P313	Positive feedback mechanism between extracellular matrix metalloproteinase inducer (EMMPRIN) and metalloproteinases – implication in the development of plaque instability.
1M.Bachem, 1V.Kreutle, 1F.Diaz, 1S.Zhou, 1Chr.Lenz, 2Ph.Lepper, 1X.Zhang, 1C.Haug
1University Hospital, Clinical Chemistry, Ulm, DE; 2University Hospital, Cardiology, Ulm, DE.

Objectives: Macrophages (Ma) within atherosclerotic lesions show an increased expression of the cell membrane glycoprotein EMMPRIN (extracellular matrix metalloproteinase inducer). Because EMMPRIN (EMM) induces the synthesis of metalloproteinases (MMPs), EMM expression might propagate the development of plaque instability. Here we studied the regulation of EMM expression in Ma and its role in the induction of MMPs in Ma and smooth muscle cells (SMC). Methods: The influence of growth factors and cytokines (IL-1ß, IL-6, IL-8, IL-10, TGFß1, TGFalpha, TNFalpha, FGF-2, PDGF, MCP-1) on EMM expression in cultured human macrophages was studied by fluorescence microscopy, Western blot, fluorescence-immunoassay and RT-PCR (LightCycler). Zymography and enzyme-immunoassays were applied to detect and quantitate MMPs. EMM was isolated from supernatants and cell lysates of macrophages by sequential chromatography. In addition recombinant EMM was produced in HEK293 cells. Results: TGFalpha, IL-6, PDGF and FGF-2 added to cultured Ma increased cell associated EMM while TNFalpha, IL-1ß and to a lesser extend TGFß1 reduced cell associated EMM but increased soluble EMM. EMM transcription was not regulated. Decreased cell associated EMM was associated with an TNFalpha and IL-1ß induced increase in MMP-9 and MMP-2. Recombinant MMP-9 and MMP-2, respectively, generated the soluble form of EMM lacking the carboxyl terminus. In the presence of the MMP-inhibitors EDTA and Ilomastat TNFalpha and IL-1ß failed to increase EMM shedding from macrophage cell surface. Addition of soluble (shedded) EMM and isolated EMM from macrophage cell lysate, respectively, increased the synthesis of MMP2 and MMP9 in cultured SMCs. Furthermore, supernatants of IL-1ß and TNFalpha stimulated Ma induced the synthesis of MMP2 and MMP9 in cultured SMCs. EMM immunodepletion in Ma supernatants resulted in a reduced stimulatory effect on MMP synthesis. Conclusion: We propose a model in which soluble EMM shedded from Ma cell surface stimulates the synthesis of MMPs in SMCs and macrophages. MMPs accelerate EMM shedding, hereby increasing soluble EMM. This positive feed back regulation is further amplified in the presence of the inflammatory cytokines IL-1ß and TNFalpha. Increased MMP synthesis induced by EMM should be relevant in the development of plaque instability.

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