Sylvester M. Black

Inhibition of the membrane attack complex of complement for induction of accommodation in the hamster‐to‐rat heart transplant model

Abstract:  Background:  To induce accommodation in the hamster‐to‐rat cardiac transplantation model, in addition to cyclosporin A (CSA) to inhibit T‐cell‐mediated graft rejection, cobra venom factor (CVF) is often used to prevent complement‐mediated graft rejection. Although it is generally assumed that CVF makes accommodation possible because it inactivates the complement membrane attack complex (MAC), it is not known which complement components must be inactivated and whether complement activation products generated by CVF are also involved in the induction of accommodation. Therefore, to investigate mechanisms by which CVF contributes to accommodation, we studied induction of accommodation of hamster hearts grafted into rats with complement deficiencies of C6; these rats cannot assemble the MAC but, in contrast to CVF, retain in their native state all complement proteins that precede the MAC.

Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-α. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis

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.

Protection of porcine endothelial cells against apoptosis with interleukin‐4

Black SM, Benson BA, Idossa D, Vercellotti GM, Dalmasso AP. Protection of porcine endothelial cells against apoptosis with interleukin‐4. Xenotransplantation 2011; 18: 343–354.

Interleukin-4 induces lipogenesis in porcine endothelial cells, which in turn is critical for induction of protection against complement-mediated injury.

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.

Open cardiac repair under direct vision: F. John Lewis and the University of Minnesota.

It was against this background of daunting skepticism, voiced by surgical giants of the 19th century, that the field of heart surgery had its beginnings. Cardiac surgery is one of the youngest of the surgical subspecialties. Terms that are now commonplace such as LVAD, CABG, artificial heart valves, cardiac transplantation, and the total artificial heart were inconceivable to the early pioneers of surgery. Entering the chest was often a treacherous venture, let alone putting stitches into the heart muscle. The earliest attempts at “cardiac surgery” essentially involved cardiac suture for penetrating trauma. The first clinically successful cardiac operation occurred in 1896 when Ludwig Rehn of Frankfurt placed three interrupted sutures in a 1.5-cm right ventricular wound in a 22 year-old-male who had been stabbed in

C. Walton Lillehei and the birth of open heart surgery.

Clarence Walton Lillehei was born in Minneapolis, Minnesota, in 1918. Lillehei graduated from the University of Minnesota in 1939 with a B.S. and proceeded to complete 2 years of medical studies. With World War II interrupting his career, Lillehei served as commanding officer of the 33rd field Mash unit of the U.S. Army Medical Corps. He was awarded the European Theatre Ribbon with five battle stars, the Bronze Arrowhead Award, and the Bronze Star for service in Italy. Upon returning to the United States, he completed surgical residency and doctoral studies at the University of Minnesota (1946 to 1950). Lillehei’s doctoral thesis, titled Role of Cardiovascular Stress in the Pathogenesis of Endocarditis and Glomerulonephritis,1 guided him in his career. In Lillehei’s study, he created large arteriovenous fistulae in canines. The canines developed endocarditis with resultant valvular vegetations. This study gave insight as to why patients with congenital heart defects, such as patent ductus arteriosus and ventricular septal defects, develop bacterial endocarditis. More importantly, Lillehei’s work underscored the importance of early correction of these defects. Lillehei later (1951) received The Theobald Smith Award in Medical Sciences from the American Medical Association for the Advancement of Science. One day after completing surgical residency (February 1950) Lillehei discovered a growth in his neck. He underwent a 12-hour operation including radical neck dissection, mediastinal exploration, and left parotidectomy for what would later be diagnosed as a lymphosarcoma. The surgical innovations of the 1950s proved crucial to the further development of what was then the fledgling field of cardiac surgery. The operating rooms of the early 1950s were without the technologies that