Colonic mucosal biopsies obtained during confocal endomicroscopy are pre-stained with fluorescein in vivo and are suitable for histologic evaluation.

BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) with intravenous infusion of fluorescein allows noninvasive, real-time in vivo visualization of gastrointestinal mucosa at ~ × 1000 magnification ("virtual biopsy"). Conventional biopsies obtained during these procedures serve as the reference and established diagnostic standard. The aim of the present study was to assess whether the standard histologic biopsies that are obtained during CLE retain fluorescein in the tissues and allow the visualization of mucosal structures without any additional staining. PATIENTS AND METHODS: CLE optical imaging of the mucosa was performed in 16 patients who were undergoing CLE colonoscopy. Standard conventional biopsies were also obtained from both normal colonic mucosa and colonic polyps. De-paraffinized mucosal sections were examined under a fluorescence microscope for the presence and distribution of fluorescein, and then underwent immunostaining for expression of vascular endothelial growth factor (VEGF). RESULTS: Standard mucosal biopsy sections from patients undergoing CLE displayed a strong fluorescence and showed well-delineated mucosal structures. In colonic adenomas, there was a 4.6-fold increased vascular permeability compared with normal mucosa (P<0.001), indicated by fluorescein leakage to the extravascular space. Immunostaining demonstrated an aberrantly increased expression of VEGF in the epithelium of colonic adenomas but not in the epithelium of normal mucosa or hyperplastic polyps. CONCLUSIONS: This study shows for the first time that standard colonic biopsies obtained during CLE retain fluorescein, show excellent delineation of mucosal structures without additional staining, allow the evaluation of mucosal microvasculature and vascular permeability, and are suitable for immunostaining.

Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing.

Angiogenesis, the formation of new capillary blood vessels, is essential not only for the growth and metastasis of solid tumors, but also for wound and ulcer healing, because without the restoration of blood flow, oxygen and nutrients cannot be delivered to the healing site. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin and ibuprofen are the most widely used drugs for pain, arthritis, cardiovascular diseases and, more recently, the prevention of colon cancer and Alzheimer disease. However, NSAIDs produce gastroduodenal ulcers in about 25% of users (often with bleeding and/or perforations) and delay ulcer healing, presumably by blocking prostaglandin synthesis from cyclooxygenase (COX)-1 and COX-2 (ref. 10). The hypothesis that the gastrointestinal side effects of NSAIDs result from inhibition of COX-1, but not COX-2 (ref. 11), prompted the development of NSAIDs that selectively inhibit only COX-2 (such as celecoxib and rofecoxib). Our study demonstrates that both selective and nonselective NSAIDs inhibit angiogenesis through direct effects on endothelial cells. We also show that this action involves inhibition of mitogen-activated protein (MAP) kinase (ERK2) activity, interference with ERK nuclear translocation, is independent of protein kinase C and has prostaglandin-dependent and prostaglandin-independent components. Finally, we show that both COX-1 and COX-2 are important for the regulation of angiogenesis. These findings challenge the premise that selective COX-2 inhibitors will not affect the gastrointestinal tract and ulcer/wound healing.

Mesalazine downregulates c-Myc in human colon cancer cells. A key to its chemopreventive action?

BACKGROUND: Dysplasia and malignant transformation of colonocytes in ulcerative colitis are associated with overexpression of c-Myc and genes regulating cell survival. 5-Aminosalicylates such as mesalazine may reduce the development of colorectal cancer in ulcerative colitis, but the mechanisms of its chemopreventive action are not clear. AIMS: To examine whether mesalazine affects the expression of c-Myc in human colon cancer cell lines. METHODS: Human colon cancer cells were treated with vehicle or mesalazine (4 mm or 40 mm). We examined: (i) mRNA expression by gene array, (ii) protein expression by Western blotting and immunohistochemistry and (iii) apoptosis by Annexin V labelling. RESULTS: Mesalazine significantly reduced expression of c-Myc mRNA and protein. CONCLUSIONS: Mesalazine downregulates gene and protein expression of c-Myc. The apoptotic and growth inhibitory effects of mesalazine are dose-dependent. Expression of c-Myc is significantly reduced by mesalazine 40 mm.

