Effect Of AGEs On MMP-9 Expression/Activity And Preliminary Study Of Underlying Mechanism

IntroductionUnder the hyperglycemic condition, macromolecules can be induced into advanced glycation end products, which deposit in the vascular wall. Recent studies indicate that AGE plays an important role in atherogenesis. However, studies on the relationship between AGE and metalloproteinase are quite few.In this study, we observed the morphological changes of mouse peritoneal macrophages in AGE conditional medium, studied the activity/ expression changes of macrophage, and explored the relationship between AGE - induced MMP activation and plaque rupture. Furthermore, we ascertained the role of isopre-noid pathway by using isoprenoid pathway inhibitor ( Statins) and its intermediate products ( GGPP).MethodsAfter preparing BSA - AGE via incubation, fluorospectrophotometry and SDS - PAGE were applied for identification. Simvastatin cytotoxicity was excluded by MTT method. Mouse peritoneal macrophages were incubated with BSA -AGE(400 mg/L)and then morphological changes were observed. Macrophages were incubated with BSA -AGE at different levels ( 0,50,100,.200,400 mg/ L)and for different intervals (0,12, 24, 36,48 h);furthermore, different levels of Simvastatin ( 5μmol/L, 50μxmol/L) were applied to observe the inhibitory effect and then GGPP(10μmol/L)was added to determine the reversal effect on Simvatatin - induced inhibition . After collecting the cell culture supernatant, MMP - 9 activity was determined by Gelatin Zymography and the expression byWestern - blot. The intensity of immunoreactive bands was determined by scanning PVDF film and measuring the optical density as arbitrary units of integrated density value (IDV). All the data were analyzed by SPSS software 11.5.ResultsAccording to fluorospectrophotometry scanning, our incubation product showed the specific double - peak of AGEs. SDS - PAGE indicated that the e-lectrophoresis rate changed as expected. MTT test excluded the possibility of Statin cytotoxicity in the MMP -9 inhibition. AGE - BSA induced morphological changes of macrophage in vitro. After treatment of AGE - BSA(0, 50, 100, 200, 400 mg/L)for48 hours, macrophage MMP-9 increased significantly in contrast to the control, showing the dose - dependent effect (n=5, P<0.05). After treatment of 400 mg/L AGE - BSA for different duration, MMP - 9 activity presented significant difference in contrast to the control, showing the time dependent effect (n =5, P <0. 05). Simvastain decreased MMP -9 activity significantly, which was reversed by GGPP.DiscussionChronic hyperglycemia can induce protein non - enzymatic glycation to form advance glycation end products, which deposit in the vascular wall. On the other hand, AGEs can produce various pathogenic effects via AGE - Rage signal pathway. In this study, BSA - AGE was with the actual level of pathological condition in human body, namely, it doesn't exceed 400 mg/L.Metalloproteinases play an important role in atherogenesis by degrading extracellular matrix. Recently, MMP - 9 becomes a hot issue in the aspect and is regarded as a crucial culminant in atherogenesis. Meanwhile, advanced glycation end product is preferentially distributed in rupture - prone area. Our experiment indicated that advanced glycation end product can activate macrophage to secrete MMP -9 in time and dose dependent pattern. It further proved that AGE - induced MMP -9 is involved in diabetic atherogenesis.Previous reports indicate that small GTPase is at the center of AGE - mediated signal transduction. Cytoplasmic small GTPase, an inactive molecule, should be acylated in order to be anchored to cell membrane, which is closely related to isoprenoid. In this study, we proved that Simvastatin (inhibitor of isoprenoid pathway ) inhibited the AGE - induced MMP - 9 activity/expression and GGPP (isoprenoid) reversed this inhibitory effect of statins, indicating the crucial role of isoprenoid pathway in the AGE -induced MMP -9 expression.In all, advanced end product can activate macrophages and enhance MMP -9 activity, which underlies the pathogenesis of diabetic complication. It also suggests inhibition of AGE production is a new therapeutic target of diabetic atherosclerosis. Our results suggest AGE - induced MMP - 9 expression is related isoprenoid pathway, but the detailed mechanism should be studied further.Conclusions1. BSA - AGE can enhance macrophage MMP - 9 activity and expression in dose and time dependent pattern.2. Simvastatin inhibits AGE - induced macrophage MMP -9 and isoprenoid intermediate (GGPP) reverses the statins'inhibitory effect, indicating that BSA- AGE induced MMP - 9 secretion is related with isoprenoid pathway.