記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Heart Failure Clinics  

DOI： 10.1016/j.hfc.2022.02.010

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Circulation Research  

Rationale: Cancer therapy can be associated with short- and long-term cardiac dysfunction. Patients with cancer often exhibit therapy-related clonal hematopoiesis (t-CH), an aggressive form of clonal hematopoiesis that can result from somatic mutations in genes encoding regulators of the DNA-damage response (DDR) pathway. Gain-of-function mutations in exon 6 of the protein phosphatase Mg2+/Mn2+ dependent 1D (PPM1D) gene are the most frequently mutated DNA-damage response gene associated with t-CH. Whether t-CH can contribute to cardiac dysfunction is unknown. Objective: We evaluated the causal and mechanistic relationships between Ppm1d-mediated t-CH and nonischemic heart failure in an experimental system. Methods and Results: To test whether gain-of-function hematopoietic cell mutations in Ppm1d can increase susceptibility to cardiac stress, we evaluated cardiac dysfunction in a mouse model where clonal hematopoiesis-associated mutations in exon 6 of Ppm1d were produced by CRISPR-Cas9 technology. Mice transplanted with hematopoietic stem cells containing the mutated Ppm1d gene exhibited augmented cardiac remodeling following the continuous infusion of Ang II (angiotensin II). Ppm1d-mutant macrophages were impaired in DDR pathway activation and displayed greater DNA damage, higher reactive oxygen species generation, and an augmented proinflammatory profile with elevations in IL (interleukin)-1β and IL-18. The administration of an NLRP3 (NLR family pyrin domain containing 3) inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d-mutated hematopoietic stem cells under conditions of Ang II-induced stress. Conclusions: A mouse model of Ppm1d-mediated t-CH was more susceptible to cardiac stress. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that t-CH involving activating mutations in PPM1D can contribute to the cardiac dysfunction observed in cancer survivors, and that anti-inflammatory therapy may have utility in treating this condition.

DOI： 10.1161/CIRCRESAHA.121.319314

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Jci Insight  

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.

DOI： 10.1172/jci.insight.135204

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Jacc Basic to Translational Science  

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.

DOI： 10.1016/j.jacbts.2019.08.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FASEB Journal  

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.—Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice. FASEB J. 33, 14147-14158 (2019). www.fasebj.org.

DOI： 10.1096/fj.201900786R

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1247/csf.16011

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Angiogenesis  

Objective: Adipose-derived regenerative cells (ADRCs) are promising cell sources for damaged tissue regeneration. The efficacy of therapeutic angiogenesis with ADRC implantation in patients with critical limb ischemia has been demonstrated in clinical studies. There are several possible mechanisms in this process such as cytokines and microRNA. Recently, cell-to-cell transfer of mitochondria gains more attention in regenerative medicine. However, the role of the mitochondrial transfer mechanism in ADRCs in the regeneration of functional tissue perfusion following ischemic injury remains unclear. In this study, we aimed to investigate whether mitochondrial transfer is a potential mechanism of therapeutic angiogenesis in ADRCs using a murine hindlimb ischemia model. Methods and results: In initial studies, the occurrence of mitochondrial transfer of ADRC to endothelial cells and macrophages in a series of pro-angiogenic effects of ADRC was demonstrated in a mouse model of hindlimb ischemia. Subsequently, we comprehensively elucidated the modes of mitochondrial transfer from ADRCs to HUVECs and macrophages mediated by Connexin43-based gap junctions and tunneling nanotubes using time-lapse confocal microscopy and cell sorting techniques. Furthermore, mitochondrial transfer from ADRCs enhanced mitochondrial biogenesis and angiogenesis in vascular endothelial cells and shifted macrophages toward the M2-phenotype. Notably, partially canceled mitochondrial transfer from ADRCs could impede the angiogenic ability of ADRCs in hind limb ischemia. Conclusions: ADRCs can protect against ischemic limbs, at least in part by mitochondrial transfer via gap junctions and tunneling of nanotubes into injured endothelial cells and macrophages. Additionally, mitochondrial transfer is a potential mechanism for therapeutic angiogenesis with ADRCs in hindlimb ischemia.

