[Show abstract][Hide abstract] ABSTRACT: Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus.
Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy.
Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines.
Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.
Neurogastroenterology and Motility 07/2011; 23(7):e309-23. · 2.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gastrointestinal stromal tumors (GIST) are related to interstitial cells of Cajal (ICC) and often contain activating stem cell factor receptor (Kit) or platelet-derived growth factor receptor alpha (Pdgfra) mutations. Kit/Pdgfra inhibitors such as imatinib mesylate have increased progression-free survival in metastatic GIST but are not curative. In mouse models we investigated whether Kit(low) ICC progenitors could represent an inherently Kit/Pdgfra inhibitor-resistant reservoir for GIST.
Isolated Kit(low)Cd44(+)Cd34(+) cells were characterized after serial cloning. The tumorigenic potential of spontaneously transformed cells was investigated in nude mice. The Kit(low)Cd44(+)Cd34(+) cells' responsiveness to Kit activation and blockade was studied by enumerating them in Kit(K641E) mice (a GIST model), in mice with defective Kit signaling, and pharmacologically.
Single isolated Kit(low)Cd44(+)Cd34(+) cells were clonogenic and capable of self-renewal and differentiation into ICC. In nude mice, spontaneously transformed cells formed malignant tumors expressing GIST markers. The Kit(low)Cd44(+)Cd34(+) cells were resistant to in vitro Kit blockade, including by imatinib, and occurred in normal numbers in mice with reduced Kit signaling. In Kit(K641E) mice, the mutant ICC stem cells were grossly hyperplastic but remained imatinib-resistant. In contrast, the cancer stem, cell-targeting drug salinomycin blocked the proliferation of Kit(low)Cd44(+)Cd34(+) cells and increased their sensitivity to imatinib.
Kit(low)Cd44(+)Cd34(+) progenitors are true stem cells for normal and hyperplastic ICC and give rise to GIST. Resistance to Kit/Pdgfra inhibitors is inherent in GIST and is caused by the native ICC stem cells' lack of dependence on Kit for survival, which is maintained after the acquisition of oncogenic Kit mutation. Cancer stem cell drugs may target these cells.
[Show abstract][Hide abstract] ABSTRACT: Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing-associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti-ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing-associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3-dimensional reconstruction, flow cytometry, quantitative RT-PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT-PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho-deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho-deficient mice were marantic and had low insulin, insulin-like growth factor-I and membrane-bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho-deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.
The Journal of Physiology 08/2010; 588(Pt 16):3101-17. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with chronic calorie insufficiency commonly suffer from upper gastrointestinal dysfunction and consequent dyspeptic symptoms, which may interfere with their nutritional rehabilitation. To investigate the relationship between gastric dysfunction and feeding behavior, we exposed mice to chronic caloric restriction and demonstrated gastric motor abnormalities in them. Gastric dysmotility is typically associated with dyspeptic symptoms but sensations cannot be directly assessed in animal models. Therefore, as an initial step toward establishing measurable correlates of postprandial symptoms in small animals, we have attempted to characterize central responses to food intake by positron emission tomography-computerized microtomography (PET-CT) in normal and calorically restricted mice. Animals consumed a standard test meal after an overnight fast before receiving 2-deoxy-2[18F]fluoro-D-glucose tracer. The same mice were also scanned in the fasting state on a separate day. We were able to bring the fed and fasting PET volume images into spatial registration with each other and with an MR-derived atlas of the mouse brain, so that the differences in uptake between the two states could be mapped quantitatively against the neuroanatomic regions of the atlas. Our approach is suitable for studying the effects of gastric dysmotilities on central responses to feeding.