Interstitial cells of Cajal: clinical relevance in pediatric gastrointestinal motility disorders.

Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca2+-activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.

Effects of Emodin on Protein Expression Related to Autophagy of Interstitial Cells of Cajal in Diabetic Rats.

This work aims to investigate the effects and mechanism of emodin in treating diabetic gastroenteropathy and colonic dysmotility in STZ + HS/HF diet induced diabetic gastroenteropathy rats. Diabetic colonic dysmotility model was established by high-fat/high-glucose (HS/HF) feeding combined with streptozotocin (STZ). Emodin was divided into high, medium and low dose groups. After eight weeks of intervention, fasting blood glucose (FBG) and body weight were measured. Gastrointestinal transmission time was evaluated. Serum vasoactive intestinal peptide (VIP) and substance P (SP) were detected. Colonic protein expression of selective autophagy adaptor proteins p62 and beclin1 were detected by immunohistochemistry. Colonic protein expression of beclin1, autophagy related gene 5 (Atg5), C-kit and p62 were detected by Western blot. After treating with emodin, gastrointestinal transmission rate was improved. The expression of serum SP was increased and serum VIP was decreased. Colonic c-kit and p62 were up-regulated. The expressions of beclin1 and Atg5 were down-regulated. Emodin can improve colonic dysmotility and promote the recovery of colonic motility and intestinal defecation in diabetic rats. Its mechanism may involved with up-regulating the expression of C-kit and P62, down-regulating the expression of Beclin1 and Atg5 in colon, which are associated with colon over-autophagy of Cajal interstitial cell (ICC).

Anoctamin 1 and c-Kit immunohistochemical study of interstitial cells of Cajal in the muscularis externa of human gastrointestinal tract.

BACKGROUND: Interstitial cells of Cajal (ICC) are widely distributed in human gastrointestinal (GI) tract, especially in the layer of muscularis externa between neurons and smooth muscles. They play a very important role of coordination of GI tract motility. The aims of this research were to study the morphology and distribution of ICC in the muscularis externa of the GI tract, using immunohistochemistry staining methods, to determine the distribution of immune reactivity of anoctamin 1 (Ano1) compared with c-Kit, and to determine if Ano1 is a reliable marker for ICC in human GI tract. MATERIALS AND METHODS: Specimens from the wall of stomach, small intestine, and colon were taken from human cadavers and processed for histological and immunohistochemical study using c-Kit and Ano1 primary antibodies. RESULTS: Interstitial cells of Cajal appeared as bipolar cells, not forming network, in both the circular and longitudinal muscle layers, while in the myenteric area they appeared as multipolar interconnected cells. They were unevenly distributed in and between the muscle layers of the muscularis externa of human GI tract. They were more numerous in the stomach followed by the colon then the small intestine, and more numerous in the myenteric area followed by the circular muscle layer then the longitudinal muscle layer, in the three organs. Our results also showed that Ano1 is a more reliable marker for human ICC than c-Kit. CONCLUSIONS: Interstitial cells of Cajal differed in morphology and were unevenly distributed between muscle layers of muscularis externa and between different parts of human GI tract.

