfused aortas (P < .01). By day 7, MT1-MMP protein was present only in the elastase-perfused aortas (P = .02). By immunohistochemistry, MT1-MMP was detectable only in the elastase-perfused group at day 7. Cleaved MMP-2 activity (P = .045) was increased in elastase-perfused aortas compared with saline perfused aortas at day 7. In rat aortic smooth muscle cells, MT-1 MMP expression increased in response to elastase (P = .02). CONCLUSION: The rodent aortic aneurysm model exhibits upregulation of MT1-MMP expression and protein with subsequent increased conversion of MMP-2 from the latent to the cleaved form.     

Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall

BACKGROUND: Current research has shed new light on the role of matrix metalloproteinase (MMP) 2 in the development of abdominal aortic aneurysms (AAAs). MMP-2 is a major protease in the wall of small aneurysms and is produced at increased levels by smooth muscle cells derived from AAAs compared with normal controls. In vivo, MMP-2 is produced as an inactive proenzyme that is activated predominantly by the cell membrane-bound enzyme, membrane type 1 matrix metalloproteinase (MT1-MMP). This study investigated the production of the MMP-2-MT1-MMP-tissue inhibitor of metalloproteinases (TIMP) 2 system within the wall of aortic aneurysms and in age-matched control arterial tissue. METHODS: Arterial tissue from four patients with aortic aneurysms and four age-matched aortic samples was examined for the production and expression of MMP-2, TIMP-2 and MT1-MMP protein using immunohistochemistry, in situ hybridization and in situ zymography. RESULTS: All components of the MMP-2-TIMP-2-MT1-MMP enzyme system were detected in the arterial wall of both aneurysm and control samples, specifically in the medial tissue. The enzymes co-localized with medial smooth muscle cells. Gelatinolytic activity was localized to elastin fibres in normal and aneurysmal aorta. CONCLUSION: The presence of MT1-MMP within the media of arterial tissue suggests a powerful pathway for the activation of MMP-2. The localization of the MMP-2-TIMP-2-MT1-MMP enzyme system to the medial layer of the arterial wall gives support to the concept that this system may play an aetiological role in the pathogenesis of AAAs.     

Decreased collagen-degrading activity could be a marker of prolonged mesangial matrix expansion

Background Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion.Methods We prepared two rat models, showing reversible or prolonged mesangial matrix expansion, induced by a single injection or two consecutive injections of anti-Thy-1.1 monoclonal antibody 1-22-3, respectively. We analyzed the glomerular expression of type I and type IV collagens; MMP-2, -9, and -13; membrane type 1-MMP (MT1-MMP); TIMP-1; and urinary type I collagen-degrading activity in both models.Results There were no differences in glomerular mRNA levels of type I and type IV collagens between the reversible and the prolonged models. MMP-9 mRNA expression and protein level was lower in the prolonged model than in the reversible one, whereas there were no differences in mRNA levels of MMP-2, -13, MT1-MMP, or TIMP-1 between the two models. Urinary type I collagen-degrading activity in the prolonged model was lower than that in the reversible one. Furthermore, there was a significant correlation between the mesangial matrix expansion and urinary type I collagen-degrading activity.Conclusions Impaired expression of MMP-9 may contribute to the development of prolonged mesangial matrix expansion. Analysis of urinary type I collagen-degrading activity may provide additional diagnostic information in mesangial proliferative glomerulonephritis.     

Local Medial Microenvironment Directs Phenotypic Modulation of Smooth Muscle Cells After Experimental Renal Transplantation

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft‐derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation‐induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low‐density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF‐BB‐stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α‐Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF‐BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.     

TGF-β1和TGF-α在膀胱癌侵袭和转移中的作用

目的 探讨转化生长因子-β（TGF-β1）和转化生长因子－α（TGF-α）对膀胱癌细胞株（EJ）生长的影响以及促进膀胱癌侵袭和转移的可能途径。方法 采用MTT、Western Blot和RT－PCR方法,观察TGF－β1和TGF－α对DJ细胞株生长以及在mRNA和蛋白水平上对金属基质蛋白酶（MMPs)和组织特异性金属蛋白酶抑制剂－2（TIMP－2）表达的影响。结果 （1）TGF-β1和TGF－α对EJ细胞生长存在抑制趋势,但差别无显著性意义。（2）TGF－β1和TGF－α作用于EJ细胞48h时 ,MMP－2、TIMP－2、MT1－MMP mRNA表达降低;TGF－β1组MMP-9 mRNA表达增加,而对照组和TGF－α组无MP－9mRNA表达。（3）当TGF－β1浓度为0．1、1．0ng/ml时,增加细胞培养上清液中MMP－2蛋白含量对TIMP－2蛋白水平无影响。当浓度为5．0、10．0ng/ml时对MMP－2无影响而降低TIMP－2表达;TGF－α在浓度为1.0、5.0、10.0ng/ml时,对MMP－2蛋白表达无影响而降低TIMP－2表达;在100．0ng/ml时增加MMP－2蛋白表达而对TIMP－2表达无影响;无论在对照组和实验组均未检测到MMP－9。结论 TGF-β1、TGF-α不能抑制EJ细胞生长,参与肿瘤侵袭和转移作用可能与调节MMPs、TIMP－2表达途径有关。