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CD2-associated protein (CD2AP) interacts with E-cadherin in a cell culture model simulating gastric restitution
T908
effects of glucose on ischemie-injured mucosa. Furthermore, addition of PGE2 (10-6 M)to indomethacin-treated tissue prevented glucose-induced reductions in TER. Glucose impairs the recovery of TER following ischemic injury in the presence of indomethacin, in an MLCK and NHE-3-dependent fashion. Because the eflecta of glucose are only seen in the presence of nidomethacin, and are reversed by PGE2, we speculate glucose and PGE2 have the same ultimate site of action dunng epithelial repair, most likely the tight junction. We have also shown that ghitamine is capable of reducing TER in repairing mucosa, implying that provision of nutrients preferentially absorbed and metabolized by entemcytes may complicate mucosal recovery following acute injury.
CD2-Associated Protein (CD2AP) Interacts with E-Cadherin in a Cell Culture Model Simulating Gastric Restitution Amra LepistO, Ham Mustonen, Sauna Lehtonen, Eero Lehtonen, Panh Puolakkainen, Eero Ktvilaakso The ttequently occurring superficial gastric mucosal injuries provoked by various exogenous and endogenous nnxae are pmnarfly healed by rapid gastric restitution, whereby the exfofiated surface epithelium is replaced hy a flattened neo-epithelium tormed by nngrating vital epithelial cells around the lesion. CD2AP is a novel adapter/scallblding protein which seems to regufate d'ymamic actni assembly and cell motility, possibly functiomng as a scaffolding protein between celt membrane proteins and the actin cytoskeleton (Lehtonen et al. AJP 283:F734,2002) This study explores the role of CD2AP in the locomotive transition between adbesive at~d migrating eeflufar states in a ceil culture model simulating gastric restitution. Special attention was paid on the pntential association of CD2AP with the adberens junction protein E-cadherin. Methods: "fhe localization oi CDLatP and E-cadherin was analyzed by imnmnncytocbemistry with cont0cal microscopy in cultured rat gastric mucosal (RGM) cells. Two models of cell detachment, migration and reattachinent were used: wounded confluent monolayer call repair model and HGF/scatter tactor-induced cell scattering model. Coimmunoprecipitation assay was used to assess the interaction between CD2AP and E-cadherin in contacting and migrating cells. Results: In stationary" confluent RGM cells CD2AP was localized perinuclearly while E-cadberin was expressed along the cell membrane. In migrating ceils, CI)2AP and E-cadherni co-localized in thread-like accunmlations in Leading edges and, later, as fingerqike extensions in cell-cell contact areas. Upon cell-cell contact with resultant cessation oi migration and torntation of confluent cell monolayer, CD2AP vanished from the contact areas at (he cell membranes and resumed the perinuclear localization. Reciprocal coimmunoprecipitatiou az~suysrevealed that CD2AP and E-cadherin interact being physically in complex in migrating and contacting RGM cel!s. Conchision: The results suggest that CD2AP, in complex with E-cadherin, may participate in the regulation and maintenance of cell migration and cell-cell adhesion during epithelial restitution.
