Abstract 33: Unlocking Reprogramming Capability: Silencing Antiplasticity Gene p63 Enhances the Reprogramming of Fibroblasts into Induced Cardiomyocytes

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Vivekkumar Patel ◽  
Austin Cooney ◽  
Elsa Flores ◽  
Vivek Singh ◽  
Megumi Mathison ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Fold Increase ◽  
Cellular Reprogramming ◽  
Embryonic Fibroblasts ◽  
Reprogramming Efficiency ◽  
Cardiac Transcription Factors

Objective: In situ cellular reprogramming of cardiac fibroblasts into (induced) cardiomyocytes (iCMs) represents a promising new potential intervention for the treatment of heart failure. Despite encouraging in vivo data in rodent myocardial infarction models, the relative resistance of human cells to reprogramming may be a significant barrier to the clinical application of this new therapy. We hypothesized that knockdown of the anti-plasticity gene p63 could therefore be used to enhance cellular reprogramming efficiency. Methods: p63 knockout (KO) murine embryonic fibroblasts (MEFs) and MEFs treated with p63 silencing shRNA were assessed for expression of the cardiomyocyte marker Cardiac Troponin T (cTnT) and pro-cardiogenic genes, with or without the treatment with known cardiac transcription factors Hand2 and Myocardin (HM). Results: After 3 wks in culture, expression of the cardiomyocyte marker cTnT (FACS) was significantly greater in p63 KO MEFs than in wild-type (WT) MEFs or WT MEFs treated with transcription factors Hand2 and Myocardin (39% ± 8%, 2.0% ± 1% and 2.7 ± 0.3%, respectively, p < 0.05). Treatment of p63 KO MEFs with Hand2 and Myocardin further increased cTnT expression up to 74% ± 3%. Treatment of WT MEFs with p63 shRNA likewise yielded a 20-fold increase in cTnT expression (qPCR) without HM and a 600-fold increase with HM when compared to non-silencing shRNA treated MEFs. Consistent with these findings, p63 KO or p63 shRNA-treated MEFs demonstrated increased expression (qPCR) of pro-cardiogenic genes Gata4, Mef2c and Tbx5 compared to naïve or non-silencing shRNA treated MEFs. After treatment with p63 shRNA, adult human epidermal cells also demonstrated increased expression of cTnT, myosin heavy chain and pro-cardiogenic genes when analyzed by qPCR. Conclusions: Downregulation of the anti-plasticity gene p63 enhances cellular reprogramming efficiency and iCM generation, as reflected in the increased expression of the cardiomyocyte marker cTnT and pro-cardiogenic genes Gata4, Mef2c and Tbx5. Use of such cellular plasticity enhancing strategies may be a useful strategy to overcome barriers to cellular reprogramming in the clinical arena.

Abstract MP12: The Mef2c Transcription Factor Regulates The Renin Cell Identity

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Omar E Guessoum ◽  
Kristyna Kupkova ◽  
Nathan Sheffield ◽  
Maria Luisa Sequeira Lopez ◽  
Roberto A Gomez
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Consensus Sequence ◽  
Renin Angiotensin System ◽  
Fold Increase ◽  
Conditional Knockout ◽  
Motif Analysis ◽  
Regulation Of Expression ◽  
Cell Identity

