Publications by authors named "Xingbo Xu"

25 Publications

  • Page 1 of 1

Mechanisms and Perspectives of Sodium-Glucose Co-transporter 2 Inhibitors in Heart Failure.

Front Cardiovasc Med 2021 10;8:636152. Epub 2021 Feb 10.

State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, China.

Heart failure (HF) is a common complication or late-stage manifestation of various heart diseases. Numerous risk factors and underlying causes may contribute to the occurrence and progression of HF. The pathophysiological mechanisms of HF are very complicated. Despite accumulating advances in treatment for HF during recent decades, it remains an intractable clinical syndrome with poor outcomes, significantly reducing the quality of life and expectancy of patients, and imposing a heavy economic burden on society and families. Although initially classified as antidiabetic agents, sodium-glucose co-transporter 2 (SGLT2) inhibitors have demonstrated reduced the prevalence of hospitalization for HF, cardiovascular death, and all-cause death in several large-scale randomized controlled clinical trials. These beneficial effects of SGLT-2 inhibitors can be attributed to multiple hemodynamic, inflammatory and metabolic mechanisms, not only reducing the serum glucose level. SGLT2 inhibitors have been used increasingly in treatment for patients with HF with reduced ejection fraction due to their surprising performance in improving the prognosis. In addition, their roles and mechanisms in patients with HF with preserved ejection fraction or acute HF have also attracted attention. In this review article, we discuss the possible mechanisms and applications of SGLT2 inhibitors in HF.
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http://dx.doi.org/10.3389/fcvm.2021.636152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902509PMC
February 2021

Optimal antithrombotic therapy after transcatheter aortic valve replacement in patients with atrial fibrillation.

Ther Adv Chronic Dis 2020 14;11:2040622320949068. Epub 2020 Oct 14.

State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Ave, Guangzhou 510515, China.

Atrial fibrillation (AF) is prevalent in patients with aortic stenosis (AS) undergoing transcatheter aortic valve replacement (TAVR). Depending on the timing of AF detection, it is usually categorized as pre-existing AF or new-onset AF. Antiplatelet therapy, rather than a vitamin K antagonist, may be considered as the primary treatment for patients without an indication for oral anticoagulants who undergo TAVR. However, the optimal postprocedural antithrombotic regimen for patients with AF undergoing TAVR remains unknown. In this review, we briefly introduce the management strategies of antithrombotic therapy and list the evidence from related studies to elucidate the optimal antithrombotic management for patients with AF undergoing TAVR.
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http://dx.doi.org/10.1177/2040622320949068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576914PMC
October 2020

CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications.

Int J Mol Sci 2020 Apr 25;21(9). Epub 2020 Apr 25.

Department of Cardiology and Pneumology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.
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http://dx.doi.org/10.3390/ijms21093038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246536PMC
April 2020

Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1.

Theranostics 2020 4;10(9):3905-3924. Epub 2020 Mar 4.

Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August University, Göttingen, Germany.

: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. : We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. TGFβ1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the promoter region in MCECs. : Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFβ-pSMAD2/3 signaling in endothelial cells. : This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.
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http://dx.doi.org/10.7150/thno.38640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086357PMC
March 2020

The axis of local cardiac endogenous Klotho-TGF-β1-Wnt signaling mediates cardiac fibrosis in human.

J Mol Cell Cardiol 2019 11 11;136:113-124. Epub 2019 Sep 11.

Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Background: Cardiovascular fibrosis is a major contributor to cardiovascular disease, the primary cause of death in patients with chronic kidney disease (CKD). We previously reported expression of endogenous Klotho in human arteries, and that CKD is a state of Klotho deficiency, resulting in vascular calcification, but myocardial expression of Klotho is poorly understood. This study aimed to further clarify endogenous Klotho's functional roles in cardiac fibrosis in patients with underlying CKD.

