Role of sulfhydryls and early vascular lesions in gastric mucosal injury

This paper reviews the recently discovered role of sulfhydryls and early vascular injury in the pathogenesis of acute gastric mucosal injury. In the rat ethanol caused a dose-dependent decrease in nonprotein sulfhydryl concentration in the gastric mucosa within 1-5 min following an intragastric dose. These biochemical changes were accompanied by increased vascular permeability in the glandular stomach as revealed by the measurement of extravasated Evans blue injected i.v. prior to the administration of ethanol. Morphologic evidence of vascular injury was provided by labelling of damaged blood vessels in the stomach following the i.v. administration of colloidal particles in the form of india ink or monastral blue. The functional and structural damage to capillaries and venules in the glandular stomach was also maximal within 1-6 min after 1 ml of 75 or 100% ethanol given orally. Pretreatment with sulfhydryl (SH) containing drugs (e.g., L-cysteine, N-acetyl-L-cysteine, cysteamine, dimercaprol) or prostaglandin (PG) F2 beta prevented the ethanol-induced increase in vascular permeability, the labelling of blood vessels with vascular tracers, and the subsequent haemorrhagic erosions. The desquamation of superficial epithelial cells, however, was not markedly modified by either SH or PG compounds. This organoprotective effect of SH and PG drugs was virtually counteracted in adrenalectomized rats that exhibited "vascular fragility". Glucocorticoid treatment restored the response of adrenalectomized animals. Thus, a SH- and glucocorticoid-sensitive early vascular injury seems to be of major significance in the pathogenesis of haemorrhagic gastric erosions and SH-containing compounds represent a new group of cytoprotective or organoprotective agents.     

肠上皮快速复原过程中的细胞信号传递: 多胺和K+通道的影响

胃肠道粘膜上皮细胞具有重要的屏障作用,可以保护次上皮组织抵御一系列的有害物质,包括过敏原、病毒以及微生物病原体。粘膜损伤后的修复有赖于上皮细胞对信号网络的调节,而这一网络系统控制着基因的表达、细胞的存活、迁移及增殖。近几年的研究结果显示,在胃肠道粘膜的修复中,多胺起到关键作用;且细胞多胺的调控是众多信号传递路径的焦点。本文简要综述了多胺在肠粘膜上皮快速复原中的功能和机制,特别是对K^ 通道活性的影响。     

Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.     

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) results from Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through Ca(2+)-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca(2+) entry (CCE) induced by Ca(2+) store depletion represents a major Ca(2+) influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca(2+)-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca(2+) currents and CCE generated by Ca(2+) influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca(2+) channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca(2+)](cyt).     

Trefoil peptides promote restitution of wounded corneal epithelial cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.     

Factors influencing the restitution of the duodenal and colonic mucosa after damage

Rapid epithelial restitution is an important protective mechanism which enables the gastrointestinal mucosa to reestablish epithelial integrity following superficial injury within hours. In this study we examined the influence of an acidic luminal pH, removal of the necrotic layer, nutrient bicarbonate, calcium and sodium desoxycholate (Na-DOC) on restitution in the rabbit duodenum in vitro and the role of Na-DOC and calcium for rapid restitution of the human colon in vitro. Transmucosal potential difference (PD), short-circuit current (lsc) were measured and resistance against passive ion flux (R) was calculated. Electrophysiological changes paralleled morphological injury but did not necessarily reflect restitution in all experiments. The extent of mucosal injury was assessed by computerized real-time morphometry. 5 hrs after luminal exposure to 10 mH HCl for 10 min residual damage (RD) was 14% in the duodenum. Luminal pH of 3.0 (RD of 30%), removal of necrotic layer at acidic luminal pH (RD of 66%), absence of bicarbonate from the serosal solution (RD of 35% at neutral luminal pH; RD of 96% at acidic luminal pH) and removal of calcium from the serosal solution (RD of 58%) impaired restitution in the duodenum. Continuous postinjury luminal Na-DOC exposure did not influence restitution in the duodenum (RD of 19%). 5 hrs after luminal exposure to 0.5 mM Na-DOC for 10 min RD was 26% in the human colon. Continuous postinjury luminal Na-DOC exposure (RD of 51%) and removal of calcium from the nutrient solution (RD of 65%) impaired restitution in the human colon. Thus we conclude that restitution of the rabbit duodenum in vitro requires a necrotic layer and bicarbonate flux to withstand acidic luminal pH, while restitution is not affected by Na-DOC. In the human colon Na-DOC inhibits restitution. Both the duodenum and colon require calcium for rapid restitution.     

