Abstract 332: Circulating CCN1 Promotes Angiogenesis via Interaction with Blood CD34+ Cells
Introduction: CCN1 is so far known as a protein of the extracellular matrix mediating proliferation, adhesion, migration and angiogenesis in an integrin-dependent manner. We recently reported that CCN1 is up-regulated under pathophysiological conditions in the cardiovascular system. Since blood CD34+ progenitor cells have been shown to promote tissue regeneration, we investigated if CCN1 induces angiogenesis by stimulating blood CD34+ cells.
Methods and Results: Circulating CCN1 was detected for the first time in human serum samples (Western blot). Rekombinant CCN1 (100 ng/mL) and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted tube-formation (Matrigel-Assay; P < 0.05; n = 4) and proliferation of endothelial cells (BrdU-incorporation; P < 0.05; n = 4). Moreover, CCN1 significantly induced angiogenesis in the context of blood CD34+ cells in vivo determined in Matrigel implanted into C57/BL6 mice as measured by the hemoglobin content (Drabkin’s reagent) and by CD31 staining (immunohistochemistry). In addition, CCN1 induced migration (Transwell cell culture inserts; 7.3 ± 1.3; P < 0.05; n = 4), transendothelial invasion (Transwell cell culture inserts; 2.3 ± 0.7; P < 0.05; n = 3) of CD34+ progenitor cells and release of various growth factors, chemokines (protein array; e.g. G-CSF, GM-CSF, CCL2, TGF-β1) and matrix metalloproteinase-9 (zymography; 2.5 ± 0.5-fold; P < 0.05; n = 6) from these cells. CD34+ progenitor cells expressed the CCN1-specific integrins αMβ2 and αVβ3 (flow cytometry) and binding of CCN1 to CD34+ progenitor cells was diminished by integrin antagonizing RGD peptides. Furthermore, CCN1 stimulated integrin-dependent signaling in CD34+ progenitor cells as shown by enhanced phosphorylation of focal adhesion kinase (immunofluorescence) and promoted their adhesion to endothelial cells which could be inhibited with RGD peptides (P < 0.01; n = 4).
Conclusion: Circulating CCN1 binds integrin-specific to CD34+ blood cells and thereby induces migration, adhesion and the release of chemokines and MMPs. Moreover, CCN1-dependent stimulation of blood CD34+ cells promotes endothelial cell proliferation and angiogenesis known to be important for tissue regeneration.