In vitro model of vasculo-angiogenesis: demonstration that bone marrow derived endothelial progenitor cells form new hybrid capillary blood vessels jointly with gastric endothelial cells.

Regeneration of blood vessels (neovascularization) is critical for tissue injury healing. The contribution of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during tissue injury healing is not fully elucidated and it is not clear whether BMD-EPCs can form new capillary blood vessels independently or jointly with fully differentiated endothelial cells (ECs). The aim of this study was to establish an in vitro model of vasculogenesis/angiogenesis by co-culture of BMD-EPCs and gastric endothelial cells (GECs) on Matrigel, examine direct interactions of these cells; and, identify the mechanisms involved. We isolated BMD-EPCs and GECs from bone marrow and stomach of rats, respectively. In these cells, we examined the expression of CD34, CD133, CD31, VEGF-R2, stromal derived factor 1 (SDF-1) and CXCR4, and, their ability to form capillary-like tubes when cultured separately or when co-cultured (1:5 ratio) on growth factor-reduced Matrigel. Fluorescence-labeled BMD-EPCs seeded alone on Matrigel formed capillary-like tubes reflecting in vitro vasculogenesis, and when co-cultured with GECs on Matrigel, formed 'hybrid' tubes containing BMD-EPCs nested between GECs thus reflecting in vitro angio-vasculogenesis. These 'hybrid' tubes were 1.5-fold wider (P < 0.001) and had more extensive (5.1-fold increase) loops (P < 0.01) at the junctions of BMD-EPCs and GECs versus tubes formed by GECs alone. GECs expressed SDF-1 that likely mediated homing of BMD-EPCs (which expressed the SDF-1 receptor, CXCR4) and their incorporation during neovascularization. BMD-EPCs can independently form capillary-like tubes on Matrigel, and when co-cultured with fully differentiated ECs on Matrigel, form capillary-like 'hybrid' tubes comprised of both cell types. Both BMD-EPCs and GECs express SDF-1 and CXCR4, which indicate direct paracrine interactions between these cells during neovascularization.

Formation of new blood vessels during gastric ulcer healing. Role of bone marrow derived endothelial progenitor cells.

Regeneration of blood vessels (neovascularization) is critical for gastric ulcer (GU) healing. The contributions of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during GU healing are not fully elucidated. Our specific aims were to determine whether in GU, BMD-EPCs are incorporated into blood vessels of GU granulation tissue jointly with ECs, thus forming hybrid vessels; or, form separate vessels consisting of only BMD-EPCs. GUs were induced in rats by serosal application of acetic acid. Vascular cast studies were performed at 7, 21 and 60 days after GU induction and tissue specimens were immunostained for CD34, CD133, VEGFR2, and SDF-1 at 14 days. Human relevance was determined using archival human GU specimens. In rat GU granulation tissue BMD-EPCs constituted 28 ± 3% of all cells lining newly formed blood vessels, and were nested between fully differentiated ECs. In rat GU granulation tissue, expression of stromal derived factor-1 (SDF-1) - the major chemoattractant for BMD-EPCs was strongly upregulated. In human GU specimens, BMD-EPCs were also present in granulation tissue constituting 34 ± 3% of all cells lining blood vessels and jointly formed hybrid vessels with differentiated ECs. Our study uncovered that BMD-EPCs incorporate into newly formed blood vessels in GU granulation tissue; and, together with ECs of pre-existing vessels, contribute to and support neovascularization through vasculogenesis. This study is the first demonstration that vasculogenesis occurs during GU healing in both humans and in rats.

Expression of nerve growth factor in rat stomach. Implications for interactions between endothelial, neural and epithelial cells.

This study was aimed to determine the expression and localization of nerve growth factor (NGF) in the gastric mucosa. Transmural gastric specimens were obtained from euthanized rats. STUDIES: 1) expression of NGF and TrkA receptor by Western blotting; 2) histological evaluation of gastric wall architecture; 3) expression of NGF using immunostaining. Immunostaining showed strong and differential expression of NGF in neural elements of gastric myenteric and submucosal plexuses; in epithelial cells: mainly in chief and progenitor cells, in enterochromaffin-like (ECL) cells; and, in endothelial cells (ECs) lining blood vessels. We concluded that NGF expression in neural elements, epithelial cells and endothelial cells of blood vessels indicated a complex local interaction between neural, epithelial and endothelial cells that regulated gastric mucosal homeostasis and, likely, the protection against gastric injury and ulcer healing.