T911 Achievement of Quality of Ulcer Healing Through The Gene Transfer of VEGF in Gastric Ulcer Models Tar Young Oh, Ju Mi Kim, SeuI Min Choi, Jae Hoon Choi, Byoung Ok Ahn, Won Bar Kim, Young Bar Kim, Ki b/)mng Lee, Jin Hung Kim, Sung Won Cho, Ki Baik Hahm Background: Based on the findings that the ulcer prevalence site in the gastro-duodenum coincide with high distribution sites of end-artery of extramural origin, defects in the angiogenesis may be the fundamental and prime factor in the etio-patbogenesis of peptic ulcer diseases. In order to prove this hypothesis, we developed the acetic acid-induced gastric ulcer models in rats and followed the changes of gastric ulcer according to groups, consisting of empty vector injected (C g-'.oup), proton pump inhibitor administered (P group), and pfasmid DNA encoding human "vq~GF100 mg/kg (VL group) and 500 mg/kg (Vll group) injected. All these agents were injected into the muscle layer of ulcer beds except the intravenous delivery of PPI (Omeprazole, iv) Methods: The rats from each group were sacrificed and the excised stomach was opened along the greater curvature. The gross and microscopic observations were done. RT-PCR was done to check the sequential changes of various growth factors involved in the ulcer repair and immunohistochemical staining and western blot analysis were done to look at pS2/TFF1, EGF, VEGF receptor, BrdU, and muein. The resistance to ulcer recurrence was measured 1L-l[3 injection after 6 months of acetic acid injection. Resuhs; Significantly increased transcripts of EGF, PDGF, pS2/TFFI, COX-2 were observed in V1. and VH groups compared to C and P groups. The mean size of ulcer and surrounding regeneration were siginificantly decreased in x/1. and VII groups, suggesting that the complete and accelerated ulcer healings were achieved after the gene transfer of VEGF and significant remodeling of previously ulcerated mucosa were observed in commitmem to significant increase in regeneration by VEGF gene transfer. Only one rat among 10 rats showed the recurrence of healed ulcer after IL-l[3 injection, whereas 100.0% and 88.8% of healed ulcers were recurred in C and P group. The BrdU positive cells and the intensity and amount of positive staining with mucin antibodies were significantly increased in VL and VH groups compared to C and P groups. Conclusion; The defects in angiogenesis may be the fundamental basis of pathogenesis of peptic ulcer disease and the gene transfer of VEGF into the ulcerated bed could achieve the quality of ulcer healing and ideal ulcer healing, necessitating its potential clinical application in the treamtent of peptic ulcer disease.
T909 Characterisation of Barrier Function in Cytokine (TNF~t and INF~y) Treated T84 Cells Guy Sander, Baby Powell Introductions and aims: A decrease in tight junction barrier tunction has been suggested to be an important pathogenic factor in Crohn's disease. Cytokines are known to decrease barrier function and induce apopt~is both in vitro and in vivo. The aims of this study were to determine whether the cytokines IFN-y or TNF-~qNF-y aft~ect TJ protein expression, apoptosis and permeability in cdonic T84 ceils. Methods: T84 (passage 30) cell permeability ~v~tsassessed by measuring flux of the paracellula maker FITC-dextran (lOkDa and 40kDa) and trans-epithelial resistance. ZO-1, Occhidin, CIaudin-1, Claudin-2, Claudine3, Cfaudin5, E-Cadherin and F-Aetin expression was determined by Western blot and con.focal microscopy. Apoptosis was detennined by Hoechst staining and counting extruded cells in the apical media. Necrotic cells were determined by trypan blue staining of intact monotayers. Results: IFN-y (100ng/ml) produced a lime (O-72hrs) dependent increase m T84 TJ pemreability. When added in combination with TNFmt (10ng/ml), IFN-',/produced an earlier decrease in TER suggestmg a synergistic eft~ct. The end point TER for both treatments was the same after 3 days (60%). IFN-y reduced an increase in the flux of 10 kDa but not 40 kDa FITC-dextran. An increase in apoptosis was detected when ceils were treated with both cytokines (60 fold) compared to IFN-y alone (4 tbld) No increase in necrotic cells was &tected Contocal analysis revealed TJ expression (ZO-1, Occludin, CI-1, C1-3 and CI-5) was maintained beneath extruding apoptotic cells TJ proteins were expressed at the apical fateml membrane bm not in the cytoplasm Apical F-Actin expression was reduced to undetectable levels in both treatment groups. Minimal changes in expression of ZO-1, Occludin, CI- 1, CI-3 and CI- 5 were detected in IFN-3, treated cells~ However, a 50 % reduction In Cl-2 RNA and protein levels was detected with IFN-',/treatment. Conclusion: We have shown that in T84 cell& IFN-~/~q'NF-c~ show a synergistic effect on barrier function by decreasing TER and increasing permeability selectively to 10kDA molecules, A barrier, although reduced, is maintained during extrusion of apoptotic cells. We speculate that the decrease in fER is due in part to both a decrease in CI-2 and an increase in apoptosis.
