Biology subjects

Yokoyama, U.

Publications and source records attributed to Yokoyama, U..

5 recordsLinked to original sources

bioRxiv2025

Mechanosensitive Stanniocalcin-1 Attenuates Pulmonary Arterial Hypertension by Suppressing Smooth Muscle Cell Proliferation

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) is driven by progressive pulmonary vascular remodeling, particularly pulmonary arterial smooth muscle cell (PASMC) proliferation. Current combination vasodilator therapies have markedly improved outcomes; however, prognosis remains poor in subgroups such as patients with respiratory comorbidities, highlighting the need for novel therapies. Elevation of intravascular hydrostatic pressure is a hallmark of IPAH, yet its direct role in PASMC pathobiology remains largely unexplored. We developed a cyclic hydrostatic pressurization culture system to model hypertensive hemodynamic stress in vitro and identify pressure-responsive mediators. MethodsPASMCs from 4 patients with IPAH were exposed to high hydrostatic pressure (70/40 mmHg, 60 bpm). Transcriptomic profiling identified differentially expressed genes, validated by qPCR. Functional studies included piezo-type mechanosensitive ion channel component 1 (PIEZO1) modulation, recombinant human stanniocalcin-1 (rhSTC1) treatment, bromodeoxyuridine (BrdU) incorporation, and western blotting for cell-cycle regulators. In vivo, chronic hypoxia-induced pulmonary hypertension was assessed in wild-type and Stc1-/- mice by hemodynamic and histological analyses, with or without intratracheal rhSTC1 administration. ResultsRNA sequencing revealed STC1 as a robustly pressure-induced gene in IPAH PASMCs. PIEZO1 activation upregulated STC1, whereas knockdown blunted this response. Elevated STC1 expression was observed in PASMCs of IPAH lung tissues, and rhSTC1 reduced PASMC proliferation and increased p-p53, p21, and p27 expression. In the chronic hypoxia model, Stc1-/- mice exhibited higher right ventricular systolic pressure (RVSP) (43.7 {+/-} 1.3 vs. 30.6 {+/-} 0.9 mmHg) and greater pulmonary arterial medial thickness (39.1 {+/-} 2.5% vs. 26.5 {+/-} 1.3%) than wild-type mice. CD68-positive macrophages were increased in Stc1-/- mice under normoxia and further elevated with hypoxia. In wild-type and Stc1-/- PAH models, intratracheal administration of rhSTC1 markedly reduced medial thickening, CD68-positive macrophage accumulation, and RVSP in both wild-type and Stc1-/- mice. ConclusionsWe demonstrate that elevated hydrostatic pressure drives STC1 expression via PIEZO1, conferring potent anti-remodeling effects in IPAH. STC1 supplementation represents a potential therapeutic strategy that addresses an urgent medical need not fulfilled by conventional therapies. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe established a novel hydrostatic pressurization system to recapitulate idiopathic pulmonary arterial hypertension (IPAH) hemodynamic conditions in vitro. C_LIO_LIStanniocalcin-1 (STC1) is a hydrostatic pressure-responsive gene in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients, induced via the mechanosensitive receptor piezo type mechanosensitive ion channel component 1 (PIEZO1). C_LIO_LIExogenous STC1 suppresses PASMC proliferation and attenuates pulmonary vascular remodeling in chronic hypoxia-induced PAH models. C_LI What Are the Clinical Implications?O_LISTC1 supplementation represents a potential therapeutic strategy for IPAH, acting through a non-vasodilatory mechanism. C_LIO_LISTC1 supplementation may offer benefit in patients with limited response to current vasodilator therapies or with comorbid respiratory disease. C_LIO_LITargeting mechanotransduction pathways could expand treatment options for pulmonary hypertension. C_LI

molecular biology↗

bioRxiv2025

The coiled-coil domain of myosin-11 as a potential biomarker to detect patients with atherosclerotic cardiovascular disease risk