DOI： 10.1007/s10456-025-10001-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oncologist  

Background While immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, they can trigger severe immune-related adverse events (irAEs). The safety and efficacy of ICI retreatment after severe irAEs remain poorly understood. Methods We conducted a retrospective analysis of 1271 patients with malignancies treated with ICIs at a university hospital in Japan between September 2014 and June 2023. We evaluated the incidence and characteristics of severe irAEs, defined as grade ≥3, and the safety and efficacy of ICI retreatment. Results Severe irAEs occurred in 222 patients (17.5%). Patients with single endocrinopathies were excluded, and 46 (28.4%) of the remaining 162 patients underwent ICI retreatment. Upon retreatment, 14 patients (30.4%) experienced recurrent or new grade ≥2 irAEs. One patient who experienced hepatotoxicity (grade 3) at initial ICI treatment developed a recurrence (grade 4). Regarding antitumor response, the objective response rate to retreatment was 28.3% (13/46), with 10.9% achieving complete and 17.4% partial response. The median duration of ICI administration after retreatment was 218 days (95% confidence interval [CI]: 84-399). At 1 year after retreatment, 15.4% (95% CI: 6.8-27.4) of patients discontinued due to irAEs, 44.4% (95% CI: 29.7-58.1) due to disease progression, 6.6% (95% CI: 1.7-16.3) completed planned treatment, and 33.4% (95% CI: 20.3-47.2) continued treatment. Conclusions ICI retreatment after severe irAEs demonstrated a manageable safety profile and promising efficacy, even in patients with grade ≥3 irAEs. ICI retreatment may be a viable option for patients with limited alternatives, particularly those showing favorable antitumor responses at initial treatment.

DOI： 10.1093/oncolo/oyaf120

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Despite the efficacy of many therapies for heart failure, it remains a leading cause of morbidity and mortality worldwide, with many patients progressing to advanced stages of the condition. Since the standard treatment for heart failure includes small-molecule drugs targeting G protein-coupled receptors (GPCRs), GPCRs are still considered novel targets for the diagnosis and treatment of cardiovascular diseases. Corticotropin-releasing hormone receptor 2, a highly expressed GPCR in cardiomyocytes, and its ligand, urocortin2 (UCN2), have been reportedly associated with cardiovascular diseases; however, their clinical significance remains unclear. In this study, a UCN2 measurement assay was developed to measure blood UCN2 levels in patients with heart failure. The assay showed that blood UCN2 values indicated a negative relationship with cardiac ejection fraction in 52 patients with heart failure. Blood UCN2 levels were not correlated with brain natriuretic peptide, a standard marker of heart failure, and were higher in patients with cardiomyopathy than in those with heart failure, suggesting that measuring blood UCN2 levels may be a novel test for assessing the pathophysiology of heart failure.

DOI： 10.1038/s41598-025-99509-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Physiological Reports  

Chronic kidney disease is a life-threatening disease worldwide. PPARα is a crucial transcriptional regulator of lipid metabolism and inflammation. Here, we examine whether a novel selective PPARα modulator, pemafibrate modulates renal injury in a model of unilateral ureteral obstruction (UUO). Administration of pemafibrate to wild-type (WT) mice led to reduction of renal dysfunction and fibrosis after UUO with accompanying increases in plasma levels of fibroblast growth factor (FGF) 21 and ketone body β-hydroxybutyrate (BHB). Treatment of WT mice with FGF21 or BHB precursor resulted in attenuation of renal fibrotic and inflammatory responses after UUO. Treatment of proximal tubular cells with FGF21 or BHB reduced expression of epithelial–mesenchymal transition markers. These findings suggest that pemafibrate could ameliorate renal damage, at least in part, by its abilities to increase the production of FGF21 and BHB.