[Electroacupuncture promotes gastrointestinal motility by activating autophagy of Cajal interstitial cells via downregulating PI3K/Akt/mTOR signaling pathway in stomach of diabetic gastro-paresis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36), "Sanyinjiao" (SP6) and "Liangmen" (ST21) on gastrointestinal motility, blood glucose content and expression of autophagy-related proteins 1 light chain 3 (LC3), p62, phosphatidyli-nositol-3 kinase (PI3K), protein kinase B (Akt), p-Akt and mammalian target protein of rapamycin (mTOR) of interstitial cells of Cajal (ICCs) in the cultured gastric antrum cells in diabetic gastroparesis (DGP) rats, so as to reveal its mechanisms underlying improvement of DGP. METHODS: A total of 45 Sprague Dawley (SD) rats were randomly divided into blank control, model, EA, medication (3-methyladenine, 3-MA) and EA+3-MA groups, with 9 rats in each group. The DGP model was established by intraperitoneal injection of 2% streptozotocin (STZ) combined with high-fat and high sugar diet for 8 weeks. The gastric emptying rate was measured by using gavage of phenol red (to measure the propelling length of the phenol red/total length of small intestine ×100%). The symptom score (mental state, coat color and luster, behavior and activity, stool traits) of rats was observed every week and the blood glucose content was measured by using a glucometer. EA (20 Hz/100 Hz, 2 mA) was applied to unilateral ST36, SP6 and ST21 alternatively for 15 min, once daily, 5 days a week for 3 weeks. Rats of the 3-MA and 3-MA+EA groups received intraperitoneal injection of 3-MA (30 mg·kg-1·d-1, 10 mg/mL), once daily, 5 days a week for 3 weeks. After 15 days' intervention, the rats were operated for gastric emptying rate test, specimen collection, isolation, and culture of primary ICCs. The expression levels of microtubule associated protein LC3, p62, PI3K, Akt, p-Akt and mTOR of ICCs of cultured gastric antrum cells were detected using Western blot, and the number of autophagosomes in ICC of gastric antrum was observed under transmission electron microscope. RESULTS: Compared with the blank control group, the symptom score, blood glucose, and the expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins were increased significantly (P<0.01), while the gastric emptying rate and ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein were significantly decreased (P<0.05, P<0.01) in the model group. In comparison with the model group, the increase of symptom score, blood glucose, and expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins and the decrease of gastric empty rate and LC3Ⅱ/LC3Ⅰ ratio and the expression level of class Ⅲ PI3K protein were all reversed in both EA and EA+3-MA groups (P<0.05, P<0.01), rather than in the 3-MA group. In addition, 3-MA also reversed modeling-induced increase of class Ⅰ PI3K, Akt, p-Akt and mTOR proteins expression (P<0.01). No significant differences were found between the EA and EA+3-MA in downregulating the levels of symptom score and blood glucose content, and in upregulating gastric empty rate(P>0.05). The effect of EA was notably superior to that of EA+3-MA in upregulating the ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein, and in downregulating the expression of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins (P<0.05, P<0.01). The findings of transmission electron microscopy showed obvious swelling, breakage of some mitochondrial cristae in the ICC cells of antrum and no autophagosomes in the model group and 3-MA group, which was milder in the damage of mitochondrial cristae and marked increase in the autophagosomes in both EA and EA+3-MA groups. CONCLUSION: EA can improve the gastrointestinal motility and symptoms in DGP rats, which may be related to its functions in downregulating PI3K/Akt/mTOR signaling to promote autophagy level of ICC.

Effect of miR-129-3p on autophagy of interstitial cells of Cajal in slow transit constipation through SCF C-kit signaling pathway.

OBJECTIVE: To explore the mechanism by which miR-129-3p affected the autophagy of interstitial cells of Cajal (ICCs) in slow transit constipation tissues through the SCF C-kit signaling pathway. METHODS: Colon samples from 20 Slow transit constipation (STC) patients who underwent total colectomy plus ileorectal anastomosis or subtotal colon resection plus anti-peristaltic rectal anastomosis were collected in our hospital. The colon of 20 non-STC patients was used as control. The control of this study was 20 patients undergoing radical surgery for colon cancer (left colon cancer) in our hospital. Fifty healthy SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. RESULTS: The mRNA expression of miR-129-3p in the STC group was lower than that in the control group (CTLR) group (P<0.05). The mRNA expression of miR-129-3p in STC group was lower than that in the NC group (P<0.05), and mRNA expression in STC+miR-129-3p group was higher than that in STC+miR-NC group (P<0.05). In the first week, the weight of dry and wet feces of the STC group was lower than that of the NC mice (P<0.05), and the weight of dry feces and wet feces of the STC group was lower than that of the NC group at the 2, 3, and 4 weeks, STC+miR-129 -3p was higher than that in the STC group (P<0.05). CONCLUSION: The increased expression of C-kit and SCF regulated by miR-129-3p contributed to the protection of interstitial cells. Knockdown of miR-129-3p expression could inhibit the activation of AKT/mTOR signaling pathway, reduce cell proliferation activity.

Newly raised anti-c-Kit antibody visualizes morphology of interstitial cells of Cajal in the developing gut of chicken embryos.

