Tsutomu Kume

Aniridia, driven by PAX6 mutations, causes aniridia-associated keratopathy (AAK), a progressive condition linked to limbal stem cell deficiency. A major hurdle to developing targeted therapies for AAK is the incomplete understanding of the molecular abnormalities in affected corneas. To address this, we leveraged Pax6± (Pax6 het) mice, a model of AAK, and applied single-cell RNA sequencing (scRNA-seq) to profile the transcriptomic changes at a single-cell resolution. ScRNA-seq of corneal/limbal tissues of wild type (WT) and Pax6 het mice were conducted. Immunostaining was performed to examine the expression of specific markers for stem cells. ScRNA-seq identified a quiescent limbal epithelial stem cell (LESC)-like cell cluster and an early transient amplifying cell (eTAC)-like cluster. An increase in the cell numbers in these two clusters in the Pax6 het mouse corneas was observed. Immunostaining detected a marked increase in markers for these two clusters including Tmem176b, Apoe, and Krt15 in the corneal epithelium of Pax6 het mice, suggesting an increase of these LESC/eTA-like cells into the corneal epithelium. The Pax6 deficiency inhibited the expression of genes involved in cell proliferation in the eTAC-like cluster as well as the expression of genes related to corneal epithelial cell fate and differentiation compared with WT mice. Our single cell transcriptome of the limbus and cornea of Pax6 het mice indicates that AAK may be due to the increase of dysfunctional stem/eTACs with defects in committing to a corneal epithelial cell fate and differentiation.

Myocarditis, an inflammatory disease of the heart muscle, is a leading cause of sudden cardiac death in young adults and a major contributor to the development of dilated cardiomyopathy. Many studies highlight immune-mediated cardiomyocyte injury as a major contributor to myocarditis progression; however, myocardial edema may also play a significant role that has been overlooked. Beyond being a passive byproduct of inflammation, edema can mechanically stress the myocardium and create a proinflammatory microenvironment that may stimulate fibrosis, stiffen the myocardium, and impair cardiac function. Myocardial edema arises from an imbalance between vascular filtration and lymphatic clearance, often triggered by disrupted endothelial junctions that increase vascular permeability. The resulting accumulation of interstitial fluid fosters sustained inflammation, fibroblast activation, and extracellular matrix (ECM) remodeling. Furthermore, recent research highlights the therapeutic potential of targeting lymphatic regeneration to enhance edema resolution, attenuate inflammation, and limit fibrotic remodeling. This review examines the mechanistic pathways by which lymphatic dysfunction in myocarditis impairs lymphatic fluid clearance, focusing on the breakdown of lymphatic integrity, cytokine-mediated suppression of lymphangiogenesis, and maladaptive lymphatic remodeling. These processes contribute to adverse ventricular remodeling and dysfunction. Given that myocardial edema may be a key mediator for these pathological changes, we also discuss how emerging imaging techniques such as cardiac magnetic resonance (CMR) have enhanced the ability to detect and quantify edema, reinforcing its clinical relevance as both a diagnostic marker and prognostic indicator in myocarditis. Understanding the mechanistic pathways linking myocardial edema to pathology in myocarditis is promising for identifying novel therapeutic interventions.

Volumetric segmentation of Schlemm’s Canal (SC) in optical coherence tomography (OCT) is time-consuming, creating a barrier to experiments studying glaucoma and the anatomy of the trabecular outflow pathways in vivo . To this end, we developed an automated segmentation tool, Schlemm’s Canal-Localization and Semantic Segmentation (SC-LSS), for the volumetric segmentation of SC in in vivo mice eyes from visible-light OCT (vis-OCT). SC-LSS first localizes the boundaries of SC and subsequently determines the boundaries of SC within the localized region. We used 324 B-scans from 16 mouse eyes for training, validation, and testing the model, and 203 additional B-scans from 16 mouse eyes to evaluate the model’s accuracy. We found that the Dice coefficient between segmentations generated by SC-LSS and manual expert graders was 0.70 ± 0.20 and that the Dice coefficient between two expert graders was 0.73 ± 0.18 (p = 0.10). Furthermore, SC-LSS captured decreases in SC size with increasing intraocular pressure, yielding a 51.5% decrease in SC size at 20 mmHg compared to 5 mmHg. SC-LSS also identified a 20.1% increase in SC size following the administration of pilocarpine. We anticipate that SC-LSS will accelerate studies on factors regulating the trabecular outflow pathways and their role in glaucoma development and management.

