enhanced-spatial-mapping-mouse-gastric-muscle-layers-using-modified

Enhanced Spatial Mapping of Mouse Gastric Muscle Layers Using a Modified Swiss Roll Technique

Spatial "omics" technologies provide unprecedented opportunities to study the biology and interactions of individual cells of the gastric neuromuscular ...

spatially-resolved-integrated-single-cell-multiomic-profiling

Spatially Resolved, Integrated Single-Cell Multiomic Profiling of the Transcriptome and Epigenomic Targets in Frozen Tissue Sections

Spatial multiomic technologies combined with single-cell profiling offer unprecedented opportunities to elucidate the molecular and cellular architecture ...

Mayo Clinic College of Medicine and Science

Gastroenteropathy causes considerable morbidity in patients with diabetes mellitus and represents a major healthcare burden. Current treatments are largely symptomatic and frequently ineffective. Development of new therapeutic options is hampered by poor understanding of the underlying pathomechanisms. Experimental studies and sparse human data indicate that diabetic gastroenteropathy is multifactorial and involves not only parasympathetic and sympathetic autonomic nerves, but also enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). ICC are mesenchymal cells that occur throughout the muscular coat of the gastrointestinal tract and provide functions critical for normal gastrointestinal motility including generation and propagation of electrical slow waves and mediation of bidirectional communication between the autonomic nervous system and smooth muscle cells. Through these functions, and in concert with other cell types of the gastrointestinal muscles, ICC support basic gastrointestinal functions such as digestion, absorption and waste removal. Loss or dysfunction of ICC in various dysmotilities and their animal models has been shown to lead to gastric dysrhythmias, gastroparesis, slow intestinal transit, impaired neuroeffector mechanisms and altered visceral afferent signalling that are considered hallmarks of diabetic gastroenteropathy. These findings and an increasing body of evidence indicating disruptions of ICC networks in diabetes suggest that the loss of ICC in this disorder is probably of functional significance and could even be a major pathogenetic factor. Future research should focus on the identification of the molecular and cellular mechanisms underlying ICC loss in diabetes and the translation of the experimental findings into treatments.

Interstitial cells of Cajal in diabetic gastroenteropathy.

Gastroenteropathy manifesting in upper gastrointestinal symptoms, delayed gastric emptying, constipation, diarrhea and fecal incontinence occurs frequently in patients with diabetes mellitus and represents a significant health care burden. Current treatments are largely symptomatic and ineffective. Better understanding of the cellular and molecular pathogenesis of these disorders is required for the development of more effective therapies. Recent advances in our understanding of the inherent, high-level complexities of the control systems that execute and regulate gastrointestinal motility, together with the utilization of new experimental models and sophisticated physiological, morphological and molecular techniques have lead to the realization that diabetic gastroenteropathies cannot be ascribed to any singular defect or dysfunction. In fact, these disorders are multifactorial and involve a spectrum of metabolic and dystrophic changes that can potentially affect all key components of motor control including the systemic autonomic and enteric nervous systems, interstitial cells of Cajal and smooth muscle cells. Candidate pathomechanisms are also varied and include imbalance between pro- and anti-oxidative factors, altered trophic stimuli to mature cells and their progenitors, and, possibly, autoimmune factors. The goal of this paper is to review the cellular changes underlying diabetic gastroenteropathies and their potential causes, with particular focus on functional interactions between various cell types. It is proposed that diabetic gastroenteropathies should be considered a form of gastrointestinal neuromuscular dystrophy rather than a "functional" disorder. Future research should identify ways to block cytotoxic factors, support the regeneration of damaged cells and translate the experimental findings into new treatment modalities.

Cellular pathogenesis of diabetic gastroenteropathy.

After allogeneic transplantation, murine stem cells (SCs) for interstitial cells of Cajal (ICCs), electrical pacemaker, and neuromodulator cells of the gut, were incorporated into gastric ICC networks, indicating in vivo immunosuppression. Immunosuppression is characteristic of bone marrow- and other non-gut-derived mesenchymal stem cells (MSCs), which are emerging as potential therapeutic agents against autoimmune diseases, including inflammatory bowel disease. Therefore, we investigated whether gut-derived ICC-SCs could also mitigate experimental colitis and studied the mechanisms of ICC-SC-mediated immunosuppression in relation to MSC-induced pathways.

Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via prostaglandin E2 secretion.

In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.

Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.

SummaryGastrointestinal stromal tumors (GISTs) represent 20%-40% of human sarcomas. Although approximately half of GISTs are cured by surgery, prognosis of advanced disease used to be poor due to the high resistance of these tumors to conventional chemo- and radiotherapy. The introduction of molecularly targeted therapy (e.g., with imatinib mesylate) following the discovery of the role of oncogenic mutations in the receptor tyrosine kinases KIT and platelet-derived growth factor α (PDGFRA) significantly increased patient survival. However, GIST cells persist in 95%-97% of imatinib-treated patients who eventually progress and die of the disease because of the emergence of clones with drug-resistant mutations. Because these secondary mutations are highly heterogeneous, even second- and third-line drugs that are effective against certain genotypes have only moderately increased progression-free survival. Consequently, alternative strategies such as targeting molecular mechanisms underlying disease persistence should be considered. We reviewed recently discovered cell-autonomous and microenvironmental mechanisms that could promote the survival of GIST cells in the presence of tyrosine kinase inhibitor therapy. We particularly focused on the potential role of adult precursors for interstitial cells of Cajal (ICCs), the normal counterpart of GISTs. ICC precursors share phenotypic characteristics with cells that emerge in a subset of patients treated with imatinib and in young patients with GIST characterized by loss of succinate dehydrogenase complex proteins and lack of KIT or PDGFRA mutations. Eradication of residual GIST cells and cure of GIST will likely require individualized combinations of several approaches tailored to tumor genotype and phenotype.

