RESUMEN
Esophageal adenocarcinoma (EAC) is still associated with poor prognosis, despite modern multi-modal therapies. New molecular markers, which control cell cycle and promote lymph node metastases or tumor growth, may introduce novel target therapies. Dickkopf-1 (DKK1) is a secreted glycoprotein that blocks the oncogenic Wnt/ß-catenin signaling and its aberrant expression has been observed in many malignancies, including EAC. In this study, we investigated the biological role of DKK1 in EAC. Analysis of DKK1 and active ß-catenin expression in human esophageal tissues confirmed a simultaneous DKK1-overexpression together with aberrant activation of ß-catenin signaling in EAC in comparison with Barrett's and healthy mucosa. To elucidate the molecular role of DKK1, the OE33 adenocarcinoma cells, which were found to overexpress DKK1, were subjected to functional and molecular assays following siRNA-mediated DKK1-knockdown. At the functional level, OE33 cell viability, proliferation, migration and invasion were significantly attenuated by the absence of DKK1. At the molecular level, neither DKK1-knockdown nor application of exogenous recombinant DKK1 were found to alter the baseline ß-catenin signaling in OE33 cells. However, DKK1-knockdown significantly abrogated downstream Akt-phosphorylation. On the other hand, the Wnt-agonist, Wnt3a, restored the Akt-phorphorylation in the absence of DKK1, without, however, being able to further stimulate ß-catenin transcription. These findings suggest that the ß-catenin transcriptional activity in EAC is independent of Wnt3a/DKK1 site-of-action and define an oncogenic function for DKK1 in this type of malignancy via distinct activation of Akt-mediated intracellular pathways and independently of Wnt-axis inhibition. Taken together, DKK1 may present a novel therapeutic target in EAC.
RESUMEN
UNLABELLED: The mechanism underlying the progression of normal esophageal mucosa to esophageal adenocarcinoma remains elusive. WNT5A is a noncanonical WNT, which mainly functions via the receptor tyrosine kinase-like orphan receptor 2 (ROR2), and has an unclear role in carcinogenesis. In this study, we aimed to determine the role of WNT5A/ROR2 signaling in esophageal adenocarcinoma. Analysis of WNT5A and ROR2 expression patterns in healthy controls, Barrett and esophageal adenocarcinoma patients' esophageal clinical specimens as well as in various esophageal cell lines demonstrated a ROR2 overexpression in esophageal adenocarcinoma tissues compared with Barrett and healthy mucosa, whereas WNT5A expression was found significantly downregulated toward esophageal adenocarcinoma formation. Treatment of esophageal adenocarcinoma OE33 cells with human recombinant WNT5A (rhWNT5A) significantly suppressed proliferation, survival, and migration in a dose-dependent fashion. rhWNT5A was found to inhibit TOPflash activity in ROR2 wild-type cells, whereas increased TOPflash activity in ROR2-knockdown OE33 cells. In addition, ROR2 knockdown alone abolished cell proliferation and weakened the migration properties of OE33 cells. These findings support an early dysregulation of the noncanonical WNT5A/ROR2 pathway in the pathogenesis of esophageal adenocarcinoma, with the loss of WNT5A expression together with the ROR2 overexpression to be consistent with tumor promotion. IMPLICATIONS: The dysregulation of WNT5A/ROR2 noncanonical WNT signaling in Barrett-associated esophageal adenocarcinoma introduces possible prognostic markers and novel targets for tailored therapy of this malignancy. Mol Cancer Res; 14(7); 647-59. ©2016 AACR.
