Chaihu Shugan Powder inhibits interstitial cells of cajal mitophagy through USP30 in the treatment of functional dyspepsia.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaihu Shugan Powder (CHSGP) has significant clinical efficacy in the treatment of functional dyspepsia (FD), but the specific mechanism requires further study. AIM OF STUDY: The aim of this study was to investigate the therapeutic effect of CHSGP on FD rats and the underlying mechanism of the effect on interstitial cells of cajal (ICC) mitophagy. MATERIALS AND METHODS: The tail-clamping stimulation method was utilized to establish an FD rat model in vivo. Gastric emptying rate and small intestinal propulsion rate test, H&E staining, and Immunohistochemistry were conducted to evaluate the therapeutic effects of CHSGP on FD rats. In vitro, the regulatory effect of CHSGP on CCCP-mediated ICC mitophagy was further investigated by CCK8, Transmission electron microscope, immunofluorescence co-staining, Quantitative polymerase chain reaction and Western blot to reveal the potential mechanisms of CHSGP inhibited ICC mitophagy. RESULTS: Animal experiments provided evidence that CHSGP promoted gastric motility, increased ICC numbers, reduced Parkin expression, and elevated USP30 expression in FD rats. In vitro, further mechanism research demonstrated that CHSGP decreased LC3Ⅱ/LC3Ⅰ、PINK1、Parkin、PHB2 protein expression and increased USP30 protein expression. Furthermore, CHSGP increased Mfn2 protein expression by suppressing activation of the PINK1/Parkin pathway when USP30 is knocked down, consequently reducing CCCP-induced ICC mitophagy. CONCLUSIONS: These results suggest that CHSGP may treat FD against CCCP-induced ICC mitophagy by the up-regulation of via PINK1/Parkin pathway.

A Study on the Effects of Muscarinic and Serotonergic Regulation by Bojanggunbi-tang on the Pacemaker Potential of the Interstitial Cells of Cajal in the Murine Small Intestine.

In traditional Korean medicine, the 16-herb concoction Bojanggunbi-tang (BGT) is used to treat various gastrointestinal (GI) diseases. In this study, we investigated the regulatory mechanism underlying the influence of BGT on the interstitial cells of Cajal (ICCs), pacemaker cells in the GI tract. Within 12 h of culturing ICCs in the small intestines of mice, the pacemaker potential of ICCs was recorded through an electrophysiological method. An increase in the BGT concentration induced depolarization and decreased firing frequency. This reaction was suppressed by cholinergic receptor muscarinic 3 (CHRM3) antagonists, as well as 5-hydroxytryptamine receptor (5HTR) 3 and 4 antagonists. Nonselective cation channel inhibitors, such as thapsigargin and flufenamic acid, along with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors, also suppressed the BGT reaction. Guanylate cyclase and protein kinase G (PKG) antagonists inhibited BGT, but adenylate cyclase and protein kinase A antagonists had no effect. In conclusion, we demonstrated that BGT acts through CHRM3, 5HTR3, and 5HTR4 to regulate intracellular Ca2+ concentrations and the PKC, MAPK, guanylate cycle, and PKG signaling pathways.

Intestinal expression patterns of transcription factors and markers for interstitial cells in the larval zebrafish.

For the digestion of food, it is important for the gut to be differentiated regionally and to have proper motor control. However, the number of transcription factors that regulate its development is still limited. Meanwhile, the interstitial cells of the gastrointestinal (GI) tract are necessary for intestinal motility in addition to the enteric nervous system. There are anoctamine1 (Ano1)-positive and platelet-derived growth factor receptor α (Pdgfra)-positive interstitial cells in mammal, but Pdgfra-positive cells have not been reported in the zebrafish. To identify new transcription factors involved in GI tract development, we used RNA sequencing comparing between larval and adult gut. We isolated 40 transcription factors that were more highly expressed in the larval gut. We demonstrated expression patterns of the 13 genes, 7 of which were newly found to be expressed in the zebrafish larval gut. Six of the 13 genes encode nuclear receptors. The osr2 is expressed in the anterior part, while foxP4 in its distal part. Also, we reported the expression pattern of pdgfra for the first time in the larval zebrafish gut. Our data provide fundamental knowledge for studying vertebrate gut regionalization and motility by live imaging using zebrafish.

