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A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac... more
A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to elimina...
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Cell therapy for treatment of ischemic heart disease has been under rigorous evaluation in recent years. Heart-derived c-Kit cells showed promising results in clinical trials with scar reduction and improved ejection fraction following... more
Cell therapy for treatment of ischemic heart disease has been under rigorous evaluation in recent years. Heart-derived c-Kit cells showed promising results in clinical trials with scar reduction and improved ejection fraction following coronary infusion. We previously developed a c-Kit lineage tracing model showing that these cells very rarely convert into de novo cardiomyocytes. To potentially reduce this rate to zero, and unequivocally evaluate cellular fusion processes in vivo, we deleted the cardiogenic transcription factors Gata4 and Gata6 in c-Kit cells using a Cre-loxP approach (Kit-Gata4/6 KO). We used the tamoxifen inducible Kit-MerCreMer allele crossed into Gata4/6 homozygous LoxP targeted background and the Rosa26-eGFP reporter, which were given tamoxifen at weaning to delete Gata4 and Gata6 in all c-Kit expressing cells and show them and their progeny as eGFP positive. Unexpectedly, we observed a greater than 10-fold increase in Kit lineage-traced cardiomyocytes in some ...
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The limited ability of mammalian adult cardiomyocytes to proliferate following an injury to the heart, such as myocardial infarction, is a major factor that results in adverse fibrotic and myocardial remodeling that ultimately leads to... more
The limited ability of mammalian adult cardiomyocytes to proliferate following an injury to the heart, such as myocardial infarction, is a major factor that results in adverse fibrotic and myocardial remodeling that ultimately leads to heart failure. The continued high degree of heart failure-associated morbidity and lethality requires the special attention of researchers worldwide to develop efficient therapeutics for cardiac repair. Recently, various strategies and approaches have been developed and tested to extrinsically induce regeneration and restoration of the myocardium after cardiac injury have yielded encouraging results. Nevertheless, these interventions still lack adequate success to be used for clinical interventions. This review highlights and discusses both cell-based and cell-free therapeutic approaches as well as current advancements, major limitations, and future perspectives towards developing an efficient therapeutic method for cardiac repair.
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Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating heart failure given the prominent role that fibrosis plays in this disorder. CFs directly convert to myofibroblasts (MFs) with injury where they mediate both... more
Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating heart failure given the prominent role that fibrosis plays in this disorder. CFs directly convert to myofibroblasts (MFs) with injury where they mediate both adaptive wound healing after acute myocardial infarction as well as long-standing fibrosis during chronic disease states. However, the fate of activated MFs after injury recovery or when an infarction scar is stabilized remains poorly understood, in part because the field has lacked a definitive strategy for identifying and tracing MFs and CFs in vivo. To address this issue we recently generated a novel mouse model that permits lineage tracing of all MFs in the heart after injury or stress stimulation, which we used to address the fate of MFs after injury resolution. MFs were lineage traced with a tamoxifen inducible periostin allele knockin of the MerCreMer cDNA (PostnMCM), with a Rosa26-eGFP dependent reporter. PostnMCM x R26-eGFP mice were trans...
Research Interests: Medicine and Circulation
Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative... more
Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.
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Background Myocardial infarction results in a large‐scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult... more
Background Myocardial infarction results in a large‐scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell–mediated cardiac regeneration, numerous recent studies using advanced lineage‐tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle. Methods and Results In vitro experiments with small interfering RNA –mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from ...