Asunto(s)
Adenocarcinoma/metabolismo , Esófago de Barrett/metabolismo , Neoplasias Esofágicas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Proteína Wnt-5a/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Esófago de Barrett/patología , Proliferación Celular/fisiología , Progresión de la Enfermedad , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
INTRODUCTION: Wnt/ß-catenin signaling activation has been reported only during the late steps of Barrett's esophagus (BE) neoplastic progression, but not in BE metaplasia, based on the absence of nuclear ß-catenin. However, ß-catenin transcriptional activity has been recorded in absence of robust nuclear accumulation. Thus, we aimed to investigate the Wnt/ß-catenin signaling in nondysplastic BE. METHODS: Esophageal tissues from healthy and BE patients without dysplasia were analyzed for Wnt target gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Esophageal squamous (EPC1-& EPC2-hTERT), BE metaplastic (CP-A), and adenocarcinoma (OE33) cell lines were characterized for Wnt activation by qRT-PCR, Western blot, and luciferase assay. Wnt activity regulation was examined by using recombinant Wnt3a and Dickkopf-1 (Dkk1) as well as Dkk1 short interfering RNA. RESULTS: Wnt target genes (AXIN2, c-MYC, Cyclin D1, Dkk1) and Wnt3a were significantly upregulated in nondysplastic BE compared with squamous mucosa. Elevated levels of dephosphorylated ß-catenin were detected in nondysplastic BE. Nuclear active ß-catenin and TOPflash activity were increased in CP-A and OE33 cells compared with squamous cells. Wnt3a-mediated ß-catenin signaling activation was abolished by Dkk1 in CP-A cells. TOPFlash activity was elevated following Dkk1 silencing in CP-A but not in OE33 cells. Dysplastic and esophageal adenocarcinoma tissues demonstrated further Dkk1 and AXIN2 overexpression. CONCLUSIONS: Despite the absence of robust nuclear accumulation, ß-catenin is transcriptionally active in nondysplastic BE. Dkk1 overexpression regulates ß-catenin signaling in BE metaplastic but not in adenocarcinoma cells, suggesting that early perturbation of Dkk1-mediated signaling suppression may contribute to BE malignant transformation.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Proteína Axina/biosíntesis , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Ciclina D1/biosíntesis , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Proteína Wnt3A/biosíntesis , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/genéticaRESUMEN
Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat's digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1ß with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial cell-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1ß-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1ß activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.
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Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Células Endoteliales/efectos de los fármacos , Esófago/irrigación sanguínea , Intestinos/irrigación sanguínea , Microvasos/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Extractos Vegetales/farmacología , Rubus , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Frutas , Humanos , Mediadores de Inflamación/metabolismo , Microvasos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Fitoterapia , Plantas Medicinales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Endothelial-mesenchymal transition (EndoMT) has been recognized as a key determinant of tumor microenvironment in cancer progression and metastasis. Endothelial cells undergoing EndoMT lose their endothelial markers, acquire the mesenchymal phenotype, and become more invasive with increased migratory abilities. Early stages of esophageal adenocarcinoma (EAC) are characterized by strong microvasculature whose impact in tumor progression remains undefined. Our aim was to determine the role of EndoMT in EAC by investigating the impact of tumor cells on normal primary human esophageal microvascular endothelial cells (HEMEC). HEMEC were either cocultured with OE33 adenocarcinoma cells or treated with IL-1ß and transforming growth factor-ß2 (TGF-ß2) for indicated periods and analyzed for EndoMT-associated changes by real-time PCR, Western blotting, immunofluorescence staining, and functional assays. Additionally, human EAC tissues were investigated for detection of EndoMT-like cells. Our results demonstrate an increased expression of mesenchymal markers [fibroblast-specific protein 1 (FSP1), collagen1α2, vimentin, α-smooth muscle actin (α-SMA), and Snail], decreased expression of endothelial markers [CD31, von Willebrand factor VIII (vWF), and VE-cadherin], and elevated migration ability in HEMEC following coculture with OE33 cells. The EndoMT-related changes were inhibited by IL-1ß and TGF-ß2 gene silencing in OE33 cells. Recombinant IL-1ß and TGF-ß2 induced EndoMT in HEMEC. Although the level of VEGF expression was elevated in EndoMT cells, the angiogenic property of these cells was diminished. In vivo, by immunostaining EndoMT-like cells were detected at the invasive front of EAC. Our findings underscore a significant role for EndoMT in EAC and provide new insights into the mechanisms and significance of EndoMT in the context of tumor progression.
