Cystathionine- -synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-β-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway. extracellular matrix; matrix metalloproteinase-9; intercellular cell adhesion molecule-1; vascular cell adhesion molecule-1; collagen type-1; hyperhomocysteinemia     

Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography

 In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation. Accepted: 15 June 1998     

Thioredoxin secreted upon ultraviolet A irradiation modulates activities of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in human dermal fibroblasts

Regulation of the balance of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (TIMP-2) by thioredoxin (Trx) was investigated in human dermal fibroblasts. Expression and secretion of Trx and Trx reductase 1 (TR1) was increased after ultraviolet (UV) A irradiation. A significant increase in proMMP-2 activity and a decrease of TIMP-2 activity in supernatants of UVA-irradiated fibroblasts were observed in gelatin and reverse zymography compared to non-irradiated fibroblasts. Removal of Trx or TR1 by immunoprecipitation diminished these changes in proMMP-2 activity. Incubation with 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB) also suppressed these changes. Incubation with recombinant Trx or TR decreased TIMP-2 activity and increased MMP-2 activity. UVA-irradiated fibroblasts, transiently transfected with a dominant-negative mutant or wild-type Trx, showed down- or upregulation of proMMP-2 activities, respectively, without significant change of protein amount. In conclusion, thioredoxin secreted by UVA irradiation is involved in the regulation of MMP-2 and TIMP-2 activities through its redox activity in human dermal fibroblasts.     

BMP-2 treatment of C3H10T1/2 mesenchymal cells blocks MMP-9 activity during chondrocyte commitment

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. We explored the underlying mechanism of BMP-2-induced chondrocyte commitment of C3H10T1/2 cells. Treating cells with exogenous BMP-2 was tied to chondrocyte commitment by inhibiting matrix metalloproteinase-9 activity (MMP-9: 92 kDa type IV collagenase/gelatinase B). Glycogen synthase kinase (GSK)-3β inhibition by its specific inhibitor blocked BMP-2-induced chondrocyte commitment by stimulating MMP-9 activity. These findings indicate that the downregulation of MMP-9 by BMP-2 is associated with chondrocyte commitment, and that the GSK-3β signaling pathway is involved in this process.     

Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells

The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.     

博莱霉素致肺间质成纤维细胞MMP-2/TIMP-1表达失衡

观察博莱霉素对肺间质成纤维细胞中基质金属蛋白酶-2（MMP-2）及组织金属蛋白酶抑制剂-1（TIMP-1）表达的影响,探讨博莱霉素引起肺纤维化的机制。体外培养肺间质成纤维细胞,并向培养基中加入博莱霉素,在作用不同时间后收集样本,采用酶谱图测定细胞培养上清液中MMP-2酶活性、ELISA测定TIMP-1量,免疫组织化学法检测细胞中MMP-2、TIMP-1的原位表达,RT-PCR法检测MMP-2和TIMP-1的mRNA水平。结果发现,博莱霉素在2h、12h促进MMP-2的分泌,24h后无促分泌作用;而2-48h,MMP-2的原位表达及mRNA均不受博莱霉素的影响;博莱霉素从12h开始促进TIMP-1及mRNA的表达,并持续至48h。结果表明博莱霉素可引起肺间质戍纤维细胞MMP-2／TIMP-1表达失衡,并可能参与肺纤维化的发生。     

Copper decreases gene expression of TNF-<Emphasis Type="Italic">α</Emphasis>, IL-10, and of matrix metalloproteinases MMP-2 and MMP-9 in isolated perfused rat livers

Copper is known to induce oxidative stress in a number of models. It was shown that many pathophysiological events were associated with oxidative stress. Further, oxidative stress can increase gene expression of cytokines and of metalloproteinases. We previously found that copper toxic effects in isolated perfused rat livers were associated with significant oxidative stress (as assessed by lipid peroxidation, protein oxidation and oxidative DNA damage, particularly at concentration of 0.03 mM of Cu²⁺ in the perfusate). Here we investigated gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10); matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in frozen liver tissue samples by the real-time PCR assay. Compared to controls, copper at concentration of 0.01 mM did not affect gene expression of TNF-α, IL-10, MMP-2 and MMP-9, whereas copper at concentration of 0.03 mM significantly decreased gene expression of all the four TNF-α, IL-10, MMP-2 and MMP-9 by 69%, 81%, 43%, and 62%, respectively. These results suggest that copper-induced oxidative stress in the isolated rat liver can lead to the suppression of gene expression. Because TNF-α and metalloproteinases are involved also in liver regeneration, the suppression of these genes by copper may be one of the mechanisms by which acute intoxication of animals and humans with copper may impair regenerative capability of the liver.     

转录因子Ets-1与MMP-9在胃癌中的表达及临床意义

目的观察转录因子Ets-1和MMP-9在胃癌组织中的表达;探讨两者在胃癌浸润转移中的作用、相互关系及意义。方法采用免疫组织化学S-P法检测97例胃癌及癌旁组织、28例正常胃黏膜组织中Ets-1和MMP-9的表达。结果胃癌组织中Ets-1和MMP-9的阳性表达率分别为74.2%(72/97)、75.3%(73/97),均明显高于癌旁组织(40.2%、18.6%)及正常胃黏膜组织(17.9%、14.3%)(P0.01);而在癌旁组织及正常胃黏膜组织中两者的表达差异无显著性(P0.05)。Ets-1和MMP-9的阳性表达率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性(P0.01)。Ets-1和MMP-9的高表达与胃癌浸润深度、淋巴结转移及临床分期有关(P0.01),与性别、年龄、肿瘤大小及肿瘤位置均无关(P0.05)。Ets-1和MMP-9的表达成正相关(r=0.700,P0.01)。结论Ets-1和MMP-9高表达与胃癌的浸润、转移密切相关;通过上调MMP-9的表达,可能是Ets-1促进胃癌浸润转移的机制之一。