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

BACKGROUND & AIMS: 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. METHODS: 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 ex vivo systems. Transformation-related protein 53 was induced with nutlin 3a and ERK1/2 signaling was activated by rhIGF-1 in the ICC-SC line. RESULTS: 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 in vivo systems. In ICC-SC cultures, rhIGF1 mitigated nutlin 3a-induced reduced ERK1/2 phosphorylation and cell growth arrest. CONCLUSIONS: 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.

Effects of Banxia Xiexin Decoction on apoptosis of interstitial cells of Cajal via regulation of MiR-451-5p: An in vivo and in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin Decoction (BXD) is a traditional Chinese medical formula applied to gastrointestinal (GI) motility disorders. Previous studies showed that miR-451-5p was down-regulated in rats with GI motility disorders induced by gastric electrical dysrhythmia. Interstitial cells of Cajal (ICCs) are pacemakers for GI motility, while loss of ICCs is responsible for GI motility disturbance. Thus, the underlying interaction mechanisms for BXD regulating ICCs apoptosis via miR-451-5p remain to be explored. AIM OF THE STUDY: In this work, the main objectives were to examine the efficacy of BXD on ICCs via miR-451-5p both in GI motility disorders rats model and in vitro, as well as the potential contributions of SCF/c-kit signaling. MATERIALS AND METHODS: Rats with gastric electrical dysrhythmia were established in male SD rats by using a single-day diet and a double fasting method (drinking diluted hydrochloric acid water during the period) for 4 weeks. The gastric slow wave (GSW) recording, RT-qPCR, and western blot were performed to examine the effects of BXD on ICCs apoptosis in rats with GED and miR-451-5p expression. In vitro assays included CCK-8, flow cytometry analysis, RT-qPCR, and western blot were applied to investigate the potential molecular mechanism of BXD on ICCs apoptosis via miR-451-5p. RESULTS: BXD promoted gastric motility, reduced ICCs apoptosis, and elevated miR-451-5p in GED rats. In addition, miR-451-5p was significantly up-regulated in ICCs after BXD treatment compared with that in ICCs with miR-451-5p inhibitor transfection. Meanwhile, high miR-451-5p expression with either BXD treatment or miRNA mimics enhanced ICCs proliferation and inhibit apoptosis. Moreover, overexpression of miR-451-5p can reverse G0/G1 arrest in ICCs by BXD treatment. Further, SCF and c-kit protein levels were detected to demonstrate that modulation of miR-451-5p by BXD treatment was involved in this signaling. CONCLUSIONS: Through this study, we demonstrated that BXD could promote ICCs proliferation and inhibit apoptosis via miR-451-5p and may involve the modulations of SCF/c-kit signaling, thus suggesting a new therapy basis for GI motility dysfunction from the perspective of modulation of ICCs apoptosis by targeting miR-451-5p.

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.

Single-cell profiling reveals various types of interstitial cells in the bladder.

Clarifying the locations, molecular markers, functions and roles of bladder interstitial cells is crucial for comprehending the pathophysiology of the bladder. This research utilized human, rat and mouse bladder single-cell sequencing, bioinformatics analysis and experimental validation. The main cell types found in human, rat and mouse bladder tissues include epithelial cells, smooth muscle cells, endothelial cells, fibroblasts, myofibroblasts, neurons and various immune cells. Our study identified two significant types of interstitial cells (PTN+ IGFBP6+ PI16 (CD364)+ CD34+ ) and myofibroblasts (STC1+ PLAT+ TNC+ ). These two types of interstitial cells are mainly located in the subepithelial lamina propria, between muscles and between muscle bundles. In the CYP (cyclophosphamide)-induced bladder injury mouse model, the interaction types and signals (MK, MIF, GDF and CXCL) of fibroblasts and myofibroblasts significantly increased compared with the normal group. However, in the aging mouse model, the signals CD34, LAMININ, GALECTIN, MK, SELPLG, ncWNT, HSPG, ICAM and ITGAL-ITGB2 of fibroblasts and myofibroblasts disappeared, but the signals PTN and SEMA3 significantly increased. Our findings identified two crucial types of interstitial cells in bladder tissue, fibroblasts and myofibroblasts, which play a significant role in normal bladder physiology, CYP-induced bladder injury and aging bladder development.