Methods And Results: Human atrial appendage specimens were collected during cardiac surgery from individuals with or without CKD. Cardiac fibrosis was quantified using trichrome staining. For endogenous Klotho functional studies, primary human cardiomyocytes (HCMs) were treated with uremic serum from CKD patients or recombinant human TGF-β1. The effects of endogenous Klotho in HCMs were studied using Klotho-siRNA and Klotho-plasmid transfection. Both gene and protein expression of endogenous Klotho are found in human heart, but decreased Klotho expression is clearly associated with the degree of cardiac fibrosis in CKD patients. Moreover, we show that endogenous Klotho is expressed by HCMs and cardiac fibroblasts (HCFs) but that HCM expression is suppressed by uremic serum or TGF-β1. Klotho knockdown or overexpression aggravates or mitigates TGF-β1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-β1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs.

Conclusions: Our data from analysis of human hearts as well as functional in vitro studies strongly suggests that the loss of cardiac endogenous Klotho in CKD patients, specifically in cardiomyocytes, facilitates intensified TGF-β1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/β-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.
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http://dx.doi.org/10.1016/j.yjmcc.2019.09.004DOI Listing
November 2019

Causal Connections From Chronic Kidney Disease to Cardiac Fibrosis.

Semin Nephrol 2018 11;38(6):629-636

Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Partner Site, Goettingen, Germany. Electronic address:

Cardiovascular disease and heart failure are the primary cause of morbidity and mortality in patients with chronic kidney disease. Because impairment of kidney function correlates with heart failure and cardiac fibrosis, a kidney-heart axis is suspected. Although our understanding of the underlying mechanisms still is evolving, the possibility that kidney-heart messengers could be intercepted offers ample reason to focus on this clinically highly relevant problem. Here, we review the current knowledge of how kidney injury causes heart failure and fibrosis.
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http://dx.doi.org/10.1016/j.semnephrol.2018.08.007DOI Listing
November 2018

Modulation of NCAM/FGFR1 signaling suppresses EMT program in human proximal tubular epithelial cells.

PLoS One 2018 1;13(11):e0206786. Epub 2018 Nov 1.

Institute of Pathology, University of Belgrade-Faculty of Medicine, Belgrade, Serbia.

Neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor 1 (FGFR1) cross-talk have been involved in epithelial-to-mesenchymal transition (EMT) process during carcinogenesis. Since EMT also contributes to maladaptive repair and parenchymal damage during renal fibrosis, we became encouraged to explore the role of NCAM/FGFR1 signaling as initiating or driving forces of EMT program in cultured human proximal tubular epithelial cells (TECs). TECs stimulated with TGF-β1 (10ng/mL) was used as an established in vitro EMT model. TGF-β1 downstream effectors were detected in vitro, as well as in 50 biopsies of different human kidney diseases to explore their in vivo correlation. NCAM/FGFR1 signaling and its modulation by FGFR1 inhibitor PD173074 (100nM) were analyzed by light microscopy, immunolabeling, qRT-PCR and scratch assays. Morphological changes associated with EMT appeared 48h after TGF-ß1 treatment and was clearly apparent after 72 hours, followed by loss of CDH1 (encoding E-Cadherin) and transcriptional induction of SNAI1 (SNAIL), SNAI2 (SLUG), TWIST1, MMP2, MMP9, CDH2 (N-Cadherin), ITGA5 (integrin-α5), ITGB1 (integrin-β1), ACTA2 (α-SMA) and S100A4 (FSP1). Moreover, at the early stage of EMT program (24 hours upon TGF-β1 exposure), transcriptional induction of several NCAM isoforms along with FGFR1 was observed, implicating a mechanistic link between NCAM/FGFR1 signaling and induction of EMT. These assumptions were further supported by the inhibition of the EMT program after specific blocking of FGFR1 signaling by PD173074. Finally, there was evidence for an in vivo TGF-β1 pathway activation in diseased human kidneys and correlation with impaired renal excretory functions. Collectively, NCAM/FGFR1 signaling appears to be involved in the initial phase of TGF-ß1 initiated EMT which can be effectively suppressed by application of FGFR inhibitor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206786PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211750PMC
April 2019

Epigenetic Regulation of Endothelial-to-Mesenchymal Transition in Chronic Heart Disease.

