Tumor-associated macrophages (TAMs) represent the most abundant immune cell population within the tumor microenvironment and play a critical role in cancer progression. However, the molecular mediators underlying TAM-driven tumor invasion remain incompletely defined. This study investigated whether ADAM9 functions as a key effector of pro-invasive TAM polarization using a canine mammary tumor model integrated with human transcriptomic datasets.

Dyslipidemia and obesity are key risk factors for cardiometabolic diseases and are also linked to osteoporosis and other bone disorders. Evidence shows lipid metabolism influences bone homeostasis largely through immune regulation. This review first explains how abnormal lipid metabolism disrupts adipogenic and osteogenic differentiation in bone marrow mesenchymal stem cells and alters adipokines like leptin and adiponectin, upsetting bone formation and resorption and leading to bone loss. It then examines the lipid-immune-bone axis. In innate immunity, high lipid levels shift macrophages from M2 to pro-inflammatory M1, increase bone-resorbing cytokines such as TNF-α and IL-1β, and trigger neutrophil senescence and lipid peroxidation with excess reactive oxygen species, all of which promote osteoclast formation and suppress bone growth. In adaptive immunity, hyperlipidemia changes T-cell metabolism, weakens Treg function, and drives Th17 differentiation; this Th17/Treg imbalance boosts osteoclasts via RANKL, IL-17, and related pathways. Meanwhile, in inflammation, B cells switch from producing OPG to releasing RANKL and G-CSF, while Breg-derived IL-10, IL-35, and TGF-β1 protect bone. The review also highlights how M1 macrophages and Th17 cells work together to worsen bone damage. Understanding these immune mechanisms could lead to new treatments for metabolic bone diseases. Despite these advances, the translation of these preclinical findings into clinical practice remains a challenge that warrants further investigation.

Severe combined immunodeficiency (SCID) pigs have become a promising large animal model for biomedical research, offering significant advantages over traditional mouse models due to their anatomical, physiological, and genetic similarities to humans. Humanized SCID pig models can potentially improve preclinical research in areas such as cancer immunotherapies, stem cell therapies, and transplantation methods, yet often lack significant lymphocyte development, including evidence of B cell and myeloid cell development. This work aims to increase the extent of humanization of the SCID pig. CRISPR guide RNAs were successfully developed for the RAG2 and IL2RG genes, and a double-knockout cell line (RAG2-/-IL2RG-/Y, RG) was established. Somatic cell nuclear transfer (SCNT) was then used to create cloned SCID fetuses, which were injected intraperitoneally with in vitro expanded human CD34+ umbilical cord cells at day 41-42 of gestation. Human leukocytes, including T, B, NK, and myeloid cell types, were detected in peripheral blood, spleen, bone marrow and within the thymus of neonatal animals using flow cytometry. Six of the twelve pigs injected had >5% human cells within the CD45+ cell thymic population. Histology of thymus tissues from multiple pigs showed substantial development of the cortex and medulla, which is absent in non-injected RG neonates. This work demonstrates an improvement in the spectrum of xenogenic immune cell lineages developed using an RG line injected with highly expanded CD34+ cells, yet functional analysis of these cell types is needed for further establishment of an in utero humanized SCID pig model.

Colorectal cancer is one of the most prevalent malignant tumors worldwide, and cancer-initiating (CI) cells or cancer stem (CS) cells are critical for tumor progression. We purified CI/CS cells from colon cancer cells utilizing a membrane filtration method. We developed membrane filters with different surface charges (zeta potentials) by blending a negatively ionized polymer [poly(vinyl alcohol-itaconic acid), PVI] or a positively ionized polymer [poly(L-lysine), PLL] into poly(lactide-co-glycolic acid) (PLG). Suspensions of HT-29 colon cancer cells and PAT-3 cells (primary colon cancer cells from a colon cancer patient in this research) were permeated through unmodified PLG filters and modified PLG filters blended with and without PVI or PLL. The cells in the filtration and recovering solutions and migrating cells from the filters after filtration were evaluated to identify the cells in each fraction and which filter enriched CI/CS cells. CI/CS cells were evaluated for (a) CD44 and CD133 (CI/CS cell markers) expression by flow cytometry and immunostaining in vitro. Cells were also (b) evaluated by a colony formation assay in vitro, (c) evaluated for carcinoembryonic antigen (CEA) production by enzyme-linked immunosorbent assay in vitro and (d) evaluated for a xenograft tumorigenicity test using NOD.CB17-Prkdcscid/NcrCrl (NOD-SCID) mice in vivo. The results revealed that the migration of colon cancer cells from positively charged PLG/PLL filters increased the number of CI/CS cells efficiently and killed 83% of NOD-SCID mice after transplantation, whereas the other fraction of cells did not kill NOD-SCID mice. The filtration method through PLG/PLL filters is effective for purifying CI/CS cells from colon cancer cells.

