Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction

Background-Autologous adipose tissue-derived mesenchymal stem cells (ATMSCs) therapy is a promising strategy to improve post-myocardial infarction outcomes. In a porcine model of acute myocardial infarction, we studied the long-term effects and the mechanisms involved in allogeneic ATMSCs administration on myocardial performance. Methods and Results-Thirty-eight pigs underwent 50 minutes of coronary occlusion; the study was completed in 33 pigs. After reperfusion, allogeneic ATMSCs or culture medium (vehicle) were intracoronarily administered. Follow-ups were performed at short (2 days after acute myocardial infarction vehicle-treated, n=10; ATMSCs-treated, n=9) or long term (60 days after acute myocardial infarction vehicle-treated, n=7; ATMSCs-treated, n=7). At short term, infarcted myocardium analysis showed reduced apoptosis in the ATMSCs-treated animals (48.6 +/- 6\% versus 55.9 +/- 5.7\% in vehicle; P=0.017); enhancement of the reparative process with up-regulated vascular endothelial growth factor, granulocyte macrophage colony-stimulating factor, and stromal-derived factor-1 alpha gene expression; and increased M2 macrophages (67.2 +/- 10\% versus 54.7 +/- 10.2\% in vehicle; P=0.016). In long-term groups, increase in myocardial perfusion at the anterior infarct border was observed both on day-7 and day-60 cardiac magnetic resonance studies in ATMSCs-treated animals, compared to vehicle (87.9 +/- 28.7 versus 57.4 +/- 17.7 mL/min per gram at 7 days; P=0.034 and 99 +/- 22.6 versus 43.3 +/- 14.7 22.6 mL/min per gram at 60 days; P=0.0001, respectively). At day 60, higher vascular density was detected at the border zone in the ATMSCs-treated animals (118 +/- 18 versus 92.4 +/- 24.3 vessels/mm(2) in vehicle; P=0.045). Cardiac magnetic resonance-measured left ventricular ejection fraction of left ventricular volumes was not different between groups at any time point. Conclusions-In this porcine acute myocardial infarction model, allogeneic ATMSCs-based therapy was associated with increased cardioprotective and reparative mechanisms and with better cardiac magnetic resonance-measured perfusion. No effect on left ventricular volumes or ejection fraction was observed.This work was supported by grants from Fundacion la Marato de TV3 (122230); Fondo de Investigacion Sanitaria Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FIS PI14/01682), (RD12/0042/0006), (RD12/0042/0047), (RD12/0019/0029) (TerCel RD16/0011/0006), CIBER Cardiovascular (CB16/11/00403) projects and Ministerio de Educacion y Ciencia (SAF2011-30067-C02-01) (SAF2014-59892). Fernaandez-Jimenez was the recipient of nonoverlapping grants from the Ministerio de Economia, Industria, y Competitividad through the Instituto de Salud Carlos III (Rio Hortega fellowship); and the Fundacion Jesus Serra, the Fundacion Interhospitalaria de Investigacion Cardiovascular (FIC), and the CNIC (FICNIC fellowship). The use of QMass software was partly supported by a scientific collaboration between the CNIC and Medis Medical Imaging Systems BV. The CNIC is supported by the Ministerio de Economia, Industria, y Competitividad (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). This work was also funded by ``la Caixa Banking Foundation, and the Generalitat de Catalunya (SGR 2014, CERCA Programme). This work has been developed in the context of AdvanceCat with the support of ACCIO (Catalonia Trade \& Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014-2020.S

CA125-Guided Diuretic Treatment Versus Usual Care in Patients With Acute Heart Failure and Renal Dysfunction

Background: The optimal diuretic treatment strategy for patients with acute heart failure and renal dysfunction remains unclear. Plasma carbohydrate antigen 125 (CA125) is a surrogate of fluid overload and a potentially valuable tool for guiding decongestion therapy. The aim of this study was to determine if a CA125-guided diuretic strategy is superior to usual care in terms of short-term renal function in patients with acute heart failure and renal dysfunction at presentation. Methods: This multicenter, open-label study randomized 160 patients with acute heart failure and renal dysfunction into 2 groups (1:1). Loop diuretics doses were established according to CA125 levels in the CA125-guided group (n = 79) and in clinical evaluation in the usual-care group (n = 81). Changes in estimated glomerular filtration rate (eGFR) at 72 and 24 hours were the co-primary endpoints, respectively. Results: The mean age was 78 ± 8 years, the median amino-terminal pro-brain natriuretic peptide was 7765 pg/mL, and the mean eGFR was 33.7 ± 11.3 mL/min/1.73m2. Over 72 hours, the CA125-guided group received higher furosemide equivalent dose compared to usual care (P = 0.011), which translated into higher urine volume (P = 0.042). Moreover, patients in the active arm with CA125 >35 U/mL received the highest furosemide equivalent dose (P <0.001) and had higher diuresis (P = 0.013). At 72 hours, eGFR (mL/min/1.73m2) significantly improved in the CA125-guided group (37.5 vs 34.8, P = 0.036), with no significant changes at 24 hours (35.8 vs 39.5, P = 0.391). Conclusion: A CA125-guided diuretic strategy significantly improved eGFR and other renal function parameters at 72 hours in patients with acute heart failure and renal dysfunction

SETD7 interacting proteins identified by mass spectrometry.

