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919 Role of CA2+ on Mouse Gastric Epithelial Repair In Vivo
by excluding a possibility of involvement of non-T cells in the parabiosis system, we cotransferred the same number of colitogenic Ly5.1(+) and Ly5.2(+) CD4(+) T cells from lamina propria (LP) of colitic CD4(+)CD45RB(high) T cell-transferred RAG-2(-/-) and colitic IL-10(-/-) mice, respectively, into RAG-2(-/-) mice. Surprisingly, the co-transferred RAG2(-/-) mice showed the vast cellular infiltration of LP CD4(+) T cells like RAG-2(-/-) mice re-transferred with the cells from the colitic RAG-2(-/-) mice alone, but without wasting like RAG-2(-/-) mice transferred with the cells from colitic IL-10(-/-) mice alone. Furthermore, both the ratio of Th1 and Th17 cells originated from IL-10(-/-) mice and the ratio of Th1 cells from colitic RAG-2(-/-) mice significantly decreased as compared to the paired RAG2(-/-) mice. Conclusions: Collectively, all the present results suggest that there exist “Th1 vs. Th17 competition” and their orchestration appeared as a merged clinical phenotype of the colitis of two different colitic mice.
gastric mucosal injury. The Msi1 dependent regulation is an important pathway in gastric epithelial regeneration.
Involvement of Nitric Oxide and Prostaglandins in the Angiotensin 1-7 Induced Acceleration of the Healing of Experimental Gastric Ulcers Michael W. Pawlik, Ryszard Sendur, Jaroslaw Biernat, Tomasz Brzozowski, Stanislaw Konturek, Wieslaw W. Pawlik We demonstrated that inhibition of angiotensin converting enzyme (ACE) or the blockade of angiotensin AT1 receptors accelerates the healing process of preexisting gastric ulcers. Recently, angiotensin 1-7 was identified as the main product of angiotensin I conversion in rat stomach wall and the increase in plasma angiotensin 1-7 levels have been found in the animals treated with ACE inhibitors. To test the hypothesis that angiotensin 1-7 is responsible for the ucer healing activity of ACE inhibitors we employed an selective angiotensin 1-7 receptor antagonist A 779 applied with or without inhibition of ACE and AT1 receptors. Seventy Wistar rats with gastric ulcers induced by serosal application of acetic acid of initial area of 30 mm2 were used. They were randomly divided into three grups and received i.p. treatment with: 1)vehicle, 2)lisinopril (10 mg/kg), 4)losartan (10 mg/kg) without or with A779 (5 μg/kg). Seven days after ulcer induction, the area of gastric ulcer (UA) and microcirculatory gastric blood flow (GBF) in the ulcer crater and margin using laser-Doppler flowmetry were measured. At day 7 after ulcer induction the UA decreased by 66% and GBF increased in gastric ulcer margin vs. intact mucosa by 34% (p< 0.05) in vehicle-control animals. In both lisinopril- and losartan-treated animals, a marked acceleration of ulcer healing was observed. In lisinopril treated group UA was decreased by 79% and GBF increased in the ulcer margin by about 53%. Administration of A 779 failed to affect the healing rate of acetic acid ulcers. However, when A779 was given to Lisinopril treated animals, this angiotensin 17 receptor antagonist almost completely reversed the healing and microcirculatory effects of lisinopril. The UA was significantly reduced by 64% in rats treated with combination of A779 and lisinopril and the ulcer size was not significantly different than that measured in vehicle-control group with gastric ulcers. The combination therapy of A779 and lisinopril significantly increased the GBF at ulcer margin vs. vehicle-control gastric mucosa by 31%. This effect of A 779 was completly abolished by concomitant administration L-NAME (5 mg/kg s.c.) which suppressed the luminal NO content, and indomethacin 2.5 mg/kg (i.p.), which inhibited the mucosal generation of PGE2 and PGI2 by 85% and 89%, respectively. We conclude that 1) activation of renin-angiotensin system delays healing of preexisting gastric ulcers, and 2)angiotensin 1-7, a potent vasodilatatory member of angiotensin family peptides may be responsible for the acceleration of ulcer healing by ACE inhibitors due to mechanism involving endogenous NO and PG such as PGE2 and PGI2.
