Intestinal Smooth Muscle Cells Research Articles

Rapid cyclic stretching induces a synthetic, proinflammatory phenotype in cultured human intestinal smooth muscle, with the potential to alter signaling to adjacent bowel cells.

Bowel smooth muscle experiences mechanical stress constantly during normal function, and pathologic mechanical stressors in disease states. We tested the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. Primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to pathologic, high frequency (1 Hz) uniaxial 3% cyclic stretch (loaded) or kept unloaded in culture for 6 hours. Total RNA sequencing, qRT-PCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. Loading induced differential expression of 4537 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle and on other cells in the bowel wall. HISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMCs into proliferative, fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

Pancreatic Tumor Organoid-Derived Factors from Cachectic Patients Disrupt Contractile Smooth Muscle Cells

Patients with pancreatic cancer often suffer from cachexia and experience gastrointestinal symptoms that may be related to intestinal smooth muscle cell (SMC) dysfunction. We hypothesized that pancreatic tumor organoids from cachectic patients release factors that perturb the SMC’s contractile characteristics. Human visceral SMCs were exposed to conditioned medium (CM) from the pancreatic tumor organoid cultures of cachectic (n = 2) and non-cachectic (n = 2) patients. Contractile proteins and markers of inflammation, muscle atrophy, and proliferation were evaluated by qPCR and Western blot. SMC proliferation and migration were monitored by live cell imaging. The Ki-67-positive cell fraction was determined in the intestinal smooth musculature of pancreatic cancer patients. CM from the pancreatic tumor organoids of cachectic patients did not affect IL-1β, IL-6, IL-8, MCP-1, or Atrogin-1 expression. However, CM reduced the α-SMA, γ-SMA, and SM22-α levels, which was accompanied by a reduced SMC doubling time and increased expression of S100A4, a Ca2+-binding protein associated with the synthetic SMC phenotype. In line with this, Ki-67-positive nuclei were increased in the intestinal smooth musculature of patients with a low versus high L3-SMI. In conclusion, patient-derived pancreatic tumor organoids release factors that compromise the contractile SMC phenotype and increase SMC proliferation. This may contribute to the frequently observed gastrointestinal motility problems in these patients.

Role of Nrf2/GPX4 mediated ferroptosis in intestinal injury in sepsis

To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe3+), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe3+ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe3+ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe3+ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.

Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility

In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145’s effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only ∼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

Salts of short-chain fatty acids increase the activity of the large conductance, Ca&lt;sup&gt;2+&lt;/sup&gt;-activated K&lt;sup&gt;+&lt;/sup&gt; channels and reduce calcium oscillations in rat GH3 cells

The short-chain fatty acids such as acetic, propionic and butyric acids are microbiota metabolites that can exert a series of physiological effects both in the intestine and other organs, including the central nervous system. The present work aimed to examine the effects of sodium acetate, propionate, and butyrate on the activity of large conductance Ca2+ activated K+ channels and calcium oscillations in rat pituitary GH3 cells. It has been shown that fatty acids under study cause a dose-dependent increase in the amplitude of total outward potassium currents and these effects are prevented by tetraethylammonium, a Ca2+ activated K+ channel blocker, indicating the involvement of Ca2+ activated K+ channels in the effects of fatty acids. It is worthy of note that fatty acids increased open probability of single channels with no changes in the amplitude and the mean channel open time. In addition, fatty acids were associated with a significant reduction in the amplitude and frequency of Ca2+ oscillations in GH3 cells. An increase in potassium conductance and a decrease in the intracellular Ca2+ level can mediate the effects of short-chain fatty acids in various excitable structures, such as a relaxation of intestinal and vascular smooth muscle cells, hyperpolarization of neurons, and the regulation of hormone and neurotransmitter release.