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Adenocarcinoma/patología , Células Endoteliales/citología , Neoplasias Esofágicas/patología , Esófago/citología , Interleucina-1beta/fisiología , Mesodermo/citología , Factor de Crecimiento Transformador beta2/fisiología , Microambiente Tumoral , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Neoplasias Esofágicas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta2/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Squamous esophageal epithelium adapts to acid reflux-mediated injury by proliferation and differentiation via signal transduction pathways. Induction of the Wnt antagonist Dickkopf-1 (Dkk1) is involved in tissue repair during inflammation and cellular injury. In this study, we aimed to identify the biological role of Dkk1 in human reflux esophagitis with respect to cell growth and regulation of Wnt signaling. Esophageal biopsies from reflux-esophagitis patients (n = 15) and healthy individuals (n = 10) were characterized in terms of Dkk1 expression. The role of Dkk1 in response to acid-mediated epithelial injury was analyzed by cellular assays in vitro utilizing squamous esophageal epithelial cell lines (EPC1-hTERT, EPC2-hTERT, and HEEC). Dkk1 was significantly overexpressed in human reflux-esophagitis tissue compared with healthy esophageal mucosa at transcriptional and translational levels. After acute and chronic acid (pH 4) exposure, esophageal squamous epithelial cell lines expressed and secreted high levels of Dkk1 in response to stress-associated DNA injury. High extracellular levels of human recombinant Dkk1 inhibited epithelial cell growth and induced cellular senescence in vitro, as demonstrated by reduced cell proliferation, G0/G1 cell cycle arrest, elevated senescence-associated ß-galactosidase activity, and upregulation of p16. Acid pulsing induced Dkk1-mediated senescence, which was directly linked to the ability of Dkk1 to antagonize the canonical Wnt/ß-catenin signaling. In healthy esophageal mucosa, Dkk1 expression was associated with low expression of transcriptionally active ß-catenin, while in reflux-esophagitis tissue, Dkk1 overexpression correlated with increased senescence-associated ß-galactosidase activity and p16 upregulation. The data indicate that, in human reflux esophagitis, Dkk1 functions as a secreted growth inhibitor by suppressing Wnt/ß-catenin signaling and promoting cellular senescence. These findings suggest a significant role for Dkk1 and cellular senescence in esophageal tissue homeostasis during reflux esophagitis.
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Senescencia Celular , Células Epiteliales/metabolismo , Esofagitis Péptica/metabolismo , Esófago/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vía de Señalización Wnt , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Puntos de Control del Ciclo Celular , Línea Celular , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Epiteliales/patología , Esofagitis Péptica/genética , Esofagitis Péptica/patología , Esófago/patología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Membrana Mucosa/patología , Interferencia de ARN , Factores de Tiempo , Transfección , Regulación hacia Arriba , Adulto Joven , beta Catenina/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
AIM: To elucidate the signaling mechanisms involved in the protective effect of EUK-207 against irradiation-induced cellular damage and apoptosis in human intestinal microvasculature endothelial cells (HIMEC). METHODS: HIMECs were irradiated and treated with EUK-207. Using hydroethidine and DCF-DA fluorescent probe the intracellular superoxide and reactive oxygen species (ROS) were determined. By real-time PCR and western blotting caspase-3, Bcl2 and Bax genes and proteins were analyzed. Proliferation was determined by [(3)H]-thymidine uptake. Immunofluorescence staining was used for translocation of p65 NFκB subunit. KEY FINDING: Irradiation increased ROS production, apoptosis, Bax, Caspase3 and NFkB activity in HIMEC and inhibited cell survival/growth/proliferation. EUK-207 restored the endothelial functions, markedly inhibited the ROS, up-regulated the Bcl2 and down-regulated Bax and prevented NFκB caspase 3 activity in HIMEC. SIGNIFICANCE: HIMEC provide a novel model to define the effect of irradiation induced endothelial dysfunction. Our findings suggest that EUK-207 effectively inhibits the damaging effect of irradiation.
