Rat heart-derived endothelial and smooth muscle cell cultures: Isolation, cloning and characterization

Abstract

This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro- and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (>P 10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered ‘hill and valley’ pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (<P 10) and late (>P 10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (>P 10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.