Protection of porcine endothelial cells against apoptosis with interleukin-4

Background: Apoptosis is crucial for tissue development and homeostasis, and insufficient apoptosis is pivotal in cancer pathogenesis. Apoptosis may also be important in tissue injury and in this case, it is of interest to induce protection against apoptosis. In organ transplantation, apoptosis has been implicated in acute vascular rejection (AVR); in xenotransplantation, the inducers of apoptosis of relevance in AVR, such as tumor necrosis factor-alpha (TNF-alpha), also cause endothelial cell (EC) activation. We have previously shown that interleukin (IL)-4 and IL-13 induced protection in porcine ECs against activation and apoptosis triggered by TNF-alpha. Now we define signaling processes activated by IL-4 in porcine ECs and mechanisms required for IL-4-induced protection against apoptosis. Methods: Porcine aortic ECs were used as primary cultures or as virus-induced immortalized cells derived from galactosyl transferase-deficient (Gal(-/-)) or wild-type pigs. ECs were stimulated with porcine IL-4, either extrinsically or transduced with recombinant adenovirus (adeno) IL-4, and analyzed using immunoblotting. Apoptosis was induced with TNF-alpha plus cycloheximide and assessed using neutral red uptake or flow cytometry. The role of various signaling proteins in IL-4-induced protection was established using pharmacologic inhibitors and siRNA downregulation of protein expression. \Results: IL-4 induced similar degrees of phosphorylation of STAT6 in all 3 types of ECs, and STAT6 was phosphorylated through Jak3. IL-4 induced phosphorylation of Bad through Jak3. Stimulation of ECs with IL-4 caused protection of ECs against apoptosis with an absolute requirement of Jak3/STAT6 activation and major participation of mammalian target of rapamycin complex 2 (mTORC2), Akt, and extracellular signal-regulated kinase 1/2. IL-4 caused no increase in EC levels of protective proteins hemoxygenase-1, inhibitor of apoptosis protein, heat shock protein 70, Bcl-2, and Bcl-xL. ECs transduced with adenoIL-4 exhibited strong and durable protection from apoptosis. Gal(-/-) ECs were as susceptible to induction of protection with IL-4 as wild-type ECs. Conclusions: IL-4 induces activation of Jak3/STAT6 and phosphorylation of Bad in porcine ECs, ultimately resulting in effective protection of the ECs from apoptosis. Delineation of downstream signals activated by IL-4 that are required for induction of protection suggests possible sites of intervention to design effective therapeutic agents. This is of interest because substances such as IL-4 have pleiotropic effects and cannot be used directly due to potential deleterious effects. Inducing resistance to apoptosis in porcine vascular endothelium may be important to facilitate xenograft survival and accommodation.