Mechanical Stress-induced Connective Tissue Growth Factor Plays a Critical Role in Intestinal Fibrosis in Crohn's-like Colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of pro-fibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I, but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMC). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends towards normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by up-regulating pro-fibrotic mediators i.e. CTGF, may play a critical role in fibrosis in CD.

Loss of alcohol dehydrogenase 1B in cancer-associated fibroblasts: contribution to the increase of tumor-promoting IL-6 in colon cancer.

BACKGROUND: Increases in IL-6 by cancer-associated fibroblasts (CAFs) contribute to colon cancer progression, but the mechanisms involved in the increase of this tumor-promoting cytokine are unknown. The aim of this study was to identify novel targets involved in the dysregulation of IL-6 expression by CAFs in colon cancer. METHODS: Colonic normal (N), hyperplastic, tubular adenoma, adenocarcinoma tissues, and tissue-derived myo-/fibroblasts (MFs) were used in these studies. RESULTS: Transcriptomic analysis demonstrated a striking decrease in alcohol dehydrogenase 1B (ADH1B) expression, a gene potentially involved in IL-6 dysregulation in CAFs. ADH1B expression was downregulated in approximately 50% of studied tubular adenomas and all T1-4 colon tumors, but not in hyperplastic polyps. ADH1B metabolizes alcohols, including retinol (RO), and is involved in the generation of all-trans retinoic acid (atRA). LPS-induced IL-6 production was inhibited by either RO or its byproduct atRA in N-MFs, but only atRA was effective in CAFs. Silencing ADH1B in N-MFs significantly upregulated LPS-induced IL-6 similar to those observed in CAFs and lead to the loss of RO inhibitory effect on inducible IL-6 expression. CONCLUSION: Our data identify ADH1B as a novel potential mesenchymal tumor suppressor, which plays a critical role in ADH1B/retinoid-mediated regulation of tumor-promoting IL-6.

Cardiovascular manifestations of inflammatory bowel diseases and the underlying pathogenic mechanisms.

Inflammatory bowel disease (IBD), consisting of ulcerative colitis and Crohn's disease, mainly affects the gastrointestinal tract but is also known to have extraintestinal manifestations because of long-standing systemic inflammation. Several national cohort studies have found that IBD is an independent risk factor for the development of cardiovascular disorders. However, the molecular mechanisms by which IBD impairs the cardiovascular system are not fully understood. Although the gut-heart axis is attracting more attention in recent years, our knowledge of the organ-to-organ communication between the gut and the heart remains limited. In patients with IBD, upregulated inflammatory factors, altered microRNAs and lipid profiles, as well as dysbiotic gut microbiota, may induce adverse cardiac remodeling. In addition, patients with IBD have a three- to four times higher risk of developing thrombosis than people without IBD, and it is believed that the increased risk of thrombosis is largely due to increased procoagulant factors, platelet count/activity, and fibrinogen concentration, in addition to decreased anticoagulant factors. The predisposing factors for atherosclerosis are present in IBD and the possible mechanisms may involve oxidative stress system, overexpression of matrix metalloproteinases, and changes in vascular smooth muscle phenotype. This review focuses mainly on 1) the prevalence of cardiovascular diseases associated with IBD, 2) the potential pathogenic mechanisms of cardiovascular diseases in patients with IBD, and 3) adverse effects of IBD drugs on the cardiovascular system. Also, we introduce here a new paradigm for the gut-heart axis that includes exosomal microRNA and the gut microbiota as a cause for cardiac remodeling and fibrosis.

Neonatal Injury Increases Gut Permeability by Epigenetically Suppressing E-Cadherin in Adulthood.

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms.

Lactobacillus rhamnosus GG increases cyclooxygenase-2 expression and prostaglandin E2 secretion in colonic myofibroblasts via a MyD88-dependent mechanism during homeostasis.

Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

Immunosuppression by Intestinal Stromal Cells.

