Publications by authors named "Soichi Sano"

40 Publications

Somatic Mosaicism in Biology and Disease.

Annu Rev Physiol 2021 Oct 12. Epub 2021 Oct 12.

Department of Cardiovascular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan; email:

Contrary to earlier beliefs, every cell in the individual is genetically different due to somatic mutations. Consequently, tissues become a mixture of cells with distinct genomes, a phenomenon termed somatic mosaicism. Recent advances in genome sequencing technology have unveiled possible causes of mutations and how they shape the unique mutational landscape of the tissues. Moreover, the analysis of sequencing data in combination with clinical information has revealed the impacts of somatic mosaicism on disease processes. In this review, we discuss somatic mosaicism in various tissues and its clinical implications for human disease. Expected final online publication date for the , Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-physiol-061121-040048DOI Listing
October 2021

Hematopoietic JAK2-mediated clonal hematopoiesis: AIM2 understand mechanisms of atherogenesis.

J Cardiovasc Aging 2021 6;1. Epub 2021 Jun 6.

Hematovascular Biology Center, Robert M, Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

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http://dx.doi.org/10.20517/jca.2021.06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360421PMC
June 2021

Employing the CRISPR-Cas System for Clonal Hematopoiesis Research.

Int J Phys Med Rehabil 2021 30;9(1). Epub 2020 Nov 30.

Department of Cardiovascular Research, University of Virginia, Charlottesville, Virginia, United States.

Clonal hematopoiesis is a state in which substantial fraction of hematopoietic stem cells acquire mutations in specific driver genes and expand in the absence of an overt hematological malignancy. Recent clinical studies have shown that clonal hematopoiesis increases likelihood of hematological malignancy and cardiovascular disease. While clinical studies have identified countless candidate driver genes associated with clonal hematopoiesis, experimental studies are required to evaluate causal and mechanistic relationships with disease processes. This task is technically difficult and expensive to achieve with traditional genetically engineered mice. The versatility and programmability of CRISPR-Cas system enables investigators to evaluate the pathogenesis of each mutation in experimental systems. Technical refinements have enabled gene editing in a cell type specific manner and at a single base pair resolution. Here, we summarize strategies to apply CRISPR-Cas system to experimental studies of clonal hematopoiesis and concerns that should be addressed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360470PMC
November 2020

The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Promotes Inflammation and Non-Ischemic Heart Failure in Mice.

Circ Res 2021 Sep 28;129(6):684-698. Epub 2021 Jul 28.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA (Y.Y., E.M.-Y., K.-D.M., N.C., A.H.P., H.O., K.H., H.D., M.A.E., M.S., Y.W., S.S., K.W.).

[Figure: see text].
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http://dx.doi.org/10.1161/CIRCRESAHA.121.319314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409899PMC
September 2021

TP53-mediated therapy-related clonal hematopoiesis contributes to doxorubicin-induced cardiomyopathy by augmenting a neutrophil-mediated cytotoxic response.

JCI Insight 2021 Jul 8;6(13). Epub 2021 Jul 8.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Therapy-related clonal hematopoiesis (t-CH) is often observed in cancer survivors. This form of clonal hematopoiesis typically involves somatic mutations in driver genes that encode components of the DNA damage response and confer hematopoietic stem and progenitor cells (HSPCs) with resistance to the genotoxic stress of the cancer therapy. Here, we established a model of TP53-mediated t-CH through the transfer of Trp53 mutant HSPCs to mice, followed by treatment with a course of the chemotherapeutic agent doxorubicin. These studies revealed that neutrophil infiltration in the heart significantly contributes to doxorubicin-induced cardiac toxicity and that this condition is amplified in the model of Trp53-mediated t-CH. These data suggest that t-CH could contribute to the elevated heart failure risk that occurs in cancer survivors who have been treated with genotoxic agents.
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http://dx.doi.org/10.1172/jci.insight.146076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410064PMC
July 2021

Murine models of clonal hematopoiesis to assess mechanisms of cardiovascular disease.

