Profile of interstitial cells of Cajal in a murine model of chagasic megacolon.

Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach's myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development.

Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal.

Aurantii Fructus Immaturus total flavonoids (AFIF) is the main effective fraction extracted from AFI, which has a good effect on promoting gastrointestinal motility. This study aimed to investigate AFIF which regulates miR-5100 to improve constipation symptoms in mice by targeting Frizzled-2 (Fzd2) to alleviate interstitial cells of Cajal (ICCs) calcium ion balance and autophagy apoptosis. The constipated mouse model was induced by an antibiotic suspension, and then treated with AFIF. RNA-seq sequencing, luciferase assay, immunofluorescence staining, transmission electron microscopy, ELISA, flow cytometry, quantitative polymerase chain reaction (PCR), and Western blot were applied in this study. The results showed that AFIF improved constipation symptoms in antibiotic-induced constipated mice, and decreased the autophagy-related protein Beclin1 levels and the LC3-II/I ratio in ICCs. miR-5100 and its target gene Fzd2 were screened as key miRNAs and regulator associated with autophagy. Downregulation of miR-5100 caused increased expression of Fzd2, decreased proliferation activity of ICCs, increased apoptotic cells, and enhanced calcium ion release and autophagy signals. After AFIF treatment, miR-5100 expression was upregulated and Fzd2 was downregulated, while autophagy-related protein levels and calcium ion concentration decreased. Furthermore, AFIF increased the levels of SP, 5-HT, and VIP, and increased the expression of PGP9.5, Sy, and Cx43, which alleviated constipation by improving the integrity of the enteric nervous system network. In conclusion, AFIF could attenuate constipation symptoms by upregulating the expression of miR-5100 and targeting inhibition of Fzd2, alleviating calcium overload and autophagic death of ICCs, regulating the content of neurotransmitters, and enhancing the integrity of the enteric nervous system network.

Characteristics of the Cajal interstitial cells and intestinal microbiota in children with refractory constipation.

BACKGROUND: Children with refractory constipation experience intense and persistent symptoms that greatly diminish their quality of life. However, the underlying pathophysiological mechanism responsible for this condition remains uncertain. Our objective was to evaluate characteristics of colonic motor patterns and interstitial cells of Cajal (ICCs) to refractory constipation children, as well as intestinal microbiota compositions. METHODS: Colonic manometry (CM) was conducted on a cohort of 30 patients with refractory constipation to assess colonic motility, and 7 of them underwent full-thickness colon biopsy specimens. Another 5 colonic specimens from nonconstipation patients were collected to identify the ICCs by immunohistochemistry. Fecal samples from 14 children diagnosed with refractory constipation and subjecting 28 age-matched healthy children to analysis using high-throughput sequencing of 16S rRNA. RESULTS: According to CM results, dividing 30 children with refractory constipation into 2 groups: normal group (n = 10) and dysmotility group (n = 20). Dysmotility subjects showed lower colonic motility. Antegrade propagating pressure waves, retrograde propagating pressure waves, and periodic colonic motor activity were common in normal subjects and rare in dysmotility subjects (32.7 ± 8.9 vs 20.7 ± 13.0/17 h, P < 0.05, 11.5 ± 2.3 vs 9.6 ± 2.3/17 h, P < 0.05, and 5.2 ± 8.9 vs 3.5 ± 6.8 cpm, P < 0.005, respectively), whereas periodic rectal motor activity was more common in dysmotility subjects (3.4 ± 4.8 vs 3.0 ± 3.1 cpm, P < 0.05). Dysmotility subjects exhibited a significantly greater number of preprandial simultaneous pressure waves compared to the normal subjects (32.3 ± 25.0 vs 23.6 ± 13.2/1 h, P < 0.005). Dysmotility subjects displayed a notable decrease in postprandial count of antegrade propagating pressure waves and high amplitude propagating pressure waves when compared to normal subjects (3.9 ± 2.9 vs 6.9 ± 3.5/1 h and 2.3 ± 1.5 vs 5.4 ± 2.9/1 h, respectively, P < 0.05). The number, distribution, and morphology of ICCs were markedly altered in refractory constipation compared children to the controls (P < 0.05). Children diagnosed with refractory constipation displayed a distinct dissimilarity in composition of their intestinal microbiota comparing with control group (P < 0.005). In genus level, Bacteroidetes represented 34.34% and 43.78% in the refractory constipation and control groups, respectively. Faecalibacterium accounted for 3.35% and 12.56%, respectively (P < 0.005). Furthermore, the relative abundances of Faecalibacterium (P < 0.005), Lachnospira (P < 0.05), and Haemophilus (P < 0.05) significantly decreased, whereas those of Parabacteroides (P < 0.05), Alistipes (P < 0.005), Prevotella_2 (P < 0.005), [Ruminococcus]_torques_group (P < 0.005), Barnesiella (P < 0.05), Ruminococcaceae_UCG-002 (P < 0.005), and Christensensenellaceae_R-7_group (P < 0.05) were markedly increased in children with refractory constipation. CONCLUSIONS: Dysmotility subjects showed lower colonic motility and an impaired postprandial colonic response. The decreased number and abnormal morphology of colonic ICCs may contribute to the pathogenesis of refractory constipation. Children with refractory constipation exhibited significant variations in microbiota composition across various taxonomic levels compared to the healthy control group. Our findings contribute valuable insights into pathophysiological mechanism underlying refractory constipation and provide evidence to support the exploration of novel therapeutic strategies for affected children.