Evans syndrome (ES) is a rare autoimmune cytopenia characterized by autoimmune hemolytic anemia and immune thrombocytopenia. Patients who are refractory to multiple immunosuppressive therapies have limited treatment options, and long-term outcomes remain poor.

Current therapeutic strategies for bone defects, including autografts, allografts, and conventional biomaterial scaffolds, are limited by donor site morbidity, immune rejection, and insufficient vascularization. Moreover, the complex inflammatory microenvironment in bone defects often impairs healing outcomes, necessitating the development of advanced biomaterials with enhanced immunomodulatory and regenerative capabilities. This study investigates the therapeutic potential of extracellular vesicles derived from Astragalus-modified calcium silicate (AstCS)-stimulated M2 macrophages (AstCSM2EVs) in bone regeneration. The AstCSM2EVs demonstrated superior immunomodulatory capabilities by effectively polarizing macrophages toward the M2 phenotype, characterized by significant downregulation of pro-inflammatory cytokines (IL-1β, TNF-α) and concurrent upregulation of anti-inflammatory mediators (IL-4, IL-10). Notably, AstCSM2EVs exhibited enhanced angiogenic potential, evidenced by increased endothelial tube formation and elevated VEGF secretion, while simultaneously promoting osteogenic differentiation of mesenchymal stem cells through upregulated expression of key markers including ALP, BSP, and OC. Mechanistic investigations revealed that AstCSM2EVs modulated these regenerative processes primarily through miR-218-5p-mediated regulation of multiple signaling pathways, including NOD-like receptor and ECM-receptor interaction pathways. In a rabbit femoral defect model, local administration of AstCSM2EVs significantly enhanced bone regeneration, demonstrated by increased bone volume fraction and improved trabecular architecture, while effectively suppressing local inflammation. These findings establish AstCSM2EVs as a promising therapeutic agent for bone regeneration, highlighting their multifaceted roles in immunomodulation, angiogenesis, and osteogenesis. This research introduces an innovative approach that combines extracellular vesicles (EV) with immunomodulatory tissue engineering strategies to improve the treatment of bone defects.

Spatio-temporal regulation of gene expression and cellular behavior is critical for ensuring developmental robustness in all multicellular organisms. The mechanisms regulating robustness are often multilayered, with underlying processes occurring at different spatio-temporal scales. Pluripotent stem cells are maintained in SAMs despite periodic differentiation of stem-cell progeny into all above-ground organs, which together form much of the biomass on Earth. Shoot apical meristems (SAMs) are often exposed to variable growth conditions in nature; therefore, stem cell maintenance must be robust to withstand these changes. We focus our review on the mechanisms that regulate the robustness of the central CLAVATA (CLV)-WUSCHEL (WUS) feedback system, highlighting insights from new studies that integrate biological experiments with mathematical modeling. These studies have revealed the importance of WUS concentration-dependent transcription of CLV3 involving cis-regulatory module and WUS-binding cofactors, CLV3 peptide processing and modifications, multiple signals converging on WUS transcriptional regulation, and WUS protein in regulating its subcellular partitioning, diffusion between adjacent cells, and degradation. We discuss mechanisms that could provide robustness to each of these processes and how their integration could provide tissue-level robustness in stem cell maintenance.

