Interstitial Cells Of Cajal Depletion Research Articles

Quantification of interstitial cells of Cajal and fibrosis during gastric per-oral endoscopic myotomy and its association with clinical outcomes.

Background and study aims Alterations to interstitial cells of Cajal (ICC) and collagen fibrosis have been implicated in the pathogenesis of gastroparesis. We aimed to evaluate the feasibility and safety of pyloric muscle sampling during gastric peroral endoscopic myotomy (G-POEM) and the association between pyloric ICC density and degree of fibrosis with clinical outcomes. Patients and methods This was a single-center prospective study of gastroparetic patients who underwent G-POEM and intraprocedural pyloric muscle biopsies between January 2022 and April 2023. ICC count was estimated using CD117 stain and trichome for collagen fibrosis. Clinical response to G-POEM was defined as an improvement of ≥ 1 point on the Gastroparesis Cardinal Symptom Index. Results Fifty-six patients (median age 60 years, 71.4% women) underwent G-POEM (100% technical success; 71.4% clinical response). ICC depletion (< 10/high-power field) and fibrosis were encountered in 70.4% and 75% of the cases, respectively. There was no difference in mean ICC count between G-POEM responders vs. non-responders (7±3.6 vs. 7.7±3.3; P = 0.9). There was no association between ICC density or degree of fibrosis with the etiology of gastroparesis, duration of symptoms, gastric emptying rate, or pyloric impedance planimetry. Patients who did not respond to G-POEM had a significantly higher degree of moderate/severe fibrosis when compared with those who responded (81.3% vs. 25%; P = 0.0002). Conclusions Pyloric muscle biopsies during G-POEM was feasible and safe. ICC depletion and pyloric muscle fibrosis are common in gastroparetic patients. The degree of fibrosis may be related to pyloric dysfunction and clinical response to G-POEM. Additional studies are needed to confirm these results.

Insulin-Like Growth Factor1 Preserves Gastric Pacemaker Cells and Motor Function in Aging via ERK1/2 Activation

Impaired gastric motor function in the elderly causes reduced food intake leading to frailty and sarcopenia. We previously found that aging-related impaired gastric compliance was mainly owing to depletion of interstitial cells of Cajal (ICC), pacemaker cells, and neuromodulator cells. These changes were associated with reduced food intake. Transformation-related protein 53-induced suppression of extracellular signal-regulated protein kinase (ERK)1/2 in ICC stem cell (ICC-SC) cell-cycle arrest is a key process for ICC depletion and gastric dysfunction during aging. Here, we investigated whether insulin-like growth factor 1 (IGF1), which can activate ERK in gastric smooth muscles and invariably is reduced with age, could mitigate ICC-SC/ICC loss and gastric dysfunction in klotho mice, a model of accelerated aging. Klotho mice were treated with the stable IGF1 analog LONG R3 recombinant human (rh) IGF1 (150 μg/kg intraperitoneally twice daily for 3 weeks). Gastric ICC/ICC-SC and signaling pathways were studied by flow cytometry, Western blot, and immunohistochemistry. Gastric compliance was assessed in exvivo systems. Transformation-related protein 53 was induced with nutlin 3a and ERK1/2 signaling was activated by rhIGF-1 in the ICC-SC line. LONG R3 rhIGF1 treatment prevented reduced ERK1/2 phosphorylation and gastric ICC/ICC-SC decrease. LONG R3 rhIGF1 also mitigated the reduced food intake and impaired body weight gain. Improved gastric function by LONG R3 rhIGF1 was verified by invivo systems. In ICC-SC cultures, rhIGF1 mitigated nutlin 3a-induced reduced ERK1/2 phosphorylation and cell growth arrest. IGF1 can mitigate age-related ICC/ICC-SC loss by activating ERK1/2 signaling, leading to improved gastric compliance and increased food intake in klotho mice.

Management of Dyspepsia and Gastroparesis in Patients with Diabetes. A Clinical Point of View in the Year 2021.

