Production and characterization of spontaneous rat heart endothelial cell lines

Endothelial cells (EC) are important regulatory cells in physiology and pathology. in vitro studies with rat EC from heart tissue are hampered by laborious isolation and purification procedures, low yield, and limited lifespan of the cells. Therefore, it is essential to obtain long-term heart EC lines that offer a more adequate in vitro system for studying rat heart EC. An ex vivo perfusion model was used to isolate EC from rat heart. Isolation and culture conditions were modified to allow spontaneous development of immortalized rat heart EC (RHEC) lines. Produced cell lines were tested for endothelial nature using a panel of markers. The selected RHEC lines were subsequently tested for a series of phenotypic and functional properties representative of EC in the context of physiologic and inflammatory functions in vivo. A series of three spontaneous RHEC lines was produced from 13 isolations from Lewis rat hearts: RHEC-3, RHEC-10, and RHEC-11. These lines were stable for more than 30 passages (RHEC-3 for more than 100). The cell lines were tumorigenic and developed hemangiomas on in vivo injection. All three lines expressed major histocompatibility complex (MHC) class I but no MHC class II. Intercellular adhesion molecule 1 was only expressed by RHEC-3. Cytokine stimulation induced vascular cell adhesion molecule 1 in RHEC-3 and RHEC-11 as well as MHC class II in all three lines in different quantities. The phenotypic characteristics of the different RHEC lines resembled the myocardial microvascular endothelium in situ. The three lines expressed angiotensin-converting enzyme, and they responded to histamine and ATP but not to thrombin and bradykinin. They constitutively produced small amounts of endothelin and high levels of tissue plasminogen activator; they produced little (after stimulation with phorbol-ester PMA) or no von Willebrand factor. The RHEC lines produced thromboxane A2 but no prostacyclin; on stimulation with arachidonic acid and A23187, they produced prostaglandin E2. Therefore, we conclude the following. 1) The described isolation and culture technique is successful for production of spontaneous stable EC lines from rat heart. 2) RHEC-3, -10, and -11 can be considered a series of different lines representative of the heterogeneity of heart microvascular endothelium in vivo. 3) The RHEC lines offer a series of valuable tools to study various heart EC functions and mechanisms in physiology and pathology.