Bone marrow-derived mesenchymal stem cells (BMSCs) are recruited into the gastric cancer (GC) microenvironment and promote progression, though the underlying mechanisms remain unclear. This study investigated how RYK-silenced BMSCs induce GC cell apoptosis, with a focus on the novel role of dihydrocapsaicin (DHC).

As an emerging biological therapeutic approach, stem cell therapy demonstrates broad application prospects in analgesia and tissue regeneration, particularly achieving significant advances in treating conditions such as spinal cord injury and intervertebral disc degeneration. In recent years, preclinical model studies have deepened our understanding of the mechanisms underlying stem cell-mediated pain relief and tissue repair, revealing their potential to regulate inflammatory responses, promote nerve regeneration, and repair damaged tissues through multiple pathways. However, the heterogeneity of preclinical models and the discrepancies between these models and clinical practice, coupled with often insufficient critical appraisal of study quality, remain critical issues requiring urgent resolution in this field. This narrative review systematically summarizes the fundamental theories and key mechanisms underlying stem cell-mediated analgesia and regeneration. It comprehensively evaluates the advantages and limitations of different animal models, critically analyzes major controversies and technical challenges in research, and identifies key directions for future studies. The literature discussed herein was identified through searches in PubMed and Web of Science databases, focusing on recent preclinical studies (primarily within the last decade) involving stem cells, pain models, and tissue regeneration. Selected studies were evaluated for their methodological rigor and contribution to mechanistic understanding. This review aims to synthesize current evidence, critically appraise preclinical models, and provide a forward-looking perspective for research on stem cell-related analgesia and regenerative mechanisms, thereby promoting further development in clinical translation.

Investigations into the anticancer stem cell (CSC) properties of rhodium complexes are extremely rare. Here we report the synthesis, characterization, photophysical properties, and in vitro anti-CSC activity of a series of cyclometalated rhodium-(III)-polypyridyl complexes 1-4. The 4,7-diphenyl-1,10-phenanthroline-bearing complex 4 exhibits activity against monolayer- and three-dimensionally cultured breast CSCs and osteosarcoma stem cells in the sub-micromolar to low micromolar range, outperforming the metallodrug cisplatin and the established anti-CSC agent salinomycin. Our results suggest that the reported rhodium-(III) scaffold, containing cyclometalated 2,2'-(phenylmethylene)-dipyridine, could be a useful synthon for the future development of anti-CSC rhodium complexes.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau ( MAPT ) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD -MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

The activation of cis-regulatory enhancers is essential for cell fate specification by driving cell type-specific gene expression. Differentiation models are widely used to study enhancer biology but the asynchronous and interdependent nature of gene regulatory changes during cell state transitions can complicate mechanistic studies. To overcome these limitations, here we develop a tamoxifen-gated system for acute enhancer activation in embryonic stem cells (ESCs) based on GRHL2, a pioneer transcription factor which naturally becomes expressed as naive ESCs differentiate into the formative ESC state. Using this system, we investigate the functional relationship between GRHL2 and the histone mono-methyltransferases KMT2C and KMT2D (KMT2C/D). GRHL2 readily binds its target sites independent of KMT2C/D. However, in the absence of KMT2C/D, there are dramatic reductions in H3K4me1/2, P300 recruitment, and H3K27ac deposition at these sites as well as diminished transcriptional activation. Still, strikingly, a basal level of active enhancer mark acquisition and transcriptional activation occurs. Consistent with these findings, during the naive to formative ESC differentiation, GRHL2 enhancer remodeling and target expression is also strongly but incompletely dependent on KMT2C/D. Together, these results define a functional co-activator relationship in which KMT2C/D act as important amplifiers of GRHL2-driven enhancer activation in ESCs and establish a rapid inducible system for dissecting the kinetics and enzymatic dependencies of pioneer transcription factor mediated enhancer remodeling.