This chapter summarizes evidence that intestinal myofibroblasts, also called intestinal stromal cells, are derived in the adult from tissue mesenchymal stem cells under homeostasis and may be replenished by bone marrow mesenchymal stromal (stem) cells that are recruited after severe intestinal injury. A comparison of mechanism of immunosuppression or tolerance by adult intestinal stromal cells (myofibroblasts) is almost identical with those reported for mesenchymal stem cells of bone marrow origin. The list of suppression mechanisms includes PD-L1 and PD-L2/PD-1 immune checkpoint pathways, soluble mediator secretion, toll-like receptor-mediated tolerance, and augmentation of Treg cells. Further, both mesenchymal stem cells and intestinal stromal cells express an almost identical repertoire of CD molecules. Lastly, others have reported that isolate intestinal stromal cells are capable of differentiating into bone and less well into chondrocyte, but not into adipocytes, a finding that we have confirmed. These findings suggest that intestinal stromal cells (myofibroblasts) are partially differentiated adult, tissue-resident stem cells which are capable of exerting immune tolerance in the intestine. Their role in repair of inflammatory bowel disease and immune suppression in colorectal cancer needs further investigation.

CD90(+) stromal cells are the major source of IL-6, which supports cancer stem-like cells and inflammation in colorectal cancer.

IL-6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90(+) innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL-6; however, their contribution to the increase in IL-6 in CRC and to tumor-promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL-6 producing CMFs is increased in CRC (C-CMFs) and they represent the major source of IL-6 in T2-T3 CRC tumors. Activity/expression of stem cell markers-aldehyde dehydrogenase and LGR5- was significantly up-regulated in colon cancer cells (SW480, Caco-2 or HT29) cultured in the presence of conditioned medium from tumor isolated C-CMFs in an IL-6 dependent manner. C-CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL-6 dependent manner. Our study suggests that CD90(+) fibroblasts/myofibroblasts may be the major source of IL-6 in T2-T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL-6 producing CAFs (a.k.a. C-CMFs) may provide a useful target for treating or preventing CRCs.

PDGF-AA mediates mesenchymal stromal cell chemotaxis to the head and neck squamous cell carcinoma tumor microenvironment.

BACKGROUND: The robust desmoplasia associated with head and neck squamous cell carcinoma (HNSCC) suggests that the tumor microenvironment may be an important component in the pathophysiology of this cancer. Moreover, the high recurrence rate and poor clinical response to chemotherapy and radiation treatment further underscores that the non-cancerous cells of the microenvironment, such as mesenchymal stromal cells (MSCs), cancer associated fibroblasts (CAFs), and pericytes, may be important in the pathophysiology of HNSCC. METHODS: Confocal microscopy and immunohistomchemistry approaches were used to identify MSCs tumor microenvironment from patients with oral cavity and oral pharyngeal squamous cell carcinoma (SCC). In vitro Boyden chamber assays and multiplex magnetic bead assays were used to measure MSC chemotaxis and to identify the chemokines secreted by JHU-011, -012, -019, three cells lines derived from patients with oral pharyngeal SCC. RESULTS: We show here that MSCs reside in the tumor microenvironment of patients with oral cavity and oral pharyngeal SCC and are recruited via paracrine mediated tumor cell secretion of (platelet derived growth factor) PDGF-AA. The MSC markers CD90+, CD105+, and gremlin-1+ were found to co-localize on cells within the tumor microenvironment in oral cavity SCC specimens distinct from α-smooth muscle actin staining CAFs. The conditioned media from JHU-011, -012, and -019 caused a significant increase in MSC migration (>60%) and invasion (>50%; p < 0.0001) compared to oral keratinocyte (OKT) controls. Tumor cell induced MSC chemotaxis appears to be mediated through paracrine secretion of PDGF-AA as inhibition of the PDGF-AA receptor, PDGFR-α but not PDGFR-ß, resulted in near arrest of MSC chemotaxis (p < 0.0001). CONCLUSIONS: Tumor microenvironment expression of PDGFR-α has been shown to correlate with a worse prognosis in patients with prostate, breast, ovarian, non-small cell lung cancer and osteosarcoma. This is the first evidence that a similar signaling paradigm may be present in HNSCC. PDGFR-α inhibitors have not been studied as adjunctive treatment options in the management of HNSCC and may prove to be an important driver of the malignant phenotype in this setting.