The lymphatic system plays central roles in the maintenance of fluid balance, clearance of macromolecules, fat absorption, and immune surveillance. Lymphatic functions vary notably across organs and physiological contexts and play important roles in numerous pathological conditions (Janardhan et al., 2023, Mehrara et al., 2023). Despite recent progress in understanding these functions, the cellular and molecular mechanisms that govern the formation and regulation of lymphatic vascular heterogeneity in various organs and tissues remain incompletely defined. This Research Topic brought together a collection of three original research articles and five review articles from diverse disciplines -developmental biology, lymphatic malformations, inflammation, pathophysiology, bioengineering, and potential treatments -to deepen our understanding of organ-specific lymphatic functions in health and disease.Vascular endothelial growth factor receptor 3 (VEGFR3) signaling in lymphatic endothelial cells (Kuonqui et al., 2025). This is a comprehensive review detailing VEGFR3 pathway regulation in lymphatic development and function. It outlines key mediators downstream of the VEGFC/VEGFR3 signaling pathway in lymphatic endothelial cells (LECs) and their functional roles in lymphatic vessel physiology and discusses the regulation of overall VEGFR3 activity in lymphatic vessels.Lymphangiocrine signaling pathways in the heart and intestine (Kurup et al., 2023).Recent evidence indicates that LECs secrete paracrine (lymphoangiocrine) molecules that are specific to organ tissues. This review article provides a comprehensive overview of the crosstalk between LECs and adjacent cells to augment tissue regeneration in cardiac and intestinal disease. The roles of lymphangiocrine factors, including Reelin, Apelin, Adenomedulin, and R-Spondin 3, in the heart and intestine in tissue maintenance and self-renewal following injury are discussed. (Davis et al., 2024). This original research article is aimed at investigating the developmental progression of functional lymphatic valve leaflets that prevent backflow to maintain a unidirectional flow.Lymphatic valve development includes four main stages (stages 1-4) of morphogenic dynamics. Ex vivo tests of valve function using isolated murine mesenteric collecting lymphatic vessels reveal that valves become functional, preventing backflow between stages 3 and 4 of development. (Fernandes et al., 2023). Somatic activating mutations in KRAS cause complex lymphatic anomalies (CLAs). However, its pathogenesis remains largely unknown. This original research article demonstrates that hyperactive KRAS signaling caused by the KRAS mutation (p.G12D) results in enlarged lymphatic vessels in mouse embryos. This mutation induces cell spindling, proliferation, and migration in vitro. Importantly, this study also reveals the therapeutic potential of targeting hyperactive KRAS signaling in lymphatic anomalies associated with somatic KRAS mutations.These articles provide insights into the various functions of lymphatic vessels in different organs and tissues and how the extracellular signaling pathways, ranging from VEGFC/VEGFR3 to lymphangiocrine factors, contribute to maintaining proper lymphatic function and tissue homeostasis.Secondary lymphedema molecular pathology (Lee and Kim, 2024). This review article provides a comprehensive overview of the pathophysiology of secondary lymphedema, a condition that arises due to injury or obstruction of the lymphatic system, and most commonly and globally, filariasis. It highlights three consequences in lymphedema: (1) chronic inflammation-mediated lymphangiogenesis, which is dependent on the VEGF-C/VEGFR axis, (2) adipocyte hypertrophy and adipose tissue deposition, and (3) tissue fibrosis. Despite surgical treatments of reconstructing the lymphatic system to facilitate lymphatic fluid drainage, there is a limitation of their effectiveness in treating already damaged lymphatic vessels.Lung lymphatics in chronic obstructive pulmonary disease (COPD) (Trivedi et al., 2024). Using in vitro and in vivo models, this original research article reveals inflammatory and fibrinogenic alterations in pulmonary lymphatics within cigarette smoke-augmented COPD models. Immunohistochemistry of human lung tissue also shows increased expression of inflammatory markers in lymphatics in COPD patients. These studies indicate that the lung LECs undergo inflammatory changes associated with cigarette smoke exposure and increased thrombin in COPD. The results of this study reinforce the evidence that lymphatic dysfunction occurs early in the pathogenesis of COPD caused by cigarette smoke, which is associated with increased thrombin and fibrin clots in lung lymph.Primary lymphatic anomalies and immune homeostasis (Pearce et al., 2023). Pearce et al. investigate how primary lymphatic anomalies (PLA), a group of developmental disorders affecting lymphatic vessels, affect immune homeostasis. Examination of both retrospective data from 177 PLA patients and a prospective cohort of 28 patients alongside 20 healthy controls, remarkably uncovers a selective reduction in circulating lymphocytes, which preferentially depletes naive CD4 + T cells. Interestingly, genital oedema and the likelihood of concomitant intestinal lymphangiectasia independently predicted CD4 + T cell depletion. The findings in this study underscore the role of lymphatic-lymphocyte interactions in immune regulation and increased circulating Tregs.These articles highlight the significance of lymphatic dysfunction and anomalies in various pathological conditions.Therapeutic approaches to central nervous system (CNS) diseases via the meningeal lymphatics (Zhang et al., 2024). This review article outlines the roles of meningeal lymphatic vessels and the glymphatic system in maintaining fluid balance, clearance of neurotoxic proteins, and immune surveillance in the brain. Importantly, dysfunction in either...

