We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection. IntroductionThe vascular endothelium has critically important physiologic functions [1] and, if injured, pathologic states such as atherosclerosis, ischemia reperfusion, and vasculopathy, as observed in vascularized allo-and xenograft rejection, may result. The vascular endothelium is directly exposed to complement, complement activation may take place in blood secondary to various physiologic and pathologic events, and activated complement may influence vascular endothelial physiology [2,3]. Among products derived from complement activation, the membrane attack complex (MAC) of human complement is known to have multiple effects on the vascular endothelium and the vasculature as a whole, including activation, production of inflammatory cytokines, expression of adhesion molecules, loss of endothelial anticoagulant properties, gain of procoagulant properties, cell retraction and detachment from the basement membrane, and cell death [2,3]. Consequently, the MAC may initiate or contribute to ischemia or to acute and chronic inflammation, becoming a major pathophysiologic mediator in vascular diseases, autoimmune diseases, and graft rejection. Cells possess at least three control mechanisms for protection against injury from the MAC. First, the membrane proteins decayaccelerating factor (CD55) and membrane cofactor protein (CD46) inhibit progression of complement activation at the C3 and C5 activation stages, and CD59 inhibits MAC assembly by blocking C9 binding to C5b-8 [4]. Second, complement proteins bound to a cell membrane may be removed from the membrane by exocytosis or endocytosis [5]. Third, nucleated cells may be in...
Interleukin (IL)-4 has been shown to induce protection in porcine vascular endothelial cells (ECs) from killing by human complement. This protection is dependent on the PI3K/Akt signaling pathway. In this study, we investigated mechanisms downstream of Akt and found that activation of the lipid biosynthesis pathway is required for protection from complement in ECs treated with IL-4. Cells incubated with IL-4 for 48 hours contained increased fatty acids and phospholipids but cholesterol was not increased when compared with medium-treated controls. The transcription factor SREBP-1, which regulates fatty acid synthesis, was found to be activated in extracts of ECs incubated with IL-4 for 6 hours. Finally, induction of protection from complement killing with IL-4 was fully prevented by the presence of the SREBP inhibitor 25-OH cholesterol. This study showed that IL-4 induces lipid biosynthesis in porcine ECs through activation of SREBP-1 and that the activation of this pathway is critical for IL-4 to induce protection of porcine ECs from killing by human complement. Further study of these mechanisms may provide new strategies for the prevention of complement-mediated vascular injury as it occurs in xenograft rejection.The vascular endothelium is critically important in physiologic functions such as maintaining an antithrombotic surface to blood flow, providing a selective barrier to plasma proteins, lymphocyte trafficking with the upregulation of adhesion molecules, and regulation of blood flow through control of vasomotor tone. Injury to the vascular endothelium can result in diseases such as atherosclerosis, ischemia-reperfusion injury, and vasculopathy as observed in vascularized allo-and xenograft rejection. Because it is in contact with blood, the vascular endothelium may be exposed to multiple mechanisms of injury including antibody, complement, oxygen radicals, direct cellular mediated cytotoxicity, and pore-forming toxins such as those released by bacteria. A common factor in injury induced by these mechanisms is the loss or disruption in plasma membrane integrity, which, if unchecked, can lead to inflammation, dysfunction, and cell death. Endothelial cells (ECs) possess survival mechanisms that enable limited repair and resistance to injury. Recently, it has been demonstrated that ECs are able to survive after limited exposure to streptolysin-O by resealing the lesions in the plasma membrane. 1,2 It has also been demonstrated that after exposure to bacterial pore-forming toxins such as aerolysin, cells are able to survive by upregulation and activation of lipid biosynthetic pathways, which then allow for membrane synthesis. 3 Antibody activation of the complement cascade plays an important role in the pathology observed in ABO-mismatch allograft and xenograft rejection. The membrane attack complex of complement may create a lesion in the plasma membrane that leads to EC activation, and in some cases ionic imbalance and even cell death. It has been observed that in some cases the METHODSPrimary cult...
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