TLR4 activation enhances the PD-L1-mediated tolerogenic capacity of colonic CD90+ stromal cells.

Signaling via programmed death ligand-1 (PD-L1) and PD-L2 is crucial for maintaining peripheral tolerance. CD90(+) myofibroblasts/fibroblasts (CMFs) are major programmed cell death-1 (PD-1) ligand-expressing cells in normal human colonic mucosa. CMFs suppress activated CD4(+) T cell proliferation via PD-1 ligands. It is not known whether signaling through TLRs contribute to the regulation PD-1 ligands on CMFs upon colonic mucosal tolerance. In this study, we demonstrated that stimulation of TLR4 on human CMFs upregulates PD-L1, but not PD-L2, and reinforces CMF-mediated suppression of CD4(+) T cell proliferation and IFN-γ production. TLR4-mediated upregulation of PD-L1 on CMFs involved NF-κB pathways and was JAK2 and MyD88 dependent. MyD88-dependent stimulation of TLR1/2 and TLR5 also upregulated PD-L1 expression on CMFs in culture. PD-L1 expression was drastically decreased in vivo in the colonic mucosa of mice devoid of MyD88. Induction of MyD88 deficiency in CMFs in fibroblast-specific MyD88 conditional knockout mice resulted in a strong increase in a mucosal IFN-γ expression concomitantly with the abrogation of PD-L1 expression in CMFs under homeostasis and epithelial injury induced by dextran sodium sulfate. Together, these data suggest that MyD88-dependent TLR stimulation of CMFs in the normal colonic mucosa may reinforce these cells' anti-inflammatory capacity and thus contribute to the maintenance of mucosal tolerance.

miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD.

OBJECTIVE: Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD. METHODS: miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells. RESULTS: The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon-γ inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses. CONCLUSIONS: Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.

Erlotinib suppresses tumorigenesis in a mouse model of colitis-associated cancer.

Colitis-associated cancer (CAC) in inflammatory bowel diseases exhibits more aggressive behavior than sporadic colorectal cancer; however, the molecular mechanisms remain unclear. No definitive preventative agent against CAC is currently established in the clinical setting. We investigated the molecular mechanisms of CAC in the azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model and assessed the antitumor efficacy of erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR). Erlotinib premixed with AIN-93 G diet at 70 or 140 parts per million (ppm) inhibited tumor multiplicity significantly by 96%, with ∼60% of the treated mice exhibiting zero polyps at 12 weeks. Bulk RNA-sequencing revealed more than a thousand significant gene alterations in the colons of AOM/DSS-treated mice, with KEGG enrichment analysis highlighting 46 signaling pathways in CAC development. Erlotinib altered several signaling pathways and rescued 40 key genes dysregulated in CAC, including those involved in the Hippo and Wnt signaling. These findings suggest that the clinically-used antitumor agent erlotinib might be repurposed for suppression of CAC, and that further studies are warranted on the crosstalk between dysregulated Wnt and EGFR signaling in the corresponding patient population.

Disruption of retinol-mediated IL-6 expression in colon cancer-associated fibroblasts: new perspectives on the role of vitamin A metabolism.

Stromal myo-/fibroblasts (MFs) account for up to 30% of lamina propria cells in the normal human colon and their number is dramatically increased in colon cancer (CRC). Fibroblasts from cancers, also known as cancer-associated fibroblasts (CAFs), differ from normal colonic MF (N-MFs) and support tumor-promoting inflammation, in part due to increased IL-6 secretion. In this editorial, we highlight recent data obtained regarding IL-6 regulation in colorectal cancer CAFs through vitamin A (retinol) metabolism, discuss current limitations in our understanding of the mechanisms leading to the CAF pro-inflammatory phenotype, and discuss potential approaches to target CAF retinoid metabolism during CRC treatment.