BackgroundAtherosclerosis leads to severe clinical complications, and biomarkers that could predict its progression are highly desired. Phenotypic changes and dysregulation of smooth muscle cells contribute substantially to both subclinical and clinical atherosclerosis. Plasma levels of myosin-11, a smooth muscle cell-specific myosin, have been reported to increase in patients with clinical manifestations of atherosclerosis, including coronary artery disease and/or peripheral artery disease. This study aimed to investigate the association between circulating myosin-11 levels and atherosclerotic cardiovascular disease (ASCVD) risk in patients without symptomatic atherosclerosis. Methods and resultsThis prospective study recruited 161 participants: 80 patients with either hypertension, dyslipidemia, or diabetes mellitus but without symptomatic atherosclerosis (ASCVD risk group); 50 patients with ischemic heart disease (IHD group); and 31 control participants (Controls). We developed rat monoclonal antibodies specific for the coiled-coil domain of myosin-11 and quantified circulating myosin-11 levels using an enzyme-linked immunosorbent assay with these antibodies. Myosin-11 levels were highest in the IHD group, followed by the ASCVD risk group. Both groups had significantly higher myosin-11 levels than Controls. The area under the receiver-operating characteristic curve of myosin-11 was 0.980 for IHD versus Controls and 0.874 for ASCVD risk versus Controls. Univariate analysis detected significant associations between myosin-11 levels and hypertension and age. The association between myosin-11 levels and hypertension remained significant after multivariate logistic regression analysis. ConclusionsCirculating myosin-11 levels are elevated in patients with ASCVD risk. Myosin-11 levels may be a useful biomarker for stratification of asymptomatic individuals with higher risk of ASCVD. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe developed a sandwich enzyme-linked immunosorbent assay (ELISA) consisting of rat monoclonal antibodies specific for the coiled-coil domain of myosin-11 to detect trace concentrations of myosin-11 in blood circulation. C_LIO_LIPlasma myosin-11 concentrations were elevated in both asymptomatic individuals with traditional risk factors for atherosclerotic cardiovascular disease and in patients with symptomatic coronary atherosclerosis. C_LIO_LIThis elevation may be a potential marker independent of other circulating biomarkers, traditional risk score, and non-invasive imaging analyses. C_LI What Are the Clinical Implications?O_LICirculating myosin-11 may serve as a potential biomarker for identifying dysregulation of arterial smooth muscle cells during the progression of atherosclerosis. C_LIO_LIFurther studies are warranted to evaluate whether myosin-11 levels, in combination with other reported biomarkers and risk scores, could aid in stratification of asymptomatic individuals with higher risk of ASCVD. C_LI

biochemistry↗

bioRxiv2024

A novel RyR2-selective stabilizer prevents and ameliorates severe arrhythmias in stress-induced ventricular tachycardia

Background and PurposeAberrant activation of the type 2 ryanodine receptor (RyR2) causes lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Developing drugs that suppress RyR2 hyperactivation may be key to novel arrhythmia treatments. This study evaluated the antiarrhythmic potential of Ryanozole, a recently developed novel RyR2 modulator with high affinity and selectivity, using CPVT mouse models harboring mutant RyR2s. Experimental approachIn vitro effects of Ryanozole were evaluated by ER Ca2+-based assay and [3H]Ryanodine binding assay using RyR2-expressing HEK293 cells. Two lines of mice with different arrhythmia severity, RyR2-R420W and -K4750Q, were employed for in vivo assessments. Intracellular Ca2+ signals were analyzed in isolated cardiomyocytes using Cal520. Antiarrhythmic effects were evaluated by ECG under catecholaminergic challenge in anesthetized mice and during spontaneous arrhythmias in conscious mice. ECG and echocardiographic parameters were evaluated before and after drug administration. Key resultsRyanozole inhibited both wild type and mutant RyR2s with similar IC50 of 15-40 nM. The inhibition was more potent at lower cytosolic Ca2+ concentrations. It suppressed Ca2+ waves and Ca2+ sparks without affecting action potential-evoked Ca2+ transients. Ryanozole effectively prevented adrenaline-induced arrhythmias and rapidly terminated spontaneous arrhythmias during daily activity. Importantly, Ryanozole did not impair cardiac conduction or contractility, unlike conventional antiarrhythmic drugs. Conclusions and implicationsRyanozole strongly suppresses RyR2 under diastolic Ca2+ conditions, thereby preventing the arrhythmogenic trigger of aberrant Ca2+ release. This mechanism likely provides potent antiarrhythmic effects while preserving cardiac function. Ryanozole is a promising therapeutic candidate treating RyR2-mediated arrhythmias, such as CPVT.

