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1.
J Biomed Sci ; 31(1): 12, 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38254097

RESUMEN

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.


Asunto(s)
Cicatriz Hipertrófica , Queloide , Factor de Crecimiento Transformador beta , Animales , Humanos , Ratones , Antígenos CD , Antígenos de Neoplasias , Cicatriz Hipertrófica/metabolismo , Fibroblastos , Queloide/metabolismo , Piel
3.
J Biomed Sci ; 26(1): 60, 2019 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-31451113

RESUMEN

BACKGROUND: Thrombomodulin (TM), an integral membrane protein, has long been known for its anticoagulant activity. Recent studies showed that TM displays multifaceted activities, including the involvement in cell adhesion and collective cell migration in vitro. However, whether TM contributes similarly to these biological processes in vivo remains elusive. METHODS: We adapted zebrafish, a prominent animal model for studying molecular/cellular activity, embryonic development, diseases mechanism and drug discovery, to examine how TM functions in modulating cell migration during germ layer formation, a normal and crucial physiological process involving massive cell movement in the very early stages of life. In addition, an in vivo assay was developed to examine the anti-hemostatic activity of TM in zebrafish larva. RESULTS: We found that zebrafish TM-b, a zebrafish TM-like protein, was expressed mainly in vasculatures and displayed anti-hemostatic activity. Knocking-down TM-b led to malformation of multiple organs, including vessels, heart, blood cells and neural tissues. Delayed epiboly and incoherent movement of yolk syncytial layer were also observed in early TM-b morphants. Whole mount immunostaining revealed the co-localization of TM-b with both actin and microtubules in epibolic blastomeres. Single-cell tracking revealed impeded migration of blastomeres during epiboly in TM-b-deficient embryos. CONCLUSION: Our results showed that TM-b is crucial to the collective migration of blastomeres during germ layer formation. The structural and functional compatibility and conservation between zebrafish TM-b and mammalian TM support the properness of using zebrafish as an in vivo platform for studying the biological significance and medical use of TM.


Asunto(s)
Estratos Germinativos/embriología , Morfogénesis , Organogénesis , Trombomodulina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Blastómeros/metabolismo , Embrión no Mamífero/embriología , Trombomodulina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
4.
J Biomed Sci ; 25(1): 14, 2018 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-29439742

RESUMEN

BACKGROUND: Thrombomodulin (TM), a transmembrane glycoprotein highly expressed in endothelial cells (ECs), is a potent anticoagulant maintaining circulation homeostasis. Under inflammatory states, TM expression is drastically reduced in ECs while vascular smooth muscle cells (VSMCs) show a robust expression of TM. The functional role of TM in VSMCs remains elusive. METHODS: We examined the role of TM in VSMCs activities in human aortic VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB). Using rat embryonic aorta-derived A7r5 VSMCs which do not express TM, the role of the chondroitin sulfate (CS) moiety of TM in VSMCs was delineated with cells expressing wild-type TM and the CS-devoid TM mutant. RESULTS: Expression of TM enhanced cell migration and adhesion/spreading onto type I collagen, but had no effect on cell proliferation. Knocking down TM with short hairpin RNA reduced PDGF-stimulated adhesion and migration of human aortic VSMCs. In A7r5 cells, TM-mediated cell adhesion was eradicated by pretreatment with chondroitinase ABC which degrades CS moiety. Furthermore, the TM mutant (TMS490, 492A) devoid of CS moiety failed to increase cell adhesion, spreading or migration. Wild-type TM, but not TMS490, 492A, increased focal adhesion kinase (FAK) activation during cell adhesion, and TM-enhanced cell migration was abolished by a function-blocking anti-integrin ß1 antibody. CONCLUSION: Chondroitin sulfate modification is required for TM-mediated activation of ß1-integrin and FAK, thereby enhancing adhesion and migration activity of VSMCs.


Asunto(s)
Adhesión Celular , Movimiento Celular , Sulfatos de Condroitina/química , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Trombomodulina/genética , Células Cultivadas , Humanos , Trombomodulina/metabolismo
5.
Immunol Cell Biol ; 95(4): 372-379, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-27808085

