Small Intestinal Proliferation Research Articles

Myeloid vitamin D receptor influences cell proliferation in small intestine

The vitamin D receptor (VDR) is a ubiquitous nuclear receptor that acts as a transcription factor when vitamin D binds with VDR. Intestinal epithelial VDR has various physiological and anatomical benefits to intestine. Interactions between intestinal epithelial cells (IEC) and immune cells are critical for gut health. Cellular proliferation through Wnt/β‐catenin signaling is a result of immune and IEC crosstalk. Wnt proteins from macrophages bind to intestinal epithelial cells and activate the Wnt/β‐catenin pathway. This pathway is critical for cell proliferation which enables food and water absorption in the small intestine. However, the importance of myeloid VDR in regulating the Wnt/β‐catenin pathway in epithelial cells is not fully understood. The objective of this study is to assess how the presence of myeloid VDR in the intestinal epithelial cells influences cellular proliferation through the Wnt/β‐catenin pathway. We hypothesize that the low level of myeloid VDR will reduce expression of Wnt/β‐catenin signaling downstream and alleviate cell proliferation in the small intestine. This experiment utilized selective deletion of myeloid VDR in an ex‐vivo and in‐vivo set‐up. For the ex‐vivo, control mice (VDRLoxP) were crossed with lysozyme (M) ‐cre mice to create specific knockout of myeloid VDR (VDRrLyz) mice. Twelve mice (6 VDRLoxP mice and 6 VDRrLyz) were sacrificed and mucosal scrapings from the ileum (small intestine) were collected for westerns. For cell culture, small intestine organoids derived from the intestinal stem cells were cultured alone or co‐cultured with macrophages (VDR+/+ or VDR−/−). After 3 days, the organoids and organoid‐macrophage co‐cultures were collected for western blots. Western blots were used to measure and compare expression of Wnt/β‐catenin signaling proteins. The VDR−/− organoid macrophage co‐culture showed significantly decreased expression of β‐catenin and PCNA (a cell proliferation marker), compared to the VDR+/+ organoid macrophage co‐culture. Our results suggest that myeloid VDR influences the Wnt/β‐catenin signaling pathway. We present a novel role of myeloid VDR signaling in mediating immune/epithelial cell cross talk in intestine.

Synbiotic promotion of epithelial proliferation by orally ingested encapsulated Bifidobacterium breve and raffinose in the small intestine of rats

We evaluated the effects of Bifidobacterium breve JCM1192(T )and/or raffinose on epithelial proliferation in the rat small and large intestines. WKAH/Hkm Slc rats (4 wk old) were fed a control diet, a diet supplemented with either encapsulated B. breve (30 g/kg diet, 1.5 x 10(7) colony-forming unit/g capsule) or raffinose (30 g/kg diet), or a diet supplemented with both encapsulated B. breve and raffinose, for 3 wk. Epithelial proliferation in the small intestine, as assessed by bromodeoxyuridine immunohistochemistry, was increased only in the B. breve plus raffinose-fed group. We determined the number of bifidobacteria in cecal contents using fluorescence in situ hybridization and confirmed the presence of ingested B. breve only in the B. breve plus raffinose-fed group. This suggests that the ingested B. breve cells used raffinose and were activated in the small intestine, where they subsequently influenced epithelial proliferation. In conclusion, we found a prominent synbiotic effect of encapsulated B. breve in combination with raffinose on epithelial proliferation in rat small intestine but not in large intestine. To our knowledge, this is the first report of a synbiotic that affects epithelial proliferation.

