Organ and tissue functions emerge from the coordinated activity of cell networks. Therapeutics that act on pathogenic cell networks, modulating their cellular interplay, follow naturally. Over several decades, our laboratory has developed a series of approaches for rewiring cell networks, culminating in a class of cell surface-directed signal converter proteins (SCPs) that do so by modulating juxtacrine and autocrine signaling in and among their nodal cells. A first such SCP has now produced encouraging clinical data for cancer immunotherapy. Yet, these early network-directed fusion proteins rest on a deliberately simplified picture: discrete end-cell types plugged into graphically tractable networks. That picture is increasingly at odds with what computational cell typing and spatiotemporal analytics, along with epigenetics, now reveal-a hyperdiverse, plastic, experience-shaped cellular landscape embedded in dynamic, multiway networks. Setting the stage for a next generation of network modulators, an extended cell differentiation synthesis, is proposed, which formalizes paracell ultradifferentiation and aging-associated differentiation phases. According to this model, cells are ever evolving, and no two cells are alike. A richer cellular ontology forces a more elaborate network ontology, with paralogous networks and their shifting subnetworks opening a concrete design space for next-generation network-directed SCP therapeutics. This exploration calls for a willingness to embrace complexity more fully and borrow freely from conceptual fields close and afar.

Liver X receptor alpha (LXRα) regulates serum cholesterol and lipoprotein levels under homeostatic conditions and in response to dietary fat. Previously, aged Lxrα[-/-] mice fed a standard diet were shown to develop phenotypes resembling early age-related macular degeneration (AMD). Herein, the hypothesis tested was that introducing a high-fat, high-cholesterol (HFC) diet in young Lxrα[-/-] mice could bypass aging and accelerate onset of AMD-like pathology. Young Lxrα[+/+] and Lxrα[-/-] mice (2 to 3 months old) were fed either a standard diet or an HFC diet for up to 2 months. Retinal function and morphology were assessed by electroretinography and fundus/optical coherence tomography imaging, histopathology by transmission electron microscopy, and retinal markers by immunostaining. Young Lxrα[-/-] mice fed an HFC diet exhibited impaired retinal function with reduced scotopic a- and b-waves, photopic b-waves, and c-waves. Although the retinal ultrastructure of control and HFC-fed Lxrα[+]/[+] mice appeared normal, HFC-fed Lxrα[-/-] mice showed retinal pigment epithelium thinning, disorganized basal infoldings, intracellular lipid droplets, thin basal laminar deposits, and Bruch membrane thickening enriched in apolipoprotein E. Increased microglia/macrophage infiltration in the retina and choroid suggested elevated local inflammation. Although an HFC diet accelerated AMD-like changes in young Lxrα[-/-] mice, these lesions were less severe than those observed in aged Lxrα[-/-] mice on a standard diet, indicating that aging remains the dominant driver of disease severity.

Despite the clinical importance of the meibomian gland (MG), the mechanisms controlling its proliferation and differentiation remain unclear. Ocular adnexal sebaceous carcinoma (SebCA), an aggressive malignancy that most often arises from the MG, lacks well-defined oncogenic drivers, but high MYC expression has been demonstrated in both tumors and primary human cell lines. This study evaluated the ongoing dependence of MYC in the MG, in vivo (murine MGs) and in vitro (three SebCA cell lines) using three MYC inhibitors (MYCi361, MYCMI6, and 10074-G5). In vivo evaluations included histopathology, morphometry, IHC analysis, and quantitative PCR. In vitro, viability, differentiation, and proliferation were evaluated; clonogenic potential was assessed by quantifying anchorage-dependent colony formation; and MYC expression was determined using enzyme-linked immunosorbent assay and quantitative PCR. Myc-inhibited murine MGs exhibited significantly reduced cytoplasmic volume with diminished proliferation and increased apoptosis, with altered lipid droplet formation relative to contralateral control eyelids. SebCA viability and clonogenicity were significantly impaired by MYC inhibition, and proliferation was significantly reduced in all three cell lines after incubation with MYCMI6. Of the three inhibitors, MYCMI6 showed the most prominent suppression of canonical MYC transcriptional targets. MYC concentration was significantly lower in cells treated with MYCi361 and 10074-G5 relative to vehicle control, and relative MYC expression was significantly up-regulated in 10074-G5-treated SebCA01 cells. MYC stability, MYC transcriptional activity, and the MYC:MAX heterodimer may represent critical regulators of proliferation and differentiation in the MG, with dysregulation contributing to oncogenic potential.