916 Activation of Energy Sensor - AMPK Reverses Impairment of Angiogenesis in Aging Gastric Mucosa. Underlying Molecular Mechanisms are: Activation of AMPK, Induction of Importin α and Activation of VEGF Gene. Amrita Ahluwalia, Vipal Gandhi, Sushrut S. Thiruvengadam, Andrzej S. Tarnawski Background: Aging gastric mucosa exhibits impaired In-Vivo angiogenesis in response to injury and endothelial cells (EC) isolated from aging gastric mucosa exhibit impaired invitro angiogenesis. The impairment of angiogenesis In-Vivo and in-vitro is associated with reduced VEGF levels. The molecular mechanisms underlying these aging-related changes are not fully elucidated. AMP-activated protein kinase (AMPK) is the cellular energy sensor that regulates cell metabolism and activates importin α, nuclear transport protein. AICAR (aminoimidazole carboxamide ribonucleotide) is an activator of AMPK. The role of AMPK in: a) regulation of angiogenesis and b) VEGF gene activation, and the therapeutic use of AICAR for aging-related impairment angiogenesis in gastric mucosa remain unexplored forming the basis for this study. Material and Methods: Gastric mucosal microvascular endothelial cells were isolated from Fisher F-344 rats, 3 months of age (young - YGEC) and 24 months of age (aging - AGEC) using anti-PECAM-1 selection and magnetic bead separation. Cells were cultured either under normoxia or hypoxia with and without 0.5 mM or 1 mM AICAR for 1, 2, 6 or 24 hours. Studies at baseline and in response to hypoxia: 1) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using an image analysis system; 2) Activation in AGEC by AICAR of AMPK, expression of importin α and activation of VEGF gene; 3) effect of AICAR treatment on angiogenesis in AGEC; and 4) the effect of downregulation of importin α in YGEC with specific siRNAs (Qiagen) on VEGF expression and angiogenesis. Results: Treatment with AICAR in AGEC significantly increased: a) the activation (phosphorylation) of AMPK by 6fold (p<0.01), b) the expression of importin α protein by 1.6-fold (p<0.01), c) the expression of VEGF mRNA by 2-fold (p<0.05) and d)reversed impairment of in-vitro angiogenesis by 1.8-fold (p<0.01). Conversely, in YGEC downregulation of importin α with specific siRNA reduced importin α and VEGF gene expression and also significantly inhibited in-vitro angiogenesis by 1.5-fold. Conclusions: 1) Activation of AMPK signaling by AICAR induces in AGEC importin α protein expression, VEGF gene activation and reverses aging-related impairment of angiogenesis. 2) Induction and activation of importin α by AICAR is a novel molecular mechanism underlying VEGF gene activation and the reversal of aging-related impairment of angiogenesis in EC. 3) AMPK signaling pathway and importin α are critical regulators of angiogenesis. 4) This study identified new pharmacological agent capable of reversing impairment of angiogenesis in aging gastric mucosa.
919 Role of CA2+ on Mouse Gastric Epithelial Repair In Vivo Eitaro Aihara, Vikram Prasad, Marshall H. Montrose Background/Aim: In response to acute gastric epithelial damage, rapid cell migration mediates repair. Although evidence shows Ca2+-dependent processes are required for repair, there have been no reports of Ca2+ mobilization occurring during the repair of gastric surface damage In Vivo. In the present study, we investigated the role of intracellular and extracellular Ca2+ in the mechanism of gastric epithelial cell migration In Vivo. Methods: Experiments used mice: C57BL/6, plasma membrane Ca2+ ATPase (PMCA) wildtype (+/+), PMCA1 knockout heterozygotes (+/-), or yellow cameleon (intracellular Ca2+ reporter fluorescent protein) transgenics. Anesthetized mice were placed supine on the stage of Zeiss LSM 510 confocal/ two-photon microscope such that a portion of the surgically exposed stomach mucosa protrudes into a chamber. In some experiments, Fura-red was added to saline superfusate solution to measure luminal Ca2+ level near the gastric surface (surface Ca2+). Gastric epithelial damage was induced by 5-10 sec exposure of 3-5 surface cells to 720 or 840 nm light, to photodamage cells. Result: Gastric epithelial damage was maximal at 2 min after photodamage, and subsequently healed within 10 min. During the repair process, intracellular Ca2+ significantly increased in surviving gastric epithelial cells around the damage area. Pretreatment with either chelerythrine (protein kinase C (PKC) inhibitor: 5 mg/kg, i.p.), U-73122 (phospholipase C (PLC) inhibitor: 30 mg/kg, i.p.), or verapamil (voltage-operated Ca2+ channel