Arterioscler Thromb Vasc Biol 2018 09;38(9):1986-1996

From the Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August University, Germany (M.S.H., X.X., E.M.Z.).

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose their properties and transform into fibroblast-like cells. This transition process contributes to cardiac fibrosis, a common feature of patients with chronic heart failure. To date, no specific therapies to halt or reverse cardiac fibrosis are available, so knowledge of the underlying mechanisms of cardiac fibrosis is urgently needed. In addition, EndMT contributes to other cardiovascular pathologies such as atherosclerosis and pulmonary hypertension, but also to cancer and organ fibrosis. Remarkably, the molecular mechanisms driving EndMT are largely unknown. Epigenetics play an important role in regulating gene transcription and translation and have been implicated in the EndMT process. Therefore, epigenetics might be the missing link in unraveling the underlying mechanisms of EndMT. Here, we review the involvement of epigenetic regulators during EndMT in the context of cardiac fibrosis. The role of DNA methylation, histone modifications (acetylation and methylation), and noncoding RNAs (microRNAs, long noncoding RNAs, and circular RNAs) in the facilitation and inhibition of EndMT are discussed, and potential therapeutic epigenetic targets will be highlighted.
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http://dx.doi.org/10.1161/ATVBAHA.118.311276DOI Listing
September 2018

High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis.

Nat Commun 2018 08 29;9(1):3509. Epub 2018 Aug 29.

German Center for Cardiovascular Research (DZHK) Partner Site, Göttingen, Germany.

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.
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http://dx.doi.org/10.1038/s41467-018-05766-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115451PMC
August 2018

Differentiation of functional endothelial cells from human induced pluripotent stem cells: A novel, highly efficient and cost effective method.

Differentiation 2016 Oct - Nov;92(4):225-236. Epub 2016 Jun 4.

Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg August University, 37075 Göttingen, Germany; DZHK (German Centre for Cardiovascular Research) Partner Site Göttingen, 37075 Göttingen, Germany. Electronic address:

Endothelial cells derived from human induced pluripotent stem cells (hiPSC- EC) are of significant value for research on human vascular development, in vitro disease models and drug screening. Here we report an alternative, highly efficient and cost-effective simple three step method (mesoderm induction, endothelial cell differentiation and endothelial cell expansion) to differentiate hiPSC directly into endothelial cells. We demonstrate that efficiency of described method to derive CD31+ and VE-Cadherin+ double positive cells is higher than 80% in 12 days. Most notably we established that hiPSC-EC differentiation efficacy depends on optimization of both mesoderm differentiation and endothelial cell differentiation steps.
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http://dx.doi.org/10.1016/j.diff.2016.05.004DOI Listing
August 2017

Hypoxia-induced endothelial-mesenchymal transition is associated with RASAL1 promoter hypermethylation in human coronary endothelial cells.

FEBS Lett 2016 04 21;590(8):1222-33. Epub 2016 Apr 21.

Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August University, Göttingen, Germany.

Cardiac fibrosis is integral in chronic heart disease, and one of the cellular processes contributing to cardiac fibrosis is endothelial-to-mesenchymal transition (EndMT). We recently found that hypoxia efficiently induces human coronary artery endothelial cells (HCAEC) to undergo EndMT through a hypoxia inducible factor-1α (HIF1α)-dependent pathway. Promoter hypermethylation of Ras-Gap-like protein 1 (RASAL1) has also been recently associated with EndMT progression and cardiac fibrosis. Our findings suggest that HIF1α and transforming growth factor (TGF)/SMAD signalling pathways synergistically regulate hypoxia-induced EndMT through both DNMT3a-mediated hypermethylation of RASAL1 promoter and direct SNAIL induction. The findings indicate that multiple cascades may be activated simultaneously to mediate hypoxia-induced EndMT.
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http://dx.doi.org/10.1002/1873-3468.12158DOI Listing
April 2016

DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition.