TGF-beta signaling pathway: its role in gastrointestinal pathophysiology and modulation of ulcer healing.

Gastrointestinal ulcer healing is a complex process, involving cell migration, proliferation, angiogenesis and extracellular matrix deposition, all ultimately leading to reconstruction of tissue architecture within the ulcer scar. These processes are controlled by growth factors, cytokines and hormones. Transforming growth factor-beta (TGF-beta), one of the multifunctional peptide growth factors, has been reported to positively regulate gastrointestinal ulcer healing. Although TGF-beta inhibits cell proliferation in a variety of cells, it induces cell migration, angiogenesis, and enhances extracellular matrix production necessary for gastrointestinal ulcer healing. TGF-beta exerts its action by binding to its transmembrane serine/threonine kinase receptors, which in turn triggers activation of various intracellular signaling pathways. Smads are intermediate effector proteins that play key roles in biological activities of TGF-beta by transmitting the signals from the cell surface directly into the nucleus and initiating transcription. New insight into the mechanisms underlying TGF-beta-Smad modulation of gastrointestinal ulcer healing will likely enhance our understanding of the mechanisms controlling the healing processes of gastrointestinal ulcers.

Nerve growth factor injected into the gastric ulcer base incorporates into endothelial, neuronal, glial and epithelial cells: implications for angiogenesis, mucosal regeneration and ulcer healing.

A previous study has demonstrated that locally administered growth factors such as epidermal growth factor, basic fibroblast growth factor and hepatocyte growth factor can accelerate healing of experimental gastric ulcers in rats. That study indicates that locally administered growth factors can exert potent biological effects resulting in enhanced gastric ulcers healing. However, the fate of injected growth factors, their retention and localization to specific cellular compartments have not been examined. In our preliminary study, we demonstrated that local injection of nerve growth factor to the base of experimental gastric ulcers dramatically accelerates ulcer healing, increases angiogenesis - new blood vessel formation, and improves the quality of vascular and epithelial regeneration. Before embarking on larger, definitive and time sequence studies, we wished to determine whether locally injected nerve growth factor is retained in gastric ulcer's tissues and taken up by specific cells during gastric ulcer healing. Gastric ulcers were induced in anesthetized rats by local application of acetic acid using standard methods; and, 60 min later fluorescein isothiocyanate-labeled nerve growth factor was injected locally to the ulcer base. Rats were euthanized 2, 5 and 10 days later. Gastric specimens were obtained and processed for histology. Unstained paraffin sections were examined under a fluorescence microscope, and the incorporation of fluorescein isothiocyanate-labeled nerve growth factor into various gastric tissue cells was determined and quantified. In addition, we performed immunostaining for S100ß protein that is expressed in neural components. Five and ten days after ulcer induction labeled nerve growth factor (injected to the gastric ulcer base) was incorporated into endothelial cells of blood vessels, neuronal, glial and epithelial cells, myofibroblasts and muscle cells. This study demonstrates for the first time that during gastric ulcer healing locally administered exogenous nerve growth factor is retained in gastric tissue and is taken up by endothelial, neural, muscle and epithelial cells. This is likely the basis for the therapeutic action of locally administered nerve growth factor and its stimulation of angiogenesis, tissue regeneration and gastric ulcer healing.

Expression of nerve growth factor, its TrkA receptor, and several neuropeptides in porcine esophagus. Implications for interactions between neural, vascular and epithelial components of the esophagus.