Ocular diseases leading to irreversible vision loss are driven by convergent pathologic processes, including chronic inflammation, oxidative stress, and angiogenesis. Despite therapeutic advances, corneal neovascularization, age-related macular degeneration, diabetic retinopathy, and glaucoma remain major causes of vision impairment. Conventional therapies for these diseases pose challenges related to nonresponsiveness and long-term inefficacy, highlighting the need for treatments that target upstream mechanisms shared across ocular pathologies. Fumaric acid esters (FAEs), particularly dimethyl fumarate (DMFu) and its active metabolite, monomethyl fumarate, have emerged as promising drug candidates because of their potent immunomodulatory and antioxidant properties. Mechanistically, DMFu activates nuclear factor erythroid 2-related factor 2 signaling to enhance antioxidant defense while suppressing NF-κB-mediated proinflammatory responses. Beyond inflammation, DMFu inhibits vascular endothelial growth factor-driven angiogenesis through metabolic rewiring in endothelial cells. This review discusses the efficacy of FAEs across various ocular pathologies and addresses the pharmacokinetics of the drugs and hurdles, such as rapid systemic hydrolysis and ocular barriers. Despite translational challenges, the multimodal mechanisms of FAEs offer a unique advantage over single-target therapies, positioning them as promising candidates for targeting convergent pathways in complex ocular diseases.

Phenotypic switch and apoptosis of corpus cavernosum smooth muscle cells (CCSMCs) contribute to cavernous nerve injury (CNI)-induced erectile dysfunction (CNI-ED). Krüppel-like factor 5 (KLF5) has been identified as a regulator of smooth muscle cell phenotype and apoptosis. Nevertheless, its function in CNI-ED remains unclarified. This work sought to investigate the involvement of KLF5 in a CNI-ED rat model. Fifteen male rats were randomized into sham, unilateral CNI, and bilateral CNI (BCNI) groups to evaluate KLF5 expression. An additional 20 male rats were randomly allocated to the following groups: sham, phosphate-buffered saline-treated BCNI, lentivirus-containing negative control shRNA-treated BCNI, and lentivirus-shRNA targeting Klf5-treated BCNI. Corresponding intracavernous injections were administered immediately after BCNI. Erectile function and histologic changes were assessed 3 weeks later. Additionally, the involvement of KLF5 in the phenotypic switch and apoptosis of hypoxic CCSMCs was investigated. CNI rats exhibited diminished erection, increased hypoxia-inducible factor-1α, KLF5, osteopontin, corporal fibrosis, and apoptosis levels, along with decreased α-smooth muscle actin expression. The severity of these alterations positively correlated with the degree of CNI. Notably, Klf5 inhibition alleviated BCNI-induced ED and corporal damage, and mitigated hypoxia-induced phenotypic switching, oxidative stress, mitochondrial dysfunction, and apoptosis in CCSMCs. Conversely, Klf5 overexpression aggravated these changes. Overall, Klf5 inhibition may prevent BCNI-induced ED and corporal alterations by suppressing phenotypic switch and mitochondrial dysfunction-dependent apoptosis in CCSMCs.

Alcohol-associated hepatitis (AH) is an acute inflammatory form of alcohol-associated liver disease. Previous studies have explored molecular mechanisms associated with AH pathogenesis through bulk liver tissue analysis; however, the heterogeneity of liver tissue and hence the spatial regulation within the AH liver microenvironment remained unaddressed. Here, an unbiased spatial proteomics analysis on the pathologic regions (PRs) of AH liver tissue is presented, including immune cell infiltration foci, lipid droplets, chicken-wire fibrosis, and fibrotic bands. Through combining a highly efficient nanodroplet processing in one pot for trace samples platform with ultrasensitive liquid chromatography-mass spectrometry, this study identified and quantified a total of 5186 unique proteins from PRs isolated in 200-μm-long × 200-μm-wide × 10-μm-thick areas. This in-depth spatial proteome coverage allowed us to discover mechanistic regulations within individual PRs, including compromised resolution of inflammation with infiltrated neutrophils at infiltration foci, increase of mitochondrial and peroxisomal fatty acid β-oxidation at lipid droplets, and differential cellular and extracellular regulations between chicken-wire fibrosis and fibrotic bands. Overall, this study demonstrated a new capability for AH research, revealed the significance of understanding spatial regulation within AH liver tissue, and further facilitated the development of therapeutic strategies at high resolution.

