Human Endothelial Cells Generated In Vitro from Circulating Progenitors Represent a Safe Tool for Targeting of Myocardial Tissue.

Various cell sources, culture protocols and characerization steps are being used in the research field of endothelial progenitors and circulating endothelial cells. We generated EPC by plating the mononuclear fraction onto a collagen I-coated well of a six-well-plate in endothelial cell medium. After 6 to 12 days, 1–10 clones with cobblestone morphology appeared and grew rapidly to confluency within a few days. The rapid proliferation of EPC decelerated after 30–40 days, remaining on the same level for additional 30 days and finally stopped after 70–80 days of culture. Immunofluorescence staining and FACS analysis revealed an endothelial phenotype: EC stained positive for vWF, CD31, CD34, KDR, CD105, CD146, bound UEA-1 lectin and took up acetylated LDL. Of note, CD14 and CD45, which are surrogate markers of monocytes and leukocytes, respectively were negative. The expression of ICAM-1 was upregulated by incubation with TNF-a. In addition, EC formed tubes on Matrigel and expressed VEGF-R1 as well as VEGF-R2-mRNA proven by RT-PCR. To gain deeper insight into the regulation of cellular senescence, we performed western blot analyses of cell cycle regulating proteins. In contrast to early passage cells, growth arrested cells from late passages up-regulated p15, p16, p21, whereas p27 and Id1 were significantly dowregulated. Moreover, late-passage cells highly expressed SA-ß-galactosidase. The obvious replicative senescence together with the regular karyogram indicates a relative safety aspect of their application in vivo. To study their homing behaviour to ischemic tissue, we established a model of myocardial infarction, which was induced by ligation of the left anterior descending coronary artery in Rowett nude rats. 5–10x10⁶ PKH red-labeled EPC were injected into the tail vein. Intramyocardial injection served as positive control, while administation of EPC without prior infarction represented the negative control. Rats were sacrificed on day 14 after the operation and cryosections of their hearts were prepared. PKH-positive cells were almost exclusively found in the infarcted left ventricle after i.v.-application, thus showing a high specifity for ischemic myocardium. The results were confirmed by immunohistochemical staining of human CD31.

In summary, EPC generated according to the described protocol represent a possible tool for safe cell-based therapy strategies.