blocker 3 mg/kg, s.c.) inhibited repair of damage, and all also inhibited the intracellular Ca2+ increase. KB-R7943 (Na+/Ca2+ exchanger inhibitor: 10 mg/kg, i.v.) had no effect. When using a luminal perfusate with no added Ca2+, extracellular surface Ca2+ measurably increased in response to photobleach-induced damage that released Ca2+ from the epithelial layer. Further, luminal perfusion of the Ca2+ chelator HEDTA (10 mM) inhibited repair of damage, while application of 10 mM Ca2+ tended to accelerate repair. When gastric surface cells of PMCA1 (+/-) mice were photodamaged, epithelial repair was slowed and the increase of gastric surface Ca2+ was diminished, compared to wildtype littermates. Conclusion: The intracellular and extracellular Ca2+ dynamics play an important role in the gastric epithelial cell restitution In Vivo. Intracellular Ca2+ is regulated by PKC, PLC and voltage operated channels and extracellular surface Ca2+ is increased via PMCA1 during repair of damage.
917 Splicing Variant Specific M-Numb Expression by Musashi-1 in Regenerating Gastric Mucosa Tetsufumi Takahashi, Hidekazu Suzuki, Kanji Tsuchimoto, Hideyuki Okano, Toshifumi Hibi An RNA-binding protein Musashi-1 (Msi1) is shown to be upregulated in the rat gastric corpus mucosa after ethanol-induced mucosal injury. Msi1 is known to bind to the 3'-UTR region of several target mRNA, especially m-Numb and p21, and post-transcriptionally regulate these genes. Of these genes, m-Numb has been reported to express during the early stage of gland formation in the chicken gastric epithelium. m-Numb has 4 splicing variants, but the role of Msi1 and Msi1 dependent regulation of each m-Numb variant in gastric mucosal regeneration is still unclear. In the present study, the process of tissue repair after ethanol-induced acute gastric mucosal injury was investigated in Msi1-knockout (KO) mice (PNAS 99:15194-9,2002), to examine the regulation of m-Numb expression by Msi1 in gastric mucosal regeneration after injury. Methods: Msi1-KO and wild-type ICR mice (male, 10 weeks old) were orally administered absolute ethanol (0.25 ml) after 18 h of food deprivation, and the stomachs were resected 1, 3, 5 h after the ethanol intoxication. mNumb mRNA expression was analyzed by quantitative PCR using specific primers for individual splicing variants. m-Numb protein expression was detected by Western blotting and immunohistochemical staining using anti-m-Numb antibody. The whole-gene expression at 1 h after the ethanol intoxication was evaluated by the microarray method. Results: Fairly weak expression of the m-Numb protein was found in the Msi1-KO mice associated with a delayed regeneration of the injured gastric mucosal epithelium. In contrast, phosphotyrosinebinding-site-conserved-type m-Numb expression was significantly upregulated at both mRNA (3.47 fold vs control, p<0.05) and protein (3.23 fold vs control, p<0.05) levels at 5 h after acute gastric mucosal injury in the wild-type mice. In human gastric mucosa, the induced m-Numb expression was also strongly detected in erosive area, which contains a regenerating epithelium. Additionally, microarray and continuous quantitative analysis showed that the mRNA expression of prostate stem cell antigen (PSCA), which expressed in gastric epithelial cells in the isthmus of the fundic glands, was significantly enhanced in wild type mice after 1 h ethanol intoxication (1.83 fold vs Msi1-KO, p<0.01). The PCSA expression in each mouse was significantly correlated with PRRL (R2=0.50, p<0.05) and PRRS (R2=0.60, p<0.05) type of m-Numb protein expression. Conclusion: Msi1 regulates the splicing specific expression of m-Numb, and following PSCA expression after acute
920 C-Jun Enhances Intestinal Epithelial Restitution After Wounding by Increasing Phospholipase C-γ1 Gene Transcription and CA2+ Signaling Peng-Yuan Wang, Jie Chen, Rao N. Jaladanki, Tongtong Zou, Lan Liu, Lan Xiao, Eric D. Strauch, Jian-Ying Wang Intestinal epithelium is constantly exposed to various noxious substances and its rapid restitution after wounding is crucial for maintenance of the epithelial integrity. c-Jun is a member of the family of AP-1 transcription factors that are implicated in many aspects of cellular functions. Recently, c-Jun was also shown to modulate cell motility, but its exact role and mechanism in the regulation of intestinal epithelial cell (IEC) migration after
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gastric mucosal injury. The Msi1 dependent regulation is an important pathway in gastric epithelial regeneration.