Resident mesenchymal cells and fibrosis

Fibrosis is a major clinical problem associated with as many as 45% of all natural deaths in developed nations.It can affect all organs and accumulating evidence indicates that fibrogenesis is not merely a bystander product of injury, but is a central pathological problem directly contributing to loss of organ function. In the majority of clinical cases, fibrogenesis is strongly associated with the recruitment of leukocytes, even in the absence of infection. Although chronic infections are a significant cause of fibrogenesis, in most cases fibrotic disease occurs in the context of sterile injury, such as microvascular disease, toxic epithelial injury or diabetes mellitus. Fibrogenesis is a direct consequence of the activation of extensive, and previously poorly appreciated, populations of mesenchymal cells in our organs which are either wrapped around capillaries and known as ‘pericytes’, or embedded in interstitial spaces between cell structures and known as resident ‘fibroblasts’. Recent fate-mapping and complementary studies in several organs indicate that these cells are the precursors of the scar-forming myofibroblasts that appear in our organs in response to injury. Here we will review the literature supporting a central role for these cells in fibrogenesis, and highlight some of the critical cell to cell interactions that are necessary for the initiation and continuation of the fibrogenic process. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Studies on the microvascularization of the digestive tract by scanning electron microscopy of vascular corrosion casts. 2. Bile duct in guinea pigs.

The microvascularization of the bile duct was studied in 20 adult guinea pigs (Cavia porcellus) using microvascular corrosion casts in the scanning electron microscope. The main supplying and draining vessels are located in the adventitial layer; from there they form the subepithelial capillary network. The microangioarchitecture of the common bile duct is reminiscent of that of the cystic and hepatic ducts. The diameter of these ducts is 1,000 microns on average. The microvascular arrangement in the bile duct suggests a probable involvement of the capillaries in the absorption of bile hormones.     

PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair, because function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. NSAIDs result in increased intestinal permeability and disease exacerbation in patients with inflammatory bowel disease (IBD). Thus we examined the reparative attributes of endogenous PGs after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial electrical resistance (TER) from 20-40 Omega.cm2 was abolished by indomethacin (Indo), whereas restitution of 40-100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with Indo, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of Indo by osmotic-driven collapse of the paracellular space, and PG-mediated recovery was significantly impaired by blockade of Cl- secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance vs. restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.     

Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers

Repair of superficial gastric mucosal injury is accomplished by the process of restitution-migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.     

Structural and functional aspects of porcine endometrial capillaries on days 13 and 15 after oestrus or mating.

The permeability of subepithelial capillaries in porcine endometrium was studied during midcycle and early pregnancy. Gilts were slaughtered on Day 13 or Day 15 of the oestrous cycle or pregnancy, 15 min after injection through the ear vein of 11.1 MBq of 125I-labelled human albumin in phosphate-buffered saline. The radioactivity of endometrial strips taken along the mesometrial and antimesometrial aspects of the uterine horn varied on average from 270 to 701 c.p.m./g and no difference (P greater than 0.05) was found between reproductive status, days of slaughter or sampling sites. The majority of the subepithelial capillaries showed ultrastructural evidence of increased vascular permeability, such as marked thinning of the capillary walls, especially on the side proximal to the epithelial basal lamina, multilayering and partial disparition of the endothelial basal lamina and abundant endothelial vesicles. Fenestrated pores were observed, but were rare. There was no obvious difference between reproductive status, days of sampling or sampling sites inside the uterus, suggesting that on Days 13-15 after oestrus the ultrastructural characteristics of porcine endometrial capillaries are little affected by the presence of attaching blastocysts and supporting the results obtained with radioactive albumin. Ferritin injected directly into a uterine artery of one gilt on Day 15 of pregnancy was carried through the capillary wall by endothelial vesicles, showing ultrastructural evidence of increased permeability.     

Na+/H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na⁺/H⁺ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ～10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pH_i and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.     

