Shibnath Ghatak, PhD
Research Assistant Professor Department of Medicine and Cell Biology, Medical University of SC
Department of Bioengineering
The primary goal of this pilot seed project is to turn the knowledge of discoveries in mechanisms of pathological responses to injury such as fibrosis into remedial therapies that propose to replace diseased tissues with new progenitor cells from endogenous, homeostatic sources. The working “remedial hypothesis” is that homeostatic, cell engraftment mechanisms exist for carrying remedial genetic “payloads” into the heart of adult mice by cells derived from the bone marrow that can modify adverse cardiac remodeling after injury and stress by blocking or attenuating fibrosis. Based on preliminary results, we will focus on periostin and NADPH oxidase 4 (NOX4) as a prototypic candidates for remediation of fibrosis via bone marrow derived, CD45+ cells that normally traffic to heart and differentiate into myofibroblasts which produce collagen, the key structural protein of fibrous connective tissues including scars. In Aim 1, we will test the remedial hypothesis that fibrosis and scarring can be inhibited by genetically modifying the potential of bone marrow cells that naturally engraft into the walls of the heart to differentiate into myofibroblasts that secrete high levels of collagen, the hallmark of fibrotic tissues. To do so, we propose to employ in vivo strategies that will utilize the Okabe EGFP mouse to trace the fate of marrow derived CD45+ cells in which all cells including those of the bone marrow, stably express EGFP. We will compare two approaches: (1) A direct approach in which CD45+ cells will be isolated from the bone marrow and directly transduced with inhibitory NOX4-shRNA or PN-shRNA “dual vector” nanoparticles, and (2) An indirect approach in which the CD45+ cells will be isolated from the bone marrow that previously had received systemic or marrow injections of the inhibitory NOX4-shRNA or PN-shRNA nanoparticles. Whether transduced directly or indirectly, the isolated “donor” (CD45+/EGFP+ cells) cells will be re-injected into 6-12 week old “recipient” wildtype mice in which fibrosis and scar formation had previously been induced by cryoablation. The advantage of two vector nanoparticle system is that it will suppress periostin or NOX4 only in bone marrow derived cells because only those cells have the potential to activate the Vav-i- cre driver. Resident cells – even if they were previously derived from CD45+ bone marrow - do not appear to retain potential to express Vav-i after their differentiation into fibroblasts 1. Thus, any amelioration of collagen production induced by cryoinjury should reflect the result of silencing periostin or NOX4 specifically in the donor CD45+/EGFP+ cells. Outcomes will be quantitatively assessed for the potential of engrafted CD45+ cells carrying nanoparticle silencing vectors (direct approach) vs. indirect systemic or bone marrow injections of the nanoparticles to inhibit both the secretion and alignment of collagen. If these studies prove to be new immature myocytes as suggested by their preliminary histological studies, we will then test the hypothesis that engrafted CD45+ cells differentiate into a myogenic lineage (vs. a fibroblastic lineage) when their PN and NOX4 synthesis was inhibited. These findings would have a strong foundation for testing other potential future regenerative therapies for fibrosis across different organs.