PRODUCTION AND CHARACTERIZATION OF SPONTANEOUS RAT-HEART ENDOTHELIAL-CELL LINES

Endothelial cells (EC) are important regulatory cells in physiology an d pathology, in vitro studies with rat EC from heart tissue are hamper ed by laborious isolation and purification procedures, low yield, and limited lifespan of the cells. Therefore, it is essential to obtain lo ng-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 a llow 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 se ries of phenotypic and functional properties representative of EC in t he 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 stabl e 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 b ut no MHC class II. Intercellular adhesion molecule 1 was only express ed by RHEC-3. Cytokine stimulation induced vascular cell adhesion mole cule 1 in RHEC-3 and RHEC-11 as well as MHC class II in all three line s in different quantities. The phenotypic characteristics of the diffe rent RHEC lines resembled the myocardial microvascular endothelium in situ. The three lines expressed angiotensin-converting enzyme, and the y responded to histamine and ATP but not to thrombin and bradykinin. T hey constitutively produced small amounts of endothelin and high level s of tissue plasminogen activator; they produced little (after stimula tion with phorbol-ester PMA) or no von Willebrand factor. The RHEC lin es produced thromboxane A2 but no prostacyclin; on stimulation with ar achidonic acid and A23187, they produced prostaglandin E2. Therefore, we conclude the following. 1) The described isolation and culture tech nique is successful for production of spontaneous stable EC lines from rat heart. 2) RHEC-3, -10, and -11 can be considered a series of diff erent lines representative of the heterogeneity of heart microvascular endothelium in vivo. 3) The RHEC lines offer a series of valuable too ls to study various heart EC functions and mechanisms in physiology an d pathology.