Human adipose tissue as a reservoir for memory CD4\u3csup\u3e+\u3c/sup\u3e T cells and HIV

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Objective: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. Design: We examined memory CD4+ T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4+ T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4+ T cells. Methods: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4+ T-cell activation and HIV production were measured by flow cytometry and ELISA. Results: AT-SVF CD3+ T cells were activated (\u3e60% CD69+) memory CD4+ and CD8+ T cells in uninfected andHIV-infected persons, but the AT-SVF CD4+/CD8+ ratiowas lower in HIV patients. HIVDNA(Gag and Env)was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4+ T-cell activation and HIV production approximately two to three-fold in synergy with gammachain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-a1b1. Adipocytes also enhanced T-cell viability. Conclusion: Adipose tissues of ART-treated patients harbour activated memory CD4+ T cells and HIV DNA. Adipocytes promote CD4+ T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis

Mutant SRF and YAP synthetic modified mRNAs drive cardiomyocyte nuclear replication

Introduction: Aging is associated with sarcopenia, myocyte loss, and dysfunction. The problem is compounded as the adult heart lacks the regenerative capacity to self-repair. Serum response factor’s (SRF’s) dual activity is essential for cell replication and heart cell differentiation. SRF interacts with cofactors, such as NKX2-5 and GATA4, which give cardiac-specific gene activity, and ETS factors such as ELK1 drive cell replication. Recently, the mutant YAP-5SA of the Hippo pathway was implicated in cardiomyocyte proliferation and growth.Aim: We hypothesized that disruption of interactions of SRF with NKX2-5 and GATA4 would lead to dedifferentiation of cardiomyocytes to a proliferative stem cell state and complement YAP-5SA to generate undifferentiated cardiomyocytes in a more primitive replicative state.Methods and results: To weaken SRF interactions with NKX2-5 and GATA4, alanine scanning mutations were generated across the SRF N-terminus of the MADS-box. One SRF mutant, SRF153(A3), was tested along with the YAP-5SA mutant, as degradable synthetic modified mRNAs (mmRNAs), in rat primary cardiomyocytes. To measure cell replication, adult cardiomyocytes were pulsed with alpha-EdU and then DAPI stained, while gene activity was assayed by RNA sequencing. To measure chromatin remodeling, Transposon 5 was used in ATAC sequencing. We observed that single and triple alanine substitutions of mutants centering over SRF-Lys154 essentially blocked myocyte differentiation, and NKX2-5 and GATA4 failed to stabilize mutated SRF DNA binding. Instead, many stem cell factors including NANOG and OCT4 were induced. SRF153(A3) does not recognize SRF response elements per ATAC sequencing and consequently induces stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. SRF153(A3) and YAP5SA mmRNA led to alpha-EDU incorporation in ~35% of the cardiomyocytes. DIAPH 3, a marker of the contractile ring during anaphase, appeared between and around replicated nuclei in three-month-old adult mouse cardiac myocytes. The combination of these synthetic mRNA increased nuclei replication with the expression of origin of replication genes, while genes associated with cardiomyocyte differentiation were down-regulated. ATAC sequencing revealed SRF153(A3) and YAP5SA mmRNA-induced chromatin remodeling of cell cycle, spindle, and growth factor genes by additive and synergistic activities.Conclusion: SRF153(A3) synthetic mmRNA and the mutant YAP-5SA mmRNA induced cardiomyocyte dedifferentiation, to nuclear replication in adult cardiac myocytes. The combinatorial use of mmRNA encoding SRF153(A3) and YAP-5SA has the potential to become a powerful clinical strategy for treating human heart disease

STEMIN and YAP5SA synthetic modified mRNAs regenerate and repair infarcted mouse hearts