PLoS One 2016 27;11(1):e0147816. Epub 2016 Jan 27.

Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen, Germany.

While phosphorus in the form of inorganic or organic phosphate is critically involved in most cellular functions, high plasma levels of inorganic phosphate levels have emerged as independent risk factor for cardiac fibrosis, cardiovascular morbidity and decreased life-expectancy. While the link of high phosphate and cardiovascular disease is commonly explained by direct cellular effects of phospho-regulatory hormones, we here explored the possibility of inorganic phosphate directly eliciting biological responses in cells. We demonstrate that human coronary endothelial cells (HCAEC) undergo an endothelial-mesenchymal transition (EndMT) when exposed to high phosphate. We further demonstrate that such EndMT is initiated by recruitment of aberrantly phosphorylated DNMT1 to the RASAL1 CpG island promoter by HDAC2, causing aberrant promoter methylation and transcriptional suppression, ultimately leading to increased Ras-GTP activity and activation of common EndMT regulators Twist and Snail. Our studies provide a novel aspect for known adverse effects of high phosphate levels, as eukaryotic cells are commonly believed to have lost phosphate-sensing mechanisms of prokaryotes during evolution, rendering them insensitive to extracellular inorganic orthophosphate. In addition, our studies provide novel insights into the mechanisms underlying specific targeting of select genes in context of fibrogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147816PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729486PMC
July 2016

High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation.

Biochem Biophys Res Commun 2016 Apr 14;472(3):459-64. Epub 2016 Jan 14.

Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Göttingen, Germany. Electronic address:

Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.077DOI Listing
April 2016

Snail Is a Direct Target of Hypoxia-inducible Factor 1α (HIF1α) in Hypoxia-induced Endothelial to Mesenchymal Transition of Human Coronary Endothelial Cells.

J Biol Chem 2015 Jul 13;290(27):16653-64. Epub 2015 May 13.

From the Departments of Cardiology and Pneumology and DZHK (German Centre for Cardiovascular Research) partner site, 37075 Göttingen, Germany

Endothelial to mesenchymal transition (EndMT) was originally described in heart development where the endocardial endothelial cells that line the atrioventricular canal undergo an EndMT to form the endocardial mesenchymal cushion that later gives rise to the septum and mitral and tricuspid valves. In the postnatal heart specifically, endothelial cells that originate from the endocardium maintain increased susceptibility to undergo EndMT as remnants from their embryonic origin. Such EndMT involving adult coronary endothelial cells contributes to microvascular rarefaction and subsequent chronification of hypoxia in the injured heart, ultimately leading to cardiac fibrosis. Although in most endothelial beds hypoxia induces tip cell formation and sprouting angiogenesis, here we demonstrate that hypoxia is a stimulus for human coronary endothelial cells to undergo phenotypic changes reminiscent of EndMT via a mechanism involving hypoxia-inducible factor 1α-induced activation of the EndMT master regulatory transcription factor SNAIL. Our study adds further evidence for the unique susceptibility of endocardium-derived endothelial cells to undergo EndMT and provides novel insights into how hypoxia contributes to progression of cardiac fibrosis. Additional studies may be required to discriminate between distinct sprouting angiogenesis and EndMT responses of different endothelial cells populations.
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http://dx.doi.org/10.1074/jbc.M115.636944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505417PMC
July 2015

Endothelial p53 deletion improves angiogenesis and prevents cardiac fibrosis and heart failure induced by pressure overload in mice.

J Am Heart Assoc 2015 Feb 24;4(2). Epub 2015 Feb 24.

Department of Cardiology and Pneumology, University Medical Center Göttingen, Germany (R.G., X.X., J.H.S., S.E.L., E.M.Z., K.S.) Division of Cardiology, Department of Medicine 2, University Medical Center Mainz, Germany (M.L.B., P.W., K.S.) German Center for Cardiovascular Research (DZHK), University of Maryland Baltimore, MD (S.E.L., E.M.Z., K.S.).

