Tamas Ordog

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

1 Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage‐independent, non‐selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. 2 Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. 3 Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. 4 Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea‐pig. 5 Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3‐dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. 6 Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.

Interstitial cells of Cajal generate electrical slow waves in the murine stomach

1 The gastric corpus and antrum contain interstitial cells of Cajal (ICC) within the tunica muscularis. We tested the hypothesis that ICC are involved in the generation and regeneration of electrical slow waves. 2 Normal, postnatal development of slow wave activity was characterized in tissues freshly removed from animals between birth and day 50 (D50). Slow wave amplitude and frequency increased during this period. Networks of myenteric ICC (IC‐MY) were present in gastric muscles at birth and did not change significantly in appearance during the period of study as imaged by confocal immunofluorescence microscopy. 3 IC‐MY networks were maintained and electrical rhythmicity developed in organ culture in a manner similar to normal postnatal development. Electrical activity was maintained for at least 48 days in culture. 4 Addition of a neutralizing antibody (ACK2) for the receptor tyrosine kinase, Kit, to the culture media caused progressive loss of Kit‐immunoreactive cells. Loss of Kit‐immunoreactive cells was associated with loss of slow wave activity. Most muscles became electrically quiescent after 3‐4 weeks of exposure to ACK2. 5 In some muscles small clusters of Kit‐immunoreactive IC‐MY remained after culturing with ACK2. These muscles displayed slow wave activity but only in the immediate regions in which Kit‐positive IC‐MY remained. These data suggest that regions without Kit‐immunoreactive cells cannot generate or regenerate slow waves. 6 After loss of Kit‐immunoreactive cells, the muscles could not be paced by direct electrical stimulation. Stimulation with acetylcholine also failed to elicit slow waves. The data suggest that the generation of slow waves is an exclusive property of IC‐MY; smooth muscle cells may not express the ionic apparatus necessary for generation of these events. 7 We conclude that IC‐MY are an essential element in the spontaneous rhythmic electrical and contractile activity of gastric muscles. This class of ICC appears to generate slow wave activity and may provide a means for regeneration of slow waves.

Heme Oxygenase-1 Protects Interstitial Cells of Cajal from Oxidative Stress and Reverses Diabetic Gastroparesis

BACKGROUND & AIMS Diabetic gastroparesis (delayed gastric emptying) is a well-recognized complication of diabetes that causes considerable morbidity and makes glucose control difficult. Interstitial cells of Cajal, which express the receptor tyrosine kinase Kit, are required for normal gastric emptying. We proposed that Kit expression is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of heme oxygenase-1 (HO-1), an important cytoprotective molecule against oxidative injury. METHODS Gastric emptying was measured in nonobese diabetic mice and correlated with levels of HO-1 expression and activity. Endogenous HO-1 activity was increased by administration of hemin and inhibited by chromium mesoporphyrin. RESULTS In early stages of diabetes, HO-1 was up-regulated in gastric macrophages and remained up-regulated in all mice that were resistant to development of delayed gastric emptying. In contrast, HO-1 did not remain up-regulated in all the mice that developed delayed gastric emptying; expression of Kit and neuronal nitric oxide synthase decreased markedly in these mice. Loss of HO-1 up-regulation increased levels of reactive oxygen species. Induction of HO-1 by hemin decreased reactive oxygen species, rapidly restored Kit and neuronal nitric oxide synthase expression, and completely normalized gastric emptying in all mice. Inhibition of HO-1 activity in mice with normal gastric emptying caused a loss of Kit expression and development of diabetic gastroparesis. CONCLUSIONS Induction of the HO-1 pathway prevents and reverses cellular changes that lead to development of gastrointestinal complications of diabetes. Reagents that induce this pathway might therefore be developed as therapeutics.

CD206-Positive M2 Macrophages That Express Heme Oxygenase-1 Protect Against Diabetic Gastroparesis in Mice

BACKGROUND & AIMS Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Progenitors of Interstitial Cells of Cajal in the Postnatal Murine Stomach

