Shannon W. Longshore

Retinoblastoma Protein (pRb), but Not p107 or p130, Is Required for Maintenance of Enterocyte Quiescence and Differentiation in Small Intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.

Growth factors: possible roles for clinical management of the short bowel syndrome.

The structural and functional changes during intestinal adaptation are necessary to compensate for the sudden loss of digestive and absorptive capacity after massive intestinal resection. When the adaptive response is inadequate, short bowel syndrome (SBS) ensues and patients are left with the requirement for parenteral nutrition and its associated morbidities. Several hormones have been studied as potential enhancers of the adaptation process. The effects of growth hormone, insulin-like growth factor-1, epidermal growth factor, and glucagon-like peptide 2 on adaptation have been studied extensively in animal models. In addition, growth hormone and glucagon-like peptide 2 have shown promise for the treatment of SBS in clinical trials in human beings. Several lesser studied hormones, including leptin, corticosteroids, thyroxine, testosterone, and estradiol, are also discussed.

Intestinal resection induces angiogenesis within adapting intestinal villi

PURPOSE Adaptive growth of the intestinal mucosa in response to massive gut loss is fundamental for autonomy from parenteral nutrition. Although angiogenesis is essential for cellular proliferation in other tissues, its relevance to intestinal adaptation is unknown. We tested the hypothesis that resection-induced adaptation is associated with new blood vessel growth. METHODS Male C57Bl/6 mice underwent either a 50% small bowel resection or a sham (transection and reanastomosis) operation. After 1, 3, or 7 days, capillary density within the intestinal villi was measured using confocal microscopy. A messenger RNA reverse-transcriptase polymerase chain reaction (RT-PCR) array was used to determine angiogenic gene expression during adaptation. RESULTS Mice that underwent small bowel resection had a significantly increased capillary density compared to sham-operated mice at postoperative day 7. This morphological alteration was preceded by significant alterations in 5 candidate genes at postoperative day 3. CONCLUSION New vessel blood growth is observed in the adapting intestine after massive small bowel loss. This response appears to follow rather than initiate the adaptive alterations in mucosal morphology that are characteristic of adaptation. A better understanding of this progress and the signaling factors involved may improve therapeutic options for children with short gut syndrome.

Extent of small bowel resection does not influence the magnitude of intestinal adaptation in the mouse

PURPOSE The magnitude of intestinal adaptation is considered to correlate with the extent of small bowel resection (SBR). However, this association has never been tested in mice. We sought to test the hypothesis that a greater SBR will induce a greater adaptation response. METHODS C57/B6 mice underwent 50% SBR, 75% SBR, or sham operation and were killed on postoperative day 7. The magnitude of adaptation was compared between 50% SBR and 75% SBR as changes in villus height, crypt depth, as well as rates of apoptosis and proliferation. RESULTS Seventy-five percent SBR led to decreased survival and increased weight loss compared with 50% SBR. The remnant ileum of both 50% SBR and 75% SBR displayed similar crypt expansion, enhanced villi, and increased apoptotic indices. Proliferation rates increased after 50% and 75% SBR equally. CONCLUSION Models of resection greater than 50% in mice result in greater morbidity and mortality and do not magnify the adaptation response to massive SBR. The use of more extreme resection models does not appear to provide added benefit for investigating mechanisms of intestinal adaptation.

p21waf1/cip1 deficiency does not perturb the intestinal crypt stem cell population after massive small bowel resection

BACKGROUND After small bowel resection (SBR), adaptation is initiated in intestinal crypts where stem cells reside. Prior studies revealed SBR-induced enterocyte proliferation requires the expression of p21(waf1/cip1). As deficient expression of p21(waf1/cip1) has been shown to result in reduced numbers of hematopoietic stem cells. We sought to test the hypothesis that p21(waf1/cip1)deficiency similarly perturbs the intestinal stem cell population after SBR. METHODS Control (n = 21; C57Bl/6) and p21(waf1/cip1)-null mice (n = 30) underwent 50% proximal SBR or sham operation. After 3 days, the ileum was harvested and the crypt stem cell population evaluated by counting crypt base columnar cells on histologic sections, determining the expression of Musashi-1 and Lgr5, and profiling the transcriptional expression of 84 known stem cell genes. RESULTS There were no significant differences in crypt base columnar cells, expression of Musashi-1 or Lgr5, or in stem cell gene expression after SBR in control mice. Furthermore, there were no differences in these markers between controls and p21(waf1/cip1)-null mice. CONCLUSION In contrast with bone marrow stem cells, the stem cell population of the gut is unaffected by deficient expression of p21(waf1/cip1). Additional mechanisms for the role of p21(waf1/cip1) in small bowel proliferation and adaptation after massive SBR must be considered.