The gut peristaltic movement, a wave-like propagation of a local contraction, is important for the transportation and digestion of ingested materials. Among three types of cells, the enteric nervous system (ENS), smooth muscle cells, and interstitial cells of Cajal (ICCs), the ICCs have been thought to act as a pacemaker, and therefore it is important to decipher the cellular functions of ICCs to further our understanding of gut peristalsis. c-Kit, a tyrosine kinase receptor, has widely been used as a marker for ICCs. Most studies with ICCs have been conducted in mammals using commercially available anti-c-Kit antibody. Recently, the chicken embryonic gut has emerged as a powerful model to study gut peristalsis. However, since the anti-c-Kit antibody for mammals does not work for chickens, cellular mechanisms by which ICCs are regulated have largely been unexplored. Here, we report a newly raised polyclonal antibody against the chicken c-Kit protein. The specificity of the antibody was validated by both western blotting analyses and immunocytochemistry. Co-immunostaining with the new antibody and anti-α smooth muscle actin (αSMA) antibody successfully visualized ICCs in the chicken developing hindgut in the circular muscle and longitudinal muscle layers. As previously shown in mice, common progenitors of ICCs and smooth muscle cells at early stages were double positive for αSMA and c-Kit, and at later stages, differentiated ICCs and smooth muscle cells exhibited only c-Kit and αSMA, respectively. A novel ICC population was also found that radially extended from the submucosal layer to the circular muscle layer. Furthermore, the new antibody delineated individual ICCs in a cleared hindgut. The antibody newly developed in this study will facilitate the study of peristaltic movement in chicken embryos.

Distribution of interstitial cells of Cajal in the Esophagus and change in distribution after thoracic trauma.

Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal tract. Acute thoracic trauma is a common and lethal cause of death due to physical trauma caused by traffic accidents. This study aimed to explore the distribution of esophageal ICCs and distribution changes observed after acute thoracic trauma. Thirty rabbits were randomly divided into a control group and two study groups. The control group animals underwent an esophagectomy. All animals in the study groups underwent right chest puncture using the Hopkinson bar technique. The study groups were subjected to esophagectomy 24 and 72 h after chest puncture. Distribution, morphology, and density of esophageal ICCs were detected using transmission electron microscopy, toluidine blue staining, and immunohistochemistry. Apoptosis of esophageal ICCs was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay. Western blotting and reverse transcription polymerase chain reaction were used to detect changes in the SCF/c-kit signaling pathway. Esophageal ICCs distribution and SCF/c-kit signal pathway decreased from the upper part to the lower part in both physiological state and after thoracic trauma. In contrast, death of ICCs increased from the upper part to the lower part, both in physiological and injured state (P < 0.05). After thoracic trauma, increased ICCs and decreased death of ICCs in all parts of the esophagus (P < 0.05) were observed. The observed distribution and changes in esophageal ICCs would have an impact on motility and motility disorders of the esophagus.

PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

BACKGROUND & AIMS: Platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons. METHODS: Gene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles. RESULTS: Adcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions. CONCLUSIONS: PIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

Long non-coding RNA MEG3 promotes tumor necrosis factor-alpha induced oxidative stress and apoptosis in interstitial cells of cajal via targeting the microRNA-21 /I-kappa-B-kinase beta axis.

Interstitial Cells of Cajal (ICC) plays a critical role in the peristaltic contractions of the gastrointestinal and urinary tract. The dysfunction and loss of ICC contributes to hypokinetic disease, such as gallstoneand ureteropelvic junction obstruction . In the present study, we identified the underlying driving molecular signals of oxidative stress and apoptosis in ICC. ICC was isolated from small intestine of Balb/c mice, and stimulated with tumor necrosis factor-alpha (TNF-α). MTT and flow cytometry were performed to assess cell viability, apoptosis, and the level of reactive oxygen species in ICC, respectively. The level of malondialdehyde, superoxide dismutase, and glutathione peroxidase in cells were measured to assess oxidative stress. The expression of inflammatory factors (interleukin, IL-1 and IL-6) and apoptosis-related proteins were detected by western blot. We observed that TNF-αinduced inflammation, oxidative stress and cell apoptosis in ICC. By using quantitative real-time PCR , we verified that the expression of long non-coding RNAMEG3 was elevated by TNF-α in ICC. Silencing MEG3 reversed inflammation, oxidative stress, and cell apoptosisin TNF-α-treated ICC. Subsequently, we confirmed that MEG3 sponged cytoprotective miR-21 to upregulate the expression of I-kappa-B-kinase beta (IKKB) and activate the nuclear factor kappa-B (NF-κB) pathway. Both miR-21 overexpression and IKKB knockdown reduced TNF-α-induced above symptoms in ICC. Taken together, we can conclude that MEG3 mediates inflammation, oxidative stress and apoptosis in TNF-α-treated ICC via the miR-21/IKKB-NF-κB axis. The study improves our understanding of the molecular mechanism of ICC reduction related diseases.

