Tomohisa Sakaue

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.

Background: Calcific aortic valve disease (CAVD) is characterized by progressive inflammatory and fibrocalcific remodeling. Although valvular interstitial cells (VICs) are generally considered to drive fibrosis and osteogenic remodeling, whether injury-activated VICs mount endogenous protective responses that preserve the valvular interstitial microenvironment and restrain calcification remains unknown. Methods: We performed spatial transcriptomic profiling of aortic valves in a mouse model of endothelial injury-induced CAVD to define early injury-responsive programs within the valvular interstitium. The cellular origin and spatial distribution of candidate protective factors were examined by immunohistochemistry and lineage tracing, and their relevance to human disease was assessed using stenotic aortic valves. The functional role of CTHRC1 was investigated using genetic Cthrc1 deficiency combined with longitudinal hemodynamic assessment, histological analysis, and spatial transcriptomic profiling. Results: Spatial transcriptomics identified Cthrc1 as a prominent component of an early injury-induced stromal response in the expanding valvular interstitium. CTHRC1 was strongly expressed in activated VICs within thickened murine valve leaflets and human stenotic aortic valves. Lineage tracing demonstrated that the expanded VIC population arose predominantly from PDGFRβ ⁺ resident interstitial cells, with minimal endothelial contribution. Despite comparable early hemodynamic responses to endothelial injury, Cthrc1 deficiency exacerbated chronic valvular calcification. Spatial profiling of Cthrc1 -deficient valves revealed pronounced interstitial accumulation of galectin-3 ⁺ foamy macrophages, accompanied by mitochondrial respiratory-chain signature loss and cell death-associated pathway activation. These findings indicate that transient CTHRC1 induction after endothelial injury defines an endogenous stromal protective response that preserves the valvular interstitial microenvironment and limits macrophage-associated tissue injury and subsequent dystrophic calcification. Conclusions: Injury-activated VICs are not merely effectors of pathological remodeling, but can engage an endogenous tissue-protective response through CTHRC1. These findings identify a previously unrecognized stromal defense mechanism linking endothelial injury to macrophage-associated inflammatory remodeling and dystrophic calcification and suggest CTHRC1-dependent stromal protection as a potential therapeutic axis for limiting CAVD progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease lacking clarity on the mechanisms linking epithelial injury to fibrotic remodeling. Here, we identify the microbiota-derived corisin as a potent, multifaceted driver of epithelial injury and pulmonary fibrosis. Leveraging targeted DNA sequencing of bronchoalveolar lavage fluid, we provide the first sequence-based identification of corisin in IPF patients and show that functional depletion of native corisin from patient bronchoalveolar lavage fluid abolishes its proapoptotic activity in alveolar epithelial cells. Synthetic corisin readily penetrates epithelial cells, localizes to mitochondria, and induces apoptosis, cellular senescence, and epithelial–mesenchymal transition, effects validated by single-cell transcriptomic analysis. High-throughput protein-interaction screening identifies the ubiquitin–proteasome system as the primary target, demonstrating that corisin enhances proteasome activity and disrupts epithelial proteostasis. Intracellular expression of native corisin recapitulates these cellular pathologies at concentrations relevant to human disease, confirming its high intrinsic potency. Most critically, transgenic mice constitutively expressing native corisin develop spontaneous, progressive pulmonary fibrosis and exhibit exacerbated injury and increased mortality following bleomycin challenge. Collectively, our findings establish corisin as a microbiota-derived effector that directly couples the collapse of epithelial proteostasis to multimechanistic cell-fate dysregulation and fibrotic remodeling, thereby defining a potent and causal microbial–epithelial axis in the pathogenesis of IPF.

Metastatic hormone-naïve prostate cancer (mHNPC) is a clinically aggressive form of prostate cancer characterized by early systemic dissemination and poor long-term outcomes; however, the intrinsic epithelial cell states present at diagnosis remain poorly defined. In this study, we performed single-cell transcriptomic profiling of diagnostic prostate biopsy specimens from five patients with treatment-naïve mHNPC using Fixed RNA Profiling. Integrated and case-specific analyses characterized epithelial heterogeneity and lineage-associated transcriptional programs. Across 17,825 high-quality single cells, epithelial heterogeneity was identified in all cases. In addition to luminal androgen receptor (AR)-dependent epithelial cells, reproducible basal-associated epithelial populations with reduced AR signaling and stem-like transcriptional features were observed across tumors. Epithelial–mesenchymal transition (EMT)-related transcriptional programs were detected across multiple epithelial states with inter-case variability without forming a distinct EMT cluster, whereas no transcriptionally discrete neuroendocrine epithelial cluster was identified at baseline. These findings demonstrate that treatment-naïve mHNPC harbors pre-existing basal-associated epithelial states that contribute to intrinsic tumor heterogeneity at diagnosis. The presence of AR-low and stem-like epithelial populations prior to systemic therapy suggests a potential role for lineage plasticity in the aggressive biological behavior of metastatic prostate cancer.

Chronic diabetic foot ulcers remain refractory to contemporary care, partly because durable repair requires reconstruction of an integrated, multicellular regenerative niche—an outcome rarely achieved without complex ex vivo manufacturing. We advance a patient-as-bioreactor paradigm in which in-body tissue architecture (iBTA) repurposes the body for in vivo manufacturing of autologous multicellular regenerative constructs (“biococktails”) using a temporary subcutaneous device. In a first-in-human exploratory study, 37 devices were successfully implanted, and the primary endpoint—generation of ≥ 1 construct meeting predefined quality criteria—was achieved in all 10 patients. A single application initiated sustained wound repair, reaching a mean 84.15% wound-area reduction at 12 weeks. Reproducible, self-assembled niches enriched for stem cell–associated programs, reparative macrophage signatures, nascent vasculature, extracellular matrix components, and broad regenerative signaling were observed. No amputations or treatment-related serious adverse events occurred. These findings establish iBTA as a scalable, patient-specific in vivo manufacturing platform for multicellular regenerative therapies.

