Immortalized bone-marrow derived pig endothelial cells

Primary cultures of porcine endothelial cells (EC) can only be maintained f or a limited number of passages. To facilitate studies of xenogeneic human anti-pig immune responses in vitro, pig microvascular bone-marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred min iature: swine, homozygous for the major histocompatibility locus, and immor talized with a modified SV40 large T vector. The resulting BM-derived (2A2) and aortic (PEDSV. 15) immortalized EC lines showed unlimited growth and E C phenotype as indicated by expression of von Willebrand Faster (VWF) and l ow density lipoprotein (LDL) receptors as well as by formation:of typical c obblestone monolayers. Ultrastructural studies revealed morphological simil arities in primary and immortalized EC. Flow cytometry analysis demonstrate d constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFN gamma. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalize d aortic EC but not in 2A2. Both EC lines expressed a number of myeloid mar kers, adhesion molecules and xenoantigens, the latter being determined by b inding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium-phosphate transfe ction, as well as by adenoviral infection. Finally, the functional similari ty between primary and immortalized EC was demonstrated in adhesion and cyt otoxicity assays. Together, these results suggest that 2A2 and PEDSV.15 rep resent valuable tools to study both human cellular and humoral immune respo nses in vitro against pig EC derived from microvascular and large vessels.