Background: Cardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure.

Methods And Results: Mice with endothelial-specific deletion of p53 (End.p53-KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53-KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53-WT controls, whereas banded hearts of End.p53-KO mice exhibited lower numbers of apoptotic endothelial and non-endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53-KO mice exhibited markedly less fibrosis compared with End.p53-WT controls, and lower mRNA levels of p53-regulated genes involved in extracellular matrix production and turnover (eg, Bmp-7, Ctgf, or Pai-1), or of transcription factors involved in controlling mesenchymal differentiation were observed.

Conclusions: Our analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy.
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http://dx.doi.org/10.1161/JAHA.115.001770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345879PMC
February 2015

Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis.

Cardiovasc Res 2015 Mar 23;105(3):279-91. Epub 2015 Jan 23.

Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August University, Robert-Koch-Str. 40, 37075 Göttingen, Germany German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Göttingen, Germany

Aims: Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis.

Methods And Results: In human coronary endothelial cells, TGFβ1 causes aberrant methylation of RASAL1 promoter, increased Ras-GTP activity, and EndMT. In end-stage failing vs. non-failing human myocardium, increased fibrosis was associated with significantly increased RASAL1 promoter methylation, decreased RASAL1 expression, increased Ras-GTP activity, and increased expression of markers of EndMT. In mice with pressure overload due to ascending aortic constriction, BMP7 significantly reduced RASAL1 promoter methylation, increased RASAL1 expression, and decreased EndMT markers as well as decreased cardiac fibrosis. The ten eleven translocation (TET) family enzyme TET3, which demethylates through hydroxymethylation, was significantly decreased in fibrotic mouse hearts, restored with BMP7, and BMP7 effects were absent in coronary endothelial cells with siRNA knockdown of TET3.

Conclusion: Our study provides proof-in-principle evidence that transcriptional suppression of RASAL1 through aberrant promoter methylation contributes to EndMT and ultimately to progression of cardiac fibrosis. Such aberrant methylation can be reversed through Tet3-mediated hydroxymethylation, which can be specifically induced by BMP7. This may reflect a new treatment strategy to stop cardiac fibrosis.
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http://dx.doi.org/10.1093/cvr/cvv015DOI Listing
March 2015

Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting.

Nat Commun 2015 Jan 23;6:6008. Epub 2015 Jan 23.

Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.

Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.
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http://dx.doi.org/10.1038/ncomms7008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354275PMC
January 2015

Endocardial fibroelastosis is caused by aberrant endothelial to mesenchymal transition.

Circ Res 2015 Feb 13;116(5):857-66. Epub 2015 Jan 13.

From the Department of Cardiology and Pneumology (X.X., F.A., E.M.Z.), Department of Nephrology and Rheumatology (B.T., M.Z.), University Medical Center of Göttingen, Georg-August University, Göttingen, Germany; Department of Cardiac Surgery, Boston Children's Hospital, Harvard Medical School, MA (I.F., I.V., P.J.d N.); Division of Matrix Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (T.Z.H., R.K., E.M.Z.); Lab Genetica Molecolare, Papa Giovanni XXIII Hospital, Bergamo, Italy (M.I.); Department of Cancer Biology and the Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston (R.K.); and DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany (E.M.Z.).

Rationale: Endocardial fibroelastosis (EFE) is a unique form of fibrosis, which forms a de novo subendocardial tissue layer encapsulating the myocardium and stunting its growth, and which is typically associated with congenital heart diseases of heterogeneous origin, such as hypoplastic left heart syndrome. Relevance of EFE was only recently highlighted through the establishment of staged biventricular repair surgery in infant patients with hypoplastic left heart syndrome, where surgical removal of EFE tissue has resulted in improvement in the restrictive physiology leading to the growth of the left ventricle in parallel with somatic growth. However, pathomechanisms underlying EFE formation are still scarce, and specific therapeutic targets are not yet known.

Objective: Here, we aimed to investigate the cellular origins of EFE tissue and to gain insights into the underlying molecular mechanisms to ultimately develop novel therapeutic strategies.