Diagnostic dilemma and challenges in management: Hirschsprung's disease, anal stenosis and reduced interstitial cells of Cajal enteric mesenchymopathy.

Hirschsprung's disease (HD) is one of the most well-known gastrointestinal motility disorders. Diagnosis and management of other lesser-known motility disorders are often challenging and tedious. We describe a teenager who was severely constipated from birth and needed intensive care admissions for life-threatening enterocolitis. She also had concomitant anal stenosis. Several rectal biopsies were unable to yield a conclusive diagnosis. Surgical level of resection had to be identified based on the motility of the bowel as determined by transit studies using oral ingestion of a milk feed labelled with Technetium-99m colloid. After completion of all operative stages, histopathological examination of the excised specimens concluded that she had short-segment HD associated with reduced interstitial cells of Cajal in the large bowel. She is currently continent, evacuating voluntarily approximately four times a day and is relieved of all her symptoms.

"M1/M2" Muscularis Macrophages Are Associated with Reduction of Interstitial Cells of Cajal and Glial Cells in Achalasia.

BACKGROUND AND AIMS: Several studies showed muscularis macrophages (MMφ) are associated with GI motility disorders. The purpose of this study was to preliminary explore the association between MMφ and achalasia. METHODS: Tissue samples of the lower esophageal sphincter (LES) high-pressure zone were obtained from 27 achalasia patients and 10 controls. Immunohistochemistry for MMφ, interstitial cells of Cajal (ICC), neuronal nitric oxide synthase (nNOS), and glial cells were conducted. Histological characteristics were compared between groups, and correlation analysis was performed. RESULTS: Fewer ICC was found in achalasia compared with controls (P = 0.018), and the level of M1 macrophages was higher than that in controls no matter in terms of the number or the proportion of M1(P = 0.026 for M1 and 0.037 for M1/MMφ). Statistical differences were found between two groups in terms of proportion of M2 and ratio of M1 to M2 (P = 0.048 for M2/ MMφ and < 0.001 for M1/M2). For the correlation analysis, significant correlations were detected between levels of nNOS, ICC, and glial cells in patients with achalasia (P = 0.026 for nNOS and ICC, 0.001 for nNOS and glial cells, 0.019 for ICC and glial cells). There were significant correlations between M2/MMφ and levels of ICC (P = 0.019), glial cells (P = 0.004), and nNOS (P = 0.135). CONCLUSION: Patients with achalasia had a higher level of M1/M2 ratio in LES and significant correlations were found between M2/MMφ and numbers of ICC and glial cells, which suggested that MMφ were probably associated with occurrence and development of achalasia.

Quantification of Interstitial Cells of Cajal in the Gastric Muscle of Patients with Gastroparesis at Per-Oral Endoscopic Pyloromyotomy: A Novel Approach for Future Research in Pathogenesis of Gastroparesis.