UNLABELLED: This study was aimed to determine the expression and localization of nerve growth factor (NGF) and several neural peptides in porcine esophagus. Transmural esophageal specimens were obtained from euthanized pigs. STUDIES: 1) histologic evaluation, 2) expressions of NGF and its tropomyosin receptor kinase A (TrkA) receptor, calcitonin generelated peptide (CGRP), neuronal nitric oxide synthase (nNOS), and neuronal enolase using immunostaining and quantification of signal distribution and intensity. Immunostaining for NGF, CGRP, nNOS and neuronal specific enolase (NSE) showed their strong and differential expression and localization in the neuronal network. NGF was strongly expressed in the majority of neurons and nerves, distribution of TrkA was complementary; its signal was 1.5-fold weaker P < 0.001 than NGF). Quantitatively the signal intensity was: CGRP > nNOS > NGF > NES > TrkA. In addition to neural structures, nNOS, NGF and TrkA were expressed in keratinocyte progenitor cells of esophageal mucosa and in endothelial cells of blood vessels. We conclude that a strong expression of NGF in majority of esophageal neurons and nerves indicates important, but previously unrecognized regulatory roles in the esophagus; 2) This study showed expression of NGF and some of the neuropeptides in neural elements, keratinocyte progenitor cells and endothelial cells of blood vessels, which indicates local interactions between neural, epithelial and endothelial cells.

Gastrointestinal mucosal regeneration: role of growth factors.

Growth factors and their receptors play important roles in cell proliferation, migration, tissue injury repair and ulcer healing. In gastric mucosa, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) by activating their common receptor, control cell proliferation. TGF-alpha predominantly plays this role under normal conditions and after acute injury, while EGF exerts its actions mainly during healing of chronic ulcers. During regeneration of injured gastric mucosa, these growth factors serve predominantly to restore the epithelial component. Other growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) serve to promote restoration of the connective tissue and microvessels (angiogenesis) in injured mucosa. During healing of chronic ulcers, a new epithelial lineage secreting EGF and other growth peptides develops and the majority of cells lining the ulcer margin overexpress the EGF receptor. Activation of the EGF receptor induces dramatic increases in MAP (Erk -1 and -2) kinase activity and phosphorylation levels. Inhibition of this signaling pathway dramatically delays ulcer healing. Granulation connective tissue, which grows under the stimulation of bFGF and VEGF is the major source for regeneration of connective tissue lamina propria and microvessels within the ulcer scar. Other growth factors such as insulin - like growth factor, keratinocyte growth factor, hepatocyte growth factor and trefoil peptides have been implicated in gastrointestinal (gastric ulcers, colitis) regeneration following injury. This paper is intended to provide an overview of the role of growth factors in gastrointestinal mucosal regeneration.

Reduced expression of basic fibroblast growth factor and its receptor mRNAs and proteins in portal hypertensive esophageal mucosa: a mechanism responsible for muscularis mucosae thinning and variceal rupture.

BACKGROUND: Basic fibroblast growth factor (bFGF) enhances cell migration, proliferation, and tissue integrity. This especially pertinent to the smooth muscle cells in which it stimulates cell proliferation and promotes their growth. The aim of this study was to determine whether expression of bFGF and its receptors (FGFR-1 and -2) is altered in portal hypertensive esophageal mucosa, especially in the muscularis mucosal layer, which constitutes a physical barrier to variceal rupture. METHODS: Portal hypertension (PHT) was produced by staged portal vein ligation. In 30 PHT rats and 30 sham-operated controls 2 weeks after operation, specimens of lower esophagus were obtained for (1) quantitative histologic assessment including thickness of epithelium and muscularis mucosae; (2) immunostaining with specific antibodies against bFGF and its receptors 1 and 2 (intensity of bFGF, FGFR-1 and FGFR-2 immunostaining in esophageal structures was measured with a video image system); and (3) expression of bFGF and FGFR-1 and -2 mRNAs was assessed with reverse transcription-polymerase chain reaction. RESULTS: The esophageal muscularis mucosae and epithelium overlaying large submucosal veins in PHT rats significatly thinner than those in controls (muscularis mucosae, 28.3 +/- 1.4 versus 52.2 +/- 8.0 micrometer, respectively, p<0.05); epithelium, 39.0+/- 7.1 versus 49.3 +/- 1.9 micrometer, respectively, p<0.05). The immunostaining intensity of bFGF and FGFR-2 was significantly reduced in PHT rats (42.1 +/- 2.3 and 71.3 +/- 6.5 units, respectively) versus controls (49.5 +/- 5.6 and 78.6 +/- 5.7 units, respectively, p< 0.05). Expressions of bFGF and FGFR-2 mRNAs in PHT esophageal mucosa were significantly reduced versus controls by 30.8% and 30.3%, respectively (p < 0.01, p 0.05). CONCLUSIONS: (1) Esophageal mucosa of PHT rats has thinner muscularis mucosae and reduced bFGF and FGFR-2 mRNAs and proteins. (2) Because bFGF stimulates smooth muscle cell proliferation and their growth, our findings can explain thinning of esophageal muscularis mucosae in PHT rats, thus indicating a possible mechanism for rupture of varices in the esophagus.

