Gibbons, Simon J.

Abstract Background Diabetic gastroparesis results in significant morbidity for patients and major economic burden for society. Treatment options for diabetic gastroparesis are currently directed at symptom control rather than the underlying disease and are limited. The pathophysiology of diabetic gastroparesis includes damage to intrinsic and extrinsic neurons, smooth muscle, and interstitial cells of C ajal ( ICC ). Oxidative damage in diabetes appears to be one of the primary insults involved in the pathogenesis of several complications of diabetes, including gastroparesis. Recent studies have highlighted the potential role of macrophages as key cellular elements in the pathogenesis of diabetic gastroparesis. Macrophages are important for both homeostasis and defense against a variety of pathogens. Heme oxygenase 1 ( HO 1), an enzyme expressed in a subset of macrophages has emerged as a major protective mechanism against oxidative stress. Activation of macrophages with high levels of HO 1 expression protects against development of delayed gastric emptying in animal models of diabetes, while activation of macrophages that do not express HO 1 are linked to neuromuscular cell injury. Targeting macrophages and HO 1 may therefore be a therapeutic option in diabetic gastroparesis. Purpose This report briefly reviews the pathophysiology of diabetic gastroparesis with a focus on oxidative damage and how activation and polarization of different subtypes of macrophages in the muscularis propria determines development of delay in gastric emptying or protects against its development.

AbstractBackgroundHeme oxygenase 1 (HO‐1) degrades heme and protects against oxidative stress. In vitro and animal models suggest that HO‐1 is beneficial in several diseases (e.g., postoperative ileus, gastroparesis, acute pancreatitis, and colitis). However, the only drugs (i.e., hemin and heme arginate) which pharmacologically upregulate HO‐1 in humans are expensive and can only be administered intravenously. Our aims were to compare the effects of placebo, aspirin, and simvastatin alone, and with α‐lipoic acid, on HO‐1 protein concentration and activity in humans.MethodsThis randomized, double‐blind, placebo‐controlled study compared the effects of three oral regimens administered for 7 days, i.e., placebo; aspirin (325 mg twice daily) and simvastatin (40 mg twice daily); aspirin, simvastatin, and the sodium salt of R‐ α‐lipoic acid (NaRLA, 600 mg three times daily) on markers of HO‐1 activation (i.e., plasma HO‐1 protein concentration and venous monocyte HO‐1 protein activity) in 18 healthy subjects (14 females). Markers of HO‐1 activation were evaluated at baseline, days 2, and 7.Key ResultsBaseline HO‐1 protein concentrations and activity were similar among the three groups. Compared to placebo, aspirin and simvastatin combined, or together with NaRLA did not affect HO‐1 protein concentration or activity at 2 or 7 days. HO‐1 protein concentrations and activity were correlated on day 7 (r = 0.75, p = 0.0004) but not at baseline and on day 2.Conclusions & InferencesAt therapeutic doses, aspirin, simvastatin, and α‐lipoic acid do not increase plasma HO‐1 protein concentration or venous monocyte HO‐1 activity in healthy humans.

Key points Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract. AbstractInterstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca2+‐activated Cl− channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca2+ transients between myenteric ICC (ICC‐MY) of small intestine. First, we found spontaneous Ca2+ transients in ICC‐MY in both Ano1 WT and knockout (KO) mice. However, Ca2+ transients within the ICC‐MY network in Ano1 KO mice were uncoordinated, while ICC‐MY Ca2+ transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca2+ transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA‐mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca2+ transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca2+ transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non‐rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca2+ transients in ICC‐MY.

