P172	Regulation of Angiopoietin-2 by shear stress involves VEGF.
C.Gryczka, W.Goettsch, H.Morawietz
Department of Vascular Endothelium and Microcirculation, University of Technology Dresden, Dresden, DE.

Background: Local differences in shear stress can modulate the endothelial phenotype. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. However, the regulation of Ang-2, especially in response to different magnitudes of shear stress, is not well understood. Therefore, we analyzed the regulation of Ang-2 by shear stress. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress (1 to 30 dyn/cm2 for up to 24 h) in a cone-and-plate viscometer. The mRNA expression of Ang-2 was determined by RT-PCR, Ang-2 protein expression by Western blot, and Ang-2 peptide release by ELISA. The role of protein kinase C (PKC) was analyzed using PKC inhibitor RO-31-8220. Analysis of Ang-2 promoter deletion constructs was performed using dual luciferase assay. Results: Ang-2 mRNA is 1.6-fold upregulated by low shear stress (1 dyn/cm2, 24 h, n=10), but downregulated to 57±10% by high shear stress (max. 30 dyn/cm2, 24 h, n=16). Ang-2 protein expression and release is induced by long-term (24 h) low shear stress (1 dyn/cm2), but downregulated by high laminar shear stress (30 dyn/cm2) as well. Downregulation of Ang-2 by high shear stress involves PKC. Furthermore, dose-dependent stimulation with VEGF (max. 50 ng/ml, 24 h) induced Ang-2 mRNA. The upregulation of Ang-2 by VEGF was inhibited by application of high shear stress (30 dyn/cm2, 24 h) (con: 100±20%, VEGF: 421±41%*, VEGF+shear stress: 227±50%#, *P<0.05 vs. con, #P<0.05 vs. VEGF, n=5). Serial deletions of the angiopoietin-2 promoter (241 to 1938 bp) were cloned into the pGL3-basic luciferase reporter gene vector. After transfection in HUVEC, human umbilical artery endothelial cells, human microvascular endothelial cells (HMEC-1) or HEK293 cells, all constructs show similar basal expression level. Initial experiments using the 985 bp-Ang-2 promoter fragment do not support a downregulation of Ang-2 on the transcriptional level by high shear stress. Conclusion: Low levels of shear stress causes upregulation, but high shear stress downregulation of Ang-2 in human endothelial cells. Our data suggest a role of PKC and VEGF in shear stress-dependent regulation of Ang-2. These data support a dose-dependent regulation of genes involved in the differentiation of endothelial cells by shear stress.

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