Overexpression of endothelin-1 mRNA and protein in portal hypertensive gastric mucosa of rats: a key to increased susceptibility to damage?

BACKGROUND: Portal hypertension predisposes gastric mucosa to increased injury by various noxious factors. Because endothelin-1 (ET-1) is a potent vasoconstrictor that enhances gastric mucosal injury, we examined ET-1 expression in the portal hypertensive (PHT) gastric mucosa and its possible role in increased mucosal susceptibility to damage. METHODS: In gastric specimens of PHT or sham-operated rats, ET-1 mRNA expression was studied by S1-nuclease protection assay and ET-1 protein by enzyme immunoassay and immunostaining. We also determined the extent of ethanol-induced gastric mucosal necrosis in PHT and sham-operated rats after administering either a placebo or FR 139317, a selective ETA receptor antagonist. RESULTS: In PHT stomachs ET-1 mRNA expression and protein concentration were significantly increased compared with sham-operated controls: mRNA expression (ET-1/glyceraldehyde-3-phosphate-dehydrogenase ratio), 0.54 +/- 0.18 versus 0.30 +/- 0.08; protein concentration, 7.36 +/- 2.21 pg/mg versus 3.93 +/- 0.40 pg/mg, respectively; both p < 0.01. Immunofluorescence signal of ET-1 protein was predominantly localized to endothelia of gastric mucosal and submucosal vessels. In PHT stomachs FR 139317 significantly reduced mucosal necrosis (percentage of necrotic area, from 24.9 +/- 5.9% to 10.8 +/- 4.0%; p < 0.01), although it had no effect on sham-operated controls. CONCLUSIONS: Portal hypertension activates the ET-1 gene with overexpression of ET-1 protein in the gastric mucosa. Protection of PHT gastric mucosa by ETA receptor antagonist against damage indicates that overexpression of ET-1 plays an important role in increased susceptibility of PHT gastric mucosa to injury.

Antacid talcid activates in gastric mucosa genes encoding for EGF and its receptor. The molecular basis for its ulcer healing action.

In previous studies [Gut 35 (1994) 896-904], we demonstrated that antacid talcid (TAL) accelerates gastric ulcer healing and provides better quality of mucosal restoration within the scar than the omeprazole (OME). However, the mechanisms of TAL-induced ulcer healing are not clear. Since growth factors promote cell proliferation, re-epithelization, angiogenesis and ulcer healing, we studied whether TAL and/or OME affect expression of epidermal growth factor (EGF) and its receptors (EGF-R) in both normal and ulcerated gastric mucosae. Rats with or without acetic acid-induced gastric ulcers (n = 64) received i.g. twice daily 1 mL of either: A) placebo (PLA); B) TAL 100 mg; or C) OME 50 mg x kg(-1) for 14 d. Studies of gastric specimens: 1) ulcer size; 2) quantitative histology; 3) expression of EGF mRNAs was determined by RT/PCR; 4) gastric sections were immunostained with antibodies against EGF and its receptors. In non-ulcerated gastric mucosa of placebo or omeprazole treated group, EGF expression was minimal, while EGF-R was localized to few cells in the mucosal proliferative zone. Gastric ulceration triggered overexpression of EGF and its receptor in epithelial cells of the ulcer margin and scar. In ulcerated gastric mucosa TAL treatment significantly enhanced (versus PLA and omeprazole) expression of EGF and EGF-R. OME treatment reduced expression of EGF in ulcerated mucosa by 55 +/- 2% (P < 0.01). It is concluded that: 1) treatment with TAL activates genes for EGF and its receptor in normal and ulcerated gastric mucosae; 2) since EGF promotes growth of epithelial cells and their proliferation and migration, the above actions of TAL provide the mechanism for its ulcer healing action and improved (versus OME) quality of mucosal restoration.

