Intestinal Subepithelial Myofibroblasts Research Articles

Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1.

The hereditary mixed polyposis syndrome (HMPS) was first described about 50 years ago in a large Ashkenazi Jewish family from St Mark’s Hospital, London. The family showed apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma in a high proportion of individuals. In the last 15 years, we have mapped the HMPS gene to chromosome 15q13.3 and identified an ancestral haplotype common to all the known HMPS families. Here, we have used genetic mapping, copy number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a large duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This mutation has no effect on SCG5 expression, but is associated with greatly increased, allele-specific GREM1 expression. Whilst GREM1 is expressed in intestinal sub-epithelial myofibroblasts in controls, HMPS patients predominantly express GREM1 in the epithelium of the large bowel. The HMPS duplication contains predicted transcriptional enhancer elements; we have shown that some of these interact with the GREM1 promoter and are capable of driving gene expression in vitro. Increased GREM1 expression is predicted to lead to reduced bone morphogenetic protein pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. The pathogenic mechanism in HMPS is extremely unusual in Mendelian cancer syndromes and highlights ectopic gene expression as a mechanism of tumorigenesis.

Single Lgr5+ Intestinal Stem Cells Co-Cultured With Intestinal Subepithelial Myofibroblasts (ISEMF) Differentiate into Absorptive and Secretory Cell Lineages Without Exogenous Growth Factors

Single Lgr5+ Intestinal Stem Cells Co-Cultured With Intestinal Subepithelial Myofibroblasts (ISEMF) Differentiate into Absorptive and Secretory Cell Lineages Without Exogenous Growth Factors

Prostaglandin E2Promotes Wound-Induced Migration of Intestinal Subepithelial Myofibroblasts via EP2, EP3, and EP4 Prostanoid Receptor Activation

Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that reside in the subepithelial region throughout the intestine. When the intestine is damaged, the migratory and mitotic responses of ISMFs are crucial for wound closure. However, their mechanism of action remains unknown. We have investigated the role of cyclooxygenase (COX) and its metabolite prostaglandin E(2) (PGE(2)) in the wound repair process of bovine ISMFs. The action of a mechanical scratch in a layer of ISMFs in cell culture elevated the levels of both COX-2 mRNA expression and PGE(2) secretion 1 and 6 h after the event. After 24 h ISMFs had migrated to and reduced the wounded area around the site of the scratch. Treatment with the COX-1/2 inhibitor indomethacin, the COX-2 inhibitor 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404), or E prostanoid receptor 2 to 4 (EP2-EP4) antagonists significantly inhibited wound repair. Conversely, inhibition of wound closure by indomethicin was reversed by treatment with PGE(2) or agonists of the receptors EP2, EP3, or EP4 but not of EP1. Although EP2 to EP4 stimulation did not influence ISMF proliferation, it did stimulate ISMF migration in the transwell cell migration assay. It is noteworthy that cell migration stimulated by EP2 and EP4 was inhibited by the tyrosine kinase receptor inhibitor genistein and also by (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid (SU6668). However, cell migration stimulated by EP3 was unaffected. Reverse transcription-polymerase chain reaction showed EP2 or EP4 stimulation elevated the level of mRNA expression for fibroblast growth factor-2, which stimulates ISMF migration. Collectively, COX-2-dependent PGE(2) secretion promotes wound healing by ISMFs. PGE(2)-EP3 signaling may directly stimulate ISMF migration. PGE(2)-EP2/4 signaling indirectly stimulates ISMF migration by elevating the level of growth factor secretion.

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Endocrine differentiation of rat enterocytes in long-term three-dimensional co-culture with intestinal myofibroblasts

The proliferation and differentiation of the small intestinal epithelium depends on the microenvironment surrounding the stem cells, such as intestinal subepithelial myofibroblasts. Although there have been many culture studies of intestinal epithelial–mesenchymal interaction, a culture which allows long-term observations has been difficult. This study investigated the influence of intestinal subepithelial myofibroblasts on the proliferation and differentiation of intestinal epithelial cells with a relatively long-term observation of 3 wk using a 3D co-culture system. Cultured rat intestinal subepithelial myofibroblasts, obtained from the duodenum, were embedded in collagen gel and cells from the rat intestinal epithelial cell line IEC-6 seeded onto it. Histologic sections of the cell-embedded gels were made and histochemical and immunohistochemical examinations were carried out in conjunction with expression analysis of the pancreatic duodenal homeobox 1 (pdx-1) transcription factor in IEC-6 cells. The IEC-6 cells showed increased proliferation and displayed characteristic endocrine features when co-cultured with rat intestinal subepithelial myofibroblasts, arranging themselves into multilayer structures and becoming cuboidal, with abundant cytoplasm and oval nuclei. Some IEC-6 cells were immunohistochemically positive for chromogranin A and glicentin. They also expressed the pdx-1 transcription factor at both the mRNA and protein levels. The number and percentage of chromogranin A-positive cells increased with culture time, whereas no increase was observed in cells cultured without rat intestinal subepithelial myofibroblasts. The present study using a long-term 3D co-culture model has obtained evidence of the participation of intestinal subepithelial myofibroblasts in enteroendocrine differentiation, supported by the expression of pdx-1 and glicentin production.