Myxomatous valve disease (MVD) is the most common form of cardiac valve disease in the developed world. A small fraction of MVD is syndromic and arises in association with matrix protein defects such as those in Marfan syndrome, but most MVD is acquired later in life through an undefined pathogenesis. The KLF2/4 transcription factors mediate endothelial fluid shear responses, including those required to create cardiac valves during embryonic development. Here we test the role of hemodynamic shear forces and downstream endothelial KLF2/4 in mature cardiac valves. We find that loss of hemodynamic forces in heterotopically transplanted hearts or genetic deletion of KLF2/4 in cardiac valve endothelium confers valve cell proliferation and matrix deposition associated with valve thickening, findings also observed in mice expressing the mutant fibrillin-1 protein known to cause human MVD. Transcriptomic and histologic analysis reveals increased monocyte recruitment and TGF-β signaling in both fibrillin-1–mutant valves and valves lacking hemodynamic forces or endothelial KLF2/4 function, but only loss of TGF-β/SMAD signaling rescued myxomatous changes. We observed reduced KLF2/4 expression and augmented SMAD signaling in human MVD. These studies identify hemodynamic activation of endothelial KLF2/4 as an environmental homeostatic regulator of cardiac valves and suggest that non-syndromic MVD may arise in association with disturbed blood flow across the aging valve.

BACKGROUND: Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2 , are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). METHODS: Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC–specific, compound Foxc1;Foxc2 mutations (ie, EC- Foxc -DKO and lymphatic EC- Foxc -DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. RESULTS: EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC- Foxc -DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC- Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC- Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC- Foxc1/c2 mutant MVs. CONCLUSIONS: Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Lymphatic vessels conduct a diverse range of activities to sustain the integrity of surrounding tissue. Besides facilitating the movement of lymph and its associated factors, lymphatic vessels are capable of producing tissue-specific responses to changes within their microenvironment. Lymphatic endothelial cells (LECs) secrete paracrine signals that bind to neighboring cell-receptors, commencing an intracellular signaling cascade that preludes modifications to the organ tissue’s structure and function. While the lymphangiocrine factors and the molecular and cellular mechanisms themselves are specific to the organ tissue, the crosstalk action between LECs and adjacent cells has been highlighted as a commonality in augmenting tissue regeneration within animal models of cardiac and intestinal disease. Lymphangiocrine secretions have been owed for subsequent improvements in organ function by optimizing the clearance of excess tissue fluid and immune cells and stimulating favorable tissue growth, whereas perturbations in lymphatic performance bring about the opposite. Newly published landmark studies have filled gaps in our understanding of cardiac and intestinal maintenance by revealing key players for lymphangiocrine processes. Here, we will expand upon those findings and review the nature of lymphangiocrine factors in the heart and intestine, emphasizing its involvement within an interconnected network that supports daily homeostasis and self-renewal following injury.

Editorial: Lymphatic system: organ specific functions in health and disease