Diabetes mellitus is a widespread disease, and represents an important public health burden worldwide. Together with cardiovascular, renal and neurological complications, many patients with diabetes present with gastrointestinal symptoms, which configure the so-called diabetic enteropathy. In this review, we will focus on upper gastrointestinal symptoms in patients with diabetes, with particular attention to dyspepsia and diabetic gastroparesis (DG). These two clinical entities share similar pathogenetic mechanisms, which include autonomic neuropathy, alterations in enteric nervous system and histological abnormalities, such as interstitial cells of Cajal depletion. Moreover, the differential diagnosis may be challenging because of overlapping clinical features. Delayed gastric emptying should be documented to differentiate between DG and dyspepsia and it can be assessed through radioactive or non-radioactive methods. The clinical management of dyspepsia includes a wide range of different approaches, above all Helicobacter pylori test and treat. As regards DG treatment, a central role is played by dietary modification and glucose control and the first-line pharmacological therapy is represented by the use of prokinetics. A minority of patients with DG refractory to medical treatment may require more invasive therapeutic approaches, including supplemental nutrition, gastric electric stimulation, pyloromyotomy and gastrectomy.

Comparison of the Status of Interstitial Cells of Cajal in the Smooth Muscle of the Antrum and Pylorus in Diabetic Male and Female Patients with Severe Gastroparesis

Females dominate in the area of gastroparesis (GP), making up to 70–80% of these patients. One attractive hypothesis is that females have less smooth muscle reserve and thus less resilience to recover from an insult. Our aim was to investigate if there are gender differences in the number of interstitial cells of Cajal (ICC) in the antral and pyloric smooth muscle of diabetic (DM) patients with severe gastroparesis refractory to standard medical management. Full thickness antral and pyloric biopsies were obtained during surgery to implant a gastric electrical stimulation system and perform a pyloroplasty. Thirty-eight DM patients (66% females, n = 25; mean age 44) who failed medical therapies provided antral biopsies. Pyloric tissue samples were also collected from 29 of these patients (65% females, n = 19). Tissues were stained with H&amp;E and c-Kit for the presence of ICC. ICC depletion was defined as less than 10 cells/HPF. In the antrum, 40% of females had significant ICC depletion, similar to 38% in males. In the pylorus, 68% of females had depletion of ICC, compared to 80% depletion in males. When combining both antral and pyloric smooth muscle regions, ICC depletion was similar in males (40%) when compared to females (38%). In diabetic patients with severe GP, females and males showed similar degrees of reduction in antral ICC, while more males had depletion of pyloric smooth muscle ICC compared to their female counterparts. Future larger studies should focus on whether differences in other smooth muscle biomarkers can be identified between males and females.

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

Background & AimsGastric 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).MethodsMice aged 1–107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription–polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference.ResultsThe klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity.ConclusionsIncreased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.

1204 Could Pyloroplasty Combined With Gastric Electrical Neurostimulation Be the Optimal Treatment for Drug Refractory Gastroparesis Based on Pyloric Pathology, Clinical and Gastric Emptying Results?

INTRODUCTION: Gastric electrical neuro-stimulation (GES) therapy for severe gastroparesis (GP) was approved under the Humanitarian Device Exemption (HDE) status in March of 2000. The lack of improvement in gastric emptying (GE) with GES generated our interest to supplement GES with a simultaneously performed pyloroplasty (PP). The aim of our investigation was to assess the long term effects of GES + PP on GE results and GP symptoms, while documenting the pathology of the pyloric smooth muscle. METHODS: Overall, 29 GP drug-refractory patients (19) diabetics (DM) with a mean 14.4 ± 7.3 years of DM, 8 idiopathic, and 2 post-vagotomy (PV) underwent surgical implantation of GES with the Heineke-Mikulicz PP. Biopsy of pyloric smooth muscle was obtained, and all samples were stained with C-Kit to identify the status of Interstitial Cells of Cajal (ICC) and trichrome for fibrosis. GP total symptoms score (TTS) of vomiting, nausea, early satiety, bloating, fullness and epigastric pain, were assessed with 5-point Liekert scale, and GE 4-hour scintigraphy test was performed before surgery and at the last follow-up visit. RESULTS: 29 patients, mean age 47 ± 14 years (range 21-78); 21 F; with mean 5.5 (1-20) years of GP symptoms were included in this observation. Overall, TSS improvement after a mean follow-up of 35 months (6-72) post-surgery was 58% (8-100) (Table 1), but 48% of GP patients improved their symptoms by &gt;65% after GES + PP, and ED visits and days of hospitalizations were significantly reduced. GE tests showed 77% (range 53-100) retention at 2 hour and 48% (15-100) at 4 hour before the therapies, and these results improved to 40% (3-94) at 2 hour, and 14% (0-58) at 4 hour. Also, 83% patients normalized their GE at 2 hour, and 62% normalized at 4 hour (less than 10% retention) (Table 2). Interestingly, GE remained the most delayed (58%) at 4 hour in PV GP patients. Pyloric ICC depletion (≤10 ICC/HPF) was present in 75% of patients, with a mean 7 ICC/HPF, while 80% of them had minimal/moderate amount of collagen fibrosis between the muscle bundles. There were no unanticipated SAE's or technical problems reported during this study. CONCLUSION: GP drug-refractory patients, responded to combining GES and PP with a significant long-term symptom improvement (60%) and accelerated gastric emptying, which normalized in the majority of patients. The depleted ICC and fibrosis in the pyloric smooth muscle justifies the addition of PP to GES, and explains its success.

Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.

Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. Bone marrow-derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT-PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1-CM, including tumor necrosis factor alpha (TNF-α). ELISA detected 0.697±0.03ngmL-1 TNF-α in M1-CM. Recombinant mouse TNF-α reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50=0.817ngmL-1 ). Blocking M1-CM TNF-α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z-VAD.fmk, n=6, P<.05). This work demonstrates that TNF-α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.

Depleted interstitial cells of Cajal and fibrosis in the pylorus: Novel features of gastroparesis

Depletion and ultrastructural changes of interstitial cells of Cajal (ICC) in the gastric body and antrum have been observed in gastroparesis. This research was performed to investigate the ICC population in the muscularis propria and fibrosis of the muscular layer of the pylorus in gastroparesis. Full thickness pyloric and antral biopsies were obtained from 17 gastroparetic and 6 non-gastroparetic controls. Biopsies were stained with C-Kit for ICC and Trichrome for collagen fibrosis. Interstitial cells of Cajal depletion in the antrum was defined as mean ICC count <10 per 20 high power fields (HPF) based on established data. The average pyloric ICC count was ≥10/HPF in the control patients. Twelve (70.5%) gastroparetic patients had pyloric ICC loss. Only five patients (29.4%) had ICC loss in the antrum. Gastric emptying (GE) was not significantly different in patients with depleted vs normal pyloric ICC. However, GE at 2 h was slower in patients with antral ICC <10/HPF compared to those with normal antral ICC populations. Collagen fibrosis was observed in the pylorus of 14 (82.3%) patients. Inclusion bodies in the muscularis propria of the pylorus were identified in four patients, all with diabetic gastroparesis. In gastroparetic patients, ICC loss in the pylorus is twice as common as in the antrum and fibrosis in the pyloric smooth muscle is nearly three times more common than the antrum. These findings can provide one explanation for pyloric dysfunction which is a contributing factor to the pathophysiology of gastroparesis.

Loss of miR-10b triggers depletion of interstitial cells of Cajal in diabetic mice

B acute and chronic hyperglycemias in diabetes are linked to gastrointestinal (GI) complications that can change normal function of motility in the gastrointestinal (GI) tract. The most common pathological abnormality in diabetic patients in the GI tract is characterized by depletion of interstitial cells of Cajal (ICCs). ICCs are pacemakers in the GI tract that regulate GI motility through spontaneous electrical slow waves. ICCs exclusively express the receptor tyrosine kinase KIT, which is required for the normal development and maintenance of ICCs. However, underlying molecular mechanisms in depletion of ICCs in diabetic patients are largely elusive. Here we report a microRNA mediated ICC depletion in the diabetic animal model. We identified miR-10b-5p as a diabetic cell marker that was dramatically diminished in the diabetic ICCs. MiR-10b-5p targets an epigenetic repressor, nuclear receptor co-repressor 2 (NCOR2), which negatively regulates expression of KIT. Expression of KIT and NCOR2 proteins was negatively related in diabetic intestine where KIT+ ICCs were found in lower concentration. ICC restricted miR-10b knockout in mice resulted in depletion of ICCs in the intestine. Our findings on KIT regulation by miR-10b-5p targeted NCOR2 offer a new insight into how ICCs is phenotypically changed and become non functional in diabetic conditions. Moreover, this study suggests miR-10b-5p may be potentially an attractive therapeutic target to restore KIT expression in diabetic ICCs of patients with GI dysmotility.

A Stochastic Algorithm for Generating Realistic Virtual Interstitial Cell of Cajal Networks.