Interleukin-1beta stimulates intestinal subepithelial myofibroblasts to release factors that promote intestinal epithelial restitution

Interleukin-1beta stimulates intestinal subepithelial myofibroblasts to release factors that promote intestinal epithelial restitution

A Multicellular Approach Forms a Significant Amount of Tissue-Engineered Small Intestine in the Mouse

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection. In this study, we transitioned the technique to a mouse model, allowing investigation of the processes involved during TESI formation through the transgenic tools available in this species. This is a necessary step toward applying the technique to human therapy. Multicellular organoid units were derived from small intestines of transgenic mice and transplanted within the abdomen on biodegradable polymers. Immunofluorescence staining was used to characterize the cellular processes during TESI formation. We demonstrate the preservation of Lgr5- and DcamKl1-positive cells, two putative intestinal stem cell populations, in proximity to their niche mesenchymal cells, the intestinal subepithelial myofibroblasts (ISEMFs), at the time of implantation. Maintenance of the relationship between ISEMF and crypt epithelium is observed during the growth of TESI. The engineered small intestine has an epithelium containing a differentiated epithelium next to an innervated muscularis. Lineage tracing demonstrates that all the essential components, including epithelium, muscularis, nerves, and some of the blood vessels, are of donor origin. This multicellular approach provides the necessary cell population to regenerate large amounts of intestinal tissue that could be used to treat short bowel syndrome.

The “cryptic” mechanism of action of glucagon-like peptide-2

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.

Abstract 3106: Intestinal mesenchymal fibroblasts promote early epithelial tumorigenesis through activation of integrin beta4 and receptor tyrosine kinases, bypassing common mutational mechanisms

Abstract Cancer cells communicate extensively with their stroma, exchanging signals that influence growth and metastasis. Intestinal sub-epithelial myofibroblasts (ISEMFs) direct organogenesis and constitute the colorectal cancer (CRC) microenvironment. ISEMFs from the mouse small intestine and colon express regional characteristics and different cytokines. In line with these regional patterns, ApcMin mice develop adenomas principally in the small intestine but not in the colon; conversely, humans develop tumors mainly in the colon and rarely in the small bowel. We hypothesize that intestinal ISEMFs influence tumor regionality through cell-cell communication. Murine ileal ISEMFs supported growth of human CRC cells markedly better than those originating in the colon in NOD/SCID xenografts and in vitro. We traced this effect to a single secreted factor, matrix metalloproteinase 9 (MMP9), which is exclusively mesenchymal in expression and substantially enriched in native ileal over colonic ISEMFs. Actively cycling intestinal epithelial progenitors and Lgr5+ stem cells showed selectively reduced bromodeoxyuridine uptake in Mmp9-/- mouse ileum, revealing that Mmp9 deficiency impairs epithelial self-renewal. Moreover, ApcMin mice developed fewer ileal adenomas upon treatment with an Mmp9 inhibitor or on the Mmp9-/- genetic background. MMP9 immunohistochemistry revealed expression in 106 of 642 (16.5%) human CRC samples; expression was confined to stromal fibroblasts and considerably enriched among CRCs that lack characteristic KRAS mutations and harbor the CpG island methylator phenotype. Gene expression in epithelial cells interacting with ileal fibroblasts pointed to activation of cytoskeletal remodeling and integrin ß4 signaling pathways, signatures absent in cells cultured with colonic ISEMFs. Biochemical assays verified that stromal MMP9 activates epithelial integrin ß4 and, in turn, ErbB and c-Met receptor tyrosine kinases (RTKs) in adjoining epithelial, including human CRC, cells. This activation is mediated by MMP9 cleavage of the integrin ligand laminin alpha4. Activation of epithelial integrin ß4 and RTKs and the paucity of KRAS mutations in CRCs with stromal MMP9 expression together suggest that potent MMP9-mediated microenvironment signals can help bypass common mutational mechanisms of constitutive growth factor pathway activation in CRC. These results define a novel and therapeutically tractable pathway of tumor-stromal interaction in early CRC pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3106. doi:10.1158/1538-7445.AM2011-3106