<p>SETD7 interacting proteins identified by mass spectrometry.</p

Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.

<p>Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.</p

The SETD7 knock-down affects the cell cycle profile of differentiating cells.

<p>(A) Cell cycle profile of undifferentiated and after 15 days of <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7). (B) Mean of the percentage of cells in each phase of the cell cycle in three independent differentiation experiments. *Differences in the percentage of cells in S-phase between shSETD7 and shSCR in differentiated cells was found significant at a p-value<0.05.</p

The SETD7 knock-down causes defects in the silencing of pluripotency genes.

<p>(A) Western blot showing the levels of OCT4 at different days during the in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (B) Immunolocalization of SOX2 (green) and OCT4 (red) expression at day 4 and day 15 of in vitro differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7) (C) Quantification of the percentage of embryoid bodies negative or positive for OCT4 staining at day 4 and day 7 of differentiation of cells treated with vehicle (DMSO), 1μM or 5μM PFI-2. (D) Immunolocalization of OCT4 (red) in embryoid bodies at day 4 and day 7 of in vitro differentiation of ES[4] treated with vehicle (DMSO) or 5μM PFI-2.</p

SETD7 is expressed at very low levels in pluripotent human cells and induced during differentiation.

<p>(A) Average rank of the top 700 most differentially expressed genes between pluripotent (iPSCs or ESCs) and fibroblasts, including those upregulated in pluripotent cells (left panel) and upregulated in fibroblasts (right panel). (B) <i>SETD7</i> mRNA levels in human ESCs grown under self-renewal conditions (UndES[4]), <i>in vitro</i> differentiated human ESCs (DifES[4]), human fibroblasts (HFF), two lines of human keratinocytes (HEK1 and HEK2) and two lines of iPSCs generated from keratinocytes ([H]KiPS4F and KiPS4F1). Mean and standard deviation of three technical replicates is shown. Induction of SETD7 mRNA levels during ES[4] differentiation was confirmed in more than four independent differentiation experiments. (C) Western blot showing SETD7 protein levels in pluripotent and somatic cells. Loading control beta actin (ACTB) is also shown. (D) Western blot showing protein levels of SETD7, AFP, OCT4 and SOX2 in under self-renewing conditions and <i>in vitro</i> differentiated human ESCs. Loading control alpha tubulin (TUBA) is also shown. One representative experiment out of three is shown. (E) Genomic visualization of the levels of H3K72me3, H3K4me3, H3K4me2, H3K36me3 and RNA polymerase II (Pol II) in the human embryonic stem cell line H1 around the <i>SETD7</i> gene according to ENCODE. A non-methylated CpG island is depicted in green. (F) Levels of H3K4me2 and H3K27me3 at <i>SETD7</i> gene promoter region (27 bp upstream of the transcription start site) in pluripotent and somatic cells determined by chromatin immunoprecipitation (ChIP) and ploted relative to the input. IgGs wer used as negative control. Bars show the mean and standard deviation of three independent immunoprecipitations.</p

The SETD7 knock-down causes a delay in differentiation.

<p>(A) mRNA levels of pluripotency factors (<i>OCT4</i>, <i>SOX2</i>, <i>NANOG</i>), differentiation genes (<i>HNF4</i> and <i>p21</i>) and <i>SETD7</i> normalized to <i>GAPDH</i> at different days during the <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA (sh28) that targets SETD7 (shSETD7). For pluripotency factors levels were plotted relative to d0 and for differentiation factors levels were plotted relative to day of maximum expression in the shSCR. Mean and standard deviation from three independent differentiation experiments is shown. (B) Gene set enrichment analysis (GSEA) of the global changes in gene expression found between shSETD7 and shSCR at day 8 of differentiation. Enrichment profile, normalized enrichment score (NES), p-value and false discovery rate (FDR) are shown for the significantly enriched gene sets from the Molecular Signatures Database BENPORATH_ES_1 (genes overexpressed in human embryonic stem cells according to 5 or more out of 20 profiling studies), HSIAO_LIVER_SPECIFIC_GENES (Liver selective genes), ESC_J1_UP_LATE.V1_UP (Genes up-regulated during late stages of differentiation of embryoid bodies from J1 embryonic stem cells) and CAHOY_ASTROGLIAL (Genes up-regulated in astroglia cells). (C) Heatmap of the expression of the 400 genes most differentially regulated by shSETD7 KD compared to shSCR and that are upregulated or downregulated more than 16 fold during differentiation determined by microarray analysis.</p