Methods And Results: By utilizing a novel EFE model of heterotopic transplantation of hearts from newborn reporter mice and by analyzing human EFE tissue, we demonstrate for the first time that fibrogenic cells within EFE tissue originate from endocardial endothelial cells via aberrant endothelial to mesenchymal transition. We further demonstrate that such aberrant endothelial to mesenchymal transition involving endocardial endothelial cells is caused by dysregulated transforming growth factor beta/bone morphogenetic proteins signaling and that this imbalance is at least in part caused by aberrant promoter methylation and subsequent transcriptional suppression of bone morphogenetic proteins 5 and 7. Finally, we provide evidence that supplementation of exogenous recombinant bone morphogenetic proteins 7 effectively ameliorates endothelial to mesenchymal transition and experimental EFE in rats.

Conclusions: In summary, our data point to aberrant endothelial to mesenchymal transition as a common denominator of infant EFE development in heterogeneous, congenital heart diseases, and to bone morphogenetic proteins 7 as an effective treatment for EFE and its restriction of heart growth.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.305629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344885PMC
February 2015

Increased concentration of circulating angiogenesis and nitric oxide inhibitors induces endothelial to mesenchymal transition and myocardial fibrosis in patients with chronic kidney disease.

Int J Cardiol 2014 Sep 6;176(1):99-109. Epub 2014 Jul 6.

Department of Cardiology and Pneumology, University Medical Center, Georg-August University, Göttingen, Germany; DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany.

Background: Sudden cardiovascular death is increased in chronic kidney disease (CKD). Experimental CKD models suggest that angiogenesis and nitric oxide (NO) inhibitors induce myocardial fibrosis and microvascular dropout thereby facilitating arrhythmogenesis. We undertook this study to characterize associations of CKD with human myocardial pathology, NO-related circulating angiogenesis inhibitors, and endothelial cell behavior.

Methods: We compared heart (n=54) and serum (n=162) samples from individuals with and without CKD, and assessed effects of serum on human coronary artery endothelial cells (HCAECs) in vitro. Left ventricular fibrosis and capillary density were quantified in post-mortem samples. Endothelial to mesenchymal transition (EndMT) was assessed by immunostaining of post-mortem samples and RNA expression in heart tissue obtained during cardiac surgery. Circulating asymmetric dimethylarginine (ADMA), endostatin (END), angiopoietin-2 (ANG), and thrombospondin-2 (TSP) were measured, and the effect of these factors and of subject serum on proliferation, apoptosis, and EndMT of HCAEC was analyzed.

Results: Cardiac fibrosis increased 12% and 77% in stage 3-4 CKD and ESRD and microvascular density decreased 12% and 16% vs. preserved renal function. EndMT-derived fibroblast proportion was 17% higher in stage 3-4 CKD and ESRD (P trend = 0.02). ADMA, ANG, TSP, and END concentrations increased in CKD. Both individual factors and CKD serum increased HCAEC apoptosis (P=0.02), decreased proliferation (P=0.03), and induced EndMT.

Conclusions: CKD is associated with an increase in circulating angiogenesis and NO inhibitors, which impact proliferation and apoptosis of cardiac endothelial cells and promote EndMT, leading to cardiac fibrosis and capillary rarefaction. These processes may play key roles in CKD-associated CV disease.
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http://dx.doi.org/10.1016/j.ijcard.2014.06.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161362PMC
September 2014

Pelota regulates the development of extraembryonic endoderm through activation of bone morphogenetic protein (BMP) signaling.

Stem Cell Res 2014 Jul 26;13(1):61-74. Epub 2014 Apr 26.