DOI： 10.14814/phy2.70135

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers in Cellular Neuroscience  

Introduction: Recent work has revealed that clonal hematopoiesis (CH) is associated with a higher risk of numerous age-related diseases, including ischemic stroke, however little is known about whether it influences stroke outcome independent of its widespread effects on cardiovascular disease. Studies suggest that leukocytes carrying CH driver mutations have an enhanced inflammatory profile, which could conceivably exacerbate brain injury after a stroke. Methods: Using a competitive bone marrow transplant model of Tet2-mediated CH, we tested the hypothesis that CH would lead to a poorer outcome after ischemic stroke by augmenting brain inflammation. Stroke was induced in mice by middle cerebral artery occlusion and neurological outcome was assessed at acute (24 h) and subacute (14 d) timepoints. Brains were collected at both time points for histological, immunofluorescence and gene expression assays. Results: Unexpectedly, Tet2-mediated CH had no effect on acute stroke outcome but led to a reduction in neurological deficits during the subacute phase. This improved neurological outcome was associated with lower levels of brain inflammation as evidenced by lower transcript levels of various inflammatory molecules alongside reduced astrogliosis. Discussion: These findings suggest that Tet2-mediated CH may have beneficial effects on outcome after stroke, contrasting with the conventional understanding of CH whereby leukocytes with driver mutations promote disease by exacerbating inflammation.

DOI： 10.3389/fncel.2024.1487867

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FASEB Journal  

Protein phosphorylation, controlled by protein kinases, is central to regulating various pathophysiological processes, including cardiac systolic function. The dysregulation of protein kinase activity plays a significant role in the pathogenesis of cardiac systolic dysfunction. While cardiac contraction mechanisms are well documented, the mechanisms underlying cardiac diastole remain elusive. This gap persists owing to the historical focus on systolic dysfunction in heart failure research. Recently, heart failure with preserved ejection fraction (HFpEF), an age-related disease characterized by cardiac diastolic dysfunction, has emerged as a major public health concern. However, its underlying mechanism remains unclear. In this study, we investigated cardiac protein kinases by analyzing the gene expression of 518 protein kinases in human tissues. We identified alpha-kinase 2 (ALPK2) as a novel cardiac-specific atypical kinase and generated tamoxifen-inducible, cardiomyocyte-specific Alpk2-knockout mice and Alpk2-overexpressing mice. Alpk2 deficiency did not affect cardiac systolic dysfunction in the myocardial infarction model or the pressure-overload-induced heart failure model. Notably, cardiomyocyte-specific Alpk2 deficiency exacerbated cardiac diastolic dysfunction induced by aging and in the HFpEF model. Conversely, Alpk2 overexpression increased the phosphorylation of tropomyosin 1, a major regulator that binds myosin to actin, and mitigated cardiac stiffness in HFpEF. This study provides novel evidence that ALPK2 represents a potential therapeutic target for cardiac diastolic dysfunction in HFpEF and age-related cardiac impairments.

DOI： 10.1096/fj.202402103R

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

DOI： 10.1038/s41467-024-52068-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Heart Failure Reviews  

Improvements in therapies for heart failure with preserved ejection fraction (HFpEF) are crucial for improving patient outcomes and quality of life. Although HFpEF is the predominant heart failure type among older individuals, its prognosis is often poor owing to the lack of effective therapies. The roles of the spleen and bone marrow are often overlooked in the context of HFpEF. Recent studies suggest that the spleen and bone marrow could play key roles in HFpEF, especially in relation to inflammation and immune responses. The bone marrow can increase production of certain immune cells that can migrate to the heart and contribute to disease. The spleen can contribute to immune responses that either protect or exacerbate heart failure. Extramedullary hematopoiesis in the spleen could play a crucial role in HFpEF. Increased metabolic activity in the spleen, immune cell production and mobilization to the heart, and concomitant cytokine production may occur in heart failure. This leads to systemic chronic inflammation, along with an imbalance of immune cells (macrophages) in the heart, resulting in chronic inflammation and progressive fibrosis, potentially leading to decreased cardiac function. The bone marrow and spleen are involved in altered iron metabolism and anemia, which also contribute to HFpEF. This review presents the concept of an interplay between the heart, spleen, and bone marrow in the setting of HFpEF, with a particular focus on extramedullary hematopoiesis in the spleen. The aim of this review is to discern whether the spleen can serve as a new therapeutic target for HFpEF.

DOI： 10.1007/s10741-024-10418-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Science  

Hematopoietic mosaic loss of Y chromosome (mLOY) is associated with increased risk of mortality and age-related diseases in men, but the causal and mechanistic relationships have yet to be established. Here, we show that male mice reconstituted with bone marrow cells lacking the Y chromosome display increased mortality and age-related profibrotic pathologies including reduced cardiac function. Cardiac macrophages lacking the Y chromosome exhibited polarization toward a more fibrotic phenotype, and treatment with a transforming growth factor b1–neutralizing antibody ameliorated cardiac dysfunction in mLOY mice. A prospective study revealed that mLOY in blood is associated with an increased risk for cardiovascular disease and heart failure–associated mortality. Together, these results indicate that hematopoietic mLOY causally contributes to fibrosis, cardiac dysfunction, and mortality in men.