Human colonic myofibroblasts promote expansion of CD4+ CD25high Foxp3+ regulatory T cells.

BACKGROUND & AIMS: Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells. METHODS: Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohn's disease, were used to assess activation of the Treg cells. RESULTS: Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-ß and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3. CONCLUSIONS: CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases.

Exosomal miR-29b of Gut Origin in Patients With Ulcerative Colitis Suppresses Heart Brain-Derived Neurotrophic Factor.

Background and Aims: While the interplay between heart and gut in inflammatory bowel disease (IBD) has previously been noted, how the inflamed gut impairs heart function remain elusive. We hypothesized that exosomal miRNAs of gut origin induce cardiac remodeling in IBD. Our aim was to identify plasma exosomal miRNAs that not only are of diagnostic value but also contribute to cardiac remodeling in patients with ulcerative colitis (UC). Methods: Plasma exosomes were isolated from UC patients and healthy control subjects and exosomal miRNAs were profiled by next-generation sequencing. Exosomal miR-29b levels in CCD841 CoN colon epithelial cells were detected by RT-qPCR. Exosomes packaged with miR-29b were incubated with H9c2 cells or administered to live mice. Results: The plasma exosomal miRNA profiles of the UC patients were significantly different from that of the controls and 20 miRNAs including miR-29b were differentially expressed. In CCD841 CoN cells, TNFα, IL-1ß, and H2O2 significantly elevated miR-29b in both the cells and their secreted exosomes (p < 0.01), suggesting that intestinal epithelium secrets exosomes rich in miR-29b in IBD. In H9c2 myoblast cells, miR-29b modulated multiple genes including brain-derived neurotrophic factor (BDNF). Epithelial cell-derived exosomes packaged with miR-29b also attenuated BDNF and increased cleaved caspase 3, suggestive of apoptosis. Furthermore, tail vein injection of engineered exosomes with high levels of miR-29b suppressed BDNF and augmented cleaved caspase 3 in the heart of adult mouse (p < 0.01). Conclusion: Plasma exosomal miRNA profile could be a novel diagnostic approach for IBD. Excessive plasma exosomal miR-29b suppresses critical proteins like BDNF in IBD, leading to cardiac impairment.

Matrix metalloproteinase 7 contributes to intestinal barrier dysfunction by degrading tight junction protein Claudin-7.

Background: Previous studies implicated matrix metalloproteinases (MMPs), such as MMP-7, in inflammatory bowel diseases (IBD) by showing increased activity during inflammation of the gut. However, the pathophysiological roles of MMP-7 have not been clearly elucidated. Methods: The expression of MMP-7 was assessed in colonic biopsies of patients with ulcerative colitis (UC), in rodents with experimental colitis, and in cell-based assays with cytokines. Wild-type and MMP-7-null mice treated with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid were used for determining the pro-inflammatory function(s) of MMP-7 in vivo. Results: MMP-7 was highly expressed in patients with UC and in rodents with experimental colitis. IL-1ß, IL-4, IL-13, TNFα, or lipopolysaccharide enhanced MMP-7 expression in human colonic epithelial cells, rat colonic smooth muscle cells, and THP-1-derived macrophages. Active MMP-7 degraded tight junction protein Claudin-7 in epithelial cells, cleaved recombinant Claudin-7 in cell-free system, and increased Caco-2 monolayer permeability. Immunostaining of colon biopsies revealed up-regulation of MMP-7 and reduction of Claudin-7 in UC patients. Compared to wild-type mice, Mmp7 -/- mice had significantly less inflammation in the colon upon DSS insult. DSS-induced alterations in junction proteins were mitigated in Mmp7 -/- mice, suggesting that MMP-7 disrupts the intestinal barrier. MMP-7 antibody significantly ameliorated colonic inflammation and Claudin-7 reduction in 2 different rodent models of colitis. Summary: MMP-7 impairs intestinal epithelial barrier by cleavage of Claudin-7, and thus aggravating inflammation. These studies uncovered Claudin-7 as a novel substrate of MMP-7 in the intestinal epithelium and reinforced MMP-7 as a potential therapeutic target for IBD.