BACKGROUND: The role of Interstitial Cells of Cajal (ICC) in the pathogenesis of gastroparesis has been suggested by previous studies due to their involvement in the transmission of neuronal signaling to the smooth muscles of the GI tract. However, studies have been limited by the inability to obtain a gastric muscle sample, since routine endoscopy can only biopsy the mucosa. We present a new technique of muscle biopsy during per-oral endoscopic pyloromyotomy (GPOEM), a novel endoscopic procedure for treatment of gastroparesis. PATIENTS AND METHODS: All enrolled patients had diagnosed gastroparesis and had biopsies of the muscular layer at the antrum/pylorus during POEM. All GPOEM procedures took place from August 2019 to December 2019. Various demographic, disease-related, and procedure-related data were collected from chart review. ICC in the biopsy specimen was examined and quantitated. RESULTS: Through this method, we readily expose the gastric muscle of 21 patients through dissection of a gastric submucosal tunnel during GPOEM and provide reliable muscle sample for ICC quantification. Average number of ICC were higher in clinical responders (88 ICC ± 63 vs. 39 ICC ± 24, p = 0.02), defined as those who experienced significant improvement in nausea and vomiting symptoms after GPOEM. CONCLUSIONS: This study provides a reliable novel biopsy method for safely biopsy gastric muscle for quantitating the number of gastric ICC in patients with gastroparesis. The number of ICC may be related to the outcome of GPOEM therapy. However, further studies with larger number of patients are needed to confirm the results.

Interstitial Cells of Cajal-Origin, Distribution and Relationship with Gastrointestinal Tumors.

The interstitial cells of Cajal (ICC) represent a particular network formed by some peculiar cells that were first described by the great neuroanatomist, S. Ramon y Cajal. Nowadays, the ICC have become a fascinating topic for scientists, arousing their curiosity; as a result, there is a vast number of published articles related to the ICC. Today, everybody widely accepts that the ICC represent the pacemaker of the gastrointestinal tract and are highly probable to be the origin cells for gastrointestinal tumors (GISTs). Recently, Cajal-like cells (ICLC) were described, which are found in different organs but with an as yet unknown physiological role that needs further study. New information regarding intestinal development indicates that the ICC (fibroblast-like and muscle-like) and intestinal muscle cells have the same common embryonic cells, thereby presenting the same cellular ultrastructure. Nowadays, there is a vast quantity of information that proves the connection of the ICC and GISTs. Both of them are known to present c-kit expression and the same ultrastructural cell features, which includes minimal myoid differentiation that is noticed in GISTs, therefore, supporting the hypothesis that GISTs are ICC-related tumors. In this review, we have tried to highlight the origin and distribution of Cajal interstitial cells based on their ultrastructural features as well as their relationship with gastrointestinal stromal tumors.

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.

The Number of Interstitial Cells of Cajal Differs Among Different Subtypes of Achalasia and is Related to Patients' Prognosis.

INTRODUCTION: Achalasia is a primary esophageal motility disorder with heterogeneous manometric subtypes and prognosis, characterized by degeneration of the esophageal myenteric plexus, and reduction in interstitial cells of Cajal (ICCs). This study aimed to explore the histopathologic characteristics of lower esophageal sphincter (LES) muscle from patients with achalasia with different subtypes and different prognosis. METHODS: We examined specimens of LES muscle from 122 patients with achalasia who underwent peroral endoscopic myotomy and from 10 control patients who underwent esophagectomy for esophageal cancer. Hematoxylin-eosin staining was performed to assess inflammation infiltration, fibrosis, and atrophy. Specific immunohistochemical staining was performed to identify ICCs and neuronal nitric oxide synthase (nNOS). RESULTS: The number of ICCs in patients with type I achalasia was significantly lower than that in patients with type II achalasia, followed by that in control patients (type I vs type II vs control group= 0.4 vs 1.2 vs 9.5; P < 0.001). The number of nNOS-positive cells was significantly lower in patients with achalasia than that in control patients (type I vs type II vs control group = 0.0 vs 0.0 vs 8.0; P < 0.001). Nonrecurrent group had significantly more ICCs than recurrent group (type I: nonrecurrent vs recurrent = 1.0 vs 0.1; P = 0.010; type II: nonrecurrent vs recurrent = 2.0 vs 0.4; P = 0.004). DISCUSSION: ICCs and nNOS-positive cells reduced significantly in LES muscle of patients with achalasia. The number of ICCs differed among different achalasia subtypes and was related to patients' clinical prognosis.

Relation between Cajal Cell Density and Radiological and Scintigraphic Outcomes in Patients with Ureteropelvic Junction Obstruction.