Efficient genome writing in mammalian cells requires robust methods for integrating large DNA payloads. The previously described method mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) enables iterative, biallelic genome rewriting in mammalian stem cells with DNA payloads exceeding 100 kb. However, the lack of standardized vectors and certain technical constraints have limited its broader adoption. Here we present an improved plasmid toolkit designed to streamline the implementation of mSwAP-In. The toolkit includes two core vectors. pLP-TK (pCTC174) is a landing-pad plasmid compatible with Golden Gate assembly of genomic homology arms and supports both mSwAP-In and the recombinase-mediated cassette exchange method Big-IN. mSwAP-In MC2v2 (pKBA135) is a versatile Big DNA assembly and delivery vector that supports Gibson-based assembly and incorporates positive, negative, and fluorescent selection markers, as well as a backbone counterselection cassette to minimize unwanted plasmid integration. The vector architecture also enables propagation in yeast and bacterial hosts, inducible plasmid copy-number amplification in standard E. coli strains, and CRISPR/Cas9-mediated payload release through preinstalled guide RNA target sites. We further characterize the FCU1/5-FC counterselection system in mouse embryonic stem cells and define conditions that minimize its bystander toxicity. Finally, we provide a set of Cas9-gRNA expression plasmids optimized for common mSwAP-In applications. Together, these reagents constitute a standardized and experimentally validated toolkit that simplifies large-scale genome writing using mSwAP-In.

Differentiation of trophoblast stem (TS) cells or progenitor cytotrophoblasts (CTBs) into multinucleated syncytiotrophoblasts (STBs) is essential for placental development. Disruption of this process contributes to major obstetrical syndromes, including fetal growth restriction and preeclampsia, and Trisomy 21. However, the chromatin mechanisms governing trophoblast stemness and differentiation remain inadequately defined. Here we identify the chromatin-associated factor PHF13, uncovered through a high-throughput microRNA target screen, as a key regulator of trophoblast cell fate. PHF13 knockout TS cells exhibited defects that ultimately resulted in loss of cell viability, whereas PHF13 knockdown promoted expression of fusion-associated genes, including ERVFRD-1 and human chorionic gonadotropin (hCG). Consistently, PHF13 depletion in BeWo trophoblast cells increased hCG expression and secretion while reducing expression of canonical stemness-associated transcription factors ELF5 and TEAD4. Integrated genomic analyses further revealed that PHF13 target genes comprise a gene regulatory network that maintains trophoblast stemness and restrains differentiation. Notably, the pluripotency-associated transcription factor THAP11 partially co-occupies genomic sites with PHF13. Together, these findings establish PHF13 as a previously unrecognized chromatin regulator of trophoblast stemness and differentiation, providing mechanistic insight into pathways critical for placental development and function.

Human induced pluripotent stem cells (hiPSC) and iPSC-differentiated neural cells, in combination with CRISPR editing, are commonly used for studying neurodevelopmental and other brain disorders. Female iPSCs undergo random X-chromosome inactivation (XCI) via epigenetic silencing by noncoding X inactive specific transcript (XIST). It is known that female iPSCs may lose XIST expression, leading to XCI erosion that affects both X-linked and autosomal gene expression. However, the effects of CRSIPR editing and neural differentiation on XCI erosion in iPSC-derived neurons and how this may confound a real-world transcriptomic analysis of differentially expressed genes (DEGs) are poorly understood. Here, leveraging bulk RNA-seq of hundreds of CRISPR-edited female iPSC lines from four donor lines for 66 genes and single-cell RNA-seq of iPSC-derived neurons of a subset of 42 edited genes, we investigated the effects of XCI erosion during CRISPR editing and in iPSC-derived neurons. We found that XCI erosion was variable in CRISPR-edited female iPSCs and largely preserved in iPSC-derived neurons. Like in iPSCs, XIST in neurons predominately influenced the expression of X-linked genes; however, its effect on autosomal genes was more pronounced in single neurons. Mechanistically, XIST epigenetically causes allelic imbalance of both X-linked and autosomal genes, with the former showing stronger allele-specific expression (ASE) bias. Notably, XIST-induced ASE bias exhibited a conserved positional pattern at loci affecting neurodevelopmental genes across different female lines and cell types. Finally, we demonstrated a confounding effect of XCI erosion on DEG analyses in iPSC-derived neurons. These results have significant implications in hiPSC modeling of neurodevelopmental and other brain disorders.

