P317	Regulation of tissue inhibitor of metalloproteinases (TIMP)-1 expression by parathyroid hormone-related protein (PTHrP) in microvascular endothelial cells.
C.Conzelmann, K.Weber, K-D.Schlüter
Justus-Liebig-Universität, Gießen, DE.

Objective: Parathyroid hormone-related protein (PTHrP) is constantly expressed in microvascular endothelial cells from which it can be released in a mechano-sensitive way. In addition, PTHrP can enter the nucleus of endothelial cells and contribute to apoptotic resistance by induction of bcl-2 expression. The present study was performed to identify other genes regulated by PTHrP. Methods: Microvascular endothelial cells were isolated from the hearts of adult rats. PTHrP expression was down-regulated by transformation of endothelial cells with antisense oligonucleotides directed against PTHrP. Expression profiles of endothelial cells transformed with either sense or antisense oligonucleotides were analyzed by gene array analysis. Regulation of genes of interest was confirmed via real time RT-PCR and immunoblot. Results: Using a gene array representing 1176 rat genes we identified TIMP-1 as a PTHrP-dependent regulated gene and, to a lesser extent, also TIMP-2. Endothelial cells stably expressed TIMP-1 and TIMP-2 with a gradual increase in TIMP-1 expression at later time points of culture when the cells were getting confluent and PTHrP enters the nucleus. Transformation of endothelial cells with antisense oligonucleotides produced an approximately 30% decrease in the expression of PTHrP, TIMP-1, and TIMP-2. Down-regulation of TIMP-1 was confirmed via immunoblotting of the protein. TGF-β-1, known to reduce PTHrP expression, mimicked the effect of PTHrP down-regulation by antisense technology. TIMP-1 and TIMP-2 were down-regulated by TGF-β-1 in a concentration-dependent way (1 and 10 ng/ml). Stimulation of α-adrenoceptors, known to increase PTHrP expression in endothelial cells, increased the expression of TIMP-1, but not that of TIMP-2. Co-transformation of endothelial cells with antisense oligonucletides directed against PTHrP significantly abolished the effect of α-adrenoceptor stimulation on TIMP-1 expression. Conclusions: A strict correlation between PTHrP expression and TIMP-1 expression was found in microvascular endothelial cells. In addition to the formerly shown anti-apoptotic effect of endogenously expressed PTHrP in endothelial cells, PTHrP may further stabilize vessels by regulating TIMP-1 expression. Under pathophysiological conditions, characterized by induction of TGF-β-1, down-regulation of PTHrP may contribute to the destabilization of vessels vice versa.

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