Hiroki Kurihara

Although aortic stenosis (AS) represents a major cardiovascular burden, the molecular mechanisms underlying aortic valve calcification remain unclear. Spatial transcriptomic analysis of injured valve leaflets identified Cthrc1 (collagen triple helix repeat containing 1) as a prominently induced factor within the valvular interstitium. CTHRC1 elevation in expanded valvular interstitial cells (VICs) of thickened murine valve leaflets was validated by immunohistochemistry, and similar CTHRC1 upregulation was confirmed in human stenotic aortic valves. Lineage-tracing analyses demonstrated that the expanded VIC population in thickened murine valve leaflets following wire injury was derived predominantly from PDGFRβ-positive interstitial cells, with little contribution from endothelial cells. Although hemodynamic alterations were comparable between Cthrc1 ^−/− and wild-type AS mice, Cthrc1 deficiency markedly exacerbated valvular calcification during the chronic phase. Further spatial transcriptomic analyses of Cthrc1 ^−/− AS mice revealed extensive accumulation of Galectin-3-positive foamy macrophages within the interstitial space of stenotic valves in Cthrc1 ^−/− mice compared with wild-type controls. Moreover, stenotic valves from Cthrc1 ^−/− mice exhibited depletion of respiratory-chain signatures and upregulation of cell death–associated pathways, suggesting that CTHRC1, transiently induced during the acute phase following valvular endothelial injury, protects against subsequent dystrophic calcification.

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox -positive pharyngeal NCCs to Hox -negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and gene-regulatory network architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox -codes. A Meis2 – Sox9 – Scx gene-regulatory network defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox -dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

Hematopoietic stem cells (HSCs) self-renew to expand their pool but often differentiate upon division. Thus, it remains unclear how HSCs maintain their stemness during or after division. Here, we show that the suppression of glutamate dehydrogenase (Glud1), a key enzyme responsible for glutamate (Glu) catabolism, supports the maintenance of HSCs during division. We find that self-renewing mouse HSCs during hematopoietic regeneration exhibited lower intracellular Glu levels compared to HSCs differentiating during ex vivo culture. We therefore focus on the negative regulation of Glu catabolic reactions as a candidate factor to maintain HSCs during division, and find that treatment with the Glud1 inhibitor, R162, maintains a subset of HSCs that would otherwise differentiate during division under normal culture conditions. This treatment successfully results in an approximately 500-fold expansion of functional mouse HSCs that maintain expression pattern of conventional stem cell markers over a 30-day culture period. Importantly, this positive effect of Glud1 inhibition is independent of suppressing cell divisions and dependent on robust JAK2-STAT signaling. Overall, our findings propose that Glud1 inhibition, coupled with robust JAK2–STAT signaling, characterizes a functional HSC state during division, enabling ex vivo expansion of mouse HSCs under conventional culture conditions.

G protein-coupled receptors (GPCRs) are dynamic allosteric proteins whose signaling properties are governed by reciprocal communication between extracellular ligand-binding sites and intracellular transducer interfaces. Although classical pharmacological models established the concept that ligand binding and G protein coupling are thermodynamically linked, recent structural, biophysical, and computational studies have revealed a far more complex picture in which GPCRs exist as ensembles of interconverting conformational states. Accumulating evidence indicates that G proteins function not only as downstream signaling effectors but also as endogenous allosteric modulators. By reshaping receptor conformational landscapes, G protein coupling can influence the structure and dynamics of orthosteric ligand-binding pockets, thereby regulating ligand affinity, binding kinetics, and receptor selectivity. These findings support a bidirectional model of GPCR signaling in which information is transmitted not only from ligand-binding sites to intracellular signaling partners but also in the reverse direction through receptor-wide allosteric networks. Disease-associated mutations of endothelin A receptor (ETAR) provide in vivo evidence that structural perturbations located far from orthosteric ligand-binding sites can alter ligand recognition through long-range allosteric communication. In addition, emerging studies of positive allosteric modulators demonstrate the therapeutic potential of selectively stabilizing ligand–receptor–G protein complexes. These observations suggest that ligand recognition, receptor activation, and transducer coupling should be viewed as integrated properties of a dynamic receptor–ligand–transducer ensemble. This perspective provides a conceptual framework that links classical GPCR pharmacology, structural biology, disease mechanisms, and next-generation drug discovery.

Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.

Coronary arteries are a critical part of the vascular system and provide nourishment to the heart. In humans, even minor defects in coronary arteries can be lethal, emphasizing their importance for survival. However, some teleosts survive without coronary arteries, suggesting that there may have been some evolutionary changes in the morphology and function of coronary arteries in the tetrapod lineage. Here, we propose that the true ventricular coronary arteries were newly established during amniote evolution through remodeling of the ancestral coronary vasculature. In mouse (Mus musculus) and Japanese quail (Coturnix japonica) embryos, the coronary arteries unique to amniotes are established by the reconstitution of transient vascular plexuses: aortic subepicardial vessels (ASVs) in the outflow tract and the primitive coronary plexus on the ventricle. In contrast, amphibians (Hyla japonica, Lithobates catesbeianus, Xenopus laevis, and Cynops pyrrhogaster) retain the ASV-like vasculature as truncal coronary arteries throughout their lives and have no primitive coronary plexus. The anatomy and development of zebrafish (Danio rerio) and chondrichthyans suggest that their hypobranchial arteries are ASV-like structures serving as the root of the coronary vasculature throughout their lives. Thus, the ventricular coronary artery of adult amniotes is a novel structure that has acquired a new remodeling process, while the ASVs, which occur transiently during embryonic development, are remnants of the ancestral coronary vessels. This evolutionary change may be related to the modification of branchial arteries, indicating considerable morphological changes underlying the physiological transition during amniote evolution.

In this paper, we develop a cell tracking method based on persistent homological figure detection technology. We apply our tracking method to 9 different time-series cell images and extract several kinds of cell movements. Being able to analyze various images with a single method allows researchers to systematically understand and compare different tracking data. Persistent homological cell tracking technology’s 9 parameters all have clear meanings. Thus, researchers can decide the parameters not by black box trial-and-error but by the purpose of their analysis. We use model data with ground truth to see our method’s performance. We compare persistent homological figure detection and cell tracking technology with Image-Pro, sure-foreground in watershed method, and cell detection methods in previous studies. We see that there are some cases where Image-Pro’s tracking stops and requires manual plots, while our method does not require manual plots. We show that our technology includes sure-foreground and has more information, and can be applied to different types of data that previously needed different methods. We also show that our technology is powerful as a detection technology by applying the technology to 5 different types of cell images.

Mutations of G protein coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here we establish p.E303K in the gene encoding the endothelin receptor type A (ET_AR/EDNRA) as a recurrent mutation causing Mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibit a partial maxillary-to-mandibular transformation, which is rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ET_AR mutations as gain-of-function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increases flexibility of this portion of the helix, thus favoring G-protein binding and leading to G-protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduces the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, that may provide the basis for drug design targeting GPCRs.