Targeting Disease Persistence in Gastrointestinal Stromal Tumors.

Nutritional interventions often fail to prevent growth failure in childhood and adolescent malnutrition and the mechanisms remain unclear. Recent studies revealed altered microbiota in malnourished children and anorexia nervosa. To facilitate mechanistic studies under physiologically relevant conditions, we established a mouse model of growth failure following chronic dietary restriction and examined microbiota in relation to age, diet, body weight, and anabolic treatment.

Altered gut microbiota in female mice with persistent low body weights following removal of post-weaning chronic dietary restriction.

Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Lepr), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc).

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC).

Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging.

Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic β cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, β cell function, transcriptional profile, and reestablishment of proline and acidic amino acid–rich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of β cell function and corresponding beneficial effects of tRF in prevention of T2DM.

Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function.

Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC.

Inhibition of EZH2 Reduces Aging-Related Decline in Interstitial Cells of Cajal of the Mouse Stomach.

The circadian clock plays a vital role in modulating the cellular immune response. However, its role in mediating pro-inflammatory diabetogenic β cell injury remains largely unexplored. Our studies demonstrate that the exposure of β cells to IL-1β-mediated inflammation alters genome-wide DNA binding of core circadian transcription factors BMAL1:CLOCK enriched for genomic sites important for cellular response to inflammation. Correspondingly, conditional deletion of  in mouse β cells was shown to impair the ability of β cells to recover from streptozotocin-mediated pro-inflammatory injury , leading to β cell failure and the development of diabetes. Further data integration analysis revealed that the β cell circadian clock orchestrates the recovery from pro-inflammatory injury by regulating transcriptional responses to oxidative stress, DNA damage, and nuclear factor κB(NF-κB)-driven inflammation. Our study suggests that the β cell circadian clock mediates β cell response and recovery from pro-inflammatory injury common to the pathogenesis of diabetes mellitus.

Core circadian transcription factor Bmal1 mediates β cell response and recovery from pro-inflammatory injury.

The carbon-13 spirulina gastric emptying breath test (GEBT) is approved to identify delayed, but not accelerated, gastric emptying (GE). We compared the utility of the GEBT to scintigraphy for diagnosing abnormal GE in patients with diabetes mellitus.

Utility of a C-Spirulina Stable Isotope Gastric Emptying Breath Test in Diabetes Mellitus.

Gastrointestinal motor functions are orchestrated by enteric neurons that display a high degree of anatomical and functional variability. Here, we present a protocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons. We describe steps for human gastric tunica muscularis preparation, immunofluorescent labeling of enteric neurons, and quantification of marker colocalization using Nikon NIS-Elements. This protocol addresses the challenges presented by the high density of neurons within enteric ganglia, overlapping fluorescence signals, background fluorescence, and potential user bias in quantification. For complete details on the use and execution of this protocol, please refer to Gao et al..

ArticleTotal : 2	Year
Publication title	2025
Publication title	2026

Article	Year
Interstitial cells of Cajal in diabetic gastroenteropathy. Neurogastroenterology and motility\| PubMed ID: 18173559	2008
Cellular pathogenesis of diabetic gastroenteropathy. Minerva gastroenterologica e dietologica\| PubMed ID: 19829287
Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via prostaglandin E2 secretion. Gastroenterology\| PubMed ID: 25637652	2015
Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1. Gastroenterology\| PubMed ID: 25865047
Targeting Disease Persistence in Gastrointestinal Stromal Tumors. Stem cells translational medicine\| PubMed ID: 25934947
Altered gut microbiota in female mice with persistent low body weights following removal of post-weaning chronic dietary restriction. Genome medicine\| PubMed ID: 27716401
Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying. Gastroenterology\| PubMed ID: 28438610
Wnt-induced, TRP53-mediated Cell Cycle Arrest of Precursors Underlies Interstitial Cell of Cajal Depletion During Aging. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 32771388
Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function. Science advances\| PubMed ID: 34910509	2021
Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice. Gastroenterology\| PubMed ID: 37597632	2023
Inhibition of EZH2 Reduces Aging-Related Decline in Interstitial Cells of Cajal of the Mouse Stomach. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 38969206	2024
Core circadian transcription factor Bmal1 mediates β cell response and recovery from pro-inflammatory injury. iScience\| PubMed ID: 39524327	2024
Utility of a C-Spirulina Stable Isotope Gastric Emptying Breath Test in Diabetes Mellitus. Neurogastroenterology and motility\| PubMed ID: 39873950	2025
Protocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons. STAR protocols\| PubMed ID: 40382773	2025

Tamas Ordog

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