BACKGROUND & AIMS Maintaining the integrity of networks of interstitial cells of Cajal (ICC) is essential to preserve orderly contractile activity and neuroregulation in the gastrointestinal tract and to restore these functions after tissue damage or surgeries. Maintenance of ICC requires insulin-dependent or insulin-like growth factor I (IGF-I)-dependent production of membrane-bound stem cell factor (SCF) and may involve regeneration from local progenitors. Our goal was to identify ICC precursors in postnatal murine gastric muscles. METHODS We used flow cytometry and immunohistochemistry to examine freshly dissected and cultured muscles for cells expressing CD34, an adhesion molecule expressed by stromal tumors; CD44, which occurs on mesenchymal stem cells; and receptors for SCF (Kit), insulin (Insr), and IGF-I (Igf1r). Slow waves were studied by intracellular recording. RESULTS In gastric muscles, we identified rare, Kit(low)CD44(+)CD34(+)Insr(+)Igf1r(+) cells resembling common embryonic precursors of ICC and smooth muscle. These putative progenitors were absent from organotypic cultures lacking mature ICC (Kit(+)CD44(+)CD34(-)Insr(-)Igf1r(-)) due to prolonged insulin/IGF-I deprivation but were rescued by IGF-I that also prevented ICC loss. Soluble SCF failed to prevent the loss of mature ICC but dramatically expanded the putative progenitors, which supported robust slow wave activity despite retaining an immature, Kit(+)CD44(+)CD34(+)Insr(+)Igf1r(+) phenotype. Differentiation of these cells into mature, network-forming ICC required IGF-I. Conversely, restoration of ICC networks by IGF-I after prolonged insulin and IGF-I deprivation required the survival of the presumed progenitors. CONCLUSIONS Kit(low)CD44(+)CD34(+)Insr(+)Igf1r(+) cells may be local progenitors for gastric ICC and stromal tumors. Loss of these cells may contribute to gastrointestinal dysmotilities.

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves

BACKGROUND & AIMS Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

Gastroparesis and Functional Dyspepsia: Excerpts from the AGA/ ANMS Meeting

Background  Despite the relatively high prevelance of gastroparesis and functional dyspepsia, the aetiology and pathophysiology of these disorders remain incompletely understood. Similarly, the diagnostic and treatment options for these two disorders are relatively limited despite recent advances in our understanding of both disorders.

Interstitial cells of Cajal in diabetic gastroenteropathy

Gastroenteropathy causes considerable morbidity in patients with diabetes mellitus and represents a major healthcare burden. Current treatments are largely symptomatic and frequently ineffective. Development of new therapeutic options is hampered by poor understanding of the underlying pathomechanisms. Experimental studies and sparse human data indicate that diabetic gastroenteropathy is multifactorial and involves not only parasympathetic and sympathetic autonomic nerves, but also enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). ICC are mesenchymal cells that occur throughout the muscular coat of the gastrointestinal tract and provide functions critical for normal gastrointestinal motility including generation and propagation of electrical slow waves and mediation of bidirectional communication between the autonomic nervous system and smooth muscle cells. Through these functions, and in concert with other cell types of the gastrointestinal muscles, ICC support basic gastrointestinal functions such as digestion, absorption and waste removal. Loss or dysfunction of ICC in various dysmotilities and their animal models has been shown to lead to gastric dysrhythmias, gastroparesis, slow intestinal transit, impaired neuroeffector mechanisms and altered visceral afferent signalling that are considered hallmarks of diabetic gastroenteropathy. These findings and an increasing body of evidence indicating disruptions of ICC networks in diabetes suggest that the loss of ICC in this disorder is probably of functional significance and could even be a major pathogenetic factor. Future research should focus on the identification of the molecular and cellular mechanisms underlying ICC loss in diabetes and the translation of the experimental findings into treatments.Abstract  Gastroenteropathy causes considerable morbidity in patients with diabetes mellitus and represents a major healthcare burden. Current treatments are largely symptomatic and frequently ineffective. Development of new therapeutic options is hampered by poor understanding of the underlying pathomechanisms. Experimental studies and sparse human data indicate that diabetic gastroenteropathy is multifactorial and involves not only parasympathetic and sympathetic autonomic nerves, but also enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). ICC are mesenchymal cells that occur throughout the muscular coat of the gastrointestinal tract and provide functions critical for normal gastrointestinal motility including generation and propagation of electrical slow waves and mediation of bidirectional communication between the autonomic nervous system and smooth muscle cells. Through these functions, and in concert with other cell types of the gastrointestinal muscles, ICC support basic gastrointestinal functions such as digestion, absorption and waste removal. Loss or dysfunction of ICC in various dysmotilities and their animal models has been shown to lead to gastric dysrhythmias, gastroparesis, slow intestinal transit, impaired neuroeffector mechanisms and altered visceral afferent signalling that are considered hallmarks of diabetic gastroenteropathy. These findings and an increasing body of evidence indicating disruptions of ICC networks in diabetes suggest that the loss of ICC in this disorder is probably of functional significance and could even be a major pathogenetic factor. Future research should focus on the identification of the molecular and cellular mechanisms underlying ICC loss in diabetes and the translation of the experimental findings into treatments.

Vascular endothelial growth factor promotes fibrosis resolution and repair in mice.

BACKGROUND & AIMS Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored. METHODS We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays. RESULTS VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9. CONCLUSIONS In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function.