T912 Activity of the Monocarboxylate Transporter 1 (MCT1) May Be Required for Cell Migration after Injury in Gastric Surface Cells Susan J. Hagen, Anna Zuk, Eiji Nakamura, Marianne Smith We recently showed that DIDS and SITS, stilbene compounds and bicarbonate transport inhibitors, block wound repair/restitution in the stomach predominantly by a mechanism that does not involve bicarbonate transport. Because DIDS/SITS can bind to other transporters and channels to inhibit cell function, it is important to identfl~z which transporter/channd ts inhibited by DIDS/SITS during restitution. Here we show that DIDS binds covalently to tire monocarboxylate transporter 1 (MCT1), a 40 kDa protein, in gastric RGM1 cells. METHODS: RGM1 cells were grown to semi-confluence and incubated for 1 hr at 37 % with 300 ~M DIDS, in the presence or absence of 5-10 mM cx-4-hydmxy-cinnimate (CHC), a drug which specifically binds to and inhibits MCTL Under these conditions, DIDS binds covalently to MCT1 and DIDS binding is inhibited by CHC in other cell systems. Cultures were washed extensively with buffer containing albumin (to complex all unbound DIDS) and then solubilized in SDS gel sample buffer. Western blots were incubated with anti.. DIDS antibody (kindly provided by Dr. Andrew Halestrap). Recover),, of a round wound was evaiuated in confluent monolayers of RGM1 cells in the presence or absence of CHC. RESULTS: DIDS, as detected by anti-D1DS antibody, bound to 2 major proteins on the surface of gastric RGM1 cells. One protein was approximately 100 kDa, which may he the Na+/I-ICO3- cotransporter. The second protein was approximately 40 kDa and was significantly reduced in intensity in the presence of CHC. This result strongly supports the conclusion that the 40 kDa band is MCT1. CHC inhibited wound repair after injury in RGM I cells. However, the viability of RGM1 ceils was reduced significantly with CHC during the time required (6 hr) to complete wound repair expenments. CONCLUSIONS: Our results suggest that DIDS binds, in addition to tire NaVHCO~ cotransporter, to MCTI in gastric surface cells. Because MCTI cotransports H ~ and monocarboxylates, like lactate, we propose that MCT1 is responsible tor H+aactate (lactic acid) efflux during wound repair. We recently* showed that aerobic glycolysis, which generates lactic acid as an end product, is required for cell migration after injury. Thus, MCT1 may play an important rote in the extrusion of lactic acid and in the regulation of intracefiufar pH in migrating surface cells after injury.
17910 Glucose and Glutamine Impair Intestinal Recovery Following lschemic Injury Jdfrey T Cole, Karen Young, Anthony Bliksfager In healthy intestinal mucosa, glucose transport vis SGLT-1 lowers transepitbelial electrical remtance (TER) via myosin light chain kinase (MLCK)-mediated anatomical opening of interepithelial tight junctions This pathway also appears to involve glucose metabolism and activation o{ NHE-3 Howewr, the eftects of these events on injured mucosa are unknown. We have recently cb.aracterized mucosal repair ~ollowing acute ischemic injury in porcine ileum, and have noted that recovery of TER is in large part mediated by prostanoid-induced closure of tight junctions We theretore postulated that application of glucose to the mucosal surface of ischemic-inlured mucosa would retard recovery of TER Following 45-minutes of iahemic ii~jury, pomine ileal mucosa was mounted in Ussmg chambers and TER was monitored as an index of recuvery of barrier function. During a 3-bout in vitro recovery period, there was a significant increase (P < 0.05) in TER, resulting in recovery of TER m kvels not significantly different from those of undamaged control mucosa. Application of glucose (]0 raM) to the mucosal suriace oi ischemm-injured mucosa had no effect, whereas application of the prostanoid synthesis inhibitor indomethacin (5 x 10-6 M) retarded recover}, of TER. However, applicatior~ ol nmcosal glucose (10raM) in the presence of indomethacin signihcantly reduced recovery of TER to levels below those of tissues treated with indomethacin alone (P < 005). Similar responses were noted after addition of mncosal ghitamine (10 mM) m the presence of indomethacin. De-treatment of isehemic-injured tissues with ML9 (10-5 M), an MLCK inhibitor, or 5-3226 (10-5 M), an NHE-3 inhibitor, prevented the
A-447
AGA
Abstracts
effects of glucose on ischemie-injured mucosa. Furthermore, addition of PGE2 (10-6 M)to indomethacin-treated tissue prevented glucose-induced reductions in TER. Glucose impairs the recovery of TER following ischemic injury in the presence of indomethacin, in an MLCK and NHE-3-dependent fashion. Because the eflecta of glucose are only seen in the presence of nidomethacin, and are reversed by PGE2, we speculate glucose and PGE2 have the same ultimate site of action dunng epithelial repair, most likely the tight junction. We have also shown that ghitamine is capable of reducing TER in repairing mucosa, implying that provision of nutrients preferentially absorbed and metabolized by entemcytes may complicate mucosal recovery following acute injury.
