Stimulation Of Epithelial Cell Proliferation Research Articles

Stachyose protects intestinal mucosal barrier via promotion of tight junction and Lactobacillus casei-drived inhibition of apoptosis in juvenile turbot, Scophthalmus maximus L.

This study aimed to elucidate the possible beneficial effects of stachyose on cottonseed meal-induced intestinal barrier damage and to investigate the underlying mechanism in turbot (Scophthalmus maximus L.). A 12-week feeding trial was conducted using four isonitrogenous and isolipidic diets: a fishmeal-based diet (FM), a diet with cottonseed meal replacing 40% fish meal protein (CM), CM supplemented with 1.4% (S1) and 2.8% (S2) stachyose. Dietary stachyose at 2.8% mitigated cottonseed meal-induced depression of the growth performance and feed utilization, improved the intestinal morphology, and decreased the D-lactate content and activity of diamine oxidase in serum. The protective effects of stachyose were associated with enhancement in expression of intestinal tight junction proteins and inhibition of apoptosis. An additional in vitro study showed that Lactobacillus casei, greatly enriched in the intestine of fish fed the S2 diet, alleviated gossypol-induced apoptosis in epithelial cells. The function of L. casei was found to be associated with the activation of Akt and stimulation of epithelial cell proliferation in the intestine. Collectively, these results provide novel insights into the regulatory role of stachyose in modulation of intestinal microbiota and mucosal barrier function, as well as a microbiome-targeted strategy to improve intestinal health of fish.

Effects of supplemental calcium gluconate embedded in a hydrogenated fat matrix on lactation, digestive, and metabolic variables in dairy cattle

There is growing evidence suggesting that by improving gut integrity and function, less energy is partitioned toward immune responses related to xenobiotic infiltration, sparing energy for productive purposes. Gluconic acid and its salts have previously shown prebiotic effects in the lower gut of nonruminant animals, where they serve as a precursor for butyrate, although evidence in ruminants is limited. Butyrate and its fermentative precursors have demonstrated multiple beneficial effects to gastrointestinal ecology, morphology, and function, such as the stimulation of epithelial cell proliferation and improvement of gut barrier function and ecology. The objective of this study was to evaluate changes in milk production, milk fatty acid composition, and fecal and blood parameters in lactating dairy cattle fed a hydrogenated fat-embedded calcium gluconate (HFCG) supplement designed to target the hindgut for calcium gluconate delivery. In addition, the effects of a compound feed processing method (i.e., incorporated into a mash or an extruded pellet) were tested to evaluate the effect of extrusion on product efficacy. Forty-five lactating Holstein cows at approximately 165 d in milk were used in a 3 × 3 Latin square consisting of three 28-d periods, during which animals were offered a basal ration mixed with 3 different compound feeds: a negative control in mash form containing no HFCG, or the HFCG supplement fed at a target rate of 16 g/d, delivered in either a mash or pelleted form. Supplementation of HFCG tended to increase yields of milk fat and fat- and energy-corrected milk. Total yields and concentrations of milk fatty acids ≥18 carbons in length tended to increase in response to HFCG. Plasma nonesterified fatty acids and milk urea increased in HFCG treatments. No differences were observed in fecal pH or fecal concentrations of volatile fatty acids, with the exception of isobutyrate, which decreased in HFCG-fed cows. Changes in milk fatty acid profile suggest that increased milk fat yield was driven by increased incorporation of preformed fatty acids, supported by increased circulating nonesterified fatty acid. Future research investigating the mode of action of HFCG at the level of the hindgut epithelium is warranted, as measured fecal parameters showed no response to treatment.

233 Efficacy of fat-embedded calcium gluconate on lactation performance in dairy cattle

Abstract Several beneficial effects have been observed in the gastrointestinal tract in response to the provision of short-chain fatty acids such as butyrate, including the improvement of gut barrier function, stimulation of epithelial cell proliferation and gastrointestinal homeostasis. By improving gut barrier integrity, it is possible that less energy would be spent mounting an immune response against infiltrating xenobiotic compounds, allowing for more energy to be partitioned towards productive activities, such as milk synthesis. Previous work has demonstrated that gluconic acid and its salts have a prebiotic effect in the lower gut in monogastric animals, where it acts as a substrate for butyrate synthesis, with new data suggesting a similar response in dairy cattle. The objective of this study was to evaluate the effects of supplementation of a fat-embedded calcium gluconate product on milk production in dairy cattle. Lactating heifers (n = 22) and cows (n = 53) were offered either a total mixed ration supplemented with 0.07% fat-embedded calcium gluconate or a negative control ration over the course of a full lactation. Milk production and dry matter intake were recorded daily, while milk composition was determined at regular intervals over the lactation. Cows receiving the supplement displayed significant (P ≤ 0.046) increases in milk and component yields, while consuming 1 kg DM/d more during lactation compared to control. Heifers receiving the supplement displayed decreased (P ≤ 0.044) yield of milk fat and fat-corrected milk, 0.09% increased milk protein content, and decreased feed efficiency. Overall, the results in multiparous animals are consistent with previous work examining calcium gluconate. Further work is needed to characterize the differential response in heifers.

Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC–ERK1/2–mTOR signaling pathway

The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

Keratinocyte growth factor gene delivery via mesenchymal stem cells protects against lipopolysaccharide-induced acute lung injury in mice.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality, and have no specific therapy. Keratinocyte growth factor (KGF) is a critical factor for pulmonary epithelial repair and acts via the stimulation of epithelial cell proliferation. Mesenchymal stem cells (MSCs) have been proved as good therapeutic vectors. Thus, we hypothesized that MSC-based KGF gene therapy would have beneficial effects on lipopolysaccharide(LPS)-induced lung injury. After two hours of intratracheal LPS administration to induce lung injury, mice received saline, MSCs alone, empty vector-engineered MSCs (MSCs-vec) or KGF-engineered MSCs (MSCs-kgf) via the tail vein. The MSCs-kgf could be detected in the recipient lungs and the level of KGF expression significantly increased in the MSCs-kgf mice. The MSC-mediated administration of KGF not only improved pulmonary microvascular permeability but also mediated a down-regulation of proinflammatory responses (reducing IL-1β and TNF-α) and an up-regulation of anti-inflammatory responses (increasing cytokine IL-10). Furthermore, the total severity scores of lung injury were significantly reduced in the MSCs-kgf group compared with the other three groups. The underlying mechanism of the protective effect of KGF on ALI may be attributed to the promotion of type II lung epithelial cell proliferation and the enhancement of surfactant synthesis. These findings suggest that MSCs-based KGF gene therapy may be a promising strategy for ALI treatment.

Protection against hyperoxia‐induced lung fibrosis by KGF‐induced MSCs mobilization in neonatal rats

MSCs have been shown to improve functional and pathological outcome in lung fibrosis. However, low in vivo cell engraftment of the transplanted cells limits their overall effectiveness. KGF (also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. However, the role of KGF in MSCs and its therapeutic effects have not been identified. In this study, we investigated the effect of KGF on MSCs and its preventive role in hyperoxia-induced fibrosis in neonatal rats. Neonatal rats exposed to normoxia or hyperoxia were randomly assigned to receive intraperitoneal injections of normal saline (PL), MSCs, or KGF pretreated MSCs on the fourth day of exposure. Our results showed that as compared to PL, while MSCs attenuated lung fibrosis, KGF pretreated MSCs exhibited enhanced preventive effect against lung fibrosis. This effect was partly attributed to enhanced mobilization of MSCs to the fibrotic lungs. In addition, the SHH signaling pathway, which is associated with the differentiation of stem cells was activated by KGF. Our data suggest that MSCs, especially KGF preconditioned MSCs, can attenuate lung fibrosis and KGF may regulate the MSCs behavior by activating SHH pathway.

Comparative analysis of the uterine and mammary gland effects of progesterone and medroxyprogesterone acetate

Objectives In combined hormone replacement therapy (HRT) progestins are used to inhibit estradiol-activated uterine epithelial cell proliferation. In comparison to estradiol-only therapy, combined HRT leads to enhanced proliferation of mammary epithelial cells. In a quantitative mouse model, we assessed the balance between uterine and undesired mammary gland effects for two progestins that are widely used in HRT, progesterone and medroxyprogesterone acetate. Study design Mice were ovariectomized and after 14 days they were treated subcutaneously with either vehicle, estradiol (100 ng) or estradiol plus increasing doses of progesterone or medroxyprogesterone acetate for three weeks. Main outcome measures Measures for progestogenic mammary gland activity were stimulation of side-branching and stimulation of epithelial cell proliferation. Progestogenic activity in the uterus was assessed by measuring inhibition of estradiol-activated uterine epithelial cell proliferation. ED 50 and ID 50 values for the distinct readouts were obtained and dissociation factors for uterine versus mammary gland activity were calculated. Results MPA demonstrated uterine activity and mitogenic activity in the mammary gland at the same doses. In contrast, progesterone showed uterine activity at doses lower than those leading to significant stimulation of epithelial cell proliferation in the mammary gland. Conclusions Progestins do not behave the same. Use of the natural hormone progesterone, but not MPA, in combined hormone therapy might offer a safety window between uterine effects and undesired proliferative activity in the mammary gland.