Calcium-Dependent Cytosolic Phospholipase A2α as Key Factor in Calcification of Subdermally Implanted Aortic Valve Leaflets.

Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.

Single-cell RNA sequencing predicts motility networks in purified human gastric interstitial cells of Cajal.

BACKGROUND: Gastrointestinal (GI) motility disorders affect millions of people worldwide, yet they remain poorly treated in part due to insufficient knowledge of the molecular networks controlling GI motility. Interstitial cells of Cajal (ICC) are critical GI pacemaker cells, and abnormalities in ICC are implicated in GI motility disorders. Two cell surface proteins, KIT and ANO1, are used for identifying ICC. However, difficulties accessing human tissue and the low frequency of ICC in GI tissues have meant human ICC are insufficiently characterized. Here, a range of characterization assays including single-cell RNA sequencing (scRNA-seq) was performed using KIT+ CD45- CD11B- primary human gastric ICC to better understand networks controlling human ICC biology. METHODS: Excess sleeve gastrectomy tissues were dissected; ICC were analyzed by immunofluorescence, fluorescence-activated cell sorting (FACSorting), real-time PCR, mass spectrometry, and scRNA-seq. KEY RESULTS: Immunofluorescence identified ANO1+ /KIT+ cells throughout the gastric muscle. Compared to the FACSorted negative cells, PCR showed the KIT+ CD45- CD11B- ICC were enriched 28-fold in ANO1 expression (p < 0.01). scRNA-seq analysis of the KIT- CD45+ CD11B+ and KIT+ CD45- CD11B- ICC revealed separate clusters of immune cells and ICC (respectively); cells in the ICC cluster expressed critical GI motility genes (eg, CAV1 and PRKG1). The scRNA-seq data for these two cell clusters predicted protein interaction networks consistent with immune cell and ICC biology, respectively. CONCLUSIONS & INFERENCES: The single-cell transcriptome of purified KIT+ CD45- CD11B- human gastric ICC presented here provides new molecular insights and hypotheses into evolving models of GI motility. This knowledge will provide an improved framework to investigate targeted therapies for GI motility disorders.

Interstitial cells of Cajal and human colon motility in health and disease.

Our understanding of human colonic motility, and autonomic reflexes that generate motor patterns, has increased markedly through high-resolution manometry. Details of the motor patterns are emerging related to frequency and propagation characteristics that allow linkage to interstitial cells of Cajal (ICC) networks. In studies on colonic motor dysfunction requiring surgery, ICC are almost always abnormal or significantly reduced. However, there are still gaps in our knowledge about the role of ICC in the control of colonic motility and there is little understanding of a mechanistic link between ICC abnormalities and colonic motor dysfunction. This review will outline the various ICC networks in the human colon and their proven and likely associations with the enteric and extrinsic autonomic nervous systems. Based on our extensive knowledge of the role of ICC in the control of gastrointestinal motility of animal models and the human stomach and small intestine, we propose how ICC networks are underlying the motor patterns of the human colon. The role of ICC will be reviewed in the autonomic neural reflexes that evoke essential motor patterns for transit and defecation. Mechanisms underlying ICC injury, maintenance, and repair will be discussed. Hypotheses are formulated as to how ICC dysfunction can lead to motor abnormalities in slow transit constipation, chronic idiopathic pseudo-obstruction, Hirschsprung's disease, fecal incontinence, diverticular disease, and inflammatory conditions. Recent studies on ICC repair after injury hold promise for future therapies.

Involvement of interstitial cells of Cajal in nicotinic acetylcholine receptor-induced relaxation of the porcine lower esophageal sphincter.