Progressive neuronal loss is a hallmark of many neurological disorders, yet the adult human brain has a limited capacity for endogenous neuronal replacement. Direct neuronal reprogramming represents an alternative strategy for generating new neurons. Microglia, the brain's resident immune cells, are uniquely positioned as candidate cellular substrates due to their abundance, self-renewal capacity, high motility, and rapid recruitment to sites of injury. Here, using live-cell imaging and electrophysiological recordings, we show that human pluripotent stem cell (hPSC)-derived primitive macrophage progenitors (PMPs) and their microglial derivatives exhibit neuronal reprogramming competence. Inducible expression of NEUROG2 in hPSC-derived PMPs drives acquisition of neuronal morphology, sequential expression of early and mature neuronal markers, organization of synaptic proteins, and functional excitability characterized by action potential firing. Single-nucleus RNA sequencing reveals a continuous, directionally ordered reprogramming trajectory marked by suppression of myeloid transcriptional programs, progression through intermediate remodeling states, and progressive activation of neuronal gene regulatory networks, consistent with a regulated lineage conversion rather than partial identity switching. Using a xenotransplantation-based human microglia chimeric brain model, we further demonstrate that inducible NEUROG2 expression reprograms donor-derived human microglia toward a neuronal identity in vivo. Together, these findings establish human microglial lineage cells as a previously unexplored substrate for neuronal reprogramming, providing a conceptual framework for microglia-based strategies aimed at neuronal replacement and neural repair.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease defined by its molecular hallmarks - amyloid beta peptide plaques and neurofibrillary Tau tangles. Despite significant progress that has been made in uncovering a large number of genetic risk factors through extensive genomic sequencing and genetic studies, the molecular mechanisms driving AD-associated pathology and cognitive decline remain poorly understood. Therefore, alongside the identification of more risk genes, it is also paramount to study how these genes function and influence each other within the cellular pathways and overall molecular networks in AD-relevant brain cell types. However, current human protein-protein interactome datasets were all generated in either yeast or generic human cell lines. Consequently, many important neuronal interactions, especially neuron-specific ones, have yet been discovered. To address this critical gap, we developed a highly scalable, high-quality interactome mapping pipeline in human excitatory neurons derived from induced pluripotent stem cells (iPSC), and generated a comprehensive, neuron-specific interactome map, named ADNeuronNet, for key AD risk genes. ADNeuronNet consists of 1,767 high-confidence interactions among 1,189 proteins and is the only dataset enriched with neuron-specific genes when compared to known protein interactions, including previous large-scale interactome maps, for the same baits in the literature. Within ADNeuronNet, we identified 1,375 novel interactions, many of which are likely neuron specific. For example, we identified a neuron-specific interactor, RIN2, for major AD risk factor BIN1 and confirmed RIN2's function in recruiting BIN1 to RAB5 positive early endosomes, a process that has been well-associated with AD etiology. Additionally, we performed quantitative interaction perturbation analyses on AD risk genes with AD-associated mutations or isoforms and identified significant changes in 99 protein interactions among 11 different protein variants. Finally, we found that subunits from the anaphase-promoting complex/cyclosome (APC/C), another novel BIN1 interactors identified by ADNeuronNet, mediated modulation of Tau-aggregation in neurons via regulation of APOE expression, uncovering a previously unrecognized BIN1-APC/C-APOE regulatory axis in AD pathobiology. In summary, these findings illustrate how our neuron-specific ADNeuronNet can be leveraged to uncover new risk gene candidates and cellular pathways that help advance our understanding of molecular mechanisms underlying AD etiology.