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Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Depuradores de Radicales Libres/farmacología , Compuestos Organometálicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de la radiación , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Intestinos/irrigación sanguínea , L-Lactato Deshidrogenasa/metabolismo , Malondialdehído/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de la radiación , Superóxido Dismutasa/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Signals from the intestinal microbiota are important for normal host physiology; alteration of the microbiota (dysbiosis) is associated with multiple disease states. We determined the effect of antibiotic-induced intestinal dysbiosis on circulating cytokine levels and severity of ischemia/reperfusion injury in the heart. Treatment of Dahl S rats with a minimally absorbed antibiotic vancomycin, in the drinking water, decreased circulating leptin levels by 38%, resulted in smaller myocardial infarcts (27% reduction), and improved recovery of postischemic mechanical function (35%) as compared with untreated controls. Vancomycin altered the abundance of intestinal bacteria and fungi, measured by 16S and 18S ribosomal DNA quantity. Pretreatment with leptin (0.12 µg/kg i.v.) 24 h before ischemia/reperfusion abolished cardioprotection produced by vancomycin treatment. Dahl S rats fed the commercially available probiotic product Goodbelly, which contains the leptin-suppressing bacteria Lactobacillus plantarum 299v, also resulted in decreased circulating leptin levels by 41%, smaller myocardial infarcts (29% reduction), and greater recovery of postischemic mechanical function (23%). Pretreatment with leptin (0.12 µg/kg i.v.) abolished cardioprotection produced by Goodbelly. This proof-of-concept study is the first to identify a mechanistic link between changes in intestinal microbiota and myocardial infarction and demonstrates that a probiotic supplement can reduce myocardial infarct size.
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Intestinos/microbiología , Metagenoma/efectos de los fármacos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Animales , Antibacterianos/farmacología , Citocinas/sangre , Agua Potable , Humanos , Intestinos/efectos de los fármacos , Leptina/sangre , Leptina/farmacología , Daño por Reperfusión Miocárdica/fisiopatología , Probióticos/uso terapéutico , Ratas , Ratas Endogámicas Dahl , Vancomicina/farmacologíaRESUMEN
Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an antiangiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used human intestinal microvascular endothelial cells (HIMEC) to define effects of radiation on these gut-specific endothelial cells. Low-level irradiation (1-5 Gy) activates NF-kappaB and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in cell cycle reentry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to endothelial proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radiosensitizing effects of curcumin on HIMEC subjected to low levels of irradiation. We have demonstrated that exposure of HIMEC to low levels of irradiation induced Akt and mTOR phosphorylation, which was attenuated by curcumin, rapamycin, LY294002, and mTOR small interference RNA (siRNA). Activation of NF-kappaB by low levels of irradiation was inhibited by curcumin, SN-50, and mTOR siRNA. Curcumin also induced apoptosis by induction of caspase-3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NF-kappaB and attenuated the effect of irradiation-induced prosurvival signaling through the PI3K/Akt/mTOR and NF-kappaB pathways in these gut-specific endothelial cells. Curcumin may be a potential radiosensitizing agent for enhanced antiangiogenic effect in colorectal cancer radiation therapy.
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Curcumina/farmacología , Células Endoteliales/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Anomalías Inducidas por Radiación/tratamiento farmacológico , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de la radiación , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Intestinos/irrigación sanguínea , Intestinos/efectos de los fármacos , Intestinos/efectos de la radiación , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Microvasos/citología , FN-kappa B/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Serina-Treonina Quinasas TORRESUMEN
The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-alpha but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-alpha/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-alpha/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-kappaB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-alpha/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-kappaB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD.