ATP induces contraction mediated by the P2Y 2 receptor in rat intestinal subepithelial myofibroblasts

Intestinal subepithelial myofibroblasts (IMFs) exist just under the epithelial membrane directly facing the mucosal microvascular capillary surface distributed in the lamina propria. In the gastrointestinal tract, ATP is released from epithelial and endothelial cells in response to mechanical stimuli. Although it has been reported that mechanical stimuli evoke synchronized Ca 2+ waves in cultured IMFs, the contractile responses by ATP stimulation have not been examined. The aim of this study was to clarify the mechanism of the contraction of IMFs in response to ATP. ATP (1–30 μM) induced contraction in a concentration-dependent manner. These contractions were inhibited by LaCl 3 (100–300 μM) and by Ca 2+-free solution (0.5 mM EGTA). Fura-2/Ca 2+ signals indicated that ATP (1–10 μM) elicited transient increases in intracellular Ca 2+ concentration ([Ca 2+] i). In addition, αβ-methylene-ATP (10, 30 and 300 μM), a broad spectrum P2X agonist at a concentration higher than 100 μM, induced neither contraction nor [Ca 2+] i rise. UTP (1–30 μM), a selective P2Y 2 and P2Y 4 agonist in rodent, induced concentration-dependent contractions and [Ca 2+] i increases, whereas ADP and UDP (10 μM) did not induce contractions. Pretreatment with suramin (30–100 μM), a relatively selective P2Y 2 antagonist, strongly inhibited ATP- and UTP-induced contractions and [Ca 2+] i increases. However, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS: 10–30 μM), a receptor antagonist for several P2X and P2Y but less effective to P2Y 2 receptor, failed to inhibit ATP- and UTP-induced contractions and [Ca 2+] i increases. By RT-PCR, mRNA expressions of the P2Y 1 and P2Y 2 receptors, but not P2Y 4 or P2Y 6, were detected in IMFs. These results suggest that ATP induces [Ca 2+] i-dependent contraction in IMFs, which is mediated through the P2Y 2 receptor.

Murine Intestinal Subepithelial Myofibloblast Cells (ISEMF) Provide Necessary Support For Lgr5-EGFP Positive And Negative Cells To Grow In A Matrigel Culture System

Introduction: Cells expressing the putative intestinal stem cell marker Lgr5 have been shown previously to generate all the terminally differentiated intestinal epithelial cell types. Intestinal subepithelial myofibroblast cells (ISEMF) are present immediately adjacent to the basement membrane and close to the basal surface of the intestinal epithelium. ISEMF have been shown to play important roles in the organogenesis and regeneration of the intestine by secreting growth factors and cytokines that promote epithelial migration, proliferation and differentiation.

Mechanism of Action of Glucagon-Like Peptide-2 to Increase IGF-I mRNA in Intestinal Subepithelial Fibroblasts

IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10(-8) m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10(-8) m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent β-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2.

Oxidative state and IL-6 production in intestinal myofibroblasts of Crohnʼs disease patients

Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology.

Amphiregulin Promotes Intestinal Epithelial Regeneration: Roles of Intestinal Subepithelial Myofibroblasts

Epidermal growth factor family plays critical roles in intestinal epithelial proliferation and differentiation. The precise function of amphiregulin (AREG), a member of the epidermal growth factor family, in intestinal biology is largely unknown. The present study attempted to address the functional roles of AREG in intestinal epithelial regeneration. Total body irradiation was performed, and intestinal regeneration was assessed in AREG knockout mice. Genetically disruption of AREG significantly impaired intestinal regeneration after radiation injury. It is known that prostaglandin E(2) (PGE(2)) exerts radio-protective and growth-stimulatory effects on intestinal epithelium. We found that PGE(2) radio-protective action did not involve AREG. However, PGE(2) growth-stimulatory effects required functional AREG. Localization of AREG expression was determined by immunohistochemistry in regenerative intestine. The immunoreactivity of AREG was predominantly localized in intestinal subepithelial myofibroblasts (ISEMF). Primary ISEMF cultures were established, and growth-stimulatory actions of ISEMF-generated AREG were demonstrated in cell coculture system. In addition, we found that the cAMP/protein kinase A pathway robustly induced AREG in cultured ISEMF. These studies suggest that AREG plays critical roles in intestinal epithelial growth. Modulation of levels of AREG by targeting ISEMF represents a novel strategy for treatment of certain intestinal disorders.