Chaihu Shugan San promotes gastric motility in rats with functional dyspepsia by regulating Drp-1-mediated ICC mitophagy.

CONTEXT: Chaihu Shugan San (CHSGS) was effective in the treatment of functional dyspepsia (FD). OBJECTIVE: To investigate the mechanism of CHSGS in FD through dynamin-related protein 1 (Drp-1)-mediated interstitial cells of cajal (ICC) mitophagy. MATERIALS AND METHODS: Forty Sprague-Dawley (SD) rats were randomly divided into control, model, mdivi-1, mdivi-1 + CHSGS and CHSGS groups. Tail-clamping stimulation was used to establish the FD model. Mdivi-1 + CHSGS and CHSGS groups were given CHSGS aqueous solution (4.8 g/kg) by gavage twice a day. Mdivi-1 (25 mg/kg) was injected intraperitoneally once every other week for 4 w. Mitochondrial damage was observed by corresponding kits and related protein expressions were assessed by Immunofluorescence and (or) Western Blot. RESULTS: Compared with the mean value of the control group, superoxide dismutase (SOD) and citrate synthase (CS) in the model group were decreased by 11% and 35%; malondialdehyde (MDA) and reactive oxygen species (ROS) were increased by 1.2- and 2.8-times; ckit fluorescence and protein expressions were decreased by 85% and 51%, co-localization expression of LC3 and voltage dependent anion channel 1 (VDAC1), Drp-1 and translocase of the outer mitochondrial membrane 20 (Tom20) were increased by 10.1- and 5.4-times; protein expressions of Drp-1, Beclin-1, and LC3 were increased by 0.5-, 1.4-, and 2.5-times whereas p62 was decreased by 43%. After mdivi-1 and (or) CHSGS intervention, the above situation has been improved. DISCUSSION AND CONCLUSION: CHSGS could improve mitochondrial damage and promote gastric motility in FD rats by regulating Drp-1-mediated ICC mitophagy.

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.

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).

Corticotropin-releasing factor receptor agonists decrease interstitial cells of Cajal in murine colon.

BACKGROUND: Peripheral corticotropin-releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin-releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC). METHODS: Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF-CRF receptor signaling on colonic ICC. RESULTS: We found that stressin1, a selective CRF receptor 1 (CRF1 ) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c-Kit, Anoctamin-1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1-treated mice. Accordingly, 2-(4-Chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1-treated mice compared to the saline-treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c-Kit expressions were also obtained from the colon of CHeS-treated mice. CONCLUSION: All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress-induced gastrointestinal motility disorder.

[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.

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.

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.

Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA+ interstitial cells are fibroblasts.

Fibroblasts are crucial to normal and abnormal organ and tissue biology, yet we lack basic insights into the fibroblasts that populate the bladder wall. Candidates may include bladder interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells), which express the fibroblast-associated marker PDGFRA along with VIM and CD34 but whose form and function remain enigmatic. By applying the latest insights in fibroblast transcriptomics, coupled with studies of gene expression, ultrastructure, and marker analysis, we observe the following: 1) that mouse bladder PDGFRA+ cells exhibit all of the ultrastructural hallmarks of fibroblasts including spindle shape, lack of basement membrane, abundant endoplasmic reticulum and Golgi, and formation of homotypic cell-cell contacts (but not heterotypic ones); 2) that they express multiple canonical fibroblast markers (including Col1a2, CD34, LY6A, and PDGFRA) along with the universal fibroblast genes Col15a1 and Pi16 but they do not express Kit; and 3) that PDGFRA+ fibroblasts include suburothelial ones (which express ACTA2, CAR3, LY6A, MYH10, TNC, VIM, Col1a2, and Col15a1), outer lamina propria ones (which express CD34, LY6A, PI16, VIM, Col1a2, Col15a1, and Pi16), intermuscular ones (which express CD34, VIM, Col1a2, Col15a1, and Pi16), and serosal ones (which express CD34, PI16, VIM, Col1a2, Col15a1, and Pi16). Collectively, our study revealed that the ultrastructure of PDFRA+ interstitial cells combined with their expression of multiple canonical and universal fibroblast-associated gene products indicates that they are fibroblasts. We further propose that there are four regionally distinct populations of fibroblasts in the bladder wall, which likely contribute to bladder function and dysfunction.NEW & NOTEWORTHY We currently lack basic insights into the fibroblasts that populate the bladder wall. By exploring the ultrastructure of mouse bladder connective tissue cells, combined with analyses of their gene and protein expression, our study revealed that PDGRA+ interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells) are fibroblasts and that the bladder wall contains multiple, regionally distinct populations of these cells.