Abstract Background There is increasing evidence for specific cellular changes in the stomach of patients with diabetic ( DG ) and idiopathic ( IG ) gastroparesis. The most significant findings are loss of interstitial cells of Cajal ( ICC ), neuronal abnormalities, and an immune cellular infiltrate. Studies done in diabetic mice have shown a cytoprotective effect of CD 206+ M2 macrophages. To quantify overall immune cellular infiltrate, identify macrophage populations, and quantify CD 206+ and i NOS + cells. To investigate associations between cellular phenotypes and ICC . Methods Full thickness gastric body biopsies were obtained from non‐diabetic controls (C), diabetic controls ( DC ), DG , and IG patients. Sections were labeled for CD 45, CD 206, Kit, i NOS , and putative human macrophage markers ( HAM 56, CD 68, and EMR 1). Immunoreactive cells were quantified from the circular muscle layer. Key Results Significantly fewer ICC were detected in DG and IG tissues, but there were no differences in the numbers of cells immunoreactive for other markers between patient groups. There was a significant correlation between the number of CD 206+ cells and ICC in DG and DC patients, but not in C and IG and a significant correlation between i NOS + cells and ICC in the DC group, but not the other groups. CD 68 and HAM 56 reliably labeled the same cell populations, but EMR 1 labeled other cell types. Conclusions & Inferences Depletion of ICC and correlation with changes in CD 206+ cell numbers in DC and DG patients suggests that in humans, like mice, CD 206+ macrophages may play a cytoprotective role in diabetes. These findings may lead to novel therapeutic options, targeting alternatively activated macrophages.

Our aim was to conduct a pilot case-control study of RNA expression profile using RNA sequencing of rectosigmoid mucosa of nine females with -diarrhea-predominant irritable bowel syndrome (IBS-D) with accelerated colonic transit and nine female healthy controls. Mucosal total RNA was isolated and purified, and next-generation pair-end sequencing was performed using Illumina TruSeq. Analysis was carried out using a targeted approach toward 12 genes previously associated with IBS and a hypothesis-generating approach. Of the 12 targeted genes tested, patients with IBS-D had decreased mRNA expression of TNFSF15 (fold change controls to IBS-D: 1.53, P = 0.01). Overall, up- and downregulated mRNA expressions of 21 genes ( P = 10−5 to 10−8; P values with false detection rates are shown) were potentially relevant to IBS-D including the following: neurotransmitters [ P2RY4 ( P = 0.001), vasoactive intestinal peptide ( VIP, P = 0.02)]; cytokines [ CCL20 ( P = 0.019)]; immune function [ C4BPA complement cascade ( P = 0.0187)]; interferon-related [ IFIT3 ( P = 0.016)]; mucosal repair and cell adhesion [trefoil protein ( TFF1, P = 0.012)], retinol binding protein [ RBP2 ( P = 0.017)]; fibronectin ( FN1, P = 0.009); and ion channel functions [guanylate cyclase ( GUCA2B, P = 0.017), PDZ domain-containing protein 3 ( PDZD3, P = 0.029)]. Ten genes associated with functions related to pathobiology of IBS-D were validated by RT-PCR. There was significant correlation in fold changes of the selected genes ( Rs = 0.73, P = 0.013). Up- or downregulation of P2RY4, GUC2AB, RBP2, FNI, and C4BPA genes were confirmed on RT-PCR, which also revealed upregulation of farnesoid X receptor ( FXR) and apical sodium-coupled bile acid transporter ( IBAT/ASBT). RNA-Seq and RT-PCR analysis of rectosigmoid mucosa in IBS-D show transcriptome changes that provide the rationale for validation studies to explore the role of mucosal factors in the pathobiology of IBS-D.

Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca 2+ oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca 2+ -activated Cl − channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca 2+ entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl − current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca 2+ channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca 2+ current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl − equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl − dynamics in ICC.

Depletion of interstitial cells of Cajal (ICC) networks is known to occur in several gastrointestinal motility disorders. Although confocal microscopy can effectively image and visualize the spatial distribution of ICC networks, current descriptors of ICC depletion are limited to cell numbers and volume computations. Spatial changes in ICC network structural properties have not been quantified. Given that ICC generate electrical signals, the organization of a network may also affect physiology. In this study, six numerical metrics were formulated to automatically determine complex ICC network structural properties from confocal images: density , thickness , hole size , contact ratio , connectivity and anisotropy . These metrics were validated and applied in proof-of-concept studies to quantitatively determine jejunal ICC network changes in mouse models with decreased ( 5-HT 2 B receptor knockout (KO)) and normal ( Ano1 KO) ICC numbers, and during post-natal network maturation. Results revealed a novel remodelling phenomenon occurring during ICC depletion, namely a spatial rearrangement of ICC and the preferential longitudinal alignment. In the post-natal networks, an apparent pruning of the ICC network was demonstrated. The metrics developed here enabled the first detailed quantitative analyses of structural changes that may occur in ICC networks during depletion and development.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.