Interstitial cells of Cajal (ICC) play a central role in coordinating normal gastrointestinal (GI) motility. Depletion of ICC numbers and network integrity contributes to major functional GI motility disorders. However, the mechanisms relating ICC structure to GI function and dysfunction remains unclear, partly because there is a lack of large-scale ICC network imaging data across a spectrum of depletion levels to guide models. Experimental imaging of these large-scale networks remains challenging because of technical constraints, and hence, we propose the generation of realistic virtual ICC networks in silico using the single normal equation simulation (SNESIM) algorithm. ICC network imaging data obtained from wild-type (normal) and 5-HT2B serotonin receptor knockout (depleted ICC) mice were used to inform the algorithm, and the virtual networks generated were assessed using ICC network structural metrics and biophysically-based computational modeling. When the virtual networks were compared to the original networks, there was less than 10% error for four out of five structural metrics and all four functional measures. The SNESIM algorithm was then modified to enable the generation of ICC networks across a spectrum of depletion levels, and as a proof-of-concept, virtual networks were successfully generated with a range of structural and functional properties. The SNESIM and modified SNESIM algorithms, therefore, offer an alternative strategy for obtaining the large-scale ICC network imaging data across a spectrum of depletion levels. These models can be applied to accurately inform the physiological consequences of ICC depletion.

Recent progress in gastric arrhythmia: pathophysiology, clinical significance and future horizons.

Gastric arrhythmia continues to be of uncertain diagnostic and therapeutic significance. However, recent progress has been substantial, with technical advances, theoretical insights and experimental discoveries offering new translational opportunities. The discoveries that interstitial cells of Cajal (ICC) generate slow waves and that ICC defects are associated with dysmotility have reinvigorated gastric arrhythmia research. Increasing evidence now suggests that ICC depletion and damage, network disruption and channelopathies may lead to aberrant slow wave initiation and conduction. Histological and high-resolution (HR) electrical mapping studies have now redefined the human 'gastric conduction system', providing an improved baseline for arrhythmia research. The application of HR mapping to arrhythmia has also generated important new insights into the spatiotemporal dynamics of arrhythmia onset and maintenance, resulting in the emergence of new provisional classification schemes. Meanwhile, the strong associations between gastric functional disorders and electrogastrography (EGG) abnormalities (e.g. in gastroparesis, unexplained nausea and vomiting and functional dyspepsia) continue to motivate deeper inquiries into the nature and causes of gastrointestinal arrhythmias. In future, technical progress in EGG methods, new HR mapping devices and software, wireless slow wave acquisition systems and improved gastric pacing devices may achieve validated applications in clinical practice. Neurohormonal factors in arrhythmogenesis also continue to be elucidated and a deepening understanding of these mechanisms may open opportunities for drug design for treating arrhythmias. However, for all translational goals, it remains to be seen whether arrhythmia can be corrected in a way that meaningfully improves organ function and symptoms in patients.

Numerical metrics for automated quantification of interstitial cell of Cajal network structural properties

Depletion of interstitial cells of Cajal (ICC) networks is known to occur in several gastrointestinal motility disorders. Although confocal microscopy can effectively image and visualize the spatial distribution of ICC networks, current descriptors of ICC depletion are limited to cell numbers and volume computations. Spatial changes in ICC network structural properties have not been quantified. Given that ICC generate electrical signals, the organization of a network may also affect physiology. In this study, six numerical metrics were formulated to automatically determine complex ICC network structural properties from confocal images: density, thickness, hole size, contact ratio, connectivity and anisotropy. These metrics were validated and applied in proof-of-concept studies to quantitatively determine jejunal ICC network changes in mouse models with decreased (5-HT2B receptor knockout (KO)) and normal (Ano1 KO) ICC numbers, and during post-natal network maturation. Results revealed a novel remodelling phenomenon occurring during ICC depletion, namely a spatial rearrangement of ICC and the preferential longitudinal alignment. In the post-natal networks, an apparent pruning of the ICC network was demonstrated. The metrics developed here enabled the first detailed quantitative analyses of structural changes that may occur in ICC networks during depletion and development.