Reduced smooth muscle-fibroblasts transformation potentially decreases intestinal wound healing and colitis-associated cancer in ageing mice

Cancer and impaired tissue wound healing with ageing are closely related to the quality of life of the elderly population. Given the increased incidence of cancer and the population ageing trend globally, it is very important to explore how ageing impairs tissue wound healing and spontaneous cancer. In a murine model of DSS-induced acute colitis and AOM/DSS-induced colitis-associated cancer (CAC), we found ageing significantly decreases intestinal wound healing and simultaneous CAC initiation, although ageing does not affect the incidence of AOM-induced, sporadic non-inflammatory CRC. Mechanistically, reduced fibroblasts were observed in the colitis microenvironment of ageing mice. Through conditional lineage tracing, an important source of fibroblasts potentially derived from intestinal smooth muscle cells (ISMCs) was identified orchestrating intestinal wound healing and CAC initiation in young mice. However, the number of transformed fibroblasts from ISMCs significantly decreased in ageing mice, accompanied by decreased intestinal wound healing and decreased CAC initiation. ISMCs-fibroblasts transformation in young mice and reduction of this transformation in ageing mice were also confirmed by ex-vivo intestinal muscular layer culture experiments. We further found that activation of YAP/TAZ in ISMCs is required for the transformation of ISMCs into fibroblasts. Meanwhile, the reduction of YAP/TAZ activation in ISMCs during intestinal wound healing was observed in ageing mice. Conditional knockdown of YAP/TAZ in ISMCs of young mice results in reduced fibroblasts in the colitis microenvironment, decreased intestinal wound healing and decreased CAC initiation, similar to the phenotype of ageing mice. In addition, the data from intestine samples derived from inflammatory bowel disease (IBD) patients show that activation of YAP/TAZ also occurs in ISMCs from these patients. Collectively, our work reveals an important role of the ageing stromal microenvironment in intestinal wound healing and CAC initiation. Furthermore, our work also identified a potential source of fibroblasts involved in colitis and CAC.

GABAergic signaling between enteric neurons and intestinal smooth muscle promotes innate immunity and gut defense in Caenorhabditis elegans

The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C.elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-β-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.

Abnormally elevated expression of ACTA2 of circular smooth muscle leads to hyperactive contraction in aganglionic segments of HSCR

BackgroundActin Alpha 2 (ACTA2) is expressed in intestinal smooth muscle cells (iSMCs) and is associated with contractility. Hirschsprung disease (HSCR), one of the most common digested tract malformations, shows peristaltic dysfunction and spasm smooth muscles. The arrangement of the circular and longitudinal smooth muscle (SM) of the aganglionic segments is disorganized. Does ACTA2, as a marker of iSMCs, exhibit abnormal expression in aganglionic segments? Does the ACTA2 expression level affect the contraction function of iSMCs? What are the spatiotemporal expression trends of ACTA2 during different developmental stages of the colon?MethodsImmunohistochemical staining was used to detect the expression of ACTA2 in iSMCs of children with HSCR and Ednrb−/− mice, and the small interfering RNAs (siRNAs) knockdown technique was employed to investigate how Acta2 affected the systolic function of iSMCs. Additionally, Ednrb−/− mice were used to explore the changes in the expression level of iSMCs ACTA2 at different developmental stages.ResultsThe expression of ACTA2 is higher in circular SM in the aganglionic segments of HSCR patients and Ednrb−/− mice than in normal control children and mice. Down regulation of Acta2 weakens the contraction ability of intestinal smooth muscle cells. Abnormally elevated expression of ACTA2 of circular smooth muscle occurs since embryonic day 15.5 (E15.5d) in aganglionic segments of Ednrb−/− mice.ConclusionsAbnormally elevated expression of ACTA2 in the circular SM leads to hyperactive contraction, which may cause the spasm of aganglionic segments in HSCR.

Stretch-Induced Down-Regulation of HCN2 Suppresses Contractile Activity.

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.

Ontogeny and Trophic Factor Sensitivity of Gastrointestinal Projecting Vagal Sensory Cell Types.