Cardiovasc Res 2021 Jun 23. Epub 2021 Jun 23.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Clonal hematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to hematopoietic stem and progenitor cells (HSPC) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leukocytes leading to a situation whereby a substantial fraction of an individual's blood cells originate from the HSPC mutant clone. Although this condition rarely progresses to a hematological malignancy, circulating blood cells bearing the mutation have the potential to affect other organ systems as they infiltrate into tissues under both homeostatic and disease conditions. Epidemiological and clinical studies have revealed that CH is highly prevalent in the elderly and is associated with an increased risk of cardiovascular disease and mortality. Recent experimental studies in murine models have assessed the most commonly mutated "driver" genes associated with CH, and have provided evidence for mechanistic connections between CH and cardiovascular disease. A deeper understanding of the mechanisms by which specific CH mutations promote disease pathogenesis is of importance, as it could pave the way for individualized therapeutic strategies targeting the pathogenic CH gene mutations in the future. Here, we review the epidemiology of CH and the mechanistic work from studies using murine disease models, with a particular focus on the strengths and limitations of these experimental systems. We intend for this review to help investigators select the most appropriate models to study CH in the setting of cardiovascular disease.
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http://dx.doi.org/10.1093/cvr/cvab215DOI Listing
June 2021

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation.

J Am Heart Assoc 2021 07 22;10(13):e019904. Epub 2021 Jun 22.

Cardiovascular Research Center School of Medicine University of Virginia Charlottesville VA.

Background A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early proinflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase-like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after transverse aortic constriction surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2 knockout mice displayed a proinflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, transverse aortic constriction surgery led to the death of all mice by day 6 that was associated with myocardial hyperinflammation and vascular leakage. Conclusions Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.
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http://dx.doi.org/10.1161/JAHA.120.019904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403294PMC
July 2021

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis.

J Vis Exp 2021 05 26(171). Epub 2021 May 26.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine; Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine;

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.
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http://dx.doi.org/10.3791/61875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8439117PMC
May 2021

Clonal haematopoiesis and cardiovascular disease: how low can you go?

Eur Heart J 2021 01;42(3):266-268

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 415 Lane Road, Charlottesville, VA 22908, USA.

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http://dx.doi.org/10.1093/eurheartj/ehaa848DOI Listing
January 2021

TET2-Loss-of-Function-Driven Clonal Hematopoiesis Exacerbates Experimental Insulin Resistance in Aging and Obesity.

Cell Rep 2020 10;33(4):108326

Boston University School of Medicine, Boston, MA, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA, USA. Electronic address:

Human aging is frequently accompanied by the acquisition of somatic mutations in the hematopoietic system that induce clonal hematopoiesis, leading to the development of a mutant clone of hematopoietic progenitors and leukocytes. This somatic-mutation-driven clonal hematopoiesis has been associated with an increased incidence of cardiovascular disease and type 2 diabetes, but whether this epidemiological association reflects a direct, causal contribution of mutant hematopoietic and immune cells to age-related metabolic abnormalities remains unexplored. Here, we show that inactivating mutations in the epigenetic regulator TET2, which lead to clonal hematopoiesis, aggravate age- and obesity-related insulin resistance in mice. This metabolic dysfunction is paralleled by increased expression of the pro-inflammatory cytokine IL-1β in white adipose tissue, and it is suppressed by pharmacological inhibition of NLRP3 inflammasome-mediated IL-1β production. These findings support a causal contribution of somatic TET2 mutations to insulin resistance and type 2 diabetes.
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http://dx.doi.org/10.1016/j.celrep.2020.108326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856871PMC
October 2020

Nitroxide-enhanced MRI of cardiovascular oxidative stress.

NMR Biomed 2020 09 9;33(9):e4359. Epub 2020 Jul 9.

Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA.

Background: In vivo imaging of oxidative stress can facilitate the understanding and treatment of cardiovascular diseases. We evaluated nitroxide-enhanced MRI with 3-carbamoyl-proxyl (3CP) for the detection of myocardial oxidative stress.

