This is an application for a K23 award for Dr. Enrico Novelli, a Hematologist, Assistant Professor in the Tenure Stream in the Department of Medicine and Director of the University of Pittsburgh Adult Sickle Cell Anemia Program. Dr. Novelli is establishing himself as an academic leader in the field of benign hematology with a specific focus on translational research in sickle cell disease and hemostasis. Dr. Novelli has prior extensive research experiences in hematology and has been successful in obtaining research awards for his studies on SCD. He has assembled a multidisciplinary team of mentors and advisors to guide his career towards independence and to assist with the completion of the research proposed in this application. This grant will be critical to achieve the following career objectives: (1) to develop expertise in laboratory methodologies crucial to the conduct of translational research in SCD; (2) to gain further in-depth knowledge of clinical trial design and biostatistics fundamental in the investigation of human disease; and (3) to become an expert in the vascular biology of SCD. He plans to complement his research activity with the completion of a Master of Science in Clinical Research sponsored by the University of Pittsburgh, with participation in scheduled training activities on grant writing and responsible conduct of research, and with attendance to international meetings in his areas of interest. By the end of the funding period of this K23 award, he will have several first-authored peer-reviewed publications and a critical mass of data that will form the core of an RO1 or other equivalent grant. The focus of this application is on the vascular biology of SCD. In SCD, mutant hemoglobin S polymerizes when deoxygenated, driving red blood cell (RBC)-dependent vaso-occlusion and hemolysis. These processes lead to platelet and hemostatic activation, pulmonary hypertension and vascular disease. Transgenic-knockout sickle (BERK) mice that express exclusively human - and ?S-globins mimic SCD in humans by displaying reduced NO bioavailability, impaired NO-mediated vascular reactivity and pulmonary hypertension. Recently, the platelet -granule protein thrombospondin-1 (TSP1) was found to potently inhibits physiologic NO signaling, via binding to the cell surface receptor CD47. Preliminary data from Dr. Novelli now demonstrate that circulating TSP1 levels are increased in the plasma of patients with SCD. Preclinical studies supporting this proposal also show that the specific TSP1-CD47 ligand receptor interaction not only inhibits NO signaling in endothelial cells, but also increases reactive oxygen species (ROS) production and endothelin A (ETA) receptor expression in smooth muscle, both canonical vasoconstrictive and mitogenic pathological signaling pathways contributing to pulmonary hypertension. Extensive preliminary data using chimera cross transplantation systems confirm that CD47 is critical in the development of PH in the transgenic sickle cell mouse. These findings inform the grant's overarching hypothesis that during platelet activation in patients with SCD, increases in circulating plasma levels of TSP1, via binding to the CD47 receptor, disrupt pulmonary vascular endothelial NO production and stimulate smooth muscle ROS production and endothelin 1 signaling, which lead to pulmonary hypertension in murine models and human subjects with SCD. This grant will also explore the hypothesis that therapeutic disruption of the TSP1-CD47 ligand-receptor interaction will both prevent and reverse pulmonary hypertension in SCD. This hypothesis will be tested via the following aims: (1) to examine for the first time whether plasma from human subjects with SCD TSP1 levels in the plasma of patients with SCD disrupt the vasodilator/vasoconstrictor balance in endothelium and smooth muscle by inhibition of NO signaling and increasing ROS and ETA levels; (2) to employ unique mutant murine models to determine the role of circulating TSP1 and CD47 in SCD-associated vasculopathy and pulmonary hypertension, and test the therapeutic efficacy of blocking TSP1-CD47 to prevent/mitigate SCD-based pulmonary hypertension; and (3) to test TSP1 levels in several large cohorts of patients with SCD and explore the correlation of TSP1 with platelet activation, pulmonary hypertension and vascular disease. This proposal is strengthened by the complementary and rich tool set used to investigate the hypothesis, the vast resources available within the Vascular Medicine Institute, a state of the art facility within the University of Pittsburgh devoted to the study of vascular biology, and captures the pioneering work of Dr. Novelli's mentors in defining hemolysis in the pathophysiology of SCD (Dr. Mark Gladwin's lab) and the TSP1-CD47 nexus as an upstream regulator of NO signaling (Dr. Jeff Isenberg's lab). Definition of this pathway will therefore present a novel therapeutic target for the vascular complications of SCD.
There is a critical need to elucidate the vascular pathology of sickle cell disease so that new therapeutic approaches can be developed for this orphan disease. The K23 funding mechanism will provide the candidate with a unique set of skills and expertise in the vascular biology of sickle cell disease, nitric oxide metabolism, measurement of oxidative stress and translation of research findings from bench to bedside.
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