Bone Marrow Stem Cells Expressing Keratinocyte Growth Factor via an Inducible Lentivirus Protects against Bleomycin-Induced Pulmonary Fibrosis

Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.

Inhibition of Peroxisome Proliferator-Activated Receptor γ Promotes Tumorigenesis Through Activation of the β-Catenin / T Cell Factor (TCF) Pathway in the Mouse Intestine

Although peroxisome proliferator-activated receptor γ (PPARγ) is strongly expressed in the intestinal epithelium, the role of PPARγ in intestinal tumorigenesis has not yet been elucidated. To address this issue, we investigated the effect of PPARγ inhibition and its mechanism on intestinal tumorigenesis using a selective antagonist, T0070907. We treated ApcMin/+ mice and carcinogen-induced colon cancer model C57BL/6 mice with T0070907 and counted the number of spontaneous polyps and aberrant crypt foci and observed cell proliferation and β-catenin protein in the colon epithelium. To investigate its mechanism, the changes of β-catenin/TCF (T cell factor) transcriptional activity and location of β-catenin induced by T0070907 were investigated in the colon cancer cell lines. T0070907 promoted polyp formation in the small intestine of ApcMin/+ mice and aberrant crypt foci in the colon of C57BL/6 mice. PPARγ inhibition promoted cell proliferation and increased expressions of the c-myc and cyclin D1 genes and the β-catenin protein in the colon epithelium. In vitro, cell proliferation was promoted, but it was inhibited by the transfection of dominant-negative Tcf4. T0070907 increased β-catenin/TCF transcriptional activity and β-catenin protein in the cytsol and nucleus, but relatively decreased it on the cell membrane. PPARγ antagonist promotes tumorigenesis in the small intestine and colon through stimulation of epithelial cell proliferation. β-Catenin contributes to the promotion of tumorigenesis by PPARγ antagonist due to activation of TCF/LEF (lymphoid enhancer factor) transcriptional factor.

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation.

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.

Acceleration of Gastric Ulcer Healing by Omeprazole in Portal Hypertensive Rats

Background: Gastric ulcer healing is delayed in patients with portal hypertension (PHT) and often responds poorly to histamine H₂ blockers. Although proton pump inhibitors are more effective anti-ulcer agents, there is little information regarding their efficacy for gastric ulcer in cases of PHT. Therefore, we investigated the effects of a proton pump inhibitor, omeprazole, on the healing of acetic-acid-induced gastric ulcer in PHT rats. Methods: Animals studied were 80 male Sprague-Dawley rats aged 7 weeks, of which half underwent two-staged portal vein ligation (PHT rats) and half underwent a sham operation (SO rats). Gastric ulcers were induced by acetic acid. Starting from day 4 after ulcer induction, rats received omeprazole or vehicle orally (50 mg/kg) for 5 or 10 days. Ulcer area, proliferating cell nuclear antigen labelling index (PCNA LI), and serum gastrin levels were recorded. Results: PHT significantly inhibited epithelial cell proliferation and delayed gastric ulcer healing as indicated by a decreased PCNA LI at the ulcer margin and almost 2-fold larger ulcer area in PHT versus SO rats 14 days after ulcer induction. Ten-day treatment with omeprazole (vs. vehicle) significantly accelerated ulcer healing to a similar extent in both PHT and SO rats. Serum gastrin levels were significantly higher in PHT rats than in SO rats following treatment with omeprazole. Omeprazole (vs. vehicle) restored the decreased PCNA LI at the ulcer margin in PHT rats to that noted in SO rats. Conclusions: In PHT rats, omeprazole accelerates gastric ulcer healing, stimulates epithelial cell proliferation at the ulcer margin, and increases serum gastrin levels. Since gastrin is a potent stimulator of gastric epithelial cell proliferation, increased serum gastrin levels may be an important factor in omeprazole-induced stimulation of epithelial cell proliferation and acceleration of gastric ulcer healing in conditions of PHT.

Stimulation of Epithelial Cell Proliferation of Isolated Distal Colon of Rats by Continuous Colonic Infusion of Ammonia or Short-Chain Fatty Acids Is Nonadditive

