Cilengitide Inhibits Proliferation and Differentiation of Human Endothelial Progenitor Cells In Vitro.

Abstract

Neo-angiogenesis plays an important role in many diseases such as cancer or rheumatoid arthritis. The existence of CD133+ hemangioblasts with dual differentiation capacity into both hematopoietic and endothelial cells was shown by several groups. Growing evidence indicate contribution of these endothelial progenitor cells (EPCs) to tumor angiogenesis in different preclinical models. Cytokines such as VEGF mobilize EPCs from the bone marrow leading to integration into the growing tumor vasculature. Several local factors including extracellular matrix proteins and cell-cell-interactions via adhesion molecules are responsible for differentiation, homing and incorporation of EPCs into tumor vessels. Our group has developed a cell culture system which allows expansion and endothelial differentiation of human CD133+ precursor cells in vitro. In the present study we analyzed the effect of the integrin inhibitor cilengitide on proliferation and differentiation of EPCs. Cilengitide is a peptide-like molecule, targeting αvβ3- and αvβ5-integrins with high affinity. Inhibition of these integrins by Cilengitide blocked angiogenesis and tumor growth in preclinical models and showed efficiency in clinical investigations. CD133+ cells were isolated from leukapheresis products by an immunomagnetic approach and subsequently cultivated for 28 days using the cytokines VEGF, SCGF and FLT3L. Afterwards EPCs were differentiated into endothelial cells by withdrawal of SCGF and FLT3L for 14 days. Expression of the target molecule αvβ3-integrin on freshly isolated CD133+ cells, 28 days ex-vivo expanded and subsequently differentiated cells was analyzed by immunofluorescence (n=3). αvβ3-integrin was detected only on 2 +/−3% of freshly isolated CD133+ stem cells, but after 4 weeks of expansion culture αvβ3-integrin was expressed on 34+/−4% of cells. When culture conditions were switched to induce endothelial differentiation, almost all adherent cells (98+/−1%) were positive for αvβ3 integrin. Of the non-adherent EPC the percentage of αvβ3-integrin positive cells was 60+/−9%. The influence of Cilengitide on the proliferation of EPCs was analysed by counting triplicates obtained from three individual donors over a period of 9 days with 0.1, 0.5, 1 μM Cilengitide and the appropriate solvent control. We could show a significant dose dependent reduction of cell proliferation in the presence of Cilengitide (p<0.0002 for each timepoint). After withdrawal of FLT3 ligand and SCGF, cell proliferation ceased and cells became adherent and showed endothelial morphology. Immunofluorescence analysis of adherent cells showed coexpression of VE-Cadherin and vWF. To analyze the effect of Cilengitide on the differentiation of EPCs, expanded cells were incubated after withdrawl of FLT3L and SCGF with solvent or 0.1, 0.5, or 1 μM Cilengitide. Adherent cells were counted after 7 and 14 days of differentiation. A dose dependent decrease in the number of adherent cells was noted at both time points (n=3, p< 0.03 for each time point). We then investigated the expression of endothelial antigens on EPCs after withdrawal of SCGF and FLT3L in the presence or absence of Cilengitide. Cilengitide caused a significant decrease in the percentage of VE-Cadherin- and vWF-positive cells at days 7 and 14 in a dose-dependent manner. In summary, we could show that Cilengitide inhibited proliferation and differentiation of EPCs.