CD2-Associated Protein (CD2AP) Interacts with E-Cadherin in a Cell Culture Model Simulating Gastric Restitution Amra LepistO, Ham Mustonen, Sauna Lehtonen, Eero Lehtonen, Panh Puolakkainen, Eero Ktvilaakso The ttequently occurring superficial gastric mucosal injuries provoked by various exogenous and endogenous nnxae are pmnarfly healed by rapid gastric restitution, whereby the exfofiated surface epithelium is replaced hy a flattened neo-epithelium tormed by nngrating vital epithelial cells around the lesion. CD2AP is a novel adapter/scallblding protein which seems to regufate d'ymamic actni assembly and cell motility, possibly functiomng as a scaffolding protein between celt membrane proteins and the actin cytoskeleton (Lehtonen et al. AJP 283:F734,2002) This study explores the role of CD2AP in the locomotive transition between adbesive at~d migrating eeflufar states in a ceil culture model simulating gastric restitution. Special attention was paid on the pntential association of CD2AP with the adberens junction protein E-cadherin. Methods: "fhe localization oi CDLatP and E-cadherin was analyzed by imnmnncytocbemistry with cont0cal microscopy in cultured rat gastric mucosal (RGM) cells. Two models of cell detachment, migration and reattachinent were used: wounded confluent monolayer call repair model and HGF/scatter tactor-induced cell scattering model. Coimmunoprecipitation assay was used to assess the interaction between CD2AP and E-cadherin in contacting and migrating cells. Results: In stationary" confluent RGM cells CD2AP was localized perinuclearly while E-cadberin was expressed along the cell membrane. In migrating ceils, CI)2AP and E-cadherni co-localized in thread-like accunmlations in Leading edges and, later, as fingerqike extensions in cell-cell contact areas. Upon cell-cell contact with resultant cessation oi migration and torntation of confluent cell monolayer, CD2AP vanished from the contact areas at (he cell membranes and resumed the perinuclear localization. Reciprocal coimmunoprecipitatiou az~suysrevealed that CD2AP and E-cadherin interact being physically in complex in migrating and contacting RGM cel!s. Conchision: The results suggest that CD2AP, in complex with E-cadherin, may participate in the regulation and maintenance of cell migration and cell-cell adhesion during epithelial restitution.