Methods: Three mouse models of cardiac oxidative stress were imaged, namely angiotensin II (Ang II) infusion, myocardial infarction (MI), and high-fat high-sucrose (HFHS) diet-induced obesity (DIO). For the Ang II model, mice underwent MRI at baseline and after 7 days of Ang II (n = 8) or saline infusion (n = 8). For the MI model, mice underwent MRI at baseline (n = 10) and at 1 (n = 8), 4 (n = 9), and 21 (n = 8) days after MI. For the HFHS-DIO model, mice underwent MRI at baseline (n = 20) and 18 weeks (n = 13) after diet initiation. The 3CP reduction rate, K , computed using a tracer kinetic model, was used as a metric of oxidative stress. Dihydroethidium (DHE) staining of tissue sections was performed on Day 1 after MI.

Results: For the Ang II model, K was higher after 7 days of Ang II versus other groups (p < 0.05). For the MI model, K , in the infarct region was significantly elevated on Days 1 and 4 after MI (p < 0.05), whereas K in the noninfarcted region did not change after MI. DHE confirmed elevated oxidative stress in the infarct zone on Day 1 after MI. After 18 weeks of HFHS diet, K was higher in mice after diet versus baseline (p < 0.05).

Conclusions: Nitroxide-enhanced MRI noninvasively quantifies tissue oxidative stress as one component of a multiparametric preclinical MRI examination. These methods may facilitate investigations of oxidative stress in cardiovascular disease and related therapies.
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http://dx.doi.org/10.1002/nbm.4359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904044PMC
September 2020

Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.

JCI Insight 2020 03 26;5(6). Epub 2020 Mar 26.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We developed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2-mutant (Tet2-mutant) bone marrow cells into nonirradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell lineages. The expansion of the Tet2-mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was a negligible impact on the yolk sac-derived CCR2- cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether, we show that Tet2-mediated hematopoiesis contributes to cardiac dysfunction in a nonconditioned setting that faithfully models human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.
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http://dx.doi.org/10.1172/jci.insight.135204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213793PMC
March 2020

Clonal Hematopoiesis: A New Step Linking Inflammation to Heart Failure.

JACC Basic Transl Sci 2020 Feb 24;5(2):196-207. Epub 2020 Feb 24.

Hematovascular Biology Center and the Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia.

Heart failure is a common disease with poor prognosis that is associated with cardiac immune cell infiltration and dysregulated cytokine expression. Recently, the clonal expansion of hematopoietic cells with acquired (i.e., nonheritable) DNA mutations, a process referred to as clonal hematopoiesis, has been reported to be associated with cardiovascular diseases including heart failure. Mechanistic studies have shown that leukocytes that harbor these somatic mutations display altered inflammatory characteristics that worsen the phenotypes associated with heart failure in experimental models. In this review, we summarize recent epidemiological and experimental evidence that support the hypothesis that clonal hematopoiesis-mediated immune cell dysfunction contributes to heart failure and cardiovascular disease in general.
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http://dx.doi.org/10.1016/j.jacbts.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046537PMC
February 2020

Somatic mosaicism: implications for the cardiovascular system.

Eur Heart J 2020 08;41(30):2904-2907

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 415 Lane Road, Box 801394, Suite 1010, Charlottesville, VA 22908, USA.

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http://dx.doi.org/10.1093/eurheartj/ehz907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7421771PMC
August 2020

-Mediated Clonal Hematopoiesis Accelerates Pathological Remodeling in Murine Heart Failure.

JACC Basic Transl Sci 2019 Oct 18;4(6):684-697. Epub 2019 Sep 18.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia.