The interstitial cells of Cajal (ICCs) play an important role in coordinated gastrointestinal motility. The present study aimed to elucidate whether or how ICCs are involved in the lower esophageal sphincter (LES) relaxation induced by stimulation of the nicotinic acetylcholine receptor. The application of 1,1-dimethyl-4-phenyl-piperazinium (DMPP; a nicotinic acetylcholine receptor agonist) induced a transient relaxation in the circular smooth muscle of the porcine LES. DMPP-induced relaxation was abolished by not only 1 µM tetrodotoxin but also the inhibition of ICC activity by pretreatment with 100 µM carbenoxolone (a gap junction inhibitor), pretreatment with 100 µM CaCCinh-A01 (an anoctamin-1 blocker acting as a calcium-activated chloride channel inhibitor), and pretreatment with Cl--free solution. However, pretreatment with 100 µM Nω-nitro-L-arginine methyl ester had little effect on DMPP-induced relaxation. Furthermore, DMPP-induced relaxation was inhibited by pretreatment with 1 mM suramin, a purinergic P2 receptor antagonist, but not by 1 µM VIP (6-28), a vasoactive intestinal peptide (VIP) receptor antagonist. Stimulation of the purinergic P2 receptor with adenosine triphosphate (ATP) induced relaxation, which was abolished by the inhibition of ICC activity by pretreatment with CaCCinh-A01. In conclusion, membrane hyperpolarization of the ICCs via the activation of anoctamin-1 plays a central role in DMPP-induced relaxation. ATP may be a neurotransmitter for inhibitory enteric neurons, which stimulate the ICCs. The ICCs act as the interface of neurotransmission of nicotinic acetylcholine receptor in order to induce LES relaxation.

Atractylenolide­1 alleviates gastroparesis in diabetic rats by activating the stem cell factor/c­kit signaling pathway.

Diabetic gastroparesis (DGP), also known as delayed gastric emptying, is a common complication of diabetes mellitus. There are numerous clinical symptoms associated with DGP, as well as high treatment costs and markedly reduced patient quality of life. However, the pathogenesis of DGP is not clear, thus effective treatment methods are yet to be established. In the present study, a DGP rat model was established in Sprague­Dawley rats by the intraperitoneal injection of streptozotocin (STZ). DGP model rats were treated with different doses of atractylenolide­1 to detect alterations in gastrointestinal function, including gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow. Compared with the DGP group, atractylenolide­1 treatment significantly reduced glycaemia and the level of glycated hemoglobin, as well as restoring gastrointestinal function. Gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow were significantly impaired in the STZ­induced group compared with the vehicle control group. Moreover, the STZ­induced group displayed downregulated expression levels of the DGP indicator KIT proto­oncogene, receptor tyrosine kinase (c­kit), as investigated by immunohistochemistry, and stem cell factor (SCF) protein, as assessed using ELISA, significantly enhanced rat interstitial cells of Cajal (ICC) apoptosis, and significantly altered levels of oxidative stress­related markers (malondialdehyde and superoxide dismutase) in the serum and gastric tissues compared with the vehicle control group. By contrast, treatment with atractylenolide­1 significantly counteracted the effects of DGP on peristalsis, inhibited apoptosis and suppressed oxidative stress by regulating the expression of heme oxygenase 1 in STZ­induced DGP model rats. Further research indicated that atractylenolide­1 regulated oxidative stress reactions and improved gastric function by activating the SCF/c­kit signaling pathway. Collectively, the results of the present study suggested that atractylenolide­1 promoted ICC survival and preserved the structure of the gastric tissue network in a DGP rat model via the SCF/c­kit signaling pathway, providing novel insights for the treatment of DGP.

Nopp140-chaperoned 2'-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity.

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at ∼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase.

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.

Colonic Motility Is Improved by the Activation of 5-HT2B Receptors on Interstitial Cells of Cajal in Diabetic Mice.

BACKGROUND & AIMS: Constipation is commonly associated with diabetes. Serotonin (5-HT), produced predominantly by enterochromaffin (EC) cells via tryptophan hydroxylase 1 (TPH1), is a key modulator of gastrointestinal (GI) motility. However, the role of serotonergic signaling in constipation associated with diabetes is unknown. METHODS: We generated EC cell reporter Tph1-tdTom, EC cell-depleted Tph1-DTA, combined Tph1-tdTom-DTA, and interstitial cell of Cajal (ICC)-specific Kit-GCaMP6 mice. Male mice and surgically ovariectomized female mice were fed a high-fat high-sucrose diet to induce diabetes. The effect of serotonergic signaling on GI motility was studied by examining 5-HT receptor expression in the colon and in vivo GI transit, colonic migrating motor complexes (CMMCs), and calcium imaging in mice treated with either a 5-HT2B receptor (HTR2B) antagonist or agonist. RESULTS: Colonic transit was delayed in males with diabetes, although colonic Tph1+ cell density and 5-HT levels were increased. Colonic transit was not further reduced in diabetic mice by EC cell depletion. The HTR2B protein, predominantly expressed by colonic ICCs, was markedly decreased in the colonic muscles of males and ovariectomized females with diabetes. Ca2+ activity in colonic ICCs was decreased in diabetic males. Treatment with an HTR2B antagonist impaired CMMCs and colonic motility in healthy males, whereas treatment with an HTR2B agonist improved CMMCs and colonic motility in males with diabetes. Colonic transit in ovariectomized females with diabetes was also improved significantly by the HTR2B agonist treatment. CONCLUSIONS: Impaired colonic motility in mice with diabetes was improved by enhancing HTR2B signaling. The HTR2B agonist may provide therapeutic benefits for constipation associated with diabetes.