Vagal sensory neurons (VSNs) located in the nodose ganglion provide information, such as stomach stretch or the presence of ingested nutrients, to the caudal medulla via specialized cell types expressing unique marker genes. Here, we leverage VSN marker genes identified in adult mice to determine when specialized vagal subtypes arise developmentally and the trophic factors that shape their growth. Experiments to screen for trophic factor sensitivity revealed that brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) robustly stimulate neurite outgrowth from VSNs in vitro Perinatally, BDNF was expressed by neurons of the nodose ganglion itself, while GDNF was expressed by intestinal smooth muscle cells. Thus, BDNF may support VSNs locally, whereas GDNF may act as a target-derived trophic factor supporting the growth of processes at distal innervation sites in the gut. Consistent with this, expression of the GDNF receptor was enriched in VSN cell types that project to the gastrointestinal tract. Last, the mapping of genetic markers in the nodose ganglion demonstrates that defined vagal cell types begin to emerge as early as embryonic day 13, even as VSNs continue to grow to reach gastrointestinal targets. Despite the early onset of expression for some marker genes, the expression patterns of many cell type markers appear immature in prenatal life and mature considerably by the end of the first postnatal week. Together, the data support location-specific roles for BDNF and GDNF in stimulating VSN growth, and a prolonged perinatal timeline for VSN maturation in male and female mice.

Antagonistic effect of mepyramine and tubocurarine on the histamine-mediated contraction of intestinal smooth muscle cells

Introduction: Histamine is an endogenous ligand which exerts its biological actions by binding to 4 subtypes of histamine receptors (HR). Its effects on the gastrointestinal tract are complex being the dominant effect contraction of muscle cells.&#x0D; Method: The aim of this study was to verify the antagonistic effect of mepyramine (10-10M - 10-7M) and tubocurarine (10-7M - 10-4M) in the contractile action of histamine (10-11M - 10-4M). in guinea pig ileum, for this purpose it was used the program Virtual Organ Bath 16 V 2.8, which allow quantify the change in strength(g) produced by smooth muscle contraction in the presence of these substances. Concentration-response curves were obtained through GraphPad Prism® graphic program. The determination of the competitive nature of the antagonist and the calculation of the affinity constant was performed through Schild method.&#x0D; Results: Mepyramine is a competitive antagonist of histamine (10-10M to 10-7M), making it a potential therapeutic agent for the treatment of irritable bowel syndrome. On the other hand, it is not understood that tubocurarine is an antagonist of histamine receptors.&#x0D; Conclusions: The results obtained provide evidence that the contractile effect of histamine on the smooth muscle of intestinal cells isolated from guinea pig is competitively antagonized by mepyramine. In addition, by calculating the pKB parameter, it was found that this compound has greater potency than other antihistamines.

Potential binding modes of the gut bacterial metabolite, 5-hydroxyindole, to the intestinal L-type calcium channels and its impact on the microbiota in rats

ABSTRACT Intestinal microbiota and microbiota-derived metabolites play a key role in regulating the host physiology. Recently, we have identified a gut-bacterial metabolite, namely 5-hydroxyindole, as a potent stimulant of intestinal motility via its modulation of L-type voltage-gated calcium channels located on the intestinal smooth muscle cells. Dysregulation of L-type voltage-gated calcium channels is associated with various gastrointestinal motility disorders, including constipation, making L-type voltage-gated calcium channels an important target for drug development. Nonetheless, the majority of currently available drugs are associated with alteration of the gut microbiota. Using 16S rRNA sequencing this study shows that, when administered orally, 5-hydroxyindole has only marginal effects on the rat cecal microbiota. Molecular dynamics simulations propose potential-binding pockets of 5-hydroxyindole in the α1 subunit of the L-type voltage-gated calcium channels and when its stimulatory effect on the rat colonic contractility was compared to 16 different analogues, ex-vivo, 5-hydroxyindole stood as the most potent enhancer of the intestinal contractility. Overall, the present findings imply a potential role of microbiota-derived metabolites as candidate therapeutics for targeted treatment of slow intestinal motility-related disorders including constipation.

NR4A1 modulates intestinal smooth muscle cell phenotype and dampens inflammation-associated intestinal remodeling.