163 Hyperglycemia-Induced Hyperplasia of Interstitial Cells of Cajal (ICC) and ICC Stem Cells (ICC-SC) Is Associated With Accelerated Gastric Emptying in Obese Diabetic LeprDb/Db Mice

Background & Aims: ICC depletion is the most common cellular change in diabetic gastroparesis. However, in up to 22% of diabetic patients with gastric symptoms, gastric emptying (GE) is accelerated, rather than delayed. The fate of ICC in these patients is unclear. Previously we reported ICC and ICC-SC hyperplasia in hyperinsulinemic, diabetic Lepr mice. Here, we studied the relationship between GE of solids and ICC/ICC-SC numbers, and investigated the contribution of insulin/IGF1-dependent Kit ligand (Kitl) expression and hyperglycemia-induced ERK MAPK activation to ICC/ICC-SC hyperplasia. Methods: Lepr (n=32) and agesexand strain-matched controls (n=30) were studied 13-65 weeks after the onset of diabetes. ICC and ICC-SC were quantified by flow cytometry. Serum insulin was measured by enzyme immunoassay. Oxidative stress was determined by measuring serum malondialdehyde (MDA) and gastric 8OHdG. GE of solids was analyzed by 13C breath test. Gene expression was studied by qRT-PCR and WB. Effects of high glucose were investigated in murine gastric ICC primary cultures, a conditionally immortalized cell line derived from murine gastric ICC (ICL2A), and in an ICC-SC line isolated from the mouse stomach (2XSCS2F10). ERKMAPK signaling was evaluated byWB and pharmacological inhibition of MAPKK with PD98059. Cell proliferation and apoptosis were determined byMTS assay andCaspase Glo-3/7 assay, respectively. Results: Leprmice had significantly higher blood glucose (median[IQR]: 515[462;577] vs. 124[115;134] mg/dL; P,0.001) and serum insulin levels. Serum MDA and gastric 8OHdG were moderately increased (2.4and 2.2-fold, respectively, P,0.001). In Lepr mice, ICC and ICC-SC increased 2.0±0.1-fold (P,0.001) and 1.5±0.3-fold (P,0.05), respectively, and GE was accelerated (T1/2: 67±10 vs. 100±11 min; P,0.05). GE was similarly accelerated in Kit mice with generalized ICC hyperplasia due to an activating Kit mutation. In Lepr mice, total/soluble Kitl mRNA and Kitl protein were reduced. High glucose (500 mg/dL) significantly increased the numbers of primary ICC and stimulated proliferation and ERK MAPK phosphorylation in ICL2A and 2XSCS2F10 cells. PD98059 (20 μM) inhibited cell proliferation induced by high glucose without affecting basal proliferation. In contrast, osmotic stress induced by mannitol had no effect on ICC numbers, cell proliferation and ERK MAPK phosphorylation. ICC and ICC-SC were resistant to apoptosis induced by hyperglycemia at levels seen in Lepr mice. Conclusions: In the absence of major oxidative stress, hyperglycemia induces ICCSC and ICC hyperplasia via the ERK MAPK pathway even in the presence of reduced Kitl signaling. ICC hyperplasia, in turn, leads to accelerated GE and may contribute to gastric symptoms in a subset of diabetic patients. Grant support: NIH DK58185, DK68055.

13 Role of local progenitors in the sustenance of networks of interstitial cells of Cajal (ICC) in the murine stomach

Depletion of ICC networks and consequent changes in electrical slow wave activity and neuromuscular neurotransmission play important roles in diabetic and idiopathic gastroparesis. We have shown that reduced insulin/IGF‐I signaling and consequent decrease in stem cell factor (SCF) production underlie the ICC depletion of diabetic gastroparesis. It is, however, unclear whether the ICC loss is caused primarily by the accelerated attrition of mature cells or their impaired regeneration. In this study we identified local ICC progenitors and studied their role in the sustenance of gastric ICC networks. We investigated the expression of CD34, a putative marker for ICC precursors, and receptors for SCF (Kit), insulin, and IGF‐I in murine gastric muscles by flow cytometry and immunohistochemistry. In freshly dissected tissues, CD34 was not detected in KitBright ICC but a small population of KitLow cells expressed high levels of CD34. These cells were oval‐shaped, had no processes, and occurred in small clusters on the serosal and submucosal surfaces of the gastric musculature and in the myenteric region. Unlike mature ICC, they expressed insulin and IGF‐I receptors. KitLowCD34+ cells were undetectable and Kit+ ICC were profoundly reduced in gastric muscles cultured in the absence of growth factors for up to 85 days. Soluble SCF added to the culture media for 32–85 days rescued KitLowCD34+ cells and caused their growth into densely packed clusters in the myenteric region or thick chords containing bipolar cells within the muscle layers. These cells had elevated Kit and reduced CD34 expression. Cultures treated in a similar manner with IGF‐I to stimulate both soluble and membrane‐bound SCF were dominated by normal, Kit+CD34– ICC networks. IGF‐I treatments were only effective when initiated within the first 2–4 weeks of culturing, although mature, Kit+ ICC were able to survive longer. In adult H‐2Kb‐tsA58 mice harbouring the temperature‐sensitive large tumour antigen encoded by a mutant strain of simian virus 40, stimulating cell proliferation by culturing at 33°C prevented the decline of gastric ICC even in the absence of exogenous growth factors. We conclude that maintenance of gastric ICC probably involves their regeneration from rare KitLowCD34+ precursors that depend on a nearly uninterrupted supply of insulin or IGF‐I. While the development of these cells into ICC can be initiated with soluble SCF, their expansion and conversion into mature, CD34– ICC also requires membrane‐bound SCF. Supported by NIH Grants DK58185 and RR16464.