Endometriosis is a chronic, estrogen-dependent gynecologic disorder characterized by the ectopic growth of endometrial tissue outside the uterus. Zinc finger protein 143 (ZNF143) is a DNA-binding transcription factor, yet its role in endometriosis remains unknown. Here, it is reported that ZNF143 is significantly up-regulated in ectopic endometrial lesions. Invivo, Pgr[c][re+]Zfp143[f/f] mice were used to achieve conditional deletion of Zfp143 in uterine epithelial and stromal cells via progesterone receptor (Pgr)-driven Cre recombination. Uterine-specific deletion of Zfp143 in this model markedly reduced lesion formation and growth. In contrast, epithelial-specific deletion using Sprr2f[c][re+]Zfp143[f/f] mice, in which Sprr2f is selectively expressed in uterine epithelial cells, showed no significant effect, indicating that ZNF143 primarily functions in endometrial stromal cells. In vitro, ZNF143 promoted stromal cell migration and invasion while knockdown of ZNF143 using siRNA transfection inhibited migration and invasion of hTERT-immortalized human endometrial stromal cells and primary human endometrial stromal cells. ZNF143 deficiency also reduced collagen deposition in ectopic lesions, implicating it in fibrotic remodeling. Transcriptomic profiling identified plasminogen activator inhibitor-1 (SERPINE1) as a key downstream effector mediating ZNF143-driven fibrosis, and SERPINE1 silencing attenuated these profibrotic effects. Collectively, these findings establish ZNF143 as a critical transcriptional regulator, especially in endometrial stroma that drives lesion progression and fibrosis in endometriosis, providing new mechanistic insights and highlighting a promising therapeutic target.

Recent advances in genetic analysis have led to further subtyping of small-cell lung carcinoma (SCLC). The major subtypes of SCLC are the ASCL1-predominant (SCLC-A), NEUROD1-predominant (SCLC-N), and POU2F3 (SCLC-P) subtypes. SCLC-A and SCLC-N tumors express chromogranin A (CHGA) and synaptophysin (SYP), but SCLC-P tumors do not. Large-cell neuroendocrine carcinoma, another type of neuroendocrine carcinoma (NEC), also frequently expresses CHGA and SYP. Because CHGA and SYP expression is controlled by a transrepressor, repressor element 1-silencing transcription factor (REST), the mechanisms underlying REST suppression in NEC were investigated, with a focus on miRNAs and epigenetics, to determine the causes of the differences in the expression of CHGA and SYP between SCLC-A/N and SCLC-P cells. The results showed that miR-375-3p, which was induced by ASCL1 and NEUROD1, repressed REST expression by binding to the 3'-untranslated region of REST mRNA. Bisulfite sequencing and experiments using a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, and chromatin immunoprecipitation-based quantitative PCR revealed that promoter/enhancer hypermethylation and histone deacetylation causes REST gene inactivation in SCLC-A/N. These phenomena were also observed in a large-cell neuroendocrine carcinoma cell line that expressed high levels of ASCL1 and NEUROD1. Taken together, these findings suggest that NEC has dual repressive effects on REST expression, resulting in strict regulation of the expression of CHGA, SYP, and other REST-controlled neuronal/neuroendocrine-specific genes.

Accurate molecular profiling from routine histopathology slides could transform clinical oncology. A Vision Transformer (ViT)-based model was developed to jointly predict the TP53 biomarker, detect 32 solid tumor types, and predict survival directly from whole slide images (WSIs). Over 11,000 primary tumor data were retrieved from the Pan-Cancer Atlas, along with corresponding somatic mutation, RNA-sequencing, and clinical outcome data. WSIs underwent tissue masking, quality control, stain normalization, patch extraction, and feature embedding using a ViT encoder. Seven task heads were developed to generate predictions for cancer type, TP53 mutation status, TP53 RNA expression levels, overall survival, progression-free interval, and their corresponding event times. Model training proceeded in two stages: initial training on tumor-only patches at multiple magnifications, followed by fine-tuning on WSIs using a content-aware strategy. Model performance was evaluated on an independent validation set of 1729 slides using evaluation metrics, including the area under the receiver operating characteristic curve, regression metrics, and the concordance index. An area under the receiver operating characteristic curve of 0.766 was found for TP53 mutation detection on an independent validation set across 32 human solid tumors. In conclusion, the ViT-based model could simultaneously infer TP53 mutation status, TP53 RNA expression levels, and tumor taxonomy directly from WSIs, supporting the existence of reproducible morphologic correlates of TP53 alterations across human cancers, whereas prognostic risk prediction remained limited.

Disruption of lysosomal homeostasis and accumulation of dysfunctional mitochondria contribute to degenerative pathologies, including age-related macular degeneration. Here, how inhibition of autophagic lysosome reformation (ALR) alters lysosomal dynamics, mitophagy, and downstream stress signaling in retinal pigment epithelial (RPE) cells was investigated, and whether these changes are pharmacologically reversible was determined. In ARPE-19 cells, ALR inhibition by nocodazole or siRNA-mediated depletion of kinesin-1 (UKHC) and dynamin-2 (DNM2) induced enlarged lysosomes with reduced degradative capacity, impaired mitophagic turnover, and accumulation of dysfunctional mitochondria. ALR blockade increased reactive oxygen species and cytosolic Ca[2+], promoted activation and mitochondrial translocation of protein kinase C, and triggered phosphorylation of glycogen synthase kinase-3β with subsequent stabilization of SNAIL, consistent with epithelial-to-mesenchymal transition. Metformin restored lysosomal homeostasis by activating AMP-activated protein kinase and enhancing transcription factor EB-dependent lysosome biogenesis, thereby improving autophagic flux, limiting reactive oxygen species/Ca[2+] accumulation, suppressing protein kinase C activation, and attenuating epithelial-to-mesenchymal transition-associated marker changes. In a sodium iodate-induced oxidative injury model, metformin preserved RPE microtubule architecture and reduced lysosomal and mitochondrial abnormalities. Although these findings rely on a prolonged monolayer culture system and an acute injury model, they support a protective role for AMP-activated protein kinase-transcription factor EB-driven lysosome restoration in RPE stress resilience and suggest lysosome-directed repurposing potential for metformin in degenerative retinal disease.

