Tbx20 function in Proepicardial organ (PEO) & epicardium derived cell differentiation and Asporin /PLAP-1 function in aortic valve calcification

Abstract

Nowadays, the problem of cardiovascular diseases is a major burden and a leading causes of morbidity and mortality in humans, globally. A proper understanding of the cellular and molecular mechanisms of heart failure has just started to provide better medical care and patient management. It has now become increasingly clear that cardiomyocyte proliferation, cardiac progenitor cell induction, development and differentiation of several cardiac cell lineages and cardiac extracellular matrix (ECM) remodeling are the dynamic processes, critical for injured myocardial repair. Proepicardial organ (PEO) is an extra cardiac villous protrusion at the venous pole of vertebrate embryonic heart that forms epicardium migrating onto the myocardium. PEO-derived epicardium and epicardium derived cells or EPDCs contribute several cardiac cell lineages including smooth muscle cells (SMs), fibroblasts, endothelial cells and cardiomyocytes (CMs). But in the developing heart, detailed gene expression pattern of several lineage specific markers within PEO progenitor cells and its epicardial derivatives are relatively unknown. Cardiomyocyte differentiation from proepicardial organ (PEO) and embryonic epicardium (eEpi) derived cells or EPDCs in developing heart, emerges a wide interest in purview of cardiac repair and regenerative medicine. Embryonic epicardium (eEpi) originates from the precursor PEO and EPDCs and contributes to several cardiac cell types including smooth muscle cells, fibroblasts, endothelial cells and also cardiomyocytes (CMs) during cardiogenesis. Using avian explant culture system, our data have yielded differential expression of several marker genes in PEO versus epicardial cells. Next, to explore the role of Wnt/β-catenin signaling in PEO and EPDCs, avian E5 epicardial cells were treated with lithium chloride (LiCl), recombinant Wnt3a (rhWnt3a) protein and Xav939. Addition of LiCl and rhWnt3a have inhibited glycogen synthase kinase 3β, stabilizing β-catenin and reversely, addition of Xav939 have inhibited Wnt/ β-catenin signaling in avian explant cultures. Interestingly avian epicardial explant cultures, treated with LiCl and rhWnt3a, show increased mRNA expression of Tbx20 concomitant with induced expression of CM lineage markers and Xav939 treated epicardial explant cultures show decreased expression Tbx20 and CM markers. In addition, Wnt signaling activation also increases the number of proliferating and sarcomeric myosin (Mf20) positive cells in eEpi explant culture. Together, these data suggest, eEpi cells as a source for CM differentiation and Wnt signaling mediator, β-catenin might play an important role in CM differentiation from eEpicells in culture. Overall, aim1of this thesis suggests the importance of Tbx20 viii | P a g e and β-catenin in regulation of CM lineage differentiation from EPDCs. Therefore, our studies will further provide a mechanistic insight into the PEO based therapies for CM support improving therapeutic and regenerative approaches following adult cardiac injury. Likewise, valvular diseases, importantly calcific aortic valve diseases (CAVDs) are also projecting serious concerns throughout the world. CAVDs account for 25% of all types of cardiac disease, causing significant number of morbidity and mortality among patients with cardiac abnormalities. Aortic valve mineralization and calcification are the key events of adult calcific aortic valve disease manifestation and functional insufficiency. Due to heavy mineralization and calcification, adult aortic valvular cusps show disorganized and dispersed stratification concomitant with deposition of calcific nodules with severely compromised adult valve function. Interestingly, shared gene regulatory pathways are identified between bone forming cells and heart valve cells during development. Asporin, a small leucine rich proteoglycan, acts to inhibit mineralization in periodontal ligament cells and is also detected in normal murine adult aortic valve leaflets with unknown function. Therefore, to understand the Asporin function in aortic cusp mineralization and calcification, adult avian aortic valvular interstitial cell culture system is established and osteogenesis has been induced in these cells successfully. Upon induction of osteogenesis, reduced expression of Asporin mRNA and increased expression of bone and osteogenesis markers are detected compared to cells maintained without osteogenic induction. Importantly, treatment with human recombinant Asporin protein reduces the mineralization level in osteogenic media induced aortic valvular interstitial cells with the concomitant decreased level of Wnt/β-catenin signaling. Overall, all these data are highly indicative that Asporin might be a novel bio-molecular target to treat patients of calcific aortic valve disease over current cusp replacement surgery.

Thus, studies of two aims in cardiovascular research area will enrich and reveal new insights which will enhance the ongoing as well as future investigations in cardiac therapeutic field