Janus kinase 2 (valine to phenylalanine at residue 617) ( ) mutations lead to myeloproliferative neoplasms associated with elevated myeloid, erythroid, and megakaryocytic cells. Alternatively these same mutations can lead to the condition of clonal hematopoiesis with no impact on blood cell counts. Here, a model of myeloid-restricted expression from lineage-negative bone marrow cells was developed and evaluated. This model displayed greater cardiac inflammation and dysfunction following permanent left anterior descending artery ligation and transverse aortic constriction. These data suggest that mutations arising in myeloid progenitor cells may contribute to cardiovascular disease by promoting the proinflammatory properties of circulating myeloid cells.
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http://dx.doi.org/10.1016/j.jacbts.2019.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834960PMC
October 2019

Cardiovascular Disease, Aging, and Clonal Hematopoiesis.

Annu Rev Pathol 2020 01 5;15:419-438. Epub 2019 Nov 5.

Hematovascular Biology Center and the Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA; email:

Traditional risk factors are incompletely predictive of cardiovascular disease development, a leading cause of death in the elderly. Recent epidemiological studies have shown that human aging is associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive advantage to a mutant cell, thus allowing for its clonal expansion, a phenomenon known as clonal hematopoiesis. Unexpectedly, these mutations have been associated with a higher incidence of cardiovascular disease, suggesting a previously unrecognized connection between somatic mutations in hematopoietic cells and cardiovascular disease. Here, we provide an up-to-date review of clonal hematopoiesis and its association with aging and cardiovascular disease. We also give a detailed report of the experimental studies that have been instrumental in understanding the relationship between clonal hematopoiesis and cardiovascular disease and have shed light on the mechanisms by which hematopoietic somatic mutations contribute to disease pathology.
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http://dx.doi.org/10.1146/annurev-pathmechdis-012419-032544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104598PMC
January 2020

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models.

J Vis Exp 2019 10 3(152). Epub 2019 Oct 3.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine;

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.
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http://dx.doi.org/10.3791/59977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249700PMC
October 2019

Wnt5a-Mediated Neutrophil Recruitment Has an Obligatory Role in Pressure Overload-Induced Cardiac Dysfunction.

Circulation 2019 08 7;140(6):487-499. Epub 2019 Jun 7.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville (Y. Wang, S.S., M.S., Y.Y., C.J., K.W.).

Background: Although the complex roles of macrophages in myocardial injury are widely appreciated, the function of neutrophils in nonischemic cardiac pathology has received relatively little attention.

Methods: To examine the regulation and function of neutrophils in pressure overload-induced cardiac hypertrophy, mice underwent treatment with Ly6G antibody to deplete neutrophils and then were subjected to transverse aortic constriction.

Results: Neutrophil depletion diminished transverse aortic constriction-induced hypertrophy and inflammation and preserved cardiac function. Myeloid deficiency of Wnt5a, a noncanonical Wnt, suppressed neutrophil infiltration to the hearts of transverse aortic constriction-treated mice and produced a phenotype that was similar to the neutropenic conditions. Conversely, mice overexpressing Wnt5a in myeloid cells displayed greater hypertrophic growth, inflammation, and cardiac dysfunction. Neutrophil depletion reversed the Wnt5a overexpression-induced cardiac pathology and eliminated differences in cardiac parameters between wild-type and myeloid-specific Wnt5a transgenic mice.

Conclusions: These findings reveal that Wnt5a-regulated neutrophil infiltration has a critical role in pressure overload-induced heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.038820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684855PMC
August 2019

Self-reactive CD4 IL-3 T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis.

J Exp Med 2019 02 22;216(2):369-383. Epub 2019 Jan 22.

Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA

Acquisition of self-reactive effector CD4 T cells is a major component of the autoimmune response that can occur during myocarditis, an inflammatory form of cardiomyopathy. Although the processes by which self-reactive T cells gain effector function have received considerable attention, how these T cells contribute to effector organ inflammation and damage is less clear. Here, we identified an IL-3-dependent amplification loop that exacerbates autoimmune inflammation. In experimental myocarditis, we show that effector organ-accumulating autoreactive IL-3 CD4 T cells stimulate IL-3R tissue macrophages to produce monocyte-attracting chemokines. The newly recruited monocytes differentiate into antigen-presenting cells that stimulate local IL-3 CD4 T cell proliferation, thereby amplifying organ inflammation. Consequently, mice resist developing robust autoimmune inflammation and myocardial dysfunction, whereas therapeutic IL-3 targeting ameliorates disease. This study defines a mechanism that orchestrates inflammation in myocarditis, describes a previously unknown function for IL-3, and identifies IL-3 as a potential therapeutic target in patients with myocarditis.
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http://dx.doi.org/10.1084/jem.20180722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363430PMC
February 2019

A dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, attenuates cardiac dysfunction after myocardial infarction independently of DPP-4.

J Pharmacol Sci 2019 Feb 18;139(2):112-119. Epub 2018 Dec 18.

Department of Pharmacology, Osaka City University Graduate School of Medicine, Osaka, Japan; Department of Internal Medicine, Takaishikamo Hospital, Osaka, Japan. Electronic address:

Dipeptidyl peptidase-4 (DPP-4) inhibitors not only improve impaired glucose tolerance in diabetes, but also have pleiotropic extra-pancreatic effects such as preconditioning effect for myocardial ischemia-reperfusion injury. Here, we investigated the anti-remodeling effects of linagliptin, a DPP-4 inhibitor, by use of DPP-4-deficient rats. After the induction of myocardial infarction (MI), Fischer 344 rats with inactivating mutation of DPP-4 were orally administrated with a DPP-4 inhibitor, linagliptin (5 mg kg·day), or vehicle in drinking water for 4 weeks. Linagliptin did not affect hemodynamic status, body weight, and infarct size. In echocardiography, linagliptin tended to improve left ventricular (LV) systolic function, and significantly improved LV diastolic function, surprisingly. Interstitial fibrosis in marginal region and macrophage infiltration were significantly lower in the linagliptin group than those in the vehicle group. Fibrosis-related gene expressions, such as collagen I and transforming growth factor-β1 (TGF-β1), and inflammation-related expressions, such as macrophage chemotactic protein 1 and matrix metalloproteinase-2 (MMP-2), were significantly suppressed in marginal area of the linagliptin-treated rats compared with the vehicle rats. The TGF-β1 and MMP-2 protein levels were attenuated by linagliptin in DPP-4-deficient cardiac fibroblasts. Linagliptin can attenuate MI-induced cardiac remodeling via a DPP-4-independent pathway.
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http://dx.doi.org/10.1016/j.jphs.2018.12.004DOI Listing
February 2019

Clonal Hematopoiesis and Its Impact on Cardiovascular Disease.

Circ J 2018 12 4;83(1):2-11. Epub 2018 Sep 4.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine.

A number of recent epidemiological studies have associated the clonal expansion of hematopoietic cells, a process referred to as clonal hematopoiesis, with increased mortality. Clonal hematopoiesis increases the risk of hematological cancer, but this overall risk cannot account for the increase in mortality in the general population. Surprisingly, these mutations have also been associated with higher rates of cardiovascular disease, suggesting a previously unrecognized link between somatic mutations in hematopoietic cells and chronic disease. Here, we review recent epidemiological and experimental studies on clonal hematopoiesis that relate to cardiovascular disease.
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http://dx.doi.org/10.1253/circj.CJ-18-0871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310086PMC
December 2018

CRISPR-Mediated Gene Editing to Assess the Roles of Tet2 and Dnmt3a in Clonal Hematopoiesis and Cardiovascular Disease.

Circ Res 2018 07 4;123(3):335-341. Epub 2018 May 4.

From the Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville (S.S., Y.W., K.W.)

Rationale: Clonal hematopoiesis has been associated with increased mortality and cardiovascular disease. This condition can arise from somatic mutations in preleukemic driver genes within hematopoietic stem/progenitor cells. Approximately 40 candidate driver genes have been identified, but mutations in only 1 of these genes, TET2 (ten-eleven translocation-2), has been shown to casually contribute to cardiovascular disease in murine models.

