Abstract

Endothelial cell replication in large and small vessels is generally thought to occur at a rate of 0.1–0.6% daily. Despite this low level of cell turnover, endothelial cells derived from a variety of bovine vessels display vigorous patterns of proliferation in vitro. This apparent paradox has not been resolved to date. We have recently determined that human endothelial cells are derived through a process of endopoiesis via a hierarchy of endothelial progenitor cells (EPCs) (Blood, 2004). We have developed a single cell proliferation assay that has resolved endopoiesis into distinct stages of progenitor cell development: 1) high proliferative potential-endothelial colony forming cells (HPP-ECFC; 2001-> 10,000 cells/colony) that replate into secondary and tertiary HPP-ECFC, 2) low proliferative potential-endothelial colony forming cells (LPP-ECFC; 51–2,000 cells/colony) that form colonies greater than 50 cells but fail to replate into LPP-ECFC, 3) endothelial clusters (EC-clusters; 2–50 cells/colony) that contain fewer than 50 cells, and 4) mature differentiated endothelial cells that are non-proliferative. We hypothesized that the proliferative behavior of the bovine vessel-derived endothelial cells was due to the presence of EPCs. We purchased bovine aortic endothelial cells (BAEC), bovine pulmonary artery endothelial cells (BPAEC), and bovine coronary artery endothelial cells (BCAEC) from a commercial vendor and cultured the cells as recommended. As predicted, the endothelial cells displayed a cobblestone morphology and ingested acetylated low density lipoprotein consistent with an endothelial phenotype. We initially plated the monolayer of cells of each type at 10, 25, or 100 cells per collagen I coated 6-well tissue culture wells and determined that cells from each artery gave rise to heterogenous colony sizes with different growth potentials during a 7 day culture. We then utilized flow cytometry to single cell sort the endothelial cells of each arterial type and determined the number of cells that divided in a 14 day culture. As depicted in the TABLE, the entire hierarchy of EPCs (similar to that determined for human adult peripheral blood and umbilical cord blood) is present in the endothelial cells isolated from the bovine vessels. Of interest, our preliminary data indicate that the frequency of the most proliferative progenitors (HPP-ECFC) is higher in the BAEC than the BPAEC or BCAEC samples. These data provide a new conceptual framework for understanding the mechanisms of endothelial replacement and/or repair of aged or damaged endothelial cells. While EPCs clearly circulate, they also engraft and reside in the vessel wall. We speculate that it is the presence of these EPCs that accounts for the ability of isolated BAEC, BPAEC, and BCAEC cells to proliferate ex vivo.

Single Cell Sort Colony Distributions

Cell Line BAEC-1 % BAEC-2 % BCAEC % BPAEC % 
Mature EC 31.33 39.33 56.67 53.67 
EC-clusters 2.00 2.33 10.00 5.00 
LPP-ECFC 5.00 9.00 12.00 11.00 
HPP-ECFC 61.67 49.33 21.33 30.33 
Total colonies 68.67 60.67 43.33 46.33 
Cell Line BAEC-1 % BAEC-2 % BCAEC % BPAEC % 
Mature EC 31.33 39.33 56.67 53.67 
EC-clusters 2.00 2.33 10.00 5.00 
LPP-ECFC 5.00 9.00 12.00 11.00 
HPP-ECFC 61.67 49.33 21.33 30.33 
Total colonies 68.67 60.67 43.33 46.33 

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