The precise molecular pathways driving fibrosis and calcification in aortic valve leaflets remain poorly defined. Here, we present the first data indicating a role for caldesmon-1 (CALD1) in calcified aortic valve disease (CAVD) pathogenesis. Analysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets revealed that CALD1 shows prominent upregulation in aortic valve stenosis (AS) cases when compared to normal subjects. Histological examination demonstrated that CALD1 protein expression is elevated in calcified AS valves and co-localises with α-smooth muscle actin (a myofibroblast biomarker) and vimentin, indicating its association with activated valvular interstitial cells (VICs). Bioinformatic analysis showed that CALD1-positive cells predominantly synthesize extracellular matrix components, including COL1A1 . Functional experiments using CALD1-depleted VICs revealed that CALD1 is required for maintaining spindle-shaped morphology, actin polymerisation, and proliferative capacity. Moreover, CALD1 loss significantly impaired osteoblast differentiation and attenuated VIC calcification. Bulk RNA-seq combined with pathway analysis demonstrated that CALD1-mediated actin polymerisation positively regulates key osteogenic and valvulopathy-related genes, including RUNX2 and ALPL . Collectively, these findings identify CALD1 as a novel regulator of VIC phenotypic plasticity and osteogenic transition during CAVD progression, providing mechanistic insight and a target for potential AS therapy.

Background: Diabetic foot ulcers (DFUs) are a severe complication of diabetes, associated with high morbidity and amputation risk. In-body tissue architecture (iBTA)-induced autologous biococktails, generated using a newly designed device (BSM1), offer a novel in vivo regenerative approach using somatic pluripotent stem cells (PSCs).Methods: This exploratory, single-arm clinical study evaluated the feasibility, safety, and efficacy of BSM1-derived biococktails in ten patients with chronic DFUs. Biococktails were fabricated through subcutaneous embedding of the BSM1 device for three weeks and applied to DFUs. The primary endpoint was successful formation of PSC-containing biococktails. Secondary endpoints included wound closure, reduction in wound area and depth, granulation, and adverse events over 12 weeks. Histology and mRNA analyses were performed to characterize biococktail components.Findings: BSM1 embedding and extraction were successful in all participants (100%). All participants completed the study without major complications. Biococktails from 37 devices were applied at an average density of 0·35 g/cm² over a mean wound area of 10·24 cm2. During the embedding period, when only standard wound care was provided, wound area and depth showed minimal change. Following application, both parameters decreased, reaching statistically significant improvement at 2 and 3 months. At 3 months, wound area decreased by 84·15 ± 30·46% (median 98·48%), while wound depth halved within 1 week and decreased continuously thereafter. Histology confirmed the presence of markers associated with PSCs, mesenchymal stem cells, M2 macrophages, myofibroblasts, capillaries, and extracellular matrix components. Gene expression profiling identified mRNAs encoding growth factors (VEGFA, HGF, PDGF, TGF-β1) and anti-inflammatory cytokines (IL-10). No amputations or severe biococktail-related adverse events were observed.Interpretation: BSM1-derived autologous biococktails represent a safe, feasible, and innovative in vivo-assembled regenerative strategy that may induce sustained wound improvement beyond standard care in patients with chronic DFUs. Larger controlled studies are warranted.

Prostate-specific membrane antigen (PSMA) protein expression is induced during prostate cancer progression and metastasis. Recently, we reported that PSMA-positive vesicles released by prostate cancer cell lines enhanced vascular endothelial cell angiogenesis and that PSMA may be involved in tumor angiogenesis. Similarly, it is known that PSMA is upregulated in peritumoral vessels in renal cell carcinoma (RCC). In this study, we investigated the significance and molecular function of PSMA in RCC. PSMA immunohistochemical staining confirmed PSMA presence only in perinephric tumor vessels, and PSMA intensity was strongly correlated with recurrence rate and venous invasion. Spatial gene expression analysis revealed that FOLH1 expression, which codes PSMA, was upregulated in tumor blood vessels around renal cancer, and that angiogenesis-related pathways were enhanced. The 10,000 g pellet fraction of the renal cancer cell lines Caki1- and ACHN-conditioned medium (CM) induced PSMA positivity in human umbilical vein endothelial cells (HUVECs) and enhanced tube formation. Mass spectrometry indicated that the 10,000 g pellet fraction contained various kinds of growth factors, like GDF15 and MYDGF. RNA sequencing showed that supplementing HUVECs with RCC cell CM-enhanced angiogenesis-related signaling pathways. Conclusively, microvesicle components secreted by RCC cells transform vascular endothelial cells into PSMA-positive cells, enhancing angiogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease–modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients’ synovium and murine ankle tissue with arthritis. As CD45⁺CD11b⁺ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell–specific Bub1 conditional knockout (Bub1^ΔLysM) mice were generated. Bub1^ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum–transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi–quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1^ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)–stimulated bone marrow–derived macrophages (BMMs) obtained from Bub1^ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1^ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1β treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis–associated bone loss through inhibition of inflammation–mediated osteoclastogenesis. Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cell–specific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.