Unstable atherosclerotic plaques of the carotid arteries are at great risk for the development of ischemic cerebrovascular events. The degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and nitric oxide induced apoptosis of vascular smooth muscle cells (VSMCs) contribute to the vulnerability of the atherosclerotic plaques. Basic fibroblast growth factor (bFGF) through its mitogenic and angiogenic properties has already been implicated in the pathogenesis of atherosclerosis. However, its role in plaque stability remains elusive. To address this issue, a panel of human carotid atherosclerotic plaques was analysed for bFGF, FGF‐receptors‐1 and ‐2 (FGFR‐1/‐2), inducible nitric oxide synthase (iNOS) and MMP‐9 expression. Our data revealed increased expression of bFGF and FGFR‐1 in VSMCs of unstable plaques, implying the existence of an autocrine loop, which significantly correlated with high iNOS and MMP‐9 levels. These results were recapitulated in vitro by treatment of VSMCs with bFGF. bFGF administration led to up‐regulation of both iNOS and MMP‐9 that was specifically mediated by nuclear factor‐κB (NF‐κB) activation. Collectively, our data demonstrate a novel NF‐κB‐mediated pathway linking bFGF with iNOS and MMP‐9 expression that is associated with carotid plaque vulnerability. 相似文献
Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients. 相似文献
It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling. 相似文献
Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance. 相似文献
Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy. 相似文献
Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α1 integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases. 相似文献
The matrix metalloproteinases (MMPs) gelatinase A (MMP‐2) and gelatinase B (MMP‐9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2‐DZ) as an extension of classic monodimensional zymography (1‐DZ) to analyse the complete pattern of isoforms and post‐translational modifications of both MMP‐9 and MMP‐2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP‐2 isoforms, and at least three different isoforms and two different states of organization of MMP‐9 (the multimeric MMP‐9 and the N‐GAL‐MMP‐9 complex) were observed. In addition, 2‐DZ revealed for the first time that all MMP‐9 and MMP‐2 isoforms actually exist in the form of charge variants: four or five variants in the N‐GAL complex, more charge variants in the case of MMP‐9; and five to seven charge variants for MMP‐2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP‐9) and phosphorylation (MMP‐2) contributed to molecular heterogeneity. The detection of charge variants of MMP‐9 and MMP‐2 in MS serum samples illustrates the power of 2‐DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases. 相似文献
Although past studies observed the changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in end‐stage heart failure (HF) patients, a consistent and clear pattern of type‐specific MMPs and/or TIMPs has yet to be further defined. In this study, proteomic approach of human protein antibody arrays was used to compare MMP and TIMP expression levels of left ventricular (LV) myocardial samples from end‐stage HF patients due to dilated cardiomyopathy (DCM) with those from age‐ and sex‐ matched non‐failing patients. Western blot analysis, immunohistochemistry and ELISA were used for validation of our results. We observed that MMP‐10 and ‐7 abundance increased, accompanied by decreased TIMP‐4 in DCM failing hearts (n= 8) compared with non‐failing hearts (n= 8). The results were further validated in a cohort of 34 end‐stage HF patients derived from three forms of cardiomyopathies. Cardiac and plasma MMP‐10 levels were positively correlated with the LV end‐diastolic dimension in this HF cohort. In addition, we observed that insulin‐like growth factor‐2 promoted MMP‐10 production in neonatal rat cardiomyocytes. In conclusion, this study demonstrated a selective up‐regulation of MMP‐10 and ‐7 along with a discordant change of TIMP‐4, and a positive correlation between MMP‐10 levels and the degree of LV dilation in end‐stage HF patients. Our findings suggest that type‐specific dysregulation of MMPs and TIMPs is associated with LV remodelling in end‐stage HF patients, and MMP‐10 may act as a novel biomarker for LV remodelling. 相似文献
Staphylococcal superantigen‐like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P‐selectin glycoprotein ligand‐1 (PSGL‐1) and matrix metalloproteinase (MMP)‐9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid‐containing glycans of MMP‐9, but the roles of sialic acids in the interaction between SSL5 and MMP‐9 are still controversial. In the present study, we prepared recombinant glutathione S‐transferase‐tagged SSL5 (GST‐SSL5) and analyzed its binding capacity to MMP‐9 by pull‐down assay after various modifications of its carbohydrate moieties. We observed that GST‐SSL5 specifically bound to MMP‐9 from a human monocytic leukemia cell line (THP‐1 cells) and inhibited its enzymatic activity in a concentration‐dependent manner. After MMP‐9 was treated with neuraminidase, its binding activity towards GST‐SSL5 was markedly decreased. Furthermore, recombinant MMP‐9 produced by sialic acid‐deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild‐type CHO‐K1 cells. Treatment of MMP‐9 with PNGase F to remove N‐glycan resulted in no significant change in the GST‐SSL5/MMP‐9 interaction. In contrast, the binding of GST‐SSL5 to MMP‐9 secreted from THP‐1 cells cultured in the presence of an inhibitor for the biosynthesis of O‐glycan (benzyl‐GalNAc) was weaker than the binding of GST‐SSL5 to MMP‐9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid‐containing O‐glycans of MMP‐9 for the interaction of MMP‐9 with GST‐SSL5. 相似文献
Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E‐cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐10, ‐12 and ‐13) and TIMPs (‐1 to ‐4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP‐1 and TIMP‐3 levels. Significant correlations were found between MMP‐3 and TIMP‐4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP‐9 and TIMP‐3 levels could be predictive of RT toxicity. For this reason, MMP‐3, MMP‐9, TIMP‐3 and TIMP‐4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT. 相似文献
Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase‐9 (MMP‐9) activity in the ischemic brain, which exacerbates blood‐brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP‐9 activity is not well understood. Here we report an important role of caveolin‐1 in mediating tPA‐induced MMP‐9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP‐9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP‐9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3‐fold increase of caveolin‐1 protein levels in endothelial cells. Interestingly, knockdown of Cav‐1 with siRNA inhibited tPA‐induced MMP‐9 mRNA up‐regulation and MMP‐9 increase in the conditioned media, but did not affect MMP‐9 decrease in cellular extracts. These results suggest that caveolin‐1 critically contributes to tPA‐mediated MMP‐9 up‐regulation, but may not facilitate MMP‐9 secretion in endothelial cells.
Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP‐2). The aim of the current research was to study the effects of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate (MMP‐2‐inhibitor‐NO‐donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP‐2, MMP‐9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP‐2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1‐10 µmol/L) resulted in a concentration‐dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP‐2. Decreased activity of intracellular MMP‐2 led to reduced degradation of MLC and improved myocyte contractility in a concentration‐dependent manner. An infusion of a MMP‐2‐inhibitor‐NO‐donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate protects heart from I/R injury. 相似文献