Purine nucleotides are essential for mammalian development 1,2 . Purine monophosphates support cell signaling and proliferation and are synthesized by cells through either de novo synthesis or a salvage pathway 3 . We previously identified a midgestational metabolic transition in mice (gestational days gd10.5-11.5) characterized by changes in purine metabolism 4 . Midgestation is a period of rapid growth for placenta and embryo, yet it remains unclear how the placental tissues expand without directly competing with the embryo for biosynthetic resources. Here, we show that this midgestational metabolic transition is associated with a marked reduction in embryonic expression of purine salvage enzymes, which constrains embryonic metabolism and leads to different strategies for purine synthesis between the placenta and embryo. Midgestation embryos are unable to engage the purine salvage pathway even when de novo purine synthesis is blocked either in vivo or in ex utero embryo culture, whereas placental tissue and trophoblasts retain the capacity to use either pathway. Disruption of de novo purine synthesis in mice causes reduced embryonic growth, impaired axial elongation, and abnormal brain and placental development, which are only partially rescued by supplementation with purine salvage precursors. In human placenta, trophoblast stem cells readily switch between the de novo and salvage pathways based on nutrient availability, and syncytiotrophoblasts (STB) preferentially rely on the salvage pathway. We identified guanosine monophosphate (GMP) as a metabolic checkpoint regulating STB differentiation, with insufficient GMP levels causing degradation of the small GTPase Rheb and failure of mTOR activation. Supplementation of purine salvage substrates restored GMP synthesis and STB differentiation in humans, but not mice. Further, in vivo measurements in humans revealed that maternal circulating hypoxanthine decreases during pregnancy and is further reduced in women with clinically small placentas, highlighting the role of hypoxanthine in supporting placental growth. These results uncover compartmentalized purine salvage between the embryo and placenta as a mechanism that limits competition for biosynthetic resources and enables coordinated growth during mammalian development.

The persistent HIV reservoir constitutes the main obstacle to curing HIV/AIDS disease. Our understanding of how non-productive HIV infections are established in primary human CD4 + T cells during the first round of infection remains, however, incomplete. In this study, we leveraged the HIV reporter virus pMorpheus-V5 to delineate cellular expression patterns that are upregulated in non-productively infected primary CD4 + T memory stem cells (T SCM ). We found that CD4 + T SCM harboring non-productive proviruses displayed a distinct transcriptomic signature comprising 118 upregulated genes. This non-productive expression profile was distinct from that of productively infected cells as well as from negative-exposed and mock-infected cells. Among the cellular genes most upregulated in CD4 + T cells harboring non-productive proviruses were CCR4-binding migratory chemokines ( CCL22, CCL17 ), tryptophan catabolic enzymes ( IDO1, KYNU ), and genes encoding cytoskeletal rearrangement proteins ( BASP1, TNFAIP2 ). Intracellular flow cytometry-based analyses confirmed that non-productively infected CD4 + T SCM cells were enriched for CCL22 and IDO1 co-expression compared to the other CD4 + memory subsets, underscoring a clear CD4 + T cell subset specificity for the upregulation of these two immune gene sets associated with non-productive infections. These findings suggest that primary human CD4 + T SCM harboring non-productive proviruses display a distinct immunoregulatory phenotype which may facilitate immune evasion and contribute to the persistence of the HIV reservoir.

Peripheral pain sensation is regulated by interactions between sensory nerves and various tissue cells. In obese patients with painful small fiber neuropathy, skin sensory nerves are often hypersensitive. While obesity is known to cause circulation-related vascular abnormalities, how these changes affect sensory dysfunction is not fully understood. In this study, we found that in a diet-induced obesity mouse model, skin capillaries become fenestrated, allowing insulin to diffuse into the avascular epidermis. This exposure triggers the production and secretion of nerve growth factor (NGF) from epidermal keratinocytes via insulin signaling with the forkhead box O1 (FOXO1) transcription factor. Elevated NGF leads to heightened sensory hypersensitivity by enhancing transient receptor potential vanilloid subtype 1 (TRPV1) in sensory nerves. Controlling capillary permeability reduces abnormal NGF expression and attenuates pain hypersensitivity. These findings nominate peripheral nerve-associated capillary permeability as a novel therapeutic target in obesity-associated sensory dysfunction.