Complement-Induced Endothelial Cell Activation by a Novel Rab5-ZFYVE21-SMURF2 Signaling Axis

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PROJECT SUMMARY Transplant arteriosclerosis (TA) is a vaso-occlusive condition characterized by the formation of stenotic lesions in arterial and microvascular beds of solid organ allografts that cause ischemic graft loss. Complement (C’) are immune proteins involved in host defense that become pathologically activated on endothelial cells (ECs) during TA. In TA, complement becomes activated by recipient-derived alloantibodies binding to donor ECs. Upon activation complement proteins self-assemble to form membrane attack complexes (MAC), heterodimers that insert into target EC surfaces as transmembranous pores. The pathologic effects of complement activation including assembly on ECs is well established, however the precise molecular basis underlying these effects remains unclear, largely due to the lack of relevant experimental models for studying MAC. To obviate this, we developed models for antibody-mediated C’ activation on ECs in vitro and in vivo. Our protocols incorporated human biospecimens in order to recapitulate TA phenotypes in patients. Using these protocols, we defined a novel signaling mechanism causing EC activation and EC-mediated immune responses promoting TA. We have since built a collaborative infrastructure to obtain myriad patient biospecimens to support our work. These patient specimens include ‘high’ panel reactive antibody sera, fresh human coronary arteries, sera and PBMCs from prospectively enrolled patients, healthy PBMCs, TA biopsies, and biopsies from patients with various complement-mediated conditions involving vascular inflammation. Collection of these specimens involved collaborative interactions with the Tissue Typing Lab, Dept of Cardiothoracic Surgery, Dept of Transplant Surgery, Apheresis Service, and various Depts of Pathology. Due to the broad and extensive use of the materials above, my laboratory is exquisitely dependent on their ongoing collection for productivity. In this supplement, we propose a strategy to offset potential losses of productivity due to a critical life event, i.e., the diagnosis of metastatic pancreatic adenocarcinoma in my father. Supplemental funding will be used to hire a research technician who will possess the requisite skillsets and availability on nights and weekends to collect patient specimens. A plan for hiring, training, evaluating, and mentoring this individual is provided along with descriptions of current lab productivity, the critical life event, and a planned return to full productivity at the end of the supplemental period. Use of supplemental funding will occur contemporaneously with various mechanisms intended to maintain productivity both during the supplemental period and include institutional commitments for FMLA, benefits, protected time, colleague support, and wellness resources.