Objective: To develop a facile system to evaluate the disease characteristics of different clonal hematopoiesis driver genes using lentivirus vector and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) methodology. Using this methodology, evaluate whether Dnmt3a (DNA [cytosine-5]-methyltransferase 3a)-a commonly occurring clonal hematopoiesis driver gene-causally contributes to cardiovascular disease.

Methods And Results: Lentivirus vectors were used to deliver Cas9 and guide RNA to introduce inactivating mutations in Tet2 and Dnmt3a in lineage-negative bone marrow cells. After implantation into lethally irradiated mice, these cells were engrafted and gave rise to labeled blood cell progeny. When challenged with an infusion of Ang II (angiotensin II), mice with inactivating mutations in Tet2 or Dnmt3a displayed greater cardiac hypertrophy, diminished cardiac function, and greater cardiac and renal fibrosis. In comparison with Tet2, inactivation of Dnmt3a did not lead to detectable expansion of the mutant hematopoietic cells during the time course of these experiments. Tet2 inactivation promoted the expression of IL (interleukin) 1β, IL-6, and Ccl5, whereas Dnmt3a inactivation promoted the expression of Cxcl1 (CXC chemokine ligand), Cxcl2, IL-6, and Ccl5 in a lipopolysaccharide-stimulated macrophage cell line.

Conclusions: Experiments using lentivirus vector/CRISPR methodology provided evidence suggesting that inactivating DNMT3A mutations in hematopoietic cells contributes to cardiovascular disease. Comparative analyses showed that inactivation of Tet2 and Dnmt3 was similar in their ability to promote Ang II-induced cardiac dysfunction and renal fibrosis in mice. However, gene-specific actions were indicated by differences in kinetics of hematopoietic stem/progenitor cell expansion and different patterns of inflammatory gene expression.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054544PMC
July 2018

Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1β/NLRP3 Inflammasome.

J Am Coll Cardiol 2018 02;71(8):875-886

Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts. Electronic address:

Background: Recent studies have shown that hematopoietic stem cells can undergo clonal expansion secondary to somatic mutations in leukemia-related genes, thus leading to an age-dependent accumulation of mutant leukocytes in the blood. This somatic mutation-related clonal hematopoiesis is common in healthy older individuals, but it has been associated with an increased incidence of future cardiovascular disease. The epigenetic regulator TET2 is frequently mutated in blood cells of individuals exhibiting clonal hematopoiesis.

Objectives: This study investigated whether Tet2 mutations within hematopoietic cells can contribute to heart failure in 2 models of cardiac injury.

Methods: Heart failure was induced in mice by pressure overload, achieved by transverse aortic constriction or chronic ischemia induced by the permanent ligation of the left anterior descending artery. Competitive bone marrow transplantation strategies with Tet2-deficient cells were used to mimic TET2 mutation-driven clonal hematopoiesis. Alternatively, Tet2 was specifically ablated in myeloid cells using Cre recombinase expressed from the LysM promoter.

Results: In both experimental heart failure models, hematopoietic or myeloid Tet2 deficiency worsened cardiac remodeling and function, in parallel with increased interleukin-1beta (IL-1β) expression. Treatment with a selective NLRP3 inflammasome inhibitor protected against the development of heart failure and eliminated the differences in cardiac parameters between Tet2-deficient and wild-type mice.

Conclusions: Tet2 deficiency in hematopoietic cells is associated with greater cardiac dysfunction in murine models of heart failure as a result of elevated IL-1β signaling. These data suggest that individuals with TET2-mediated clonal hematopoiesis may be at greater risk of developing heart failure and respond better to IL-1β-NLRP3 inflammasome inhibition.
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http://dx.doi.org/10.1016/j.jacc.2017.12.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828038PMC
February 2018

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.

Science 2017 02 19;355(6327):842-847. Epub 2017 Jan 19.

Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA.

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of -mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient () mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic mutations in blood cells play a causal role in atherosclerosis.
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http://dx.doi.org/10.1126/science.aag1381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542057PMC
February 2017

Follistatin-like 1 promotes cardiac fibroblast activation and protects the heart from rupture.