Introduction: The Renin-Angiotensin-System is essential to maintain blood pressure and fluid electrolyte homeostasis. Because precise regulation of expression and release of renin is critical for survival, understanding the molecular regulation of the renin cell identity is a vital area of study. Advances in epigenetics have enabled finer dissection of chromatin factors which maintain the identity of the renin cell. By studying genes with heightened accessibility profiles that are unique to the JG cell, we now have the capacity to unravel the determinants of the renin cell identity. Hypothesis: That transcription factors central to the governance of renin cell identity can be identified through the Assay for Transposase Accessible Chromatin (ATAC-seq) differential accessibility analysis. Methods: Native renin cell ATAC-seq was compared to existing ENCODE ATAC-seq datasets from 40 other cell types to define regions/peaks which characterize the JG program. Peaks with high intensity and ≥2-fold increase in signal were selected for Motif analysis to search for transcription factors (TFs) whose consensus sequence is enriched in those regions. Identified TFs were then selected for validation by in-situ hybridization and conditional deletion in renin cells. Results: 1) The Mef2c transcription factor was identified as having a consensus sequence in regulatory regions unique to the JG cell. It has clear expression in RNA-seq of renin cells (65 transcripts per million, n=3) and a predicted binding site in the renin gene. These results were validated by in-situ hybridization where signal localized at the JG area was detected in concordance with our in-silico results. 2) We generated Mef2c conditional knockout animals using our Ren1d-Cre mouse to study the effect in renin expression and identity. These mice displayed reduced renin immunostaining at the JG area and a 40% reduction in renin mRNA expression by qPCR from kidney cortices relative to wild-type (n=2, preliminary data). Conclusions: Our studies identified Mef2c as a TF target which likely has an essential role in maintaining and preserving renin cell identity. Experiments involving transcriptomics and epigenomics are ongoing to understand the changes wrought by Mef2c deletion in renin cells.

Abstract 34: Acetylation of GATA 4 Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Vivek P Singh ◽  
Megumi Mathison ◽  
Jaya P Pinnamaneni ◽  
Deepthi Sanagasetti ◽  
Narasimhaswamy S Belaguli ◽  
...  
Keyword(s):  
Ventricular Function ◽  
Troponin T ◽  
Novel Mutation ◽  
Cardiac Regeneration ◽  
Negative Control ◽  
Direct Reprogramming ◽  
Wild Type ◽  
Reprogramming Efficiency

Objective: Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown. Methods: In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes in vitro and accordingly ventricular function after myocardial infarction in rat, in vivo . Results: We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression in vitro , echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%]. Conclusions: Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs), in vitro and in vivo, and provide new insight into the molecular mechanism underlying cardiac regeneration.

Abstract P493: Histone Reader PHF7 Cooperates With The SWI/SNF Complex At Cardiac Super Enhancers To Promote Direct Reprogramming

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Glynnis A Garry ◽  
Svetlana Bezprozvannaya ◽  
Huanyu Zhou ◽  
Hisayuki Hashimoto ◽  
Kenian Chen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cardiac Fibroblasts ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Direct Reprogramming ◽  
Ischemic Disease ◽  
Treat Heart Failure ◽  
Cardiac Transcription Factors ◽  
Adult Fibroblasts

Ischemic heart disease is the leading cause of death worldwide. Direct reprogramming of resident cardiac fibroblasts (CFs) to induced cardiomyocytes (iCLMs) has emerged as a potential therapeutic approach to treat heart failure and ischemic disease. Cardiac reprogramming was first achieved through forced expression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT); our laboratory found that Hand2 (GHMT) and Akt1 (AGHMT) markedly enhanced reprogramming efficiency in embryonic and postnatal cell types. However, adult mouse and human fibroblasts are resistant to reprogramming due to staunch epigenetic barriers. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the epigenetic reader PHF7 as the most potent activating factor. We validated the findings of this screen and found that PHF7 augmented reprogramming of adult fibroblasts ten-fold. Mechanistically, PHF7 localized to cardiac super enhancers in fibroblasts by reading H3K4me2 marks, and through cooperation with the SWI/SNF complex, increased chromatin accessibility and transcription factor binding at these multivalent enhancers. Further, PHF7 recruited cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieved efficient reprogramming through these mechanisms in the absence of Gata4. Collectively, these studies highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

scholarly journals The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Antje M. Wengner ◽  
Simon C. Pitchford ◽  
Rebecca C. Furze ◽  
Sara M. Rankin
Keyword(s):  
Bone Marrow ◽  
Intraperitoneal Injection ◽  
Fold Increase ◽  
In Situ Perfusion ◽  
Acute Peritonitis ◽  
Femoral Bone Marrow ◽  
Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

scholarly journals Fibroblast transition to an endothelial “trans” state improves cell reprogramming efficiency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Megumi Mathison ◽  
Deepthi Sanagasetti ◽  
Vivek P. Singh ◽  
Aarthi Pugazenthi ◽  
Jaya Pratap Pinnamaneni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cardiac Fibroblasts ◽  
Fold Increase ◽  
Differentiation Potential ◽  
Neonatal Cardiomyocytes ◽  
Cell Transdifferentiation ◽  
Reprogramming Efficiency ◽  
Novel Strategy ◽  
Human Cardiac Fibroblasts

AbstractFibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p < 0.01). Cardiac fibroblasts treated with ETV2 demonstrated increased endothelial cell markers, and when then treated with GMT yielded greater prevalence of cells expressing cardiomyocyte markers including cTnT than did fibroblasts treated with GMT or ETV2 (10.3% ± 0.2% vs 1.7% ± 0.06% and 0.6 ± 0.03, p < 0.01). Rat cardiac fibroblasts treated with GMT + ETV2 demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes, whereas cells treated with GMT or ETV2 alone failed to contract in co-culture experiments. Human cardiac fibroblasts treated with ETV2 and then GMT likewise demonstrated greater prevalence of cTnT expression than did cells treated with GMT alone (2.8-fold increase, p < 0.05). Cardiac fibroblast transitioning through a trans-endothelial state appears to enhance cardio-differentiation by enhancing fibroblast plasticity.

Abstract 37: Mir-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Towards a Cardiomyocyte-like Fate by Directly Repressing Specificity Protein 1 (Sp1)

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Vivek P Singh ◽  
Megumi Mathison ◽  
Vivekkumar Patel ◽  
Deepthi Sanagasetti ◽  
Lina Yang ◽  
...  
Keyword(s):  
Porcine Model ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Cellular Reprogramming ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts ◽  
Spontaneous Contractions ◽  
Specificity Protein

Objective: Transdifferentiation of cardiac fibroblasts into cardiomyocyte-like cells (iCMs) represents a promising strategy in treating human heart disease. However, despite encouraging reprogramming data obtained from rodent models, the transgenes used in those models are not sufficient to reprogram human cells. We therefore sought to optimize a cocktail of factors which can efficiently transdifferentiate human cells. A porcine model was included to test its potential as a surrogate for human reprogramming studies. Methods: Lentivirus expressing Gata4 (G), Mef2c (M), Tbx5 (T), Hand2 (H), Myocardin (My), or two microRNAs miR-590, miR-199, were transduced into cultured porcine cardiac fibroblasts (PCFs) and human cardiac fibroblasts (HCFs) in different combinations. Two weeks after transduction, qRT-PCR, immunofluorescence (IF), and FACS assays were performed, and the efficiency of iCM production was evaluated by the expression of cardiomyocyte markers cardiac troponin T (cTnT) and α-sarcomeric actinin. Results: Combined G, M, T treatment alone was insufficient to transdifferentiate PCFs or HCFs into iCMs, despite the capability of GMT transduction to transdifferentiate up to 7% of treated rat cardiac fibroblasts, as assessed by FACS analysis for cTNT. However, the addition of H, My and miR-590 to GMT resulted in the transdifferentiation of approximately 5% of HCFs and PCFs, as measured by cTnT expression. IF analysis likewise demonstrated high expression of cTNT and α-actinin in these cells. Importantly, the transdifferentiated PCFs exhibited spontaneous contractions when co-cultured with murine cardiomyocytes. qPCR showed that administration of GMT plus either miR-590 or HMy upregulated cardiac genes MYH6 and TNNT2, and downregulated the fibroblast genes Collagen I and Collagen III. Mir-590 also directly suppressed Sp1, a fibrosis inducer and putative inhibitor of cellular reprogramming in the heart. Conclusions: Our data suggest that the porcine model can serve as an appropriate surrogate for human fibroblasts reprogramming studies. Enhanced transdifferentiation associated with miR-590-mediated repression of Sp1 suggests a novel pathway that may be targetable to enhance cardiac cellular reprogramming clinically.