Stricture formation is a common complication of Crohn's disease (CD), driven by enhanced deposition of extracellular matrix (ECM) and expansion of the intestinal smooth muscle layers. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor that exhibits anti-proliferative effects in smooth muscle cells (SMCs). We hypothesized that NR4A1 regulates intestinal SMC proliferation and muscle thickening in the context of inflammation. Intestinal SMCs isolated from Nr4a1+/+ and Nr4a1-/- littermates were subjected to shotgun proteomic analysis, proliferation, and bioenergetic assays. Proliferation was assessed in the presence and absence of NR4A1 agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP). In vivo, we compared colonic smooth muscle thickening in Nr4a1+/+ and Nr4a1-/- mice using the chronic dextran sulfate sodium (DSS) model of colitis. Second, SAMP1/YitFc mice (a model of spontaneous ileitis) were treated with Csn-B and small intestinal smooth muscle thickening was assessed. SMCs isolated from Nr4a1-/- mice exhibited increased abundance of proteins related to cell proliferation, metabolism, and ECM production, whereas Nr4a1+/+ SMCs highly expressed proteins related to the regulation of the actin cytoskeleton and contractile processes. SMCs isolated from Nr4a1-/- mice exhibited increased proliferation and alterations in cellular metabolism, whereas activation of NR4A1 attenuated proliferation. In vivo, Nr4a1-/- mice exhibited increased colonic smooth muscle thickness following repeated cycles of DSS. Activating NR4A1 with Csn-B, in the context of established inflammation, reduced ileal smooth muscle thickening in SAMP1/YitFc mice. Targeting NR4A1 may provide a novel approach to regulate intestinal SMC phenotype, limiting excessive proliferation that contributes to stricture development in CD.

The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Nintedanib regulates intestinal smooth muscle hyperplasia and phenotype in vitro and in TNBS colitis in vivo

Chronic inflammation of the human intestine in Crohn’s disease (CD) causes bowel wall thickening, which typically progresses to stricturing and a recurrent need for surgery. Current therapies have limited success and CD remains idiopathic and incurable. Recent evidence shows a key role of intestinal smooth muscle cell (ISMC) hyperplasia in stricturing, which is not targeted by current anti-inflammatory therapeutics. However, progression of idiopathic pulmonary fibrosis, resembling CD in pathophysiology, is controlled by the tyrosine kinase inhibitors nintedanib (NIN) or pirfenidone, and we investigated these drugs for their effect on ISMC. In a culture model of rat ISMC, NIN inhibited serum- and PDGF-BB-stimulated growth and cell migration, and promoted the differentiated phenotype, while increasing secreted collagen. NIN did not affect signaling through PDGF-Rβ or NFκB but did inhibit cytokine-induced expression of the pro-inflammatory cytokines IL-1β and TNFα, supporting a transcriptional level of control. In TNBS-induced colitis in mice, which resembles CD, NIN decreased ISMC hyperplasia as well as expression of TNFα and IL-1β, without effect in control animals. NIN also inhibited growth of human ISMC in response to human serum or PDGF-BB, which further establishes a broad range of actions of NIN that support further trial in human IBD.

Construction and identification of an immortalized rat intestinal smooth muscle cell line.

Although intestinal smooth muscle cells (ISMCs) play an important role in the remodeling of the intestinal structure, considerably less is known about the molecular mechanisms that mediate the development and growth of ISMCs. A possible reason for this lag is the lack of cell lines that recapitulate ISMCs in vivo. In this study, we separated the primary ISMCs from the rat intestinal tract and integrated the Simian Vacuolating virus 40 Large T antigen (SV40 LT) gene into the genome of the primary ISMC to construct an immortalized cell line named ISMC-Hc. ISMC-Hc proliferated persistently without any signs of senescence up to 50 passages and without neoplasticity. Analysis of the genome isolated from ISMC-Hc confirmed that the SV40LT gene recombined in the genome, and mRNA reverse transcription PCR suggested that SV40LT could be expressed normally. In addition, ISMC-Hc had few morphological differences compared with the primary ISMC. Furthermore, ISMC-Hc showed the expression of the specific protein markers (alpha-smooth muscle actin and desmin) through immunofluorescence analysis. Further studies showed that ISMC-Hc had enhanced contractility and expressed the glial-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor after co-stimulation with IL-1 beta and TNF-alpha. ISMC-Hc showed characteristics similar to that of primary ISMC and can be used as an in vitro model to explore the cellular and molecular mechanisms of ISMC. Additionally, the immortalized ISMCs could help investigate the basic functional mechanisms of intestinal diseases.