Dietary fibers accelerate colonic epithelial cell proliferation at least in part by modulating bacterial metabolism in the large intestine. Ammonia and short-chain fatty acids (SCFA) are major metabolites of hindgut bacteria and are believed to affect epithelial cell kinetics of the colon. However, the effect of luminal ammonia itself and the possible interaction of ammonia with SCFA on colonic epithelial cell proliferation have not yet been studied. The colon of rats was surgically isolated and continuously administered infusates with saline, ammonia, SCFA or both into the isolated colon for 7 d in a two-way factorial design. On d 7, vincrystine sulfate was administered intravenously to cause metaphase arrest. The activity of epithelial cell proliferation in the distal colon was estimated by using a stathmokinetic method and by histologic examination. The crypt size was significantly larger in rats given infusates containing SCFA than in rats given infusates without SCFA. Infusion of ammonia or SCFA significantly stimulated colonic epithelial cell proliferation compared with the saline infusion. Infusion of both ammonia and SCFA resulted in accumulated mitoses per crypt that did not differ from the other three infusions although the value tended to be lower than when SCFA alone were infused. Thus, stimulation of epithelial cell proliferation by ammonia and SCFA is not additive, and the interaction between them should be considered when the effects of dietary fibers on gut epithelial proliferation are investigated.

Tumor necrosis factor alpha-induced stimulation of neoplastic progression of preneoplastic, hyperplastic alveolar nodule line C4

Lymphocytic infiltrates of the mouse mammary preneoplastic, hyperplastic alveolar nodule (HAN) line C4 have elevated reactivity which correlates positively with the progression of HAN to mammary adenocarcinoma. In this study we investigated the hypothesis that the immunoregulatory mechanisms of HAN infiltrating lymphocytes (HILs) on mammary neoplastic progression are mediated, at least in part, by tumor necrosis factor alpha (TNF alpha). C4 HAN epithelial cells express mRNA for both the p55-60 receptor and the p75-80 TNF alpha receptors. High levels of both TNF alpha and TNF beta are expressed by HILs, whereas only TNF alpha is expressed by the C4 HAN epithelial cells. Treatment of C4 HAN bearers with TNF alpha in vivo decreases the latency period and enhances the frequency of HAN progression to tumor. Proliferation of monolayer cultures of epithelial cells from mammary glands of normal and C4 HAN-bearing mice, as well as C4 tumor cells, is enhanced by TNF alpha. Growth of normal mammary cells in 3-dimensional collagen cultures is also significantly stimulated by TNF alpha. Our results suggest that stimulation of epithelial cell proliferation by HIL-produced TNF alpha is one mechanism responsible for the 'immune stimulation' of neoplastic progression in the HAN model.

Clonal growth of human prostatic epithelial cells is stimulated by fibroblasts

The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.

Progesterone effects on deoxyribonucleic acid synthesis in normal mouse mammary glands.

Current in vivo and in vitro data indicate that progesterone (P) is required for epithelial proliferation in mammary glands of adult mice. P receptors (PgR) in adult mammary glands are under estrogenic regulation. By contrast in immature, pubertal 5-week-old mice, PgR are present only in low concentration and cannot be increased by estrogen (E). Having identified two different mammary gland developmental states with differing PgR compositions, the purpose of the present studies was to investigate the roles of E-dependent and -independent PgR in epithelial cell proliferation. DNA histoautoradiography was used to quantitate the effect of P on DNA synthesis; hormonal effects on mammary gland morphology were also assessed. The results demonstrate the P, rather than E, has a major role in stimulating epithelial DNA synthesis in the adult mammary gland. The morphological correlate of P's stimulatory effect on cell proliferation was increased ductal side-branching. We postulate that P is acting via E-dependent PgR and that one important way in which E promotes epithelial cell proliferation in adult tissue is by its ability to increase PgR concentration. In the immature mammary gland E rather than P is primarily responsible for stimulation of epithelial cell proliferation at this developmental stage. It appears likely that the absence of a predominant, P-mediated cell proliferative effect is related to the lack of E-dependent PgR.

Postprandial stimulation of epithelial cell proliferation in defunctioned colon of rats is not caused by gastrin

Rats were submitted, at random, to either a diverting colostomy alone or to antrectomy with a colostomy. After a 48-h fasting period, animals from each group were refed, whereas control animals were kept fasting. Animals were killed at 0, 6, 12, 18, and 24 h after the time of refeeding. In vitro labeling of colon mucosa with [3H]thymidine and autoradiography were performed to determine the proliferative parameters in the colonic crypts, and scintillation counts on mucosal scrapings were used for the estimation of mucosal deoxyribonucleic acid synthetic activity. Refeeding increased the labeling index (p < 0.01), mitotic index (p < 0.01), and mucosal deoxyribonucleic acid synthesis activity (p < 0.01) in the proximal colon as well as in the defunctioned distal segment. Despite suppression of the postprandial rise in serum gastrin (p < 0.01), antrectomy did not abolish the proliferative reaction in any colonic segment. These data confirm the existence of a potent stimulant of colonic cell proliferation that is released systemically after feeding. They indicate that gastrin is not the responsible stimulant and that another, yet unknown, physiological factor is involved.