O212	The angiogenic factor CCN1 promotes progenitor cell adhesion and migration: potential role in cardiovascular regeneration.
K.Grote, G.Salguero, M.Dangers, M.Ballmaier, B.Schieffer
Medizinische Hochschule Hannover, Hannover, DE.

Introduction: We recently showed that the expression of the angiogenic factor CCN1 is dramatically up-regulated under pathophysiological conditions, e.g. myocardial infarction, pressure overload, vascular strain and in atherosclerotic plaques. CCN1 is described as an extracellular matrix binding protein promoting proliferation, adhesion, migration and neovascularization through cell type-specific binding to different integrins on mainly vascular cells. Since hematopoietic progenitor cells (HPCs) are involved in cardiovascular tissue repair we hypothesized that CCN1 promotes tissue regenerative effects involving adhesion and migration of HPCs. Methods and Results: The angiogenic potency of recombinant CCN1 synthesized in the Baculovirus expression system was demonstrated in Matrigel assays. CCN1 stimulated both initiation and continuity of tubular network formation by endothelial cells (P<0.005, n=4). Additionally, CCN1 induced the proliferation of endothelial cells (1.9±0.11-fold, P<0.05, n=4) as measured by BrdU incorporation. The expression of CCN1 specific integrins (e.g. α5 and β2) on CD34+ HPCs from the peripheral blood of G-CSF treated normal donors was demonstrated by PCR and flow cytometry. CCN1 (100 ng/ml) promoted both the cell-matrix adhesion of HPCs (2.5±0.57-fold, P<0.005, n=4) and the cell-cell adhesion of HPCs to an endothelial monolayer (1.5±0.02-fold, P<0.05, n=4) which could be blocked by integrin specific RGD peptide antagonists. Moreover, CCN1 induced the migration of HPCs (8.1±1.2-fold, P<0.005, n=4) using permeable transwell cell culture inserts (5 µm pore size). Furthermore, CCN1 stimulation of HPCs led to a significant release of pro-angiogenic factors (e.g. G-CSF, GM-CSF, CCL2 and TGF-β1) after 24 h as determined by protein array. Zymography analysis revealed a CCN1 induced increase in MMP-9 release from HPCs (2.5±0.4-fold, P<0.05, n=6) at 24 h. Conclusion: CCN1 stimulates not only endothelial tube formation and proliferation but in addition HPC adhesion, migration and their release of pro-angiogenic factors. We recently showed that CCN1 is up-regulated under various pathophysiological conditions pointing to a potential role for CCN1 in cardiovascular tissue regeneration.

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