A20 INTESTINAL SMOOTH MUSCLE AND FIBROSTENOSIS: TARGETING NR4A1 TO MODULATE PROLIFERATIVE SIGNALLING

BackgroundFibrostenotic Crohn’s Disease (CD), presenting with intestinal fibrosis and stricture formation, has a substantial impact on patient quality of life. Given our poor understanding of its etiology, we lack viable preventative and therapeutic methods. While much focus has been on the fibrotic component, recent studies have implicated the role of intestinal smooth muscle cell (SMC) hyperplasia/hypertrophy in stricture formation. These data suggest targeting SMC proliferation may provide benefit for fibrostenotic CD patients. NR4A1 (nuclear receptor subfamily 4 group A member 1) is an orphan nuclear receptor that has shown to regulate inflammation in experimental models of colitis and dampen SMC proliferation and fibrotic signalling in intestinal and non-intestinal systems. Thus, we sought to characterize the role of NR4A1 in regulating proliferative signalling in intestinal SMCs to determine whether it could be a therapeutic target for fibrostenotic CD.AimsTo determine how NR4A1 regulates intestinal SMC proliferative responses to mitogenic signalling.MethodsPrimary intestinal SMCs were isolated from the colonic tissue of Nr4a1+/+ and Nr4a1-/- mice. A commercially available human colonic SMC line was also used. EdU incorporation assays were used to quantify the relative proliferation of Nr4a1+/+ and Nr4a1-/- SMCs in their basal or stimulated state (with platelet-derived growth factor (PDGF)-BB). In addition, NR4A1 was activated using selective agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP). Differences in PDGF-BB-induced intracellular signalling was determined using western blotting of phosphorylated proteins after stimulation. Quantification of PDGF receptor transcript expression in Nr4a1+/+ and Nr4a1-/- SMCs was done using qPCR. Finally, immunofluorescence was used to determine the localization of NR4A1 when stimulated with Csn-B, 6-MP, and/or PDGF-BB.ResultsThe proliferation assays showed that Nr4a1-/- SMCs exhibit greater proliferation at baseline and when stimulated with PDGF-BB, compared to Nr4a1+/+ SMCs. However, this was not associated with any differences in the intracellular signalling directly downstream of PDGF receptor activation. Specifically, there were no differences in the intensity and temporal characteristics of Akt- and Erk1/2-phosphorylation between Nr4a1+/+ and Nr4a1-/- SMCs. Interestingly, Nr4a1-/- SMCs had less expression of Pdgfrb, the gene encoding PDGF receptor beta, when compared to Nr4a1+/+ SMCs. However, no changes in receptor expression were observed when SMCs were stimulated with Csn-B and 6-MP.ConclusionsWe show that NR4A1 regulates basal and PDGF-BB-induced SMC proliferation, without directly altering the intracellular signalling cascades induced by receptor activation. Our data supports NR4A1 as a target to control the aberrant SMC proliferation that contributes to fibrostenosis.Funding AgenciesCIHR

Magnolol and honokiol target TRPC4 to regulate extracellular calcium influx and relax intestinal smooth muscle