Contribution of Interstitial Cells of Cajal to Gastrointestinal Stromal Tumor Risk.

BACKGROUND Gastrointestinal stromal tumors (GISTs), which originate from interstitial cells of Cajal (ICCs), are one of most common mesenchymal tumors of the gastrointestinal tract. This study explored the impact of ICCs and immunological markers on GIST risk. MATERIAL AND METHODS A total of 122 patients diagnosed with GISTs who underwent surgery were recruited for the study. Demographic and clinical information, including modified NIH criteria, sex, age, tumor site, and tumor size, of all patients were collected. GIST risk was assessed using the modified NIH risk classification for primary GISTs. Paraffin-embedded GIST specimens were evaluated by hematoxylin-eosin staining and ICCs immunohistochemistry. RESULTS According to the modified NIH criteria, most GIST cases (44 cases, 36.07%) were at very low risk. Females had greater incidence of high-risk GISTs (P<0.05). The mean age at GIST diagnosis was 58.69±9.90 years and had no impact on GIST risk (P>0.05). Most GISTs were located in the stomach (87 cases, 71.73%), and the size of the tumors varied (0.5-20 cm). CD117/c-kit and CD34 were specific immuno-markers for ICCs and GIST. Most patients with GIST were CD117-positive (115 cases, 94.26%), 111 cases (90.98%) were CD34-positive, and 109 cases (89.34%) were positive for both CD117/c-kit and CD34. With increasing GIST risk, CD117 (also named c-k0it) and CD34 expression levels increased, as well as the number of ICCs (all P<0.05). CONCLUSIONS ICCs have a great impact on GISTs incidence. CD117/c-kit and CD34 expression, as well ICCs levels, appear to affect GIST risk.

Relationships between serum electrolyte concentrations and ileus: A joint clinical and mathematical modeling study.

AIM: Prolonged postoperative ileus (PPOI) occurs in around 15% of patients after major abdominal surgery, posing a significant clinical and economic burden. Significant fluid and electrolyte changes may occur peri-operatively, potentially contributing to PPOI; however, this association has not been clearly elucidated. A joint clinical-theoretical study was undertaken to evaluate peri-operative electrolyte concentration trends, their association with ileus, and predicted impact on bioelectrical slow waves in interstitial cells of Cajal (ICC) and smooth muscle cells (SMC). METHODS: Data were prospectively collected from 327 patients undergoing elective colorectal surgery. Analyses were performed to determine associations between peri-operative electrolyte concentrations and prolonged ileus. Biophysically based ICC and SMC mathematical models were adapted to evaluate the theoretical impacts of extracellular electrolyte concentrations on cellular function. RESULTS: Postoperative day (POD) 1 calcium and POD 3 chloride, sodium were lower in the PPOI group (p < 0.05), and POD3 potassium was higher in the PPOI group (p < 0.05). Deficits beyond the reference range in PPOI patients were most notable for sodium (Day 3: 29.5% ileus vs. 18.5% no ileus, p = 0.04). Models demonstrated an 8.6% reduction in slow-wave frequency following the measured reduction in extracellular NaCl on POD5, with associated changes in cellular slow-wave morphology and amplitude. CONCLUSION: Low serum sodium and chloride concentrations are associated with PPOI. Electrolyte abnormalities are unlikely to be a primary mechanism of ileus, but their pronounced effects on cellular electrophysiology predicted by modeling suggest these abnormalities may adversely impact motility recovery. Resolution and correction of electrolyte abnormalities in ileus may be clinically relevant.

miR-10b-5p Rescues Diabetes and Gastrointestinal Dysmotility.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic ß cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. METHODS: We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of ß cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Krüppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.