EMBO Mol Med 2016 08 1;8(8):949-66. Epub 2016 Aug 1.

Department of Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA

Follistatin-like 1 (Fstl1) is a secreted protein that is acutely induced in heart following myocardial infarction (MI). In this study, we investigated cell type-specific regulation of Fstl1 and its function in a murine model of MI Fstl1 was robustly expressed in fibroblasts and myofibroblasts in the infarcted area compared to cardiac myocytes. The conditional ablation of Fstl1 in S100a4-expressing fibroblast lineage cells (Fstl1-cfKO mice) led to a reduction in injury-induced Fstl1 expression and increased mortality due to cardiac rupture during the acute phase. Cardiac rupture was associated with a diminished number of myofibroblasts and decreased expression of extracellular matrix proteins. The infarcts of Fstl1-cfKO mice displayed weaker birefringence, indicative of thin and loosely packed collagen. Mechanistically, the migratory and proliferative capabilities of cardiac fibroblasts were attenuated by endogenous Fstl1 ablation. The activation of cardiac fibroblasts by Fstl1 was mediated by ERK1/2 but not Smad2/3 signaling. This study reveals that Fstl1 is essential for the acute repair of the infarcted myocardium and that stimulation of early fibroblast activation is a novel function of Fstl1.
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http://dx.doi.org/10.15252/emmm.201506151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967946PMC
August 2016

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization.

Proc Natl Acad Sci U S A 2016 May 9;113(21):6011-6. Epub 2016 May 9.

Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys(520) (mouse Cys(533)). In addition, an HIF-1α Cys(520) serine mutant is resistant to 2-AAPA-induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys(520) promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.
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http://dx.doi.org/10.1073/pnas.1524198113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889374PMC
May 2016

miR-410 and miR-495 Are Dynamically Regulated in Diverse Cardiomyopathies and Their Inhibition Attenuates Pathological Hypertrophy.

PLoS One 2016 21;11(3):e0151515. Epub 2016 Mar 21.

Department of Biology, Program in Cell and Molecular Biology, Boston University, Boston, Massachusetts, United States of America.

Noncoding RNAs have emerged as important modulators in cardiac development and pathological remodeling. Recently, we demonstrated that regulation of the Gtl2-Dio3 noncoding RNA locus is dependent on the MEF2 transcription factor in cardiac muscle, and that two of its encoded miRNAs, miR-410 and miR-495, induce robust cardiomyocyte proliferation. Given the possibility of manipulating the expression of these miRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, it is important to determine whether the Gtl2-Dio3 noncoding RNAs are regulated in cardiac disease and whether they function downstream of pathological cardiac stress signaling. Therefore, we examined expression of the above miRNAs processed from the Gtl2-Dio3 locus in various cardiomyopathies. These noncoding RNAs were upregulated in all cardiac disease models examined including myocardial infarction (MI) and chronic angiotensin II (Ang II) stimulation, and in the cardiomyopathies associated with muscular dystrophies. Consistent with these observations, we show that the Gtl2-Dio3 proximal promoter is activated by stress stimuli in cardiomyocytes and requires MEF2 for its induction. Furthermore, inhibiting miR-410 or miR-495 in stressed cardiomyocytes attenuated the hypertrophic response. Thus, the Gtl2-Dio3 noncoding RNA locus is a novel marker of cardiac disease and modulating the activity of its encoded miRNAs may mitigate pathological cardiac remodeling in these diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801331PMC
August 2016

Secreted Frizzled-related Protein 5 Diminishes Cardiac Inflammation and Protects the Heart from Ischemia/Reperfusion Injury.

J Biol Chem 2016 Feb 2;291(6):2566-75. Epub 2015 Dec 2.