Reduced Stem Cell Factor Links Smooth Myopathy and Loss of Interstitial Cells of Cajal in Murine Diabetic Gastroparesis

Diabetic gastroparesis involves neuropathy, myopathy, and depletion of interstitial cells of Cajal (ICC), which may cause dysrhythmias and impaired neural control. Most murine gastric ICC depend on stem cell factor (SCF) signaling but can also be maintained with insulin or insulin-like growth factor-I (IGF-I). We investigated whether SCF could mediate the actions of insulin and IGF-I. Expression of insulin receptor, IGF-I receptor, and SCF was studied in gastric muscles and purified ICC by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The effects of insulin/IGF-I deficiency on SCF, ICC, smooth muscle, and neurons were investigated in nonobese diabetic mice and organotypic cultures by immunohistochemistry, microarrays, and/or quantitative RT-PCR. ICC in organotypic cultures were also studied after immunoneutralization of endogenous SCF. Insulin and IGF-I receptors were detected in smooth-muscle cells and myenteric neurons but not in ICC. Cell-surface expression of SCF was only found in smooth-muscle cells. ICC depletion in diabetes was accompanied by smooth-muscle atrophy and reduced SCF, whereas neuron-specific gene expression remained unchanged. In organotypic cultures, prevention of ICC loss by insulin or IGF-I was paralleled by rescue of smooth-muscle cells and SCF expression but not of myenteric neurons. Immunoneutralization of endogenous SCF caused ICC depletion closely resembling that elicited by insulin/IGF-I deficiency. Reduced insulin/IGF-I signaling in diabetes may lead to ICC depletion and its consequences by causing smooth-muscle atrophy and reduced SCF production. Thus, myopathy may play a more central role in diabetic gastroenteropathies than previously recognized.

Reduced Insulin and IGF-I Signaling, not Hyperglycemia, Underlies the Diabetes-Associated Depletion of Interstitial Cells of Cajal in the Murine Stomach

Damage to interstitial cells of Cajal (ICC), pacemakers, and mediators of neuromuscular neurotransmission in the gastrointestinal tract contributes to the pathogenesis of diabetic gastroenteropathy in both patients and animal models. ICC depletion in diabetes may result from chronic hyperglycemia or lost/ineffective insulin signaling. Because independent control of insulin and glucose concentrations is difficult in chronic in vivo studies, we used long-term organotypic cultures to address this problem. Murine gastric muscles were cultured in normoglycemic or hyperglycemic basal media with or without insulin or IGF-I for 1-3 months, the time required for gastroparesis and ICC damage to develop in diabetic mice. ICC were assessed by c-Kit immunohistochemistry and quantitative analysis of c-kit expression. Electrical pacemaking was studied by intracellular recording of slow waves. ICC survived for at least 34 days in unsupplemented normoglycemic media, but their networks, c-kit expression, and slow waves were profoundly reduced after 68 days. These changes could be entirely prevented by insulin or IGF-I supplementation. ICC networks were completely resistant to hyperglycemia for at least 72 days. Thus, hyperglycemia is unlikely to be responsible for the diabetes-associated depletion of ICC. In contrast, maintenance of ICC requires insulin or IGF-I, which are reduced or ineffective in diabetes.