INTRODUCTION: In this study, we aimed to investigate the correlation between Cajal cell density and preoperative and postoperative radiological and scintigraphic parameters in ureteropelvic junction obstruction (UPJO). METHODS: The study group consisted of 41 renal units (38 consecutive patients; 13 female and 25 male) surgically treated for UPJO. UPJ specimens from patients were immuno-stained with CD117 (c-kit) antibody for interstitial Cajal cells (ICCs). The relation between Cajal cell density and preoperative and postoperative radiological and scintigraphic parameters was evaluated. RESULTS: The mean age of the patients was 8.52 ± 8.86 (0-35) years. The density of Cajal cells was defined in 2 groups for convenient analysis as 0-5 cells (low) in 19 (46.3%) patients and >5 cells (moderate-high) in 22 (53.6%). There was significant difference between the preoperative and postoperative anteroposterior diameters of the related kidneys in both Cajal groups (p = 0.001-low, p = 0.000-moderate-high) independent of Cajal cell density. Regression in hydronephrosis postoperatively was determined in both Cajal groups (77.8%-low, 64.7%-moderate-high); however, there was no difference between them (p = 0.39). Preoperative T1/2 was significantly longer in the low Cajal group (p = 0.02). Postoperative T1/2 decreased in both low (p = 0.000) and moderate-high (p = 0.001) Cajal groups, but no difference was found between them (p = 0.24). There was significant improvement in the kidney differential function after surgery in the low Cajal density group (p = 0.015) while there was no correlation between the scintigraphic success or improvement and Cajal cell density (p = 0.51). DISCUSSION/CONCLUSION: ICC deficiency/density could not be shown as a predictive factor for the determination of success rate of pyeloplasty. Despite the lack of any evidence for the degree of deficiency as an indicator for the severity of obstruction and prediction of surgical success, further studies are needed for confirmation.

Interaction between the Renin-Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis.

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

Gastric Electrical Pacing Reduces Apoptosis of Interstitial Cells of Cajal via Antioxidative Stress Effect Attributing to Phenotypic Polarization of M2 Macrophages in Diabetic Rats.

BACKGROUND: Gastric electrical pacing (GEP) could restore interstitial cells of Cajal in diabetic rats. M2 macrophages contribute to the repair of interstitial cells of Cajal injury though secreting heme oxygenase-1 (HO-1). The aim of the study is to investigate the effects and mechanisms of gastric electrical pacing on M2 macrophages in diabetic models. METHODS: Sixty male Sprague-Dawley rats were randomized into control, diabetic (DM), diabetic with the sham GEP (DM+SGEP), diabetic with GEP1 (5.5 cpm, 100 ms, 4 mA) (DM+GEP1), diabetic with GEP2 (5.5 cpm, 300 ms, 4 mA) (DM+GEP2), and diabetic with GEP3 (5.5 cpm, 550 ms, 4 mA) (DM+GEP3) groups. The apoptosis of interstitial cells of Cajal and the expression of macrophages were detected by immunofluorescence technique. The expression levels of the Nrf2/HO-1 and NF-κB pathway were evaluated using western blot analysis or immunohistochemical method. Malonaldehyde, superoxide dismutase, and reactive oxygen species were tested to reflect the level of oxidative stress. RESULTS: Apoptosis of interstitial cells of Cajal was increased in the DM group but significantly decreased in the DM+GEP groups. The total number of macrophages was almost the same in each group. In the DM group, M1 macrophages were increased and M2 macrophages were decreased. However, M2 macrophages were dramatically increased and M1 macrophages were reduced in the DM+GEP groups. Gastric electrical pacing improved the Nrf2/HO-1 pathway and downregulated the phosphorylation of NF-κB. In the DM group, the levels of malonaldehyde and reactive oxygen species were elevated and superoxide dismutase was lowered, while gastric electrical pacing reduced the levels of malonaldehyde and reactive oxygen species and improved superoxide dismutase. CONCLUSION: Gastric electrical pacing reduces apoptosis of interstitial cells of Cajal though promoting M2 macrophages polarization to play an antioxidative stress effect in diabetic rats, which associates with the activated Nrf2/HO-1 pathway and the phosphorylation of NF-κB pathway.

Functional Dyspepsia and Gastroparesis in Tertiary Care are Interchangeable Syndromes With Common Clinical and Pathologic Features.