Involvement of Nitric Oxide and Prostaglandins in the Angiotensin 1-7 Induced Acceleration of the Healing of Experimental Gastric Ulcers Michael W. Pawlik, Ryszard Sendur, Jaroslaw Biernat, Tomasz Brzozowski, Stanislaw Konturek, Wieslaw W. Pawlik We demonstrated that inhibition of angiotensin converting enzyme (ACE) or the blockade of angiotensin AT1 receptors accelerates the healing process of preexisting gastric ulcers. Recently, angiotensin 1-7 was identified as the main product of angiotensin I conversion in rat stomach wall and the increase in plasma angiotensin 1-7 levels have been found in the animals treated with ACE inhibitors. To test the hypothesis that angiotensin 1-7 is responsible for the ucer healing activity of ACE inhibitors we employed an selective angiotensin 1-7 receptor antagonist A 779 applied with or without inhibition of ACE and AT1 receptors. Seventy Wistar rats with gastric ulcers induced by serosal application of acetic acid of initial area of 30 mm2 were used. They were randomly divided into three grups and received i.p. treatment with: 1)vehicle, 2)lisinopril (10 mg/kg), 4)losartan (10 mg/kg) without or with A779 (5 μg/kg). Seven days after ulcer induction, the area of gastric ulcer (UA) and microcirculatory gastric blood flow (GBF) in the ulcer crater and margin using laser-Doppler flowmetry were measured. At day 7 after ulcer induction the UA decreased by 66% and GBF increased in gastric ulcer margin vs. intact mucosa by 34% (p< 0.05) in vehicle-control animals. In both lisinopril- and losartan-treated animals, a marked acceleration of ulcer healing was observed. In lisinopril treated group UA was decreased by 79% and GBF increased in the ulcer margin by about 53%. Administration of A 779 failed to affect the healing rate of acetic acid ulcers. However, when A779 was given to Lisinopril treated animals, this angiotensin 17 receptor antagonist almost completely reversed the healing and microcirculatory effects of lisinopril. The UA was significantly reduced by 64% in rats treated with combination of A779 and lisinopril and the ulcer size was not significantly different than that measured in vehicle-control group with gastric ulcers. The combination therapy of A779 and lisinopril significantly increased the GBF at ulcer margin vs. vehicle-control gastric mucosa by 31%. This effect of A 779 was completly abolished by concomitant administration L-NAME (5 mg/kg s.c.) which suppressed the luminal NO content, and indomethacin 2.5 mg/kg (i.p.), which inhibited the mucosal generation of PGE2 and PGI2 by 85% and 89%, respectively. We conclude that 1) activation of renin-angiotensin system delays healing of preexisting gastric ulcers, and 2)angiotensin 1-7, a potent vasodilatatory member of angiotensin family peptides may be responsible for the acceleration of ulcer healing by ACE inhibitors due to mechanism involving endogenous NO and PG such as PGE2 and PGI2.
