Browsing by Author "Chakraborty, Santanu"

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    Asporin function in aortic endothelial cell mineralization and calcification
    (2019-02-14) Chakraborty, Santanu
    Worldwide, calcific aortic valve disease is one of the leading causes 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. Moreover, addition of human recombinant Asporin protein also reduces the mineralization level in cultured adult aortic valve cells with induced osteogenesis. Therefore, we have identified Asporin as a natural anti-calcific molecule with a therapeutic potential in the treatment of human calcific aortic valve disease.
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    Investigation of stress induced Tbx20 function in autophagy in heart with comparative analyses of extracellular matrix remodeling with multiple stress inductions in cultured cardiac cell line
    Chakraborty, Santanu
    Tbx20, a T-box transcription factor, is known to be crucial for cardio genesis and murine models with Tbx20 knockout fail to survive beyond E10.5. Tbx20 mutations correspond with severe congenital heart defects including valvulogenesis and septal defects. 1–3 All T-box proteins have a DNA binding domain. The T-box domain of Tbx20 consists of 180 amino acid residues and Tbx20 has a binding affinity to T/2 site [(5’-…AGGTGTGA…-3’ over the consensus T-site 5’-…TCACACCT…-3’. 25 Tbx20 promotes cardiomyocyte proliferation via the Bmp2/pSmad1/5/8 and PI3K/AKT/GSK3β/β-catenin signaling pathways.4 Tbx20 has also been found to act as a cardio-protectant against oxidative stress and downregulation of Tbx20 has been linked to increased apoptosis in cultured rat cardiomyocytes.5 Further, the cardio-protective role of Tbx20 under ROS and hypoxic conditions in the H9c2 cell line was reported. 6 Tbx20 is known to interact with and induce other transcription factors like Nkx2.5 and Gata4 7,8 which are also important for cardiogenesis, maintaining cardiac homeostasis and promoting the expression of Troponin-I and myosin heavy chain protein. Autophagy is an evolutionarily conserved catabolic phenomenon that recycles cellular components to provide for bioenergetics and fuel for cellular survival under stress conditions. At the basal level, the goal of the autophagic machinery is to maintain cellular and organ homeostasis. This is achieved by providing catabolites like fatty acids and amino acids which in turn serve as substrates for many metabolic processes. The cargo (can be organelles, protein aggregates, lipids, cellular proteins) to be degraded is sent to lysosomes via autophagosomes whereby fusion of lysosomes with the latter forms autolysosomes and the cargo is thereafter degraded by lysosomal hydrolases. Autophagy can also be activated under stress conditions like nutrient scarcity/ caloric restriction, ROS (Reactive Oxygen Species) accumulation, ER (Endoplasmic Reticulum) stress, and mitochondrial damage where autophagy serves as a substrate recycling machinery remove protein aggregates and provides much-needed ATP for the survival of cells. 9–11Autophagy has been known to be a critical factor in the survival of neonates postpartum wherein it was found that Atg5 knockout mice models failed to survive after birth. Furthermore, in mice that survived the brief period of nutrient deprivation postpartum (which is the usual scenario), massive upregulation of cardiac autophagy was observed giving away the importance of autophagy in pro-survival while also bringing cardiac autophagy to the centre.12 Impaired and altered autophagy has been an underlying cause of cardiac diseases like AMI (acute myocardial infarction), Ischemic heart disease (IHD) and cardiomyopathy. A similar role of autophagy has been found in acute myocardial infarction wherein inhibition of autophagy enlarges the infarct zone and decreases the ATP content of cardiomyocytes and there have been theories that augmenting autophagy would be a therapeutic approach in restoring the cellular integrity and cardiac functioning in these case13–17 Aging is perhaps one of the greatest risk factors responsible for failing hearts. In fact, aged individuals with no underlying cardiac condition show poor cardiac functionality, diastolic function and left ventricular dilatation. 