Institute of Human Genetics, University of Göttingen, D-37073 Göttingen, Germany. Electronic address:

Pelota (Pelo) is ubiquitously expressed, and its genetic deletion in mice leads to embryonic lethality at an early post-implantation stage. In the present study, we conditionally deleted Pelo and showed that PELO deficiency did not markedly affect the self-renewal of embryonic stem cells (ESCs) or their capacity to differentiate in teratoma assays. However, their differentiation into extraembryonic endoderm (ExEn) in embryoid bodies (EBs) was severely compromised. Conversely, forced expression of Pelo in ESCs resulted in spontaneous differentiation toward the ExEn lineage. Failure of Pelo-deficient ESCs to differentiate into ExEn was accompanied by the retained expression of pluripotency-related genes and alterations in expression of components of the bone morphogenetic protein (BMP) signaling pathway. Further experiments have also revealed that attenuated activity of BMP signaling is responsible for the impaired development of ExEn. The recovery of ExEn and down-regulation of pluripotent genes in BMP4-treated Pelo-null EBs indicate that the failure of mutant cells to down-regulate pluripotency-related genes in EBs is not a result of autonomous defect, but rather to failed signals from surrounding ExEn lineage that induce the differentiation program. In vivo studies showed the presence of ExEn in Pelo-null embryos at E6.5, yet embryonic lethality at E7.5, suggesting that PELO is not required for the induction of ExEn development, but rather for ExEn maintenance or for terminal differentiation toward functional visceral endoderm which provides the embryos with growth factors required for further development. Moreover, Pelo-null fibroblasts failed to reprogram toward induced pluripotent stem cells (iPSCs) due to inactivation of BMP signaling and impaired mesenchymal-to-epithelial transition. Thus, our results indicate that PELO plays an important role in the establishment of pluripotency and differentiation of ESCs into ExEn lineage through activation of BMP signaling.
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http://dx.doi.org/10.1016/j.scr.2014.04.011DOI Listing
July 2014

The roles of DAZL in RNA biology and development.

Wiley Interdiscip Rev RNA 2014 Jul-Aug;5(4):527-35. Epub 2014 Apr 8.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

RNA-binding proteins play an important role in the regulation of gene expression by modulating translation and localization of specific messenger RNAs (mRNAs) during early development and gametogenesis. The DAZ (Deleted in Azoospermia) family of proteins, which includes DAZ, DAZL, and BOULE, are germ cell-specific RNA-binding proteins that are implicated in translational regulation of several transcripts. Of particular importance is DAZL, which is present in vertebrates and arose from the duplication of the ancestral BOULE during evolution. Identification of DAZL target mRNAs and characterization of the RNA-binding sequence through in vitro binding assays and crystallographic studies revealed that DAZL binds to GUU triplets in the 3' untranslated region of target mRNAs. Although there is compelling evidence for the role of DAZL in translation stimulation of target mRNAs, recent studies indicate that DAZL can also function in translational repression and transport of specific mRNAs. Furthermore, apart from the well-characterized function of DAZL in gametogenesis, recent data suggest its role in early embryonic development and differentiation of pluripotent stem cells toward functional gametes. In light of the mounting evidence for the role of DAZL in various cellular and developmental processes, we summarize the currently characterized biological functions of DAZL in RNA biology and development.
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http://dx.doi.org/10.1002/wrna.1228DOI Listing
March 2015

Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis.

J Am Soc Nephrol 2014 May 30;25(5):905-12. Epub 2014 Jan 30.

Departments of Nephrology and Rheumatology, and

Methylation of CpG island promoters is an epigenetic event that can effectively silence transcription over multiple cell generations. Hypermethylation of the Rasal1 promoter contributes to activation of fibroblasts and progression of kidney fibrosis. Here, we explored whether such causative hypermethylation could be reversed through endogenous mechanisms and whether such reversal of hypermethylation is a constituent of the antifibrotic activity of bone morphogenic protein 7 (BMP7). We show that successful inhibition of experimental kidney fibrosis through administration of BMP7 associates with normalization of Rasal1 promoter hypermethylation. Furthermore, this reversal of pathologic hypermethylation was achieved specifically through Tet3-mediated hydroxymethylation. Collectively, our findings reveal a new mechanism that may be exploited to facilitate therapeutic DNA demethylation to reverse kidney fibrosis.
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http://dx.doi.org/10.1681/ASN.2013070723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005308PMC
May 2014

Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells.