540 Intestinal Subepithelial Myofibroblasts of Ulcerative Colitis Patients Express Increased WNT5A Levels That Enhance Proliferation in an Autocrine Manner

Hepatic bile salt secretion is mediated primarily by the bile salt export pump (BSEP), an ABC transporter on the canalicular membrane of the hepatocyte. Defects in bile salt secretion lead to the profound consequences of intrahepatic cholestasis, sometimes culminating in liver failure. Studies on the short term regulation of BSEP have implicated cAMP and other signaling pathways, but have yielded contradictory results regarding calcium. In order to clarify the role of calcium in BSEP regulation, we examined pericanalicular calcium signaling mediated by the inositol 1,4,5-trisphosphate receptor type 2 (InsP3R2), which is the principal calcium release channel in hepatocytes and is concentrated in the peri-canalicular domain. BSEP and InsP3R2 expression and function were examined in rat liver and in rat hepatocytes in collagen sandwich culture. Using confocal immunofluorescence microscopy, InsP3R2 and BSEP were both found in the region of the canalicular membrane. BSEP activity was examined bymonitoring the kinetics of secretion of CGamF, a fluorescent BSEP substrate, into the sealed canalicular vacuoles of hepatocytes in sandwich culture. CGamF secretion was significantly inhibited by either (1) treating hepatocytes with the calcium chelator BAPTA for 30 minutes; (2) inducing redistribution of InsP3R2 away from the canalicular region by depleting the plasma membrane of cholesterol with 5 mM methyl-beta-cyclodextrin for 30 minutes; (3) knocking down InsP3R2 expression using a specific siRNA; or (4) knocking down BSEP expression using BSEP-specific siRNA as a positive control for the secretion assay. Moreover, knockdown of InsP3R2 significantly decreased bile salt secretion without altering BSEP expression or localization, as confirmed by immunofluorescence microscopy. In separate studies, basal bile salt secretion was reduced in livers from InsP3R2 KO mice, despite maintenance of BSEP localization and expression, as assessed by immunofluorescence microscopy and western blot, respectively. These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion via post-translational regulation of BSEP.We postulate this regulationmay involvemodulating insertion of BSEP into the canalicular membrane .

S16 Intestinal subepithelial myofibroblasts of ulcerative colitis patients express increased Wnt5a levels that enhances proliferation in an autocrine manner

S16 Intestinal subepithelial myofibroblasts of ulcerative colitis patients express increased Wnt5a levels that enhances proliferation in an autocrine manner

Intestinal Subepithelial Myofibroblasts Demonstrate Cox-2 Expression in Early Stages of Epithelial Proliferation in Tissue Engineered Small Intestine

Introduction: Children with short bowel syndrome suffer severe morbidity and mortality. Tissue engineered small intestine (TESI) may serve as a viable replacement. In previous work, we have generated TESI in Lewis rats and Yorkshire swine, and TESI successfully rescued a model of short bowel syndrome in Lewis rats. We recently transitioned to a mouse model in order to investigate the mechanisms by which TESI forms. Intestinal subepithelial myofibroblasts (ISEMFs) are a specific population of cells involved in intestinal epithelial proliferation, differentiation, inflammation, and neoplasia.

Isolation and Characterization of Bovine Intestinal Subepithelial Myofibroblasts

Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that exist under the epithelium of intestines. Primarily isolated ISMFs from rodents have been applied to experiments. However, due to the size of their intestines, the available cell number is limited. Thus, we attempted to isolate ISMFs from bovine colon as an alternative material. After detachment of smooth muscle and epithelial layers, colonic mucosa was explanted. After 2-week incubation, α-SMA (+) / vimentin (+) / desmin (−) ISMFs were harvested and applied for experiments. First we examined the effect of cell passage on morphology and proliferation activity of bovine ISMFs. Although 3rd and 7th passage bovine ISMFs did not exhibit any changes, 11th passage ISMFs showed rounded enlarged shape and lost proliferation potential. On the contrary, rat ISMFs displayed the above senescent changes at earlier passage (passage 4). In intracellular Ca2+ concentration measurement, bioactive substances (0.3 – 1µ M ATP, 0.1 – 1µM serotonin, 10 – 100 nM endothelin-1, and 1 – 10 nM bradykinin) dose-dependently induced an increase in intracellular Ca2+ concentration in bovine ISMFs (passage 3 and 7). However, at passage 11, impairment in intracellular Ca2+ responses was observed. Thus, bovine ISMFs might be a novel useful tool with long life span and good cellular responses to investigate physiological/pathophysiological roles of ISMFs.

Collagen XVI induces formation of focal contacts on intestinal myofibroblasts isolated from the normal and inflamed intestinal tract

In Crohn's disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell–matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin α1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of α1β1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell–matrix interaction preferentially through recruitment of α1ß1 integrin into the tips of the focal adhesion contacts.