Trefoil factor 2 requires Na/H exchanger 2 activity to enhance mouse gastric epithelial repair

Trefoil factor (TFF) peptides are pivotal for gastric restitution after surface epithelial damage, but TFF cellular targets that promote cell migration are poorly understood. Conversely, Na/H exchangers (NHE) are often implicated in cellular migration but have a controversial role in gastric restitution. Using intravital microscopy to create microscopic lesions in the mouse gastric surface epithelium and directly measure epithelial restitution, we evaluated whether TFFs and NHE isoforms share a common pathway to promote epithelial repair. Blocking Na/H exchange (luminal 10 μm 5-(N-ethyl-N-isopropyl) amiloride or 25 μm HOE694) slows restitution 72-83% in wild-type or NHE1(-/-) mice. In contrast, HOE694 has no effect on the intrinsically defective gastric restitution in NHE2(-/-) mice or TFF2(-/-) mice. In TFF2(-/-) mice, NHE2 protein is reduced 23%, NHE2 remains localized to apical membranes of surface epithelium, and NHE1 protein amount or localization is unchanged. The action of topical rat TFF3 to accelerate restitution in TFF2(-/-) mice was inhibited by AMD3100 (CXCR4 receptor antagonist). Furthermore, rat TFF3 did not rescue restitution when NHE2 was inhibited [TFF2(-/-) mice +HOE694, or NHE2(-/-) mice]. HOE694 had no effect on pH at the juxtamucosal surface before or after damage. We conclude that functional NHE2, but not NHE1, is essential for mouse gastric epithelial restitution and that TFFs activate epithelial repair via NHE2.     

Profiles of eicosanoid production by superficial and proliferative colonic epithelial cells and sub-epithelial colonic tissue

The profile of cyclooxygenase and lipoxygenase products in normal rat colonic epithelium and subepithelium was examined. Colons were thoroughly perfused to eliminate contamination with blood. Two preparations of colonic epithelium were employed. The first consisted of intact colonic crypts and epithelial sheets. The second yielded single cell suspensions of superficial versus proliferative epithelial cells. Lipoxygenase product formation by colonic epithelium as measured by hydroxyeicosatetraenoic acid (HETE) and leukotriene B4 (LTB4) production (5-HETE greater than 12-HETE greater than 15-HETE greater than LTB4) accounted for 58% of the total colonic production of these moieties, whereas epithelium accounted for only 20% of total colonic protein. By contrast, prostaglandin (PG) E2 and PGF2 alpha production occurred predominantly (greater than 97%) in the subepithelial layers. The present studies also demonstrate markedly higher levels of accumulation of lipoxygenase products in proliferative versus superficial epithelial cells, whereas prostaglandin accumulation was greater in superficial cells. Previous studies have supported a role for lipoxygenase and cyclooxygenase products in the control of colonic secretion, inflammatory cell infiltration and proliferative activity. The present results raise the possibility that the striking differences in the sites of production of these products within the colon has functional implications.     

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase–3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase–3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H₂O₂ and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.     

Epidermal growth factor receptor-dependent PI3K-activation promotes restitution of wounded human gastric epithelial monolayers

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.     

Are all "cytoprotective" drugs gastroprotective?

The effect of three--structurally different--groups of compounds was compared against gastric mucosal damages induced by ethanol or prostaglandin (PG) synthesis inhibitors, as well as against carrageenan-induced inflammation. All the compounds studied--SH-compounds (cysteamine, 2,3-dimercaptosuccinic acid, D,L-penicillamine), SH-blocking N-ethylmaleimide (NEM) and morphine-exerted dose-dependent inhibition on carrageenan edema test and against ethanol-induced gastric damage. Mucosal lesions induced by PG synthesis inhibitors (indomethacin 20 mg/kg, naproxen 75 mg/kg, piroxicam 60 mg/kg) were inhibited by drugs studied when the compounds were given before the damaging agents. However, when the drugs were injected 1 h after the inhibitors of PG synthesis opposite actions were observed; SH-compounds exerted protective, while NEM (2 mg/kg p.o.) and morphine (5 mg/kg p.o.) aggravating action. The results suggest that: 1. SH-compounds might have therapeutic importance in the treatment of gastric damage induced by prostaglandin synthesis inhibitors. 2. Reconsideration of the use of the term "cytoprotection" is necessary, since "cytoprotective" agents may aggravate mucosal damage in other ulcer model.