T911 Achievement of Quality of Ulcer Healing Through The Gene Transfer of VEGF in Gastric Ulcer Models Tar Young Oh, Ju Mi Kim, SeuI Min Choi, Jae Hoon Choi, Byoung Ok Ahn, Won Bar Kim, Young Bar Kim, Ki b/)mng Lee, Jin Hung Kim, Sung Won Cho, Ki Baik Hahm Background: Based on the findings that the ulcer prevalence site in the gastro-duodenum coincide with high distribution sites of end-artery of extramural origin, defects in the angiogenesis may be the fundamental and prime factor in the etio-patbogenesis of peptic ulcer diseases. In order to prove this hypothesis, we developed the acetic acid-induced gastric ulcer models in rats and followed the changes of gastric ulcer according to groups, consisting of empty vector injected (C g-'.oup), proton pump inhibitor administered (P group), and pfasmid DNA encoding human "vq~GF100 mg/kg (VL group) and 500 mg/kg (Vll group) injected. All these agents were injected into the muscle layer of ulcer beds except the intravenous delivery of PPI (Omeprazole, iv) Methods: The rats from each group were sacrificed and the excised stomach was opened along the greater curvature. The gross and microscopic observations were done. RT-PCR was done to check the sequential changes of various growth factors involved in the ulcer repair and immunohistochemical staining and western blot analysis were done to look at pS2/TFF1, EGF, VEGF receptor, BrdU, and muein. The resistance to ulcer recurrence was measured 1L-l[3 injection after 6 months of acetic acid injection. Resuhs; Significantly increased transcripts of EGF, PDGF, pS2/TFFI, COX-2 were observed in V1. and VH groups compared to C and P groups. The mean size of ulcer and surrounding regeneration were siginificantly decreased in x/1. and VII groups, suggesting that the complete and accelerated ulcer healings were achieved after the gene transfer of VEGF and significant remodeling of previously ulcerated mucosa were observed in commitmem to significant increase in regeneration by VEGF gene transfer. Only one rat among 10 rats showed the recurrence of healed ulcer after IL-l[3 injection, whereas 100.0% and 88.8% of healed ulcers were recurred in C and P group. The BrdU positive cells and the intensity and amount of positive staining with mucin antibodies were significantly increased in VL and VH groups compared to C and P groups. Conclusion; The defects in angiogenesis may be the fundamental basis of pathogenesis of peptic ulcer disease and the gene transfer of VEGF into the ulcerated bed could achieve the quality of ulcer healing and ideal ulcer healing, necessitating its potential clinical application in the treamtent of peptic ulcer disease.
T909 Characterisation of Barrier Function in Cytokine (TNF~t and INF~y) Treated T84 Cells Guy Sander, Baby Powell Introductions and aims: A decrease in tight junction barrier tunction has been suggested to be an important pathogenic factor in Crohn's disease. Cytokines are known to decrease barrier function and induce apopt~is both in vitro and in vivo. The aims of this study were to determine whether the cytokines IFN-y or TNF-~qNF-y aft~ect TJ protein expression, apoptosis and permeability in cdonic T84 ceils. Methods: T84 (passage 30) cell permeability ~v~tsassessed by measuring flux of the paracellula maker FITC-dextran (lOkDa and 40kDa) and trans-epithelial resistance. ZO-1, Occhidin, CIaudin-1, Claudin-2, Claudine3, Cfaudin5, E-Cadherin and F-Aetin expression was determined by Western blot and con.focal microscopy. Apoptosis was detennined by Hoechst staining and counting extruded cells in the apical media. Necrotic cells were determined by trypan blue staining of intact monotayers. Results: IFN-y (100ng/ml) produced a lime (O-72hrs) dependent increase m T84 TJ pemreability. When added in combination with TNFmt (10ng/ml), IFN-',/produced an earlier decrease in TER suggestmg a synergistic eft~ct. The end point TER for both treatments was the same after 3 days (60%). IFN-y reduced an increase in the flux of 10 kDa but not 40 kDa FITC-dextran. An increase in apoptosis was detected when ceils were treated with both cytokines (60 fold) compared to IFN-y alone (4 tbld) No increase in necrotic cells was &tected Contocal analysis revealed TJ expression (ZO-1, Occludin, CI-1, C1-3 and CI-5) was maintained beneath extruding apoptotic cells TJ proteins were expressed at the apical fateml membrane bm not in the cytoplasm Apical F-Actin expression was reduced to undetectable levels in both treatment groups. Minimal changes in expression of ZO-1, Occludin, CI- 1, CI-3 and CI- 5 were detected in IFN-3, treated cells~ However, a 50 % reduction In Cl-2 RNA and protein levels was detected with IFN-',/treatment. Conclusion: We have shown that in T84 cell& IFN-~/~q'NF-c~ show a synergistic effect on barrier function by decreasing TER and increasing permeability selectively to 10kDA molecules, A barrier, although reduced, is maintained during extrusion of apoptotic cells. We speculate that the decrease in fER is due in part to both a decrease in CI-2 and an increase in apoptosis.