scholarly journals Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors

2012 ◽  
Vol 23 (18) ◽  
pp. 3511-3521 ◽  
Author(s):  
Ryuga Ishii ◽  
Daisuke Kami ◽  
Masashi Toyoda ◽  
Hatsune Makino ◽  
Satoshi Gojo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Human Fibroblasts ◽  
Direct Conversion ◽  
Cellular Reprogramming ◽  
Irreversible Processes ◽  
Cell Based Therapy ◽  
Decidual Cells ◽  
Cell Conversion

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. We hypothesized that combinatorial expression of chondrocyte-specific transcription factors could directly convert human placental cells into chondrocytes. Starting from a pool of candidate genes, we identified a combination of only five genes (5F pool)—BCL6, T (also called BRACHYURY), c-MYC, MITF, and BAF60C (also called SMARCD3)—that rapidly and efficiently convert postnatal human chorion and decidual cells into chondrocytes. The cells generated expressed multiple cartilage-specific genes, such as Collagen type II α1, LINK PROTEIN-1, and AGGRECAN, and exhibited characteristics of cartilage both in vivo and in vitro. Expression of the endogenous genes for T and MITF was initiated, implying that the cell conversion is due to not only the forced expression of the transgenes, but also to cellular reprogramming by the transgenes. This direct conversion system from noncartilage tissue to cartilaginous tissue is a substantial advance toward understanding cartilage development, cell-based therapy, and oncogenesis of chondrocytes.

scholarly journals Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiexue Ma ◽  
Xiangzhen Ding ◽  
Zhiying Li ◽  
Sheng Wang
Keyword(s):  
Protease Inhibitor ◽  
Molecular Farming ◽  
Expression System ◽  
Fold Increase ◽  
Protective Effects ◽  
Competitive Effects ◽  
Inoculum Dose ◽  
Plant Expression Systems

Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.

scholarly journals Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes

1997 ◽  
Vol 325 (3) ◽  
pp. 801-810 ◽  
Author(s):  
Angela CLERK ◽  
Peter H. SUGDEN
Keyword(s):  
Transcription Factors ◽  
Okadaic Acid ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Western Blots ◽  
Hyperosmotic Shock ◽  
Nuclear Extracts

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro,extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.

scholarly journals In vivo combinatory gene therapy synergistically promotes cardiac function and vascular regeneration following myocardial infarction

2020 ◽  
Vol 11 ◽  
pp. 204173142095341
Author(s):  
Sunghun Lee ◽  
Bong-Woo Park ◽  
Yong Jin Lee ◽  
Kiwon Ban ◽  
Hun-Jun Park
Keyword(s):  
Myocardial Infarction ◽  
Gene Therapy ◽  
Transcription Factors ◽  
Heart Function ◽  
Cardiac Fibroblasts ◽  
Capillary Density ◽  
Cardiac Repair ◽  
Vascular Regeneration ◽  
Intramyocardial Delivery

Since myocardial infarction (MI) excessively damage the myocardium and blood vessels, the therapeutic approach for treating MI hearts should simultaneously target these two major components in the heart to achieve comprehensive cardiac repair. Here, we investigated a combinatory platform of ETV2 and Gata4, Mef2c and Tbx5 (GMT) transcription factors to develop a strategy that can rejuvenate both myocardium and vasculatures together in MI hearts. Previously ETV2 demonstrated significant effects on neovascularization and GMT was known to directly reprogram cardiac fibroblasts into cardiomyocytes under in vivo condition. Subsequently, intramyocardial delivery of a combination of retroviral GMT and adenoviral ETV2 particles into the rat MI hearts significantly increased viable myocardium area, capillary density compared to ETV2 or GMT only treated hearts, leading to improved heart function and reduced scar formation. These results demonstrate that this combinatorial gene therapy can be a promising approach to enhance the cardiac repair in MI hearts.

Sign in / Sign up

Export Citation Format

Share Document