NSAIDs inhibit the activation of egr-1 gene in microvascular endothelial cells. A key to inhibition of angiogenesis?

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin (IND), ibuprofen and newer cyclooxygenase-2 selective NSAIDs (e.g. celecoxib) delay gastric ulcer healing partly through the inhibition of angiogenesis, but the molecular mechanisms involved are not fully elucidated. Effective angiogenesis is required for ulcer healing to supply oxygen and nutrients to the healing site. The early growth response factor (Egr-1) is a transcription factor, which is rapidly activated by a variety of extracellular signals or tissue injury and is important for angiogenesis to occur. This study aimed to determine whether indomethacin (IND) and/or the selective COX-2 inhibitor, NS-398, interfere with egr-1 gene expression in human microvascular endothelial cells (HMVEC) in response to vascular endothelial growth factor (VEGF) stimulation. HMVEC were treated with 0.5 mM IND or 100 microM NS-398 for 16 h, and then VEGF (10 ng/ml) or vehicle was added. Egr-1 mRNA and protein expression levels were determined by RT-PCR and Western-blotting, respectively. VEGF treatment caused a significant elevation of Egr-1 mRNA (261+/-21%, P<0.001) and protein expression (174+/-15%, P<0.01) vs. vehicle. IND pre-treatment significantly inhibited VEGF-induced Egr-1 mRNA expression by 29+/-4% (P<0.01) and protein expression by 41+/-8% (P<0.05). NS-398 inhibited VEGF-induced Egr-1 mRNA and protein expression by 23+/-3% and 35+/-4%, respectively (both P<0.01). Since transcriptional activation of egr-1 is responsible for expression of proteins involved in proliferation of endothelial cells essential for angiogenesis, these results provide a new mechanism for NSAIDs' interference with angiogenesis.

Role of protein kinase a pathway in epidermal growth factor-induced liver cell repair.

To gain a better understanding of the mechanisms that control the repair process in the injured liver, the actions of epidermal growth factor (EGF) and protein kinase A (PKA) were studied. Normal rat liver cells (clone 9) were grown to confluence. Standardized excisional wounds were made with a razor blade. The extent of hepatocyte migration into the wound was measured and determined at specific time intervals using a computerized digital analyzing system. Immunostaining of F-actin was performed with a fluorescein-labeled phalloidin. EGF significantly stimulated liver cell migration, whereas specific EGF-neutralizing antibody inhibited the EGF-induced migration. Agents that activate PKA at different stages of the PKA activation pathway, including 3-isobutyl-1-methylxanthine (IBMX), forskolin, and cholera toxin, inhibited EGF-induced migration. EGF triggered formation of actin stress fibers. PKA-activating agents inhibited actin stress fiber formation and stretching of cells at the wound margin. The following conclusions were drawn: (1) In excisional wounds of hepatocyte monolayers, both EGF and PKA exert action on actin microfilaments, which are stretched by EGF and inhibited by PKA; (2) the enhanced repair of wounded hepatocyte monolayers by EGF is blocked by activation of the PKA pathway at various levels; and (3) these actions of EGF and PKA indicate their important regulatory roles in controlling the rate of hepatocyte migration and restitution following the creation of excisional wounds.

Portal hypertension triggers local activation of inducible nitric oxide synthase gene in colonic mucosa.