Ethnopharmacological relevanceMagnolia officinalis Cortex (M. officinalis) is a classical traditional Chinese medicine (TCM) widely used to treat digestive system diseases. It effectively regulates gastrointestinal motility to improve abdominal pain, abdominal distension and other symptoms. Magnolol (MAG) and honokiol (HON) are the main pharmacodynamic components responsible for the gastrointestinal activity of M. officinalis. Aim of the studyThe transient receptor potential (TRP) family is highly expressed in the gastrointestinal tract and participates in the regulation of gastrointestinal motility, visceral hypersensitivity, visceral secretion and other physiological activities. In this study, the calcium-lowering mechanisms of MAG and HON contributing to the smooth muscle relaxation associated with TRP are discussed. Materials and methodsThe relaxation smooth muscle effects of MAG and HON were tested by the isolated intestine tone tests. A synthetic MAG probe (MAG-P) was used to target fishing for their possible target. The distribution of MAG on the smooth muscle was identified by a molecular tracer based on chemical biology. Ca2+ imaging and dual-luciferase reporter assays were used to determine the effects on the target proteins. Finally, the calcium-mediating effects of MAG and HON on smooth muscle cells and TRPC4-knockdown cells were compared to verify the potential mechanism. ResultsAfter confirming the smooth muscle relaxation in the small intestine induced by MAG and HON, the relaxation effect was identified mainly due to the downregulation of intracellular calcium by controlling external calcium influx. Although MAG and HON inhibited both TRPV4 and TRPC4 channels to reduce calcium levels, the inhibitory effect on TRPC4 channels is an important mechanism of their smooth muscle relaxation effect, since TRPC4 is widely expressed in the small intestinal smooth muscle cells. ConclusionsThe inhibition of MAG and HON on TRPC4 channels contributes to the relaxation of intestinal smooth muscle.

The Involvement of Glial Cell-Derived Neurotrophic Factor in Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are chronic inflammatory gastrointestinal diseases characterized by dysregulation of the intestinal epithelial barrier (IEB) and intermittent relapses. Recent data show that the glial cell line-derived neurotrophic factor (GDNF) promotes IEB function and wound healing. Apart from protective effects of GDNF on enteric nervous system and IEB, an immunomodulatory role has been assumed. However, it is inconsistent whether GDNF levels are increased or decreased in the inflamed colon of patients with IBD. Furthermore, GDNF is 1 of 3 protein markers associated with relapse in a prospective cohort study in IBD patients with clinically and endoscopically quiescent disease. Additionally, not only enteric glial cells (EGCs), but also intestinal smooth muscle cells and enterocytes synthesize GDNF in significant amounts; in addition, its receptors are expressed in intestinal neurons, EGCs, immune cells and epithelial cells, which points to a potential auto- or paracrine signaling loop between some of these cells. Whether GDNF is involved in IBD-associated fibrosis and colitis-associated colorectal cancer remains to be confirmed. In this review we aim to summarize and discuss the current knowledge on the effects of GDNF and its potential role in the contribution to the pathogenesis of IBD.

Endothelial cell-derived SSAO can increase MLC20 phosphorylation in VSMCs.

Vascular hyporesponsiveness in the shock decompensation period is an important factor leading to death. Myosin light chain 20 (MLC20) is the main effector protein that regulates vascular reactivity. However, whether the change in semicarbazide-sensitive amine oxidase (SSAO) expression during hypoxia can change the MLC20 phosphorylation level, and its underlying mechanism were not clear. The amine oxidase copper containing 3 (AOC3) overexpressing adenovirus vector was constructed and transfected into rat intestinal microvascular endothelial cells (RIMECs) to overexpress SSAO, and the RIMECs were co-cultured with rat intestinal microvascular smooth muscle cells (RIMSCs). The changes in SSAO/inducible nitric oxide synthase (iNOS)/Rho associate coiled-coil containing protein kinase 1 (ROCK1) expression levels and MLC20 phosphorylation level were detected. Here we found that the increased SSAO by AOC3 overexpression can decrease the iNOS expression level and its activity after hypoxia. In addition, RIMSCs co-cultured with RIMECs overexpressed with AOC3 gene had significantly higher ROCK1 protein level and MLC20 phosphorylation level than RIMSCs co-cultured with normal RIMECs. Our study demonstrated that SSAO overexpression can significantly inhibit iNOS activity, promote RhoA/ROCK pathway activation, and increase the phosphorylation level of MLC20, which might be the potential mechanism in relieving the vascular hyporesponsiveness during shock decompensation.