Effect of Esophageal Ligation on Small Intestinal Development in Normal and Growth‐retarded Fetal Rabbits

The uninterrupted passage of amniotic fluid through the gastrointestinal tract is hypothesized to influence both intestinal and overall fetal somatic development. The effect of in utero esophageal ligation (EL) and therefore the exclusion of AF on somatic growth, small intestinal (SI) morphology and proliferation, and the expression of the glucose transporter sodium-glucose cotransporter 1 (SGLT-1) in both normal and intrauterine growth-retarded (IUGR) fetal rabbits were evaluated. Thirteen pregnant New Zealand white rabbits underwent surgery on day 24 of their normal 31-day gestation. Ipsilateral normal and IUGR fetuses underwent EL; the contralateral normal and IUGR fetuses underwent cervical exploration only forming 4 study groups (control-normal, control-IUGR, EL-normal and EL-IUGR). Rabbits were killed on day 31. Small intestinal villus height was measured, and epithelial cell proliferation was deter mined by proliferating cell nuclear antigen staining. Sodium-glucose cotransporter 1 messenger RNA (mRNA) and protein expressions were analyzed. Statistical analysis was performed using 2-way analysis of variance. Esophageal ligation reduced fetal weight in IUGR by 15% and in normal by 10%. Villus height was significantly reduced in IUGR versus normal in both control and EL (control, P = 0.01; EL, P = 0.05). Intrauterine growth-retarded fetuses had reduced SI proliferation versus normal in both control and EL. Sodium-glucose cotransporter 1 mRNA production in EL fetuses was equal to control fetuses. Esophageal ligation-normal and EL-IUGR fetuses exhibited reduced protein levels and decreased staining for SGLT-1 in villus enterocytes. Amniotic fluid exclusion by in utero EL reduced fetal weight. Small intestinal proliferation was not affected by EL. Although SGLT-1 mRNA and protein were produced in all 4 groups, exposure of the fetal gastrointestinal tract to amniotic fluid appears necessary for proper brush border expression of nutrient transporter proteins.

Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1.

Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.

Transport and steady-state accumulation of putrescine in brush-border membrane vesicles of rabbit small intestine.

Absorption of polyamines from the lumen is essential for cell proliferation in small intestine but also in other rapidly growing body tissues and tumors. Intestinal uptake of polyamines is thought to involve one or more transport systems, but the characteristics of these systems have not yet been clearly elucidated. Because high levels of putrescine have been identified in intestinal lumen, we explored kinetic, physiochemical, and structural features of uptake of this diamine across rabbit intestinal brush-border membrane vesicles (IBBMV) prepared by CaCl2 or MgCl2 precipitation procedure. Initial rates of putrescine influx were measured during 5-min incubations at 25 or 37 degrees C (optimal temperature) for concentrations of 0.45-145 microM. At both temperatures, kinetics of putrescine transport fitted a model with a single Michaelis-Menten uptake component plus a nonsaturable uptake component. At 37 degrees C, the kinetic parameters for the saturable component of putrescine uptake, Km,app and Vmax,app, were 16.8 +/- 4.7 microM and 19.9 +/- 2.8 pmol.mg protein-1.min-1, respectively. The value of the constant for the nonsaturable component of putrescine uptake (P = 0.45 +/- 0.06 x 10(-8) l.mg protein-1.s-1) suggested this component represented essentially nonspecific binding of putrescine to IBBMV. Cadaverine, spermidine, and spermine were competitive inhibitors of putrescine transport, with inhibition constants equal to 47, 117, and 219 microM, respectively. When effects of a variety of alkyldiamines and structural analogues of polyamines (1 mM) on influx of 5.6 microM putrescine were compared, cadaverine, methylglyoxal bis(guanylhydrazone) (MGBG), and cyclic derivatives of MGBG were found to exhibit the highest inhibitory potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor Does Not Act as a Primary Cue for Inducing Developmental Changes in Suckling Mouse Jejunum