BACKGROUND: The aim of this study was to clarify the pathophysiology of functional dyspepsia (FD), a highly prevalent gastrointestinal syndrome, and its relationship with the better-understood syndrome of gastroparesis. METHODS: Adult patients with chronic upper gastrointestinal symptoms were followed up prospectively for 48 weeks in multi-center registry studies. Patients were classified as having gastroparesis if gastric emptying was delayed; if not, they were labeled as having FD if they met Rome III criteria. Study analysis was conducted using analysis of covariance and regression models. RESULTS: Of 944 patients enrolled during a 12-year period, 720 (76%) were in the gastroparesis group and 224 (24%) in the FD group. Baseline clinical characteristics and severity of upper gastrointestinal symptoms were highly similar. The 48-week clinical outcome was also similar but at this time 42% of patients with an initial diagnosis of gastroparesis were reclassified as FD based on gastric-emptying results at this time point; conversely, 37% of patients with FD were reclassified as having gastroparesis. Change in either direction was not associated with any difference in symptom severity changes. Full-thickness biopsies of the stomach showed loss of interstitial cells of Cajal and CD206+ macrophages in both groups compared with obese controls. CONCLUSIONS: A year after initial classification, patients with FD and gastroparesis, as seen in tertiary referral centers at least, are not distinguishable based on clinical and pathologic features or based on assessment of gastric emptying. Gastric-emptying results are labile and do not reliably capture the pathophysiology of clinical symptoms in either condition. FD and gastroparesis are unified by characteristic pathologic features and should be considered as part of the same spectrum of truly "organic" gastric neuromuscular disorders. CLINICALTRIALS. GOV IDENTIFIER: NCT00398801, NCT01696747.

Gastric ablation as a novel technique for modulating electrical conduction in the in vivo stomach.

Gastric motility is coordinated by underlying bioelectrical "slow wave" activity. Slow wave dysrhythmias are associated with motility disorders, including gastroparesis, offering an underexplored potential therapeutic target. Although ablation is widely used to treat cardiac arrhythmias, this approach has not yet been trialed for gastric electrical abnormalities. We hypothesized that ablation can create localized conduction blocks and modulate slow wave activation. Radiofrequency ablation was performed on the porcine serosa in vivo, encompassing a range of parameters (55-85°C, adjacent points forming a line, 5-10 s/point). High-resolution electrical mapping (16 × 16 electrodes; 6 × 6 cm) was applied to define baseline and acute postablation activation patterns. Tissue damage was evaluated by hematoxylin and eosin and c-Kit stains. Results demonstrated that RF ablation successfully induced complete conduction block and a full thickness lesion in the muscle layer at energy doses of 65-75°C for 5-10 s/point. Gastric ablation may hold therapeutic potential for gastric electrical abnormalities in the future.NEW & NOTEWORTHY This study presents gastric ablation as a new method for modulating slow wave activation and propagation in vivo, by creating localized electrical conduction blocks in the stomach, validated by high-resolution electrical mapping and histological tissue analysis. The results define the effective energy dose range for creating conduction blocks, while maintaining the mucosal and submucosal integrity, and demonstrate the electrophysiological effects of ablation. In future, gastric ablation can now be translated toward disrupting dysrhythmic slow wave activation.

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.

Morphological damage in Sertoli, myoid and interstitial cells in a mouse model of mucopolysaccharidosis type I (MPS I).

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by a mutation in the IDUA gene, which codes α-L-iduronidase (IDUA), a lysosomal hydrolase that degrades two glycosaminoglycans (GAGs): heparan sulfate (HS) and dermatan sulfate (DS). GAGs are macromolecules found mainly in the extracellular matrix and have important signaling and structural roles which are essential to the maintenance of cell and tissue physiology. Nondegraded GAGs accumulate in various cell types, which characterizes MPS I as a multisystemic progressive disease. Many tissues and vital organs have been described in MPS I models, but there is a lack of studies focused on their effects on the reproductive tract. Our previous studies indicated lower sperm production and morphological damage in the epididymis and accessory glands in male MPS I mice, despite their ability to copulate and to impregnate females. Our aim was to improve the testicular characterization of the MPS I model, with a specific focus on ultrastructural observation of the different cell types that compose the seminiferous tubules and interstitium. We investigated the testicular morphology of 6-month-old male C57BL/6 wild-type (Idua+/+) and MPS I (Idua-/-) mice. We found vacuolated cells widely present in the interstitium and important signs of damage in myoid, Sertoli and Leydig cells. In the cytoplasmic region of Sertoli cells, we found an increased number of vesicles with substrates under digestion and a decreased number of electron-dense vesicles similar to lysosomes, suggesting an impaired flux of substrate degradation. Conclusions: Idua exerts an important role in the morphological maintenance of the seminiferous tubules and the testicular interstitium, which may influence the quality of spermatogenesis, having a greater effect with the progression of the disease.