916 Activation of Energy Sensor - AMPK Reverses Impairment of Angiogenesis in Aging Gastric Mucosa. Underlying Molecular Mechanisms are: Activation of AMPK, Induction of Importin α and Activation of VEGF Gene. Amrita Ahluwalia, Vipal Gandhi, Sushrut S. Thiruvengadam, Andrzej S. Tarnawski Background: Aging gastric mucosa exhibits impaired In-Vivo angiogenesis in response to injury and endothelial cells (EC) isolated from aging gastric mucosa exhibit impaired invitro angiogenesis. The impairment of angiogenesis In-Vivo and in-vitro is associated with reduced VEGF levels. The molecular mechanisms underlying these aging-related changes are not fully elucidated. AMP-activated protein kinase (AMPK) is the cellular energy sensor that regulates cell metabolism and activates importin α, nuclear transport protein. AICAR (aminoimidazole carboxamide ribonucleotide) is an activator of AMPK. The role of AMPK in: a) regulation of angiogenesis and b) VEGF gene activation, and the therapeutic use of AICAR for aging-related impairment angiogenesis in gastric mucosa remain unexplored forming the basis for this study. Material and Methods: Gastric mucosal microvascular endothelial cells were isolated from Fisher F-344 rats, 3 months of age (young - YGEC) and 24 months of age (aging - AGEC) using anti-PECAM-1 selection and magnetic bead separation. Cells were cultured either under normoxia or hypoxia with and without 0.5 mM or 1 mM AICAR for 1, 2, 6 or 24 hours. Studies at baseline and in response to hypoxia: 1) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using an image analysis system; 2) Activation in AGEC by AICAR of AMPK, expression of importin α and activation of VEGF gene; 3) effect of AICAR treatment on angiogenesis in AGEC; and 4) the effect of downregulation of importin α in YGEC with specific siRNAs (Qiagen) on VEGF expression and angiogenesis. Results: Treatment with AICAR in AGEC significantly increased: a) the activation (phosphorylation) of AMPK by 6fold (p<0.01), b) the expression of importin α protein by 1.6-fold (p<0.01), c) the expression of VEGF mRNA by 2-fold (p<0.05) and d)reversed impairment of in-vitro angiogenesis by 1.8-fold (p<0.01). Conversely, in YGEC downregulation of importin α with specific siRNA reduced importin α and VEGF gene expression and also significantly inhibited in-vitro angiogenesis by 1.5-fold. Conclusions: 1) Activation of AMPK signaling by AICAR induces in AGEC importin α protein expression, VEGF gene activation and reverses aging-related impairment of angiogenesis. 2) Induction and activation of importin α by AICAR is a novel molecular mechanism underlying VEGF gene activation and the reversal of aging-related impairment of angiogenesis in EC. 3) AMPK signaling pathway and importin α are critical regulators of angiogenesis. 4) This study identified new pharmacological agent capable of reversing impairment of angiogenesis in aging gastric mucosa.
919 Role of CA2+ on Mouse Gastric Epithelial Repair In Vivo Eitaro Aihara, Vikram Prasad, Marshall H. Montrose Background/Aim: In response to acute gastric epithelial damage, rapid cell migration mediates repair. Although evidence shows Ca2+-dependent processes are required for repair, there have been no reports of Ca2+ mobilization occurring during the repair of gastric surface damage In Vivo. In the present study, we investigated the role of intracellular and extracellular Ca2+ in the mechanism of gastric epithelial cell migration In Vivo. Methods: Experiments used mice: C57BL/6, plasma membrane Ca2+ ATPase (PMCA) wildtype (+/+), PMCA1 knockout heterozygotes (+/-), or yellow cameleon (intracellular Ca2+ reporter fluorescent protein) transgenics. Anesthetized mice were placed supine on the stage of Zeiss LSM 510 confocal/ two-photon microscope such that a portion of the surgically exposed stomach mucosa protrudes into a chamber. In some experiments, Fura-red was added to saline superfusate solution to measure luminal Ca2+ level near the gastric surface (surface Ca2+). Gastric epithelial damage was induced by 5-10 sec exposure of 3-5 surface cells to 720 or 840 nm light, to photodamage cells. Result: Gastric epithelial damage was maximal at 2 min after photodamage, and subsequently healed within 10 min. During the repair process, intracellular Ca2+ significantly increased in surviving gastric epithelial cells around the damage area. Pretreatment with either chelerythrine (protein kinase C (PKC) inhibitor: 5 mg/kg, i.p.), U-73122 (phospholipase C (PLC) inhibitor: 30 mg/kg, i.p.), or verapamil (voltage-operated Ca2+ channel blocker 3 mg/kg, s.c.) inhibited repair of damage, and all also inhibited the intracellular Ca2+ increase. KB-R7943 (Na+/Ca2+ exchanger inhibitor: 10 mg/kg, i.v.) had no effect. When using a luminal perfusate with no added Ca2+, extracellular surface Ca2+ measurably increased in response to photobleach-induced damage that released Ca2+ from the epithelial layer. Further, luminal perfusion of the Ca2+ chelator HEDTA (10 mM) inhibited repair of damage, while application of 10 mM Ca2+ tended to accelerate repair. When gastric surface cells of PMCA1 (+/-) mice were photodamaged, epithelial repair was slowed and the increase of gastric surface Ca2+ was diminished, compared to wildtype littermates. Conclusion: The intracellular and extracellular Ca2+ dynamics play an important role in the gastric epithelial cell restitution In Vivo. Intracellular Ca2+ is regulated by PKC, PLC and voltage operated channels and extracellular surface Ca2+ is increased via PMCA1 during repair of damage.