Loss of vascular endothelial growth factor (VEGF) signaling profoundly impairs vascular growth during development. In contrast, once established in adulthood, blood vessels become largely quiescent and less dependent on VEGF signaling, providing the rationale for the clinical use of VEGF inhibitors to selectively target tumor angiogenesis. In this study, blood vessels in the epiphysis-the ends of long bones-retained an unexpected degree of plasticity in adult and even aged mice. With tamoxifen-induced, endothelium-specific deletion of Vegf receptor (Vegfr)-2, the principal mediator of Vegf signaling, marked vascular regression was found in the epiphysis, with growth plate cartilage expansion after epiphyseal ischemia. Consistently, local bone strength was significantly reduced with endothelial Vegfr2 depletion in adult mice. With epiphyseal vascular regression, bone mineral density loss was exacerbated in an ovariectomy-induced mouse model of osteoporosis. Collectively, these findings advance the understanding of angiogenic-osteogenic coupling in adulthood and provide a mechanistic framework for the vulnerability of the femoral head and neck to fractures in the elderly.

Intestinal stricture affects 2.9% to 57% of necrotizing enterocolitis (NEC) cases, primarily involving the terminal ileum and colon. Although inflammation-driven fibrosis underlies stricture development, key molecular mechanisms remain unclear. Up-regulation of REGγ was previously observed in human NEC specimens and murine models, correlating with inflammation severity. Here, elevated REGγ in human strictures correlated with fibrotic severity. Single-cell RNA sequencing and immunofluorescence confirmed REGγ enrichment in fibroblasts. In vitro, REGγ silencing suppressed transforming growth factor-β1- or tumor necrosis factor (TNF)-α-induced fibroblast activation, proliferation, and migration, accompanied by reduced Smad3 phosphorylation and nuclear translocation. Mechanistically, TNF-α enhanced REGγ transcription via NF-κB p65 binding to its promoter. NF-κB inhibition attenuated TNF-α-induced fibrotic responses, rescued by REGγ overexpression. In murine fibrosis models, REGγ deficiency reduced inflammation, collagen deposition, and phosphorylated Smad3. These findings establish REGγ as a key mediator of inflammation-fibrosis crosstalk in post-NEC intestinal strictures and a promising therapeutic target.

Pulmonary granulomas serve as a major obstacle in tuberculosis treatment. Low-dose radiotherapy (LDRT) emerged as a promising intervention for broad-spectrum inflammatory conditions after the COVID-19 pandemic. The therapeutic potential in mitigating pulmonary granuloma formation and progression was investigated by using a murine model induced by Mycobacterium tuberculosis-derived trehalose-6,6'-dimycolate. LDRT (0.5 Gy) significantly reduced fluorine-18-fluorodeoxyglucose uptake, lung index, and granuloma burden, while alleviating hypoxia and down-regulating hypoxia-inducible factor-1α and cell death markers (LC3B, p62, BNIP3, and caspase-3). This improvement correlated with reduced infiltration of leukocytes, macrophages, and monocytes. Mechanistically, LDRT suppressed interferon-β while enhancing IL-10 and transforming growth factor-β, independent of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. This response was mediated by a transient DNA damage response driven by the ataxia-telangiectasia mutated (ATM) protein involving phosphatidylinositol 3-kinase and ERK activation, which suppressed the pyrimidine salvage marker Tk1. Pharmacologic inhibition of ATM or its downstream effectors abrogated the LDRT-induced cytokine modulation. Notably, ATM activation with GJ071 oxalate reproduced the anti-inflammatory profile in vitro and effectively alleviated granuloma pathology in vivo, mimicking the therapeutic efficacy of LDRT. Collectively, these findings show that LDRT mitigates granuloma pathology by modulating hypoxic and inflammatory microenvironments through ATM-dependent signaling characterized by the down-regulation of interferon-β, establishing a mechanistic rationale for targeting the ATM pathway as a novel host-directed therapeutic strategy for tuberculosis.