18–20Accumulation of protein aggregates, misfolded proteins, a poor balance between ROS and anti-oxidants, mitochondrial derangements, attenuated expression of Sirtuins (a class of NAD+ dependent deacetylating enzymes) especially Sirtuin 1, 3 (Sirt 1,3), GSk-3 contribute to cardiac aging.21–23 As such, impaired and poor levels of autophagy are prevalent in aging hearts while apoptotic levels are on a surge. Augmenting autophagy by either calorific restriction or Rapamycin (Rap) administration has shown improvement in cardiac functioning and improved life longevity.24–26 Here in this study for the first time, we demonstrate the role of Tbx20 as a potential candidate to induce anti-senescence-like characteristics in the aging mice population. Autophagy induced expression of Tbx20 activates GSK-3 and transcription factors Nkx2.5, Gata4 and Sirt1 after subjection to starvation (Strv) and rapamycin (Rap) treatment in both in-vivo (BALB/c mice) and in-vitro (H9c2 cell line) model systems. The upregulation of Nkx2.5 and Gata4 following autophagy induction is indicative of progression towards progenitor like cardiomyocyte characteristics, while activation of Sirt1 and GSK3 suggests an anti-aging/ senescence since Sirtuin1 is closely linked with aging, is known to be a mediator of caloric restriction and Sirt1 transgenic mice prevent early mortality. With pre-existing knowledge of the expression of Sirt1 and GSK-3 under autophagy conditions along with the cardioprotective roles they play, these two were chosen as possible candidates that could interact with Tbx20. Further, Tbx20 loss of function (LOF) assay in the H9c2 cell line validated Tbx20-dependent expression of Sirt1, GSK-3, Nkx2.5 and Gata4. On the other hand, ECM remodeling in heart or cardiac remodeling remains an important factor in the pathophysiology of cardiovascular diseases.27,28 Collagen-I forms the major component of the matrix interstitium of the myocardium in addition to Collagen-III, fibronectin, proteoglycans, tissue inhibitors of matrix metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). The three stages of cardiac remodeling following cardiac injury are inflammatory, proliferative and maturation phases leading to a mature scar formation.29 The preliminary stages of ECM remodeling are necessary as it prevents rupture of the ventricular wall, however, exacerbated ECM remodeling leads to progressive fibrosis in the heart and cardiac malfunctioning.30,31 The MMPs (zinc-dependent proteases) are involved in the turnover of matrix proteins like Collagen.32 Adamts4, a member of Adamts family is an important MMP. Adamts4, also is a disintegrin with thrombospondin like motifs.33,34 The mode of action of Adamts4 is by binding to ECM proteins and thereafter cleaving ECM proteoglycans like aagrecan, versican, brevican in addition to regulating Collagen turnover.35,36 Adamts4 modulates the pathophysiology of osteoarthritis through degradation of matrix proteoglycans and eventually lead to cartilage degradation which manifests as degenerative osteoarthritis.37,38 Besides osteoarthritis, Adamts4, has also been linked with cancer and angiogenesis where its role remains controversial. Some studies report it to be an indicator of early-stage cancer like in cases of colorectal cancer, others findings suggest that its mutated and truncated fragments may suppress tumour growth through inhibition of angiogenesis. 39,40 However, the involvement of Adamts4 in cardiac remodeling is relatively less known. Only a few studies have shown the involvement of Adamts4 in atherosclerotic plaque development.41 and recent studies have shown elevated expression of both Adamts4 and Adamts1 in patients with acute aortic dissection and coronary artery disease.42,43 To decipher the molecular cascade of Adamts4 induction and associated signaling pathway, cultured H9c2 cells were used for in vitro experiments. Adamts4 expression was induced in H9c2 cells following hypoxia (Hyp) and ROS and Hyperglycaemic stress inductions. Additionally, Adamts4 expression was manipulated by siRNA-mediated loss of function and TGF- inhibitor studies with SB431542/ALKI treatment in-vitro to evaluate the hierarchy and dependency on TGF- signaling. TGF- is a known marker for inflammatory and fibrotic responses following pathological stress like Myocardial Infarction, ischemia and reperfusion (I/R) injury.44–48 Ultimately, ADAMTS4 expression was also assessed in patients with cardiac diseases namely Dilated Cardiomyopathy (DCM) and MI. Overall, this study focusses on the role of Tbx20 under stress conditions and ECM remodeling.
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    Tbx20 function in Proepicardial organ (PEO) & epicardium derived cell differentiation and Asporin /PLAP-1 function in aortic valve calcification
    Chakraborty, Santanu
    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