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Inhibidores de la Angiogénesis/farmacología , Enfermedad de Crohn/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Talidomida/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Enfermedad de Crohn/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Selectina E/metabolismo , Células Endoteliales/citología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Intestinos/irrigación sanguínea , Leucocitos/citología , Lipopolisacáridos/farmacología , Microvasos/citología , FN-kappa B/metabolismo , Neovascularización Patológica/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Endothelial activation and surface expression of cell adhesion molecules (CAMs) is critical for binding and recruitment of circulating leukocytes in tissues during the inflammatory response. Endothelial CAM expression plays a critical role in the intestinal microvasculature in inflammatory bowel disease (IBD), as blockade of leukocyte alpha4-integrin binding by gut endothelial CAM ligands has therapeutic benefit in IBD. Mechanisms underlying expression of vascular cell adhesion molecule (VCAM)-1, a ligand for alpha4-integrin in primary cultures of human intestinal microvascular endothelial cells (HIMEC) has not been defined. We investigated the effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) inhibitors on VCAM-1 expression and function in HIMEC. CAM expression was assessed and HIMEC-leukocyte adhesion was visualized under static and flow conditions. Western blotting and in vitro kinase assays were used to assess Akt and MAPK activation. Nuclear factor-kappaB (NF-kappaB) activation and nuclear translocation of its p65 subunit were determined. Tumor necrosis factor (TNF)-alpha/lipopolysaccharide (LPS)-induced VCAM-1 expression in HIMEC was suppressed by Akt small-interfering RNA, curcumin, and inhibitors of NF-kappaB (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-294002). VCAM-1 induction was partially suppressed by p44/42 MAPK (PD-098059) but unaffected by c-Jun NH2-terminal kinase (SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-kappaB activity and prevented nuclear translocation of the p65 NF-kappaB subunit following TNF-alpha/LPS. At physiological shear stress, curcumin attenuated leukocyte adhesion to TNF-alpha/LPS-activated HIMEC monolayers. In conclusion, curcumin inhibited the expression of VCAM-1 in HIMECs through blockade of Akt, p38 MAPK, and NF-kappaB. Curcumin may represent a novel therapeutic agent targeting endothelial activation in IBD.
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Curcumina/farmacología , Células Endoteliales/efectos de los fármacos , Fármacos Gastrointestinales/farmacología , Intestinos/irrigación sanguínea , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular , Células Cultivadas , Células Endoteliales/enzimología , Humanos , Inmunoglobulinas/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Microvasos/efectos de los fármacos , Microvasos/enzimología , Mucoproteínas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The molecular mechanisms governing the biology and pathobiology of esophageal squamous mucosa in health and disease are not completely understood. Earlier genome-wide expression study of normal-looking esophageal squamous mucosa has shown differential expression of the Wingless-type MMTV integration site family (Wnt) modulators Dickkopf (Dkk) homologs among healthy individuals and patients with reflux esophagitis and Barrett metaplasia suggesting that the Wnt pathway may be involved in esophageal mucosal biology. STUDY: Seven full-thickness human donor esophagi were cryosectioned for immunohistochemical analysis, and lamina propria (LP), basal (BC), intermediate (IC), and superficial (SC) cells were also dissected by laser-capture microdissection for real-time polymerase chain reaction. RESULTS: Wnt1, 2b, and 3a were expressed primarily in BC, Wnt3, and 5b in LP, and Wnt5a in IC. Frizzled 1, low-density lipoprotein receptor-related protein 6, secreted frizzled-related protein 1, T-cell-specific transcription factor 3, and dishevelled 3 were expressed highest in LP decreasing precipitously medially toward SC. Dkk1 predominantly expressed in SC was more than 100-folds greater than other layers (P<0.001). Dkk4 was expressed primarily in SC but Dkk3 was opposite with greatest expression in LP. Immunohistochemical analysis showed Wnt1 and 3a in BC, Wnt5a in IC and SC, Dkk1 predominantly in SC, Dkk4 in SC and IC, and Dkk3 and SFRP1 in LP and BC
Asunto(s)