Introduction: The adult heart lacks the regenerative capacity to self-repair. Serum response factor (SRF) is essential for heart organogenesis, sarcomerogenesis, and contractility. SRF interacts with co-factors, such as NKX2.5 and GATA4, required for cardiac specified gene activity. ETS factors such as ELK1 interact with SRF and drive cell replication. To weaken SRF interactions with NKX2.5 and GATA4, one mutant, SRF153(A3) named STEMIN, did not bind CArG boxes, yet induced stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. The mutant YAP5SA of the Hippo pathway also promotes cardiomyocyte proliferation and growth.Aim: Infarcted adult mouse hearts were injected with translatable STEMIN and YAP5SA mmRNA to evaluate their clinical potential,Methods and Results: Mice were pulsed one day later with alpha-EDU and then heart sections were DAPI stained. Replicating cells were identified by immuno-staining against members of the DNA replisome pathway that mark entry to S phase of the cell cycle. Echocardiography was used to determine cardiac function following infarcts and mRNA treatment. To monitor cardiac wall repair, microscopic analysis was performed, and the extent of myocardial fibrosis was analyzed for immune cell infiltration. Injections of STEMIN and YAP5SA mmRNA into the left ventricles of infarcted adult mice promoted a greater than 17-fold increase in the DAPI stained and alpha-EDU marked cardiomyocyte nuclei, within a day. We observed de novo expression of phospho-histone H3, ORC2, MCM2, and CLASPIN. Cardiac function was significantly improved by four weeks post-infarct, and fibrosis and immune cell infiltration were diminished in hearts treated with STEMIN and YAP5SA mmRNA than each alone.Conclusion: STEMIN and YAP5SA mmRNA improved cardiac function and myocardial fibrosis in left ventricles of infarcted adult mice. The combinatorial use of mmRNA encoding STEMIN and YAP5SA has the potential to become a powerful clinical strategy to treat human heart disease

Serum response factor orchestrates nascent sarcomerogenesis and silences the biomineralization gene program in the heart

Our conditional serum response factor (SRF) knockout, Srf Cko, in the heart-forming region blocked the appearance of rhythmic beating myocytes, one of the earliest cardiac defects caused by the ablation of a cardiac-enriched transcription factor. The appearance of Hand1 and Smyd1, transcription and chromatin remodeling factors; Acta1, Acta2, Myl3, and Myom1, myofibril proteins; and calcium-activated potassium-channel gene activity (KCNMB1), the channel protein, were powerfully attenuated in the SrfCKO mutant hearts. A requisite role for combinatorial cofactor interactions with SRF, as a major determinant for regulating the appearance of organized sarcomeres, was shown by viral rescue of SRF-null ES cells with SRF point mutants that block cofactor interactions. In the absence of SRF genes associated with biomineralization, GATA-6, bone morphogenetic protein 4 (BMP4), and periostin were strongly up-regulated, coinciding with the down regulation of many SRF dependent microRNA, including miR1, which exerted robust silencer activity over the induction of GATA-6 leading to the down regulation of BMP4 and periostin

Novel phosphorylation target in the serum response factor MADS box regulates α-actin transcription

Serum response factor (SRF) is a phosphoprotein that regulates skeletal and cardiac α-actin gene transcription. Myotonic dystrophy protein kinase (DMPK), a muscle- and neuron-restricted kinase, enhanced SRF-mediated promoter activity of the skeletal and cardiac α-actin genes in C2C12 myoblasts as well as in nonmyogenic cells. DMPK phosphorylated SRF in vitro in the αI coil of the DNA-binding domain in the MADS box, a highly conserved region required for DNA binding, dimerization, and co-activator interaction in COS and CV1 cells. Threonine 159 in the MADS box αI coil was a specific phosphorylation target in vitro as well as in vivo of both DMPK and protein kinase C-α. Substitution of threonine 159 with the nonphosphorylatable residue alanine markedly diminished activation of the cardiac α-actin promoter in the presence of kinase, while its substitution with aspartic acid, to introduce a negative charge and mimic phosphorylation, restored activation completely. Phosphorylation of the MADS box may constitute a novel mechanism for regulation of SRF-dependent actin gene transcription

Association of TCF7L2 variation with single islet autoantibody expression in children with type 1 diabetes.

The transcription factor 7-like 2 (TCF7L2) gene has the strongest genetic association with type 2 diabetes. TCF7L2 also associates with latent autoimmune diabetes in adults, which often presents with a single islet autoantibody, but not with classical type 1 diabetes