T912 Activity of the Monocarboxylate Transporter 1 (MCT1) May Be Required for Cell Migration after Injury in Gastric Surface Cells Susan J. Hagen, Anna Zuk, Eiji Nakamura, Marianne Smith We recently showed that DIDS and SITS, stilbene compounds and bicarbonate transport inhibitors, block wound repair/restitution in the stomach predominantly by a mechanism that does not involve bicarbonate transport. Because DIDS/SITS can bind to other transporters and channels to inhibit cell function, it is important to identfl~z which transporter/channd ts inhibited by DIDS/SITS during restitution. Here we show that DIDS binds covalently to tire monocarboxylate transporter 1 (MCT1), a 40 kDa protein, in gastric RGM1 cells. METHODS: RGM1 cells were grown to semi-confluence and incubated for 1 hr at 37 % with 300 ~M DIDS, in the presence or absence of 5-10 mM cx-4-hydmxy-cinnimate (CHC), a drug which specifically binds to and inhibits MCTL Under these conditions, DIDS binds covalently to MCT1 and DIDS binding is inhibited by CHC in other cell systems. Cultures were washed extensively with buffer containing albumin (to complex all unbound DIDS) and then solubilized in SDS gel sample buffer. Western blots were incubated with anti.. DIDS antibody (kindly provided by Dr. Andrew Halestrap). Recover),, of a round wound was evaiuated in confluent monolayers of RGM1 cells in the presence or absence of CHC. RESULTS: DIDS, as detected by anti-D1DS antibody, bound to 2 major proteins on the surface of gastric RGM1 cells. One protein was approximately 100 kDa, which may he the Na+/I-ICO3- cotransporter. The second protein was approximately 40 kDa and was significantly reduced in intensity in the presence of CHC. This result strongly supports the conclusion that the 40 kDa band is MCT1. CHC inhibited wound repair after injury in RGM I cells. However, the viability of RGM1 ceils was reduced significantly with CHC during the time required (6 hr) to complete wound repair expenments. CONCLUSIONS: Our results suggest that DIDS binds, in addition to tire NaVHCO~ cotransporter, to MCTI in gastric surface cells. Because MCTI cotransports H ~ and monocarboxylates, like lactate, we propose that MCT1 is responsible tor H+aactate (lactic acid) efflux during wound repair. We recently* showed that aerobic glycolysis, which generates lactic acid as an end product, is required for cell migration after injury. Thus, MCT1 may play an important rote in the extrusion of lactic acid and in the regulation of intracefiufar pH in migrating surface cells after injury.
17910 Glucose and Glutamine Impair Intestinal Recovery Following lschemic Injury Jdfrey T Cole, Karen Young, Anthony Bliksfager In healthy intestinal mucosa, glucose transport vis SGLT-1 lowers transepitbelial electrical remtance (TER) via myosin light chain kinase (MLCK)-mediated anatomical opening of interepithelial tight junctions This pathway also appears to involve glucose metabolism and activation o{ NHE-3 Howewr, the eftects of these events on injured mucosa are unknown. We have recently cb.aracterized mucosal repair ~ollowing acute ischemic injury in porcine ileum, and have noted that recovery of TER is in large part mediated by prostanoid-induced closure of tight junctions We theretore postulated that application of glucose to the mucosal surface of ischemic-inlured mucosa would retard recovery of TER Following 45-minutes of iahemic ii~jury, pomine ileal mucosa was mounted in Ussmg chambers and TER was monitored as an index of recuvery of barrier function. During a 3-bout in vitro recovery period, there was a significant increase (P < 0.05) in TER, resulting in recovery of TER m kvels not significantly different from those of undamaged control mucosa. Application of glucose (]0 raM) to the mucosal suriace oi ischemm-injured mucosa had no effect, whereas application of the prostanoid synthesis inhibitor indomethacin (5 x 10-6 M) retarded recover}, of TER. However, applicatior~ ol nmcosal glucose (10raM) in the presence of indomethacin signihcantly reduced recovery of TER to levels below those of tissues treated with indomethacin alone (P < 005). Similar responses were noted after addition of mncosal ghitamine (10 mM) m the presence of indomethacin. De-treatment of isehemic-injured tissues with ML9 (10-5 M), an MLCK inhibitor, or 5-3226 (10-5 M), an NHE-3 inhibitor, prevented the
A-447
AGA
Abstracts