Epitelio/metabolismo , Esófago/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Membrana Mucosa/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Quimiocinas , Esófago/citología , Receptores Frizzled/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Rayos Láser , Ligandos , Proteínas de la Membrana/metabolismo , Microdisección , Reacción en Cadena de la Polimerasa , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Human esophageal epithelial cells play a key role in esophageal inflammation in response to acidic pH during gastroesophageal reflux disease (GERD), increasing secretion of IL-6 and IL-8. The mechanisms underlying IL-6 and IL-8 expression and secretion in esophageal epithelial cells after acid stimulation are not well characterized. We investigated the role of PKC, MAPK, and NF-kappaB signaling pathways and transcriptional regulation of IL-6 and IL-8 expression in HET-1A cells exposed to acid. Exposure of HET-1A cells to pH 4.5 induced NF-kappaB activity and enhanced IL-6 and IL-8 secretion and mRNA and protein expression. Acid stimulation of HET-1A cells also resulted in activation of MAPKs and PKC (alpha and epsilon). Curcumin, as well as inhibitors of NF-kappaB (SN-50), PKC (chelerythrine), and p44/42 MAPK (PD-098059) abolished the acid-induced expression of IL-6 and IL-8. The JNK inhibitor SP-600125 blocked expression/secretion of IL-6 but only partially attenuated IL-8 expression. The p38 MAPK inhibitor SB-203580 did not inhibit IL-6 expression but exerted a stronger inhibitory effect on IL-8 expression. Together, these data demonstrate that 1) acid is a potent inducer of IL-6 and IL-8 production in HET-1A cells; 2) MAPK and PKC signaling play a key regulatory role in acid-mediated IL-6 and IL-8 expression via NF-kappaB activation; and 3) the anti-inflammatory plant compound curcumin inhibits esophageal activation in response to acid. Thus IL-6 and IL-8 expression by acid may contribute to the pathobiology of mucosal injury in GERD, and inhibition of the NF-kappaB/proinflammatory cytokine pathways may emerge as important therapeutic targets for treatment of esophageal inflammation.
Asunto(s)
Antiinflamatorios/farmacología , Curcumina/farmacología , Células Epiteliales/efectos de los fármacos , Esófago/efectos de los fármacos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , Antracenos/farmacología , Benzofenantridinas/farmacología , Línea Celular , Activación Enzimática , Células Epiteliales/enzimología , Células Epiteliales/inmunología , Esófago/enzimología , Esófago/inmunología , Flavonoides/farmacología , Humanos , Concentración de Iones de Hidrógeno , Imidazoles/farmacología , Interleucina-6/genética , Interleucina-8/genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/enzimología , Membrana Mucosa/inmunología , FN-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Telomerasa/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Regulación hacia ArribaAsunto(s)
Enfermedades Inflamatorias del Intestino/fisiopatología , Neovascularización Patológica/fisiopatología , Enfermedades Vasculares/fisiopatología , Animales , Caveolina 1/fisiología , Modelos Animales de Enfermedad , Endotelio Vascular/fisiopatología , Humanos , Leucocitos/fisiología , Ratones , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Tissue remodeling and mesenchymal cell accumulation accompanies chronic inflammatory disorders involving joints, lung, vasculature, and bowel. Chronic inflammation may alter DNA-mismatch repair (MMR) systems in mesenchymal cells, but is not defined in Crohn's disease (CD) and its associated intestinal remodeling and stricture formation. We determined whether DNA-MMR alteration plays a role in the pathogenesis of CD tissue remodeling. Control and CD bowel tissues were used to generate primary cultures of muscularis mucosa myofibroblasts, which were assessed directly or following stimulation with TNF-alpha/LPS or H2O2. MutS homolog (MSH)2, MSH3, and MSH6 expression in tissues and myofibroblasts was determined. Immunohistochemical staining revealed an increased expression of MSH2 in CD muscularis mucosa and submucosal tissues compared with controls or uninvolved CD tissue, and MSH2 expression was increased in CD myofibroblasts compared with control cells. TNF-alpha/LPS and H2O2 further enhanced MSH2 expression in both control and CD cells, which were decreased by simvastatin. There were no significant changes in MSH3 and MSH6 expression. Proliferating cell nuclear antigen and Ki67 staining of CD tissue revealed increased proliferation in the muscularis mucosa and submucosa of chronically inflamed tissues, and enhanced proliferation was seen in CD myofibroblasts compared with controls. Simvastatin reversed the effects of inflammatory stress on the DNA-MMR and inhibited proliferation of control and CD myofibroblasts. Gene silencing with MSH2 siRNA selectively decreased CD myofibroblast proliferation. These data demonstrate a potential role for MSH2 in the pathogenesis of nonneoplastic mesenchymal cell accumulation and intestinal remodeling in CD chronic inflammation.