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We have previously demonstrated that iron overload in hepatic reticuloendothelial system cells (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in patients with nonalcoholic fatty liver disease... more
We have previously demonstrated that iron overload in hepatic reticuloendothelial system cells (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Recruited myeloid‐derived macrophages have gained a pivotal position as drivers of NASH progression and fibrosis. In this study, we used bone marrow‐derived macrophages (BMDM) from C57Bl6 mice as surrogates for recruited macrophages and examined the effect of iron on macrophage polarization. Treatment with iron (ferric ammonium citrate, FAC) led to increased expression levels of M1 markers: CCL2, CD14, iNOS, IL‐1β, IL‐6, and TNF‐α; it also increased protein levels of CD68, TNF‐α, IL‐1β, and IL‐6 by flow cytometry. This effect could be reversed by desferrioxamine, an iron chelator. Furthermore, iron loading of macrophages in the presence of IL‐4 led to the down‐regulation of M2 markers: arginase‐1, Mgl‐1, and M2‐specific transcriptional regulator, KLF4. Iron loading of macrophages with IL‐4 also resulted in reduced phosphorylation of STAT6, another transcriptional regulator of M2 activation. Dietary iron overload of C57Bl6 mice led to hepatic macrophage M1 activation. Iron overload also stimulated hepatic fibrogenesis. Histologic analysis revealed that iron overload resulted in steatohepatitis. Furthermore, NAFLD patients with hepatic RES iron deposition had increased hepatic gene expression levels of M1 markers, IL‐6, IL‐1β, and CD40 and reduced gene expression of an M2 marker, TGM2, relative to patients with hepatocellular iron deposition pattern. We conclude that iron disrupts the balance between M1/M2 macrophage polarization and leads to macrophage‐driven inflammation and fibrogenesis in NAFLD.
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Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and previous we have shown that these two effectors are also needed for mitochondrial-dependent cellular necrosis (Karch... more
Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and previous we have shown that these two effectors are also needed for mitochondrial-dependent cellular necrosis (Karch et al., 2013). Here we show that mouse embryonic fibroblasts deficient in Bax/Bak1 are resistant to the third major form of cell death associated with autophagy through a mechanism involving lysosome permeability. Indeed, specifically targeting Bax only to the lysosome restores autophagic cell death in Bax/Bak1 null cells. Moreover, a monomeric-only mutant form of Bax is sufficient to increase lysosomal membrane permeability and restore autophagic cell death in Bax/Bak1 double-deleted mouse embryonic fibroblasts. Finally, increasing lysosomal permeability through a lysomotropic detergent in cells devoid of Bax/Bak1 restores autophagic cell death, collectively indicting that Bax/Bak integrate all major forms of cell death through direct effects on me...
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Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted... more
Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted extracellular matrix proteins produced by proliferating and differentiating fibroblasts. Here, we employed 3 different mouse lineage-tracing models and stage-specific gene profiling to phenotypically analyze and classify resident cardiac fibroblast dynamics during myocardial infarction injury and stable scar formation. Fibroblasts were activated and highly proliferative, reaching a maximum rate within 2 to 4 days after infarction injury, at which point they expanded 3.5-fold and were maintained long term. By 3 to 7 days, these cells differentiated into myofibroblasts that secreted abundant extracellular matrix proteins and expressed smooth muscle α-actin to structurally support the necrotic area. By 7 to 10 days, myofibroblasts lost proliferative abilit...
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Research Interests: Inflammation, Membrane Proteins, Oxidative Stress, Humans, Liver, and 12 moreFemale, Male, Iron, Iron Overload, Middle Aged, Adult, Tumor necrosis factor-alpha, Gene Expression Regulation, real time polymerase chain reaction, non alcoholic fatty liver disease, Cardiovascular medicine and haematology, and Inflammation Mediators
Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction (MI) and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell type in terms of... more
Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction (MI) and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell type in terms of their origins and functional effects in vivo. Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen-inducible Cre for cellular lineage-tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Lineage tracing with four additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin(+) myofibroblasts reduces collagen production and scar formation after MI. Periostin-traced myofibroblasts also revert back to a less-activated state upon injury resolution. Our results define the myofibroblast ...