SummaryThe direct influence of epidermal growth factor (EGF) on the differentiation and proliferation of small intestine was studied in organ culture. Eight‐day‐old mouse small intestine was cultured during 2 days in serum‐free Leibovitz L‐15 medium alone or supplemented with EGF (50, 100, and 500 ng/ml) either at room temperature or at 37°C. Brush border membrane hydrolytic activities, namely, sucrase, lactase, glucoamylase, trehalase, maltase, and alkaline phosphatase, were assayed in the intestinal tissue as well as in the culture medium. None of the brush border enzymic activities was affected by the addition of EGF to the culture medium. This lack of effect is not temperature dependent since it occurred both at room temperature and at 37°. The addition of hydrocortisone (10–6M) to the culture medium induced the appearance of sucrase activity and increased the activity of the other brush border enzymes. The simultaneous addition of EGF with hydrocortisone did not influence the response of the intestinal explants to hydrocortisone. The deoxyribonucleic acid (DNA) content was determined while DNA synthesis was evaluated by the incorporation of (3H)‐thymidine. The addition of EGF did not affect DNA content or (3H)‐thymidine incorporation into DNA either at room temperature or at 37°. The EGF binding to epithelial cells did not significantly vary throughout the culture period and a down‐regulation process occurred in presence of EGF. These observations strongly suggest that EGF does not act as a primary cue for inducing developmental changes in suckling mouse small intestine. It is proposed that EGF induces a systemic reaction in vivo that then influences the neonatal small intestine.

Epidermal growth factor does not act as a primary cue for inducing developmental changes in suckling mouse jejunum.

The direct influence of epidermal growth factor (EGF) on the differentiation and proliferation of small intestine was studied in organ culture. Eight-day-old mouse small intestine was cultured during 2 days in serum-free Leibovitz L-15 medium alone or supplemented with EGF (50, 100, and 500 ng/ml) either at room temperature or at 37 degrees C. Brush border membrane hydrolytic activities, namely, sucrase, lactase, glucoamylase, trehalase, maltase, and alkaline phosphatase, were assayed in the intestinal tissue as well as in the culture medium. None of the brush border enzymic activities was affected by the addition of EGF to the culture medium. This lack of effect is not temperature dependent since it occurred both at room temperature and at 37 degrees C. The addition of hydrocortisone (10(-6) M) to the culture medium induced the appearance of sucrase activity and increased the activity of the other brush border enzymes. The simultaneous addition of EGF with hydrocortisone did not influence the response of the intestinal explants to hydrocortisone. The deoxyribonucleic acid (DNA) content was determined while DNA synthesis was evaluated by the incorporation of (3H)-thymidine. The addition of EGF did not affect DNA content or (3H)-thymidine incorporation into DNA either at room temperature or at 37 degrees C. The EGF binding to epithelial cells did not significantly vary throughout the culture period and a down-regulation process occurred in presence of EGF. These observations strongly suggest that EGF does not act as a primary cue for inducing developmental changes in suckling mouse small intestine. It is proposed that EGF induces a systemic reaction in vivo that then influences the neonatal small intestine.

Effect of two intestinal resections separated by time on cell proliferation in the rat small intestine

An attempt was made to force the mucosal cell renewal system into a maximal proliferative state by performing single intestinal resections followed 7 days later by second resections. Changes in several parameters of the compensatory response were assessed 14 days after the last surgical procedure in transected, singly resected, and doubly resected animals. A second resection of sufficient magnitude and at a proper time with respect to the first resection, yielded a compensatory response greater than that achieved with a single resection as measured by crypt cell incorporation of tritiated thymidine. Provided the total amount of tissue removed in the two procedures was constant, the amount of tissue taken as a primary or secondary stimulus did not change the ultimate level of significantly increased cell proliferation in the small intestine of the rat after two resections. Intracellular adenosine 3′,5′-cyclic monophosphate values for resected animals were significantly lower than those found in other treatment groups. However, no significant difference in guanosine 3′,4′-cyclic monophosphate levels was found among the treatment groups.

Comparative analysis of cell renewal in the gastrointestinal tract of newborn hamster

Comparative rates of cell proliferation in different areas of gastrointestinal tract of newborn hamster were estimated by observing the appearance of repetitive waves of 3HTdR labeled mitoses. The proliferation of cells was also studied by constructing theoretical models to simulate the appearance in time of the repetitive waves. In each model, the cycle durations of individual cells or groups of cells in the proliferating population, and the fractional contribution of each group to the total population were known. The contributions of the population elements were summated, to indicate descriptive characteristics of the repetitive proliferative cycles. The data indicated more rapid proliferation in small intestine than in stomach and colon, and suggested additional similarities and perturbations in the proliferative cycle of cells in these areas.