Intestinal dysmotility after bowel resection in rats is associated with decreased ghrelin and vimentin expression and loss of intestinal cells of Cajal.

This study provides novel insight into the mechanisms of intestinal dysmotility following massive small bowel resection. We show that 2 wk after bowel resection in rats, impaired intestinal motility was associated with loss of interstitial cells of Cajal (ICC; downregulation of transmembrane member 16A (TMEM16A) and c-kit expression) as well as with decreased vimentin, desmin, and ghrelin levels. Impaired intestinal motility led to a decrease in final body weight, suggesting less effective nutrient absorption. The purpose of this study was to evaluate the mechanisms of intestinal motility in a rat model of short bowel syndrome (SBS). Rats were divided into three groups: Sham rats underwent bowel transection; SBS-NSI rats underwent a 75% bowel resection and presented with normal intestinal size (NSI) at euthanasia and hypermotility patterns; SBS-DYS showed dysmotile (DYS) enlarged intestine and inhibited motility patterns. Animals were euthanized after 2 wk. Illumina's digital gene expression (DGE) analysis was used to determine the intestinal motility-related gene expression profiling in mucosal samples. Intestinal motility-related and ICC genes and protein expression in intestinal muscle layer were determined using real-time PCR, Western blotting, and immunohistochemistry. Gastrointestinal tract motility was studied by microcomputer tomography. From 10 Ca2+ signaling pathway-related genes, six genes in jejunum and seven genes in ileum were downregulated in SBS vs. Sham animals. Downregulation of TMEM16A mRNA and protein was confirmed by real-time PCR. Rapid intestinal transit time in SBS-NSI rats correlated with a mild decrease in TMEM16A, c-kit, and vimentin mRNA and protein expression (vs/. Sham animals). SBS-DYS rats demonstrated enlarged intestinal loops and delayed small intestinal emptying (on imaging studies) that were correlated with marked downregulation in TMEM16A, c-kit, vimentin, and ghrelin mRNA and protein levels compared with the other two groups. In conclusion, 2 wk following massive bowel resection in rats, impaired intestinal motility was associated with decreased vimentin and ghrelin gene and protein levels as well as loss of ICC (c-kit and TMEM16A).NEW & NOTEWORTHY This study provides novel insight into the mechanisms of intestinal dysmotility following massive small bowel resection. We show that 2 weeks after bowel resection in rats, impaired intestinal motility was associated with loss of interstitial cells of Cajal (downregulation of TMEM 16A, and c-kit expression) as well as with decreased vimentin, desmin, and ghrelin levels. Impaired intestinal motility led to decrease in final body weight, suggesting less effective nutrient absorption.

Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification.

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.

Intestinal proinflammatory macrophages induce a phenotypic switch in interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) are pacemaker cells in the intestine, and their function can be compromised by loss of C-KIT expression. Macrophage activation has been identified in intestine affected by Hirschsprung disease-associated enterocolitis (HAEC). In this study, we examined proinflammatory macrophage activation and explored the mechanisms by which it downregulates C-KIT expression in ICCs in colon affected by HAEC. We found that macrophage activation and TNF-α production were dramatically increased in the proximal dilated colon of HAEC patients and 3-week-old Ednrb-/- mice. Moreover, ICCs lost their C-KIT+ phenotype in the dilated colon, resulting in damaged pacemaker function and intestinal dysmotility. However, macrophage depletion or TNF-α neutralization led to recovery of ICC phenotype and restored their pacemaker function. In isolated ICCs, TNF-α-mediated phosphorylation of p65 induced overexpression of microRNA-221 (miR-221), resulting in suppression of C-KIT expression and pacemaker currents. We also identified a TNF-α/NF-κB/miR-221 pathway that downregulated C-KIT expression in ICCs in the colon affected by HAEC. These findings suggest the important roles of proinflammatory macrophage activation in a phenotypic switch of ICCs, representing a promising therapeutic target for HAEC.