Stem Cell Res 2013 Nov 30;11(3):1045-59. Epub 2013 Jul 30.

Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.

Pluripotency is maintained by both known and unknown transcriptional regulatory networks. In the present study, we have identified Zfp819, a KRAB-zinc finger protein, as a novel pluripotency-related factor and characterized its role in pluripotent stem cells. We show that Zfp819 is expressed highly in various types of pluripotent stem cells but not in their differentiated counterparts. We identified the presence of non-canonical nuclear localization signals in particular zinc finger motifs and identified them as responsible for the nuclear localization of Zfp819. Analysis of the Zfp819 promoter region revealed the presence of a transcriptionally active chromatin signature. Moreover, we confirmed the binding of pluripotency-related factors, Oct4, Sox2, and Nanog to the distal promoter region of Zfp819, indicating that the expression of this gene is regulated by a pluripotency transcription factor network. We found that the expression of endogenous retroviral elements (ERVs) such as Intracisternal A Particle (IAP) retrotransposons, Long Interspersed Nuclear Elements (LINE1), and Short Interspersed Nuclear Elements (SINE B1) is significantly upregulated in Zfp819-knockdown (Zfp819_KD) cells. In line with the activation of ERVs, we observed the occurrence of spontaneous DNA damage in Zfp819_KD cells. Furthermore, we tested whether Zfp819 can interact with KAP1, a KRAB-associated protein with a transcriptional repression function, and found the interaction between these two proteins in both in vitro and in vivo experiments. The challenging of Zfp819_KD cells with DNA damaging agent revealed that these cells are inefficient in repairing the damaged DNA, as cells showed presence of γH2A.X foci for a prolonged time. Collectively, our study identified Zfp819 as a novel pluripotency-related factor and unveiled its function in genomic integrity maintenance mechanisms of mouse embryonic stem cells.
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http://dx.doi.org/10.1016/j.scr.2013.07.006DOI Listing
November 2013

Mouse Dazl and its novel splice variant functions in translational repression of target mRNAs in embryonic stem cells.

Biochim Biophys Acta 2013 May 6;1829(5):425-35. Epub 2013 Jan 6.

University of Goettingen, Goettingen, Germany.

Dazl (deleted in azoospermia-like) is an RNA binding protein that is important for germ cell differentiation in vertebrates. In the present study, we report the identification of a novel Dazl isoform (Dazl_Δ8) that results from alternative splicing of exon8 of mouse Dazl. We observed the expression of Dazl_Δ8 in various pluripotent cell types, but not in somatic cells. Furthermore, the Dazl_Δ8 splice variant was expressed along with the full-length isoform of Dazl (Dazl_FL) throughout male germ-cell development and in the ovary. Sub-cellular localization studies of Dazl_Δ8 revealed a diffused cytoplasmic and large granular pattern, which is similar to the localization patterns of Dazl_FL protein. In contrast to the well documented translation stimulation function in germ cells, overexpression and downregulation studies of Dazl isoforms (Dazl_FL and Dazl_Δ8) revealed a role for Dazl in the negative translational regulation of Mvh, a known target of Dazl, as well as Oct3/4 and Sox2 in embryonic stem cells (ESCs). In line with these observations, a luciferase reporter assay with the 3'UTRs of Oct3/4 and Mvh confirmed the translational repressive role of Dazl isoforms in ESCs but not in germ cells derived cell line GC-1. Further, we identified several putative target mRNAs of Dazl_FL and Dazl_Δ8 in ESCs through RNA-binding immunoprecipitation followed by whole genome transcriptome analysis. Collectively, our results show a translation repression function of Dazl in pluripotent stem cells.
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http://dx.doi.org/10.1016/j.bbagrm.2012.12.010DOI Listing
May 2013

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

PLoS One 2011 25;6(7):e22413. Epub 2011 Jul 25.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022413PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143132PMC
December 2011