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The aim of this study was to determine the effect of iron overload in the development of nonalcoholic steatohepatitis (NASH) in a genetically obese mouse model (leprdb/db). Leptin-receptor deficient mice were fed a normal or an iron-... more
The aim of this study was to determine the effect of iron overload in the development of nonalcoholic steatohepatitis (NASH) in a genetically obese mouse model (leprdb/db). Leptin-receptor deficient mice were fed a normal or an iron- supplemented chow for 8 weeks and switched to normal chow for 8 weeks. All dietary iron (DI)-fed mice developed hepatic iron overload predominantly in the reticuloendothelial system (RES). Hepatocellular ballooning injury was observed in the livers of 85% of DI mice, relative to 20% of chow-fed leprdb/db. Hepatic malonyldialdehyde levels and mRNA levels of antioxidant genes (Nrf2, Gpx1 and Hmox1) were significantly increased in the DI mice. Hepatic mRNA levels of mitochondrial biogenesis regulators Pgc1α, Tfam, Cox4 and Nrf1 were diminished in the DI mice. In addition, gene expression levels of cytokines (Il6, Tnfα) and several innate and adaptive immune cell markers such as Tlr4, Inos, CD11c, CD4, CD8 and Ifnγ were significantly increased in livers of ...
Research Interests: Physiology, Obesity, Biology, Oxidative Stress, Innate immunity, and 12 moreMedicine, Liver, Reactive Oxygen Species, Mice, Adaptive Immunity, Animals, non alcoholic fatty liver disease (NAFLD), Medical Physiology, Iron Overload, Leptin Receptors, Malondialdehyde, and non alcoholic fatty liver disease
Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function where it regulates the adaptive endoplasmic... more
Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function where it regulates the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins such as BiP (Grp78) and Atf6α, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, as were the T3R and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wildtype Thbs4 and the calcium-binding mutant, interacted with Atf6α, induced an adaptive ER stress response, and caused expansio...
Research Interests: Biology, Endoplasmic Reticulum Stress, Medicine, Transgenic Mice, Signal Transduction, and 12 moreBiological Sciences, Molecular and cellular biology, Mice, Animals, Endoplasmic Reticulum, Glycoproteins, Rats, Heat Shock Proteins, Golgi Apparatus, Thrombospondins, Protein Transport, and Medical and Health Sciences
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Research Interests: Biopsy, Humans, Liver Cirrhosis, Liver, Female, and 15 moreMale, Temperature, Iron, Iron Overload, Hemochromatosis, Aged, Middle Aged, Fatty Liver, Adult, Magnetic Fields, chronic hepatitis C, Predictive value of tests, Cohort Studies, non alcoholic fatty liver disease, and Cardiovascular medicine and haematology
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Divalent metal-ion transporter-1 (DMT1) is a widely expressed, iron-preferring membrane transport protein. Animal models establish that DMT1 plays indispensable roles in intestinal nonheme-iron absorption and iron acquisition by erythroid... more
Divalent metal-ion transporter-1 (DMT1) is a widely expressed, iron-preferring membrane transport protein. Animal models establish that DMT1 plays indispensable roles in intestinal nonheme-iron absorption and iron acquisition by erythroid precursor cells. Rare mutations in human DMT1 result in severe microcytic-hypochromic anemia. When we express DMT1 in RNA-injected Xenopus oocytes, we observe rheogenic Fe(2+) transport that is driven by the proton electrochemical potential gradient. In that same preparation, DMT1 also transports cadmium and manganese but not copper. Whether manganese metabolism relies upon DMT1 remains unclear but DMT1 contributes to the effects of overexposure to cadmium and manganese in some tissues. There exist at least four DMT1 isoforms that arise from variant transcription of the SLC11A2 gene. Whereas these isoforms display identical functional properties, N- and C-terminal variations contain cues that direct the cell-specific targeting of DMT1 isoforms to discrete subcellular compartments (plasma membrane, endosomes, and lysosomes). An iron-responsive element (IRE) in the mRNA 3'-untranslated region permits the regulation of some isoforms by iron status, and additional mechanisms by which DMT1 is regulated are emerging. Natural-resistance-associated macrophage protein-1 (NRAMP1)-the only other member of the mammalian SLC11 gene family-contributes to antimicrobial function by extruding from the phagolysosome divalent metal ions (e.g. Mn(2+)) that may be essential cofactors for bacteria-derived enzymes or required for bacterial growth. The principal or only intestinal nonheme-iron transporter, DMT1 is a validated therapeutic target in hereditary hemochromatosis (HHC) and other iron-overload disorders.