Pathophysiology of motility dysfunction in bowel obstruction: role of stretch-induced COX-2.

In gastrointestinal conditions such as bowel obstruction, pseudo-obstruction, and idiopathic megacolon, the lumen of affected bowel segments is distended and its motility function impaired. Our hypothesis is that mechanical stretch of the distended segments alters gene expression of cyclooxygenase-2 (COX-2), which impairs motility function. Partial obstruction was induced with a silicon band in the distal colon of rats for up to 7 days, and wild-type and COX-2 gene-deficient mice for 4 days. Mechanical stretch was mimicked in vitro in colonic circular muscle strips and in primary culture of colonic circular smooth muscle cells (SMC) with a Flexercell system. The rat colonic circular muscle contractility was significantly decreased in the distended segment oral to obstruction, but not in the aboral segment. This change started as early as day 1 and persisted for at least 7 days after obstruction. The expression of COX-2 mRNA and protein increased dramatically also in the oral, but not aboral, segment. The upregulation of COX-2 expression started at 12 h and the effect persisted for 7 days. At 24 h after obstruction, the COX-2 mRNA level in the oral segment increased 26-fold compared with controls. This was not accompanied by any significant increase of myeloperoxidase or inflammatory cytokines. Immunohistochemical studies showed that COX-2 was selectively induced in the colonic SMC. In vitro stretch of colonic muscle strips or cultured SMC drastically induced COX-2 expression. Incubation of circular muscle strips from obstructed segment with COX-2 inhibitor NS-398 restored the contractility. The impairment of muscle contractility in obstructed colon was attenuated in the COX-2 gene-deficient mice. In conclusion, mechanical stretch in obstruction induces marked expression of COX-2 in the colonic SMC, and stretch-induced COX-2 plays a critical role in the suppression of smooth muscle contractility in bowel obstruction.

Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development.

BACKGROUND & AIMS: Intestinal stem cells (ISCs) are regulated by the mesenchymal environment via physical interaction and diffusible factors. We examined the role of Indian hedgehog (Ihh) in mesenchymal organization and the mechanisms by which perturbations in epithelial-mesenchymal interactions affect ISC fate. METHODS: We generated mice with intestinal epithelial-specific disruption of Ihh. Gross and microscopic anatomical changes were determined using histologic, immunohistochemical, and in situ hybridization analyses. Molecular mechanisms were elucidated by expression profiling and in vitro analyses. RESULTS: Deletion of intestinal epithelial Ihh disrupted the intestinal mesenchymal architecture, demonstrated by loss of the muscularis mucosae, deterioration of the extracellular matrix, and reductions in numbers of crypt myofibroblasts. Concurrently, the epithelial compartment had increased Wnt signaling, disturbed crypt polarity and architecture, defective enterocyte differentiation, and increased and ectopic proliferation that was accompanied by increased numbers of ISCs. Mechanistic studies revealed that Hh inhibition deregulates bone morphogenetic protein signaling, increases matrix metalloproteinase levels, and disrupts extracellular matrix proteins, fostering a proliferative environment for ISCs and progenitor cells. CONCLUSIONS: Ihh regulates ISC self-renewal and differentiation. Intestinal epithelial Ihh signals to the mesenchymal compartment to regulate formation and proliferation of mesenchymal cells, which in turn affect epithelial proliferation and differentiation. These findings provide a basis for analyses of the role of the muscularis mucosae in ISC regulation.