Asunto(s)
Proliferación Celular , Enfermedad de Crohn/enzimología , Fibroblastos/enzimología , Obstrucción Intestinal/etiología , Intestinos/enzimología , Proteína 2 Homóloga a MutS/metabolismo , Adulto , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Proteínas de Unión al ADN/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Humanos , Peróxido de Hidrógeno/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inmunohistoquímica , Obstrucción Intestinal/enzimología , Obstrucción Intestinal/genética , Obstrucción Intestinal/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Lipopolisacáridos/farmacología , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga de MutS , Antígeno Nuclear de Célula en Proliferación/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Simvastatina/farmacología , Timidina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
AIMS: Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart. METHODS AND RESULTS: We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 microg/kg iv in vivo. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial K(ATP) channels (5-HD), and sarcolemmal K(ATP) channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 microg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period. CONCLUSION: A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and K(ATP) channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.
Asunto(s)
Apoptosis/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Aturdimiento Miocárdico/tratamiento farmacológico , Trombopoyetina/uso terapéutico , Animales , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Técnicas In Vitro , Janus Quinasa 2/fisiología , Canales KATP/fisiología , Masculino , Infarto del Miocardio/patología , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/fisiología , Trombopoyetina/farmacología , Factores de TiempoRESUMEN
Nitric oxide (.NO) generation from conversion of l-arginine to citrulline by nitric oxide synthase isoforms plays a critical role in vascular homeostasis. Loss of .NO is linked to vascular pathophysiology and is decreased in chronically inflamed gut blood vessels in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). Mechanisms underlying decreased .NO production in IBD gut microvessels are not fully characterized. Loss of .NO generation may result from increased arginase (AR) activity, which enzymatically competes with nitric oxide synthase for the common substrate l-arginine. We characterized AR expression in IBD microvessels and endothelial cells and its contribution to decreased .NO production. AR expression was assessed in resected gut tissues and human intestinal microvascular endothelial cells (HIMEC). AR expression significantly increased in both ulcerative colitis and Crohn's disease microvessels and submucosal tissues compared with normal. TNF-alpha/lipopolysaccharide increased AR activity, mRNA and protein expression in HIMEC in a time-dependent fashion. RhoA/ROCK pathway, a negative regulator of .NO generation in endothelial cells, was examined. The RhoA inhibitor C3 exoenzyme and the ROCK inhibitor Y-27632 both attenuated TNF-alpha/lipopolysaccharide-induced MAPK activation and blocked AR expression in HIMEC. A significantly higher AR activity and increased RhoA activity were observed in IBD submucosal tissues surrounding microvessels compared with normal control gut tissue. Functionally, inhibition of AR activity decreased leukocyte binding to HIMEC in an adhesion assay. Loss of .NO production in IBD microvessels is linked to enhanced levels of AR in intestinal endothelial cells exposed to chronic inflammation in vivo.