DOI： 10.1126/science.abn3100

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cardiovascular Research  

Clonal haematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to haematopoietic stem and progenitor cells (HSPCs) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leucocytes 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 haematological 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.

DOI： 10.1093/cvr/cvab215

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Aging  

Cellular metabolism and environmental interactions generate molecular damage affecting all levels of biological organization. Accumulation of this damage over time is thought to have a central role in the aging process. Insufficient attention has been paid to the role of molecular damage in aging-related phenotypes, particularly in humans, in part because of the difficulty in measuring its various forms. Recently, omics approaches have been developed that begin to address this challenge, because they can assess a sizable proportion of age-related damage at the level of small molecules, proteins, RNA, DNA, organelles and cells. This Review describes the concept of molecular damage in aging and discusses its diverse aspects from theoretical models to experimental approaches. Measurement of multiple types of damage enables studies of the role of damage in aging and lays a foundation for testing interventions that reduce the burden of molecular damage, thereby targeting aging.

DOI： 10.1038/s43587-021-00150-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Jci Insight  

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.

DOI： 10.1172/jci.insight.146076

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Visualized Experiments  

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.

DOI： 10.3791/61875

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：公益社団法人 日本薬理学会  

<p>Dopamine (DA) activates MAPK via PKA/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R)in the nucleus accumbens (NAc), thereby regulating reward-related behavior.However, howMAPKregulates reward-relatedlearning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we performed proteomic analysis using affinity beads coated with CREB-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identified more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We found that MAPK phosphorylated Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impaired cocaine-induced place preference, which was rescued by Npas4-WT but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory. (Funahashi et al., Cell Reports, 2019)</p>

DOI： 10.1254/jpssuppl.93.0_1-yia-26

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Circulation  

Background: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. Methods: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1^flox/flox and Pkn2^flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. Results: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. Conclusions: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

DOI： 10.1161/CIRCULATIONAHA.119.041019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Jacc Basic to Translational Science  

Janus kinase 2 (valine to phenylalanine at residue 617) (JAK2^V617F) 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 JAK2^V617F 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 JAK2^V617Fmutations arising in myeloid progenitor cells may contribute to cardiovascular disease by promoting the proinflammatory properties of circulating myeloid cells.

DOI： 10.1016/j.jacbts.2019.05.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Visualized Experiments  

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.

DOI： 10.3791/59977

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Circulation  

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.

DOI： 10.1161/CIRCULATIONAHA.118.038820

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.freeradbiomed.2017.10.068

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.freeradbiomed.2017.10.050

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca²⁺ waves, which are delivered from the growing axon to the cell body. These Ca²⁺ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca²⁺ wave is responsible for neuronal polarization.

DOI： 10.1038/s41467-017-00044-2

Open Access

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Current Protocols in Cell Biology  

Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology.

DOI： 10.1002/cpcb.8

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本細胞生物学会  

Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinasemediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophyrelated gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.

DOI： 10.1247/csf.16011

Open Access

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PubMed

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Cell Biology  

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.

DOI： 10.1083/jcb.201412008

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Cardiology  

DOI： 10.1016/j.ijcard.2015.03.379

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本細胞生物学会  

Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ ROCK2 was confirmed to be a novel substrate of PKA both in vitro and in vivo. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.

DOI： 10.1247/csf.14014

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CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本細胞生物学会  

Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pinl-WW, CHEK2-FHA, and DLG1-GK Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.

DOI： 10.1247/csf.15004

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PubMed

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Hypertension Research  

DOI： 10.1038/hr.2013.101

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J-GLOBAL

記述言語：英語   会議種別：口頭発表（一般）  

国名：アメリカ合衆国  

記述言語：英語   会議種別：ポスター発表  

開催地：Boston, Massachusetts   国名：アメリカ合衆国  

記述言語：英語   会議種別：口頭発表（一般）