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.

The hyperpolarization-activated, cyclic nucleotide-gated channel resides on myocytes in mouse bladders and contributes to adrenergic-induced detrusor relaxation.

Control of urinary continence is predicated on sensory signaling about bladder volume. Bladder sensory nerve activity is dependent on tension, implicating autonomic control over detrusor myocyte activity during bladder filling. Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are known contributors to bladder control, but their mechanism of action is not well understood. The lack of a definitive identification of cell type(s) expressing HCN in the bladder presents a significant knowledge gap. We recently reported a complete transcriptomic atlas of the C57BL/6 mouse bladder showing the dominant HCN paralog in mouse bladder, Hcn1, is limited to a subpopulation of detrusor smooth myocytes (DSMs). Here, we report details of these findings, along with results of patch-clamp experiments, immunohistochemistry, and functional myobath/tension experiments in bladder strips. With the use of a transgenic mouse expressing fluorescence-tagged α-smooth muscle actin, our data confirmed location and function of DSM HCN channels. Despite previous associations of HCN with postulated bladder interstitial cells, neither evidence of specific interstitial cell types nor an association of nonmyocytes with HCN was discovered. We confirm that HCN activation participates in reducing sustained (tonic) detrusor tension via cAMP, with no effect on intermittent (phasic) detrusor activity. In contrast, blockade of HCN increases phasic activity induced by a protein kinase A (PKA) blocker or a large-conductance Ca2+-activated K+ (BK) channel opener. Our findings, therefore, suggest a central role for detrusor myocyte HCN in regulating and constraining detrusor myocyte activity during bladder filling.

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.

Transcriptome profiling of subepithelial PDGFRα cells in colonic mucosa reveals several cell-selective markers.

Subepithelial platelet-derived growth factor receptor alpha (PDGFRα)+ cells found in the colonic mucosal tissue come in close contact with epithelial cells, immune cells, neurons, capillaries, and lymphatic networks. Mucosal subepithelial PDGFRα+ cells (MuPαC) are important regulators in various intestinal diseases including fibrosis and inflammation. However, the transcriptome of MuPαC has not yet been elucidated. Using Pdgfra-eGFP mice and flow cytometry, we isolated colonic MuPαC and obtained their transcriptome data. In analyzing the transcriptome, we identified three novel, and selectively expressed, markers (Adamdec1, Fin1, and Col6a4) found in MuPαC. In addition, we identified a unique set of MuPαC-enriched genetic signatures including groups of growth factors, transcription factors, gap junction proteins, extracellular proteins, receptors, cytokines, protein kinases, phosphatases, and peptidases. These selective groups of genetic signatures are linked to the unique cellular identity and function of MuPαC. Furthermore, we have added this MuPαC transcriptome data to our Smooth Muscle Genome Browser that contains the transcriptome data of jejunal and colonic smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and smooth muscle resident PDGFRα+ cells: (https://med.unr.edu/physio/transcriptome). This online resource provides a comprehensive reference of all currently known genetic transcripts expressed in primary MuPαC in the colon along with smooth muscle resident PDGFRα cells, SMC, and ICC in the murine colon and jejunum.

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.