917 Splicing Variant Specific M-Numb Expression by Musashi-1 in Regenerating Gastric Mucosa Tetsufumi Takahashi, Hidekazu Suzuki, Kanji Tsuchimoto, Hideyuki Okano, Toshifumi Hibi An RNA-binding protein Musashi-1 (Msi1) is shown to be upregulated in the rat gastric corpus mucosa after ethanol-induced mucosal injury. Msi1 is known to bind to the 3'-UTR region of several target mRNA, especially m-Numb and p21, and post-transcriptionally regulate these genes. Of these genes, m-Numb has been reported to express during the early stage of gland formation in the chicken gastric epithelium. m-Numb has 4 splicing variants, but the role of Msi1 and Msi1 dependent regulation of each m-Numb variant in gastric mucosal regeneration is still unclear. In the present study, the process of tissue repair after ethanol-induced acute gastric mucosal injury was investigated in Msi1-knockout (KO) mice (PNAS 99:15194-9,2002), to examine the regulation of m-Numb expression by Msi1 in gastric mucosal regeneration after injury. Methods: Msi1-KO and wild-type ICR mice (male, 10 weeks old) were orally administered absolute ethanol (0.25 ml) after 18 h of food deprivation, and the stomachs were resected 1, 3, 5 h after the ethanol intoxication. mNumb mRNA expression was analyzed by quantitative PCR using specific primers for individual splicing variants. m-Numb protein expression was detected by Western blotting and immunohistochemical staining using anti-m-Numb antibody. The whole-gene expression at 1 h after the ethanol intoxication was evaluated by the microarray method. Results: Fairly weak expression of the m-Numb protein was found in the Msi1-KO mice associated with a delayed regeneration of the injured gastric mucosal epithelium. In contrast, phosphotyrosinebinding-site-conserved-type m-Numb expression was significantly upregulated at both mRNA (3.47 fold vs control, p<0.05) and protein (3.23 fold vs control, p<0.05) levels at 5 h after acute gastric mucosal injury in the wild-type mice. In human gastric mucosa, the induced m-Numb expression was also strongly detected in erosive area, which contains a regenerating epithelium. Additionally, microarray and continuous quantitative analysis showed that the mRNA expression of prostate stem cell antigen (PSCA), which expressed in gastric epithelial cells in the isthmus of the fundic glands, was significantly enhanced in wild type mice after 1 h ethanol intoxication (1.83 fold vs Msi1-KO, p<0.01). The PCSA expression in each mouse was significantly correlated with PRRL (R2=0.50, p<0.05) and PRRS (R2=0.60, p<0.05) type of m-Numb protein expression. Conclusion: Msi1 regulates the splicing specific expression of m-Numb, and following PSCA expression after acute
920 C-Jun Enhances Intestinal Epithelial Restitution After Wounding by Increasing Phospholipase C-γ1 Gene Transcription and CA2+ Signaling Peng-Yuan Wang, Jie Chen, Rao N. Jaladanki, Tongtong Zou, Lan Liu, Lan Xiao, Eric D. Strauch, Jian-Ying Wang Intestinal epithelium is constantly exposed to various noxious substances and its rapid restitution after wounding is crucial for maintenance of the epithelial integrity. c-Jun is a member of the family of AP-1 transcription factors that are implicated in many aspects of cellular functions. Recently, c-Jun was also shown to modulate cell motility, but its exact role and mechanism in the regulation of intestinal epithelial cell (IEC) migration after
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918