From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and

Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 KO and WT mice were subjected to ischemia/reperfusion (I/R). Although Sfrp5-KO mice exhibited no detectable phenotype when compared with WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis, and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression when compared with WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.
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http://dx.doi.org/10.1074/jbc.M115.693937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742726PMC
February 2016

Percutaneous Carbon Dioxide Treatment Using a Gas Mist Generator Attenuates the Development of Right Ventricular Dysfunction in Monocrotaline-induced Pulmonary Hypertensive Rats.

Osaka City Med J 2015 Jun;61(1):31-41

Background: Highly concentrated carbon dioxide (GO2) is useful for treating ischemic diseases. Therefore, we investigated whether treatment with a few micrometers of CO2 molecules, atomized by two fluid nozzles (CO2 mist), could attenuate the development of right ventricular (RV) dysfunction in pulmonary hypertensive rats.

Methods: Six-week-old male Wistar rats were divided into three groups: one that received injected saline; a second that received subcutaneous monocrotaline (MCT; 60 mg/kg) without treatment (PH-UT) group; and a third that received MCT with CO2 mist treatment (PH-CM) after MCT administration. The lower body of each rat was encased in a polyethylene bag, filled with the designated gaseous agent via a gas mist generator, for 30 minutes daily. Hemodynamics and cardiac function were measured at 28 days after beginning MCT administration. Protein levels were measured by western blotting.

Results: Rats that received MCT without treatment began to die within 3-4 weeks of the initial administration. However, treatment with CO2 mist extended the survival period of rats in that group. At 28 days after MCT administration, the hemodynamic status, such as the blood pressure and heart rate, involved with left ventricular function, of rats in the PH-UT group were similar to those of rats in the PH-CM group. However, MCT-induced RV weight and RV dysfunction were significantly attenuated by treatment with CO2 mist. Both RV phosphorylated endothelial nitric oxide synthase and heat shock protein 72 levels increased significantly in the PH-CM group, compared to the PH-UT group.

Conclusions: Percutaneous CO2 mist therapy may alleviate RV dysfunction in patients with pulmonary hypertension.
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June 2015

Repeated remote ischemic conditioning attenuates left ventricular remodeling via exosome-mediated intercellular communication on chronic heart failure after myocardial infarction.

Int J Cardiol 2015 Jan 30;178:239-46. Epub 2014 Oct 30.

Department of Pharmacology, Osaka City University Medical School, Osaka, Japan; Department of Education, Shitennoji University, Habikino, Japan.

Background: Remote ischemic conditioning (RIC) by repeated treatment of transient limb ischemia is a clinically applicable method for protecting the heart against injury at the time of reperfusion. In this study, we investigated the effects of repeated RIC on cardiac dysfunction after myocardial infarction (MI).

Methods And Results: At 4weeks after MI, rats were separated into the untreated (UT) group or the RIC-treated group. RIC treatment was performed by 5cycles of 5min of bilateral hindlimb ischemia and 5min of reperfusion once a day for 4weeks. Despite comparable MI size, left ventricular (LV) ejection fraction (LVEF) was significantly improved in the RIC group compared with the UT group. Furthermore, the LVEF in the RIC group was improved, although not significantly, after treatment. RIC treatment also prevented the deterioration of LV diastolic function. MI-induced LV interstitial fibrosis in the boundary region and oxidant stress were significantly attenuated by RIC treatment. MicroRNA-29a (miR-29a), a key regulator of tissue fibrosis, was highly expressed in the exosomes and the marginal area of the RIC group. Even in the differentiated C2C12-derived exosomes, miR-29a expression was significantly increased under hypoxic condition. As well as miR-29a, insulin-like growth factor 1 receptor (IGF-1R) was highly expressed both in the exosomes and remote non-infarcted myocardium of the RIC group. IGF-1R expression was also increased in the C2C12-derived exosomes under hypoxic conditions.

Conclusions: Repeated RIC reduces adverse LV remodeling and oxidative stress by MI. Exosome-mediated intercellular communication may contribute to the beneficial effect of RIC treatment.
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http://dx.doi.org/10.1016/j.ijcard.2014.10.144DOI Listing
January 2015
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