Recently a new clinical entity "portal hypertensive colopathy" has been reported. It involves vascular abnormalities and bleeding. Because nitric oxide may mediate these changes, we studied whether portal hypertension affects nitric oxide synthase in portal hypertensive colonic mucosa. In portal hypertensive and sham-operated rats the following studies were done: (1) colonic mucosal blood flow, (2) quantitative histologic examination, (3) reverse transcription-polymerase chain reaction for nitric oxide synthase mRNA, (4) nitric oxide synthase activity assay, and (5) immunostaining for nitric oxide synthase. In portal hypertensive rats, colonic mucosal blood flow and the number of submucosal veins were significantly increased in comparison to sham-operated rats. The mRNA expression and enzyme activity for inducible nitric oxide synthase (but not constitutive nitric oxide synthase) were significantly increased in portal hypertensive rats. Fluorescence signal intensity for inducible nitric oxide synthase in endothelia of mucosal and submucosal veins was significantly higher in portal hypertensive rats than in sham-operated rats. Portal hypertension activates inducible nitric oxide synthase gene and protein in colonic mucosal vessels. The excess of nitric oxide generated by overexpressed inducible nitric oxide synthase may play an important role in the development of vascular and hemodynamic abnormalities characterizing portal hypertensive colopathy.

Tumor necrosis factor-alpha regulates inducible nitric oxide synthase gene expression in the portal hypertensive gastric mucosa of the rat.

Increased expression of both nitric oxide synthase (NOS) and tumor necrosis factor-alpha (TNF-alpha) have been implicated in the hyperdynamic circulation of portal hypertension. Since overexpression of these proteins would affect gastric mucosal defenses, which are impaired in portal hypertension, we examined the expression and interrelationships of TNF-alpha and NOS in the gastric mucosa of portal hypertensive rats. Following staged portal vein ligation, gastric strips from portal hypertensive rats were incubated in organ culture medium with or without TNF-alpha antibody. The expression of TNF-alpha and NOS mRNAs was assessed by reverse transcription-polymerase chain reaction (RT-PCR) at baseline and after 1, 2, and 6 hours of incubation. RT-PCR demonstrated a threefold increase in inducible NOS mRNA and a 50% increase in TNF-alpha mRNA expression at baseline in portal hypertensive animals as compared to sham-operated animals. In tissue incubated with TNF-alpha neutralizing antibody, inducible NOS mRNA expression was significantly decreased by 40%, 70%, and 80% after 1, 2, and 6 hours, respectively. Since increased TNF-alpha and NOS production could potentially impair gastric mucosal defenses, our findings suggest a major role for these proteins in the development of portal hypertensive gastropathy.

New insights into impairment of mucosal defense in portal hypertensive gastric mucosa.

Portal hypertension (PHT) increases susceptibility of the gastric mucosa to injury. The aim of this study was to investigate whether PHT affects rat gastric mucosal defense mechanisms in vivo at the pre-epithelial, epithelial, and/or post-epithelial levels. PHT was produced in rats by staged portal vein ligation and sham-operated (SO) rats served as controls. The gastric mucosa was exposed, chambered, and continuously superfused with buffers under in vivo microscopy. We measured gastric mucosal gel layer thickness, surface epithelial cell intracellular pH (pHi), mucosal blood flow, and mucosal/serosal oxygenation. In PHT rats, gastric mucosal gel layer thickness was significantly reduced (88 +/- 16 microm in PHT rats vs. 135 +/- 25 microm in SO rats; P <0.0001), and the surface epithelial cell pHi was significantly decreased (6.80 +/- 0.11 in PHT rats vs. 7.09 +/- 0.21 in SO rats; P <0.01). Although total gastric mucosal blood flow was significantly increased in PHT rats by 72% (P <0.05), the oxygenation of the gastric mucosal surface was decreased by 42% (P <0.05) compared with SO rats. PHT impairs pre-epithelial (mucosal gel layer thickness), epithelial (pHi), and post-epithelial (maldistribution of blood flow) components of the gastric mucosal barrier. These findings can explain the increased susceptibility of portal hypertensive gastric mucosa to injury.

Eicosanoids and the stomach.

Eicosanoids are arachidonic acid derivatives that include prostaglandins, thromboxanes, and leukotrienes. During the last three decades, it has become evident that these bioactive lipids play a pivotal role in gastric physiology. The goal of the present review is to describe their involvement in the normal regulation of gastric secretion and gastric motility, as well as in gastric mucosal defense. Their role in gastric mucosal mitogenesis, apoptosis, inflammation, and immune modulatory responses is also discussed.