pharmacology and toxicology↗

bioRxiv2023

Spatiotemporal EP4-fibulin-1 expression is associated with vascular intimal hyperplasia

AimsCyclooxygenase-2- and microsomal prostaglandin E synthase-1-derived prostaglandin E2 (PGE2) are involved in vascular intimal hyperplasia (IH). Although extensive studies have revealed the roles of PGE2 receptors (EPs) in IH, spatiotemporal EP expressions and downstream targets have not been fully elucidated. In this study, we focused on EP4 and investigated its role in vascular IH. Methods and ResultsWe generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found prominent EP4 expression in the proliferative neointima 2 weeks after femoral artery wire injury. Expression of EP4 were returned to the baseline level 4 weeks after vascular injury (VI). Injury-induced IH was diminished in vascular smooth muscle cell (VSMC)-specific EP4 heterozygous deficient mice (Ptger4fl/+;SM22-Cre) 2 and 4 weeks after VI compared to SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-specific EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). Systemic EP4 antagonist administration reduced VI-induced IH in wild-type mice. We investigated the role of extracellular matrix proteins, as downstream regulated targets of EP4. Stimulation of EP4 increased mRNA and protein levels of fibulin-1 (a multifunctional glycoprotein) in Ptger4-Tg VSMCs. Fibulin-1C or -1D recombinant proteins increased VSMC proliferation, whereas proliferation was decreased in fibulin-1-deficient VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6-8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was upregulated by EP4 stimulation, and fibulin-1 and ECM1 proteins additively enhanced VSMC proliferation. Similar to EP4 expression, both fibulin-1 and ECM1 were abundantly expressed in the neointima 2 weeks after VI. Furthermore, injury-induced IH was attenuated in VSMC-specific fibulin-1 deletion mice (Fbln1fl/fl;SM22-Cre) compared to Fbln1fl/fl. ConclusionsEP4 was upregulated in proliferative IH, and EP4-induced fibulin-1 cooperated with ECM1 to promote IH through VSMC proliferation. The calcium binding EGF-like modules 6-8 of fibulin-1 are indicated to regulate cell proliferation. A Translational PerspectiveRecent advances in drug-eluting stents have significantly contributed to the reduction of vascular IH. However, the detailed mechanism underlying IH after stenting remains to be elucidated. We found that prostaglandin E2-EP4-induced fibulin-1 plays a role in IH through VSMC proliferation. It is well recognized that prostaglandin E2 plays a role in IH, but inhibition of cyclooxygenase-2 has side effects such as thrombogenesis. Because EP4 and fibulin-1 were upregulated specifically in the neointima after vascular injury, oral or local administration of an EP4 antagonist or the downregulation of fibulin-1 would be potential therapeutic strategies to restrain IH.

physiology↗

bioRxiv2020

Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

TRPM7 channel activity is negatively regulated by intracellular Mg2+. We previously reported that TRPM7 was inhibited by oxidative stress due to an enhancement of the inhibition by intracellular Mg2+. In the present study, we aimed to clarify the precise mechanism underlying the TRPM7 inhibition by oxidative stress induced by hydrogen peroxide (H2O2). Site-directed mutagenesis on full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. When C1809 or C1813 was mutated, full-length TRPM7 was not expressed on the plasma membrane. We, therefore, developed a novel approach in which the full-length TRPM7 is functionally reconstituted by co-expressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate individual proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than in full-length TRPM7. Co-expression of M7cd and M7kd attenuated the current inhibition by intracellular Mg2+, and the current was sensitive to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. A similar current reconstitution was observed when M7cd was co-expressed with the kinase inactive mutant M7kd-K1645R. Thus, it is suggested that the kinase activity is not essential for the reconstitution. Co-expression of M7cd and M7kd carrying a mutation at C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was also observed with M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation together with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by M7kd, and the cysteines in the zinc-binding motif might play a role in the oxidative stress response of TRPM7.

physiology↗