Asunto(s)
Arginasa/metabolismo , Endotelio/fisiopatología , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/enzimología , ADP Ribosa Transferasas/farmacología , Amidas/farmacología , Arginina/farmacología , Toxinas Botulínicas/farmacología , Moléculas de Adhesión Celular/fisiología , Enfermedad de Crohn/fisiopatología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Óxido Nítrico/biosíntesis , Proteínas Serina-Treonina Quinasas/fisiología , Piridinas/farmacología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Valina/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
OBJECTIVES: Esophageal mucosal response to acid exposure varies from minimal changes to erosions/ulcerations and Barrett's metaplasia. While differences in acid contact time have been suggested, the reason for these different responses is not completely understood. The aim of this study was to identify and compare gene expression differences between normal distal and proximal squamous esophageal mucosa (SM) in esophagitis patients with that of healthy controls and Barrett's patients. METHODS: Gene microarray was performed on laser-capture microdissected epithelial cells isolated from biopsy specimens followed by real-time PCR. The effect of acidic pH (pH 4.5) on Dickkopf Homolog 1 (Dkk-1) expression in the human esophageal epithelial cell line (Het-1A) was determined. RESULTS: Gene microarray analysis demonstrated that the upregulation of five genes in the distal compared with the proximal SM in esophagitis patients was greater than the healthy controls and Barrett's patients. Overexpression of Dkk-1 and Dkk-4 was further confirmed by real-time PCR. Dkk-1 and Dkk-4 mRNA levels in the distal SM of the esophagitis patients were 7.0- and 3.1-fold higher, respectively, than in the distal SM of the Barrett's patients and 4.1- and 4.1-fold higher than in healthy controls, respectively. Dkk-1 protein expression in the distal esophagitis SM was also higher than the Barrett's patients and healthy controls. Acidic pH exposure of Het-1A cells resulted in Dkk-1 upregulation at the level of both mRNA and protein. CONCLUSIONS: Dkk-1 and Dkk-4 may potentially be involved in the development of different injuries in response to pathological gastroesophageal acid reflux.
Asunto(s)
Esófago de Barrett/genética , Esofagitis/genética , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Adolescente , Adulto , Análisis de Varianza , Western Blotting , Estudios de Casos y Controles , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration).
Asunto(s)
Células Endoteliales/metabolismo , Esófago/irrigación sanguínea , Proteínas del Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Supervivencia Celular/fisiología , Fosfatidilinositol 3-Quinasa Clase I , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Esófago/citología , Regulación de la Expresión Génica/genética , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP27 , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Concentración de Iones de Hidrógeno , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Fosforilación , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Transducción de Señal/fisiología , Porcinos , Factores de Transcripción/metabolismoRESUMEN
Vascular cell adhesion molecule-1 (VCAM-1), a key receptor for the leukocyte-associated integrin (VLA4), is a crucial mediator of leukocyte adhesion and has co-stimulatory functions in inflammation at various organ sites. Specifically, VCAM-1/VLA4 interactions have been shown to play important roles in the setting of cutaneous immune responses, such as psoriatic lesions in humans and acute Graft-versus-Host-Disease in mice. VCAM-1 is generally expressed on activated endothelial cells in inflamed tissues, mediating endothelium-leukocyte interactions, leading to leukocyte diapedesis to the site of inflammation. We report novel and unexpected membrane expression of VCAM-1 in the basal squamous epithelial strata of the normal human esophagus and distinct patterns of epithelial expression in esophageal pathology. To further delineate the differential expression patterns of VCAM-1 in the esophageal epithelium, we examined specimens from squamous cell carcinoma (SCC), adenocarcinoma, and Barrett's columnar cell metaplasia. VCAM-1 was strongly expressed in squamous cell carcinoma, but not adenocarcinoma nor columnar epithelia in Barrett's esophagus. VCAM-1 expression was focally accentuated at sites characteristic of microscopic tumor invasion in SCC, pointing to a potential role of VCAM-1 in the development of metastasis. In addition, in vitro immunofluorescence studies using OE21 cells, an esophageal squamous epithelial cell line, displayed distinct VCAM-1 immunoreactivity confined to mitotic and dividing cells. Cell cycle arrest caused a significant decrease in VCAM-1 immunoreactivity in OE21 cells. These data suggest a previously unappreciated role for VCAM-1 in esophageal squamous epithelial homeostasis and pathology.