RESUMEN

The leukocyte adhesion cascade involves multiple events that efficiently localize circulating leukocytes into the injured sites to mediate inflammatory responses. From rolling to firm adhesion, the interactions between adhesion molecules have pivotal roles in increasing the avidity of leukocytes to endothelial cells. Thrombomodulin (TM), an essential anticoagulant protein in the vasculature, is also expressed on leukocytes. We previously demonstrated that Lewisy (Ley), a specific ligand of TM, is upregulated in inflamed endothelium and is involved in leukocyte adhesion. The current study aimed to investigate whether leukocyte-expressed TM promotes cell adhesion by interacting with Ley. Using human monocytic THP-1 cells as an in vitro cell model, we showed that TM increases THP-1 cell adhesion to inflamed endothelium as well as to Ley-immobilized surface. When THP-1 adhered to activated endothelium and Ley-immobilized surface, the TM distribution became polarized. Addition of soluble Ley to a suspension of THP-1 cells with TM expression triggered an increase in the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which enabled THP-1 to adhere firmly to intercellular adhesion molecule (ICAM)-1 by activating ß2 integrins. In vivo, macrophage infiltration and neointima formation following arterial ligation-induced vascular injury were higher in wild-type TM (TMflox/flox) than in myeloid-specific TM-deficient (LysMcre/TMflox/flox) mice. Taken together, these results suggest a novel function for TM as an adhesion molecule in monocytes, where it enhances cell adhesion by binding Ley, leading to ß2 integrin activation via p38 MAPK.


Asunto(s)
Células Endoteliales/inmunología , Inflamación/inmunología , Monocitos/inmunología , Neointima/inmunología , Trombomodulina/metabolismo , Animales , Antígenos CD18/metabolismo , Adhesión Celular , Modelos Animales de Enfermedad , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Ligandos , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Transducción de Señal , Células THP-1 , Trombomodulina/agonistas , Trombomodulina/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
6.
J Immunol ; 194(4): 1905-15, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25609841

RESUMEN

CD14, a multiligand pattern-recognition receptor, is involved in the activation of many TLRs. Thrombomodulin (TM), a type I transmembrane glycoprotein, originally was identified as an anticoagulant factor that activates protein C. Previously, we showed that the recombinant TM lectin-like domain binds to LPS and inhibits LPS-induced inflammation, but the function of the recombinant epidermal growth factor-like domain plus serine/threonine-rich domain of TM (rTMD23) in LPS-induced inflammation remains unknown. In the current study, we found that rTMD23 markedly suppressed the activation of intracellular signaling pathways and the production of inflammatory cytokines induced by LPS. The anti-inflammatory activity of rTMD23 was independent of activated protein C. We also found that rTMD23 interacted with the soluble and membrane forms of CD14 and inhibited the CD14-mediated inflammatory response. Knockdown of CD14 in macrophages suppressed the production of inflammatory cytokines induced by LPS, and rTMD23 inhibited LPS-induced IL-6 production in CD14-knockdown macrophages. rTMD23 suppressed the binding of LPS to macrophages by blocking the association between monocytic membrane-bound TM and CD14. The administration of rTMD23 in mice, both pretreatment and posttreatment, significantly increased the survival rate and reduced the inflammatory response to LPS. Notably, the serine/threonine-rich domain is essential for the anti-inflammatory activity of rTMD23. To summarize, we show that rTMD23 suppresses the LPS-induced inflammatory response in mice by targeting CD14 and that the serine/threonine-rich domain is crucial for the inhibitory effect of rTMD23 on LPS-induced inflammation.


Asunto(s)
Inflamación/inmunología , Receptores de Lipopolisacáridos/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Trombomodulina/inmunología , Animales , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Estructura Terciaria de Proteína , Proteínas Recombinantes/inmunología , Resonancia por Plasmón de Superficie , Venas Umbilicales
7.
Arterioscler Thromb Vasc Biol ; 35(11): 2412-22, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26338301

RESUMEN

OBJECTIVE: Thrombomodulin (TM), a glycoprotein constitutively expressed in the endothelium, is well known for its anticoagulant and anti-inflammatory properties. Paradoxically, we recently found that monocytic membrane-bound TM (ie, endogenous TM expression in monocytes) triggers lipopolysaccharide- and gram-negative bacteria-induced inflammatory responses. However, the significance of membrane-bound TM in chronic sterile vascular inflammation and the development of abdominal aortic aneurysm (AAA) remains undetermined. APPROACH AND RESULTS: Implicating a potential role for membrane-bound TM in AAA, we found that TM signals were predominantly localized to macrophages and vascular smooth muscle cells in human aneurysm specimens. Characterization of the CaCl2-induced AAA in mice revealed that during aneurysm development, TM expression was mainly localized in infiltrating macrophages and vascular smooth muscle cells. To investigate the function of membrane-bound TM in vivo, transgenic mice with myeloid- (LysMcre/TM(flox/flox)) and vascular smooth muscle cell-specific (SM22-cre(tg)/TM(flox/flox)) TM ablation and their respective wild-type controls (TM(flox/flox) and SM22-cre(tg)/TM(+/+)) were generated. In the mouse CaCl2-induced AAA model, deficiency of myeloid TM, but not vascular smooth muscle cell TM, inhibited macrophage accumulation, attenuated proinflammatory cytokine and matrix metalloproteinase-9 production, and finally mitigated elastin destruction and aortic dilatation. In vitro TM-deficient monocytes/macrophages, versus TM wild-type counterparts, exhibited attenuation of proinflammatory mediator expression, adhesion to endothelial cells, and generation of reactive oxygen species. Consistently, myeloid TM-deficient hyperlipidemic mice (ApoE(-/-)/LysMcre/TM(flox/flox)) were resistant to AAA formation induced by angiotensin II infusion, along with reduced macrophage infiltration, suppressed matrix metalloproteinase activities, and diminished oxidative stress. CONCLUSIONS: Membrane-bound TM in macrophages plays an essential role in the development of AAA by enhancing proinflammatory mediator elaboration, macrophage recruitment, and oxidative stress.


Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Aortitis/metabolismo , Membrana Celular/metabolismo , Macrófagos Peritoneales/metabolismo , Trombomodulina/metabolismo , Angiotensina II , Animales , Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/inmunología , Aortitis/inducido químicamente , Aortitis/genética , Aortitis/inmunología , Cloruro de Calcio , Membrana Celular/inmunología , Células Cultivadas , Quimiotaxis , Modelos Animales de Enfermedad , Elastina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Interferencia de ARN , Estudios Retrospectivos , Transducción de Señal , Trombomodulina/deficiencia , Trombomodulina/genética , Factores de Tiempo , Transfección
8.
Diabetologia ; 57(2): 424-34, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24317792

RESUMEN

AIMS/HYPOTHESIS: Chronic inflammatory processes have been increasingly shown to be involved in the pathogenesis of diabetes and diabetic nephropathy. Recently, we demonstrated that a lectin-like domain of thrombomodulin (THBD), which is known as THBD domain 1 (THBDD1) and which acts independently of protein C activation, neutralised an inflammatory response in a mouse model of sepsis. Here, therapeutic effects of gene therapy with adeno-associated virus (AAV)-carried THBDD1 (AAV-THBDD1) were tested in a mouse model of type 2 diabetic nephropathy. METHODS: To assess the therapeutic potential of THBDD1 and the mechanisms involved, we delivered AAV-THBDD1 (10(11) genome copies) into db/db mice and tested the effects of recombinant THBDD1 on conditionally immortalised podocytes. RESULTS: A single dose of AAV-THBDD1 improved albuminuria, renal interstitial inflammation and glomerular sclerosis, as well as renal function in db/db mice. These effects were closely associated with: (1) inhibited activation of the nuclear factor κB (NF-κB) pathway and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome; (2) promotion of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation; and (3) suppression of mitochondria-derived apoptosis in the kidney of treated mice. CONCLUSIONS/INTERPRETATION: AAV-THBDD1 gene therapy resulted in improvements in a model of diabetic nephropathy by suppressing the NF-κB-NLRP3 inflammasome-mediated inflammatory process, enhancing the NRF2 antioxidant pathway and inhibiting apoptosis in the kidney.


Asunto(s)
Antioxidantes/farmacología , Proteínas Portadoras/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/metabolismo , Terapia Genética , Inflamasomas/metabolismo , FN-kappa B/metabolismo , Trombomodulina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/inmunología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/inmunología , Terapia Genética/métodos , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR
9.
Blood ; 119(5): 1302-13, 2012 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-22101897

RESUMEN

Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM-TM domain 1 (rTMD1)-in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.


Asunto(s)
Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/farmacología , Trombomodulina/química , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/patología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Células Endoteliales de la Vena Umbilical Humana/trasplante , Humanos , Lectinas/química , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Trombomodulina/metabolismo , Trombomodulina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto
10.
FASEB J ; 27(11): 4520-31, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23943648

RESUMEN

Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a KD of 0.1-0.3 µM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.


Asunto(s)
Neovascularización Fisiológica , Plasminógeno/metabolismo , Trombomodulina/metabolismo , Animales , Células CHO , Movimiento Celular , Cricetinae , Cricetulus , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Microdominios de Membrana/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteolisis , Piel/irrigación sanguínea , Trombomodulina/química , Trombomodulina/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Cicatrización de Heridas
11.
Arterioscler Thromb Vasc Biol ; 33(10): 2366-73, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23950139

RESUMEN

OBJECTIVE: The N-terminal lectin-like domain (domain 1 [D1]) of thrombomodulin (TM) is known to have an anti-inflammatory function. We previously showed that recombinant TM domain 1 (rTMD1) interacts with a carbohydrate molecule, Lewis Y (Le(y)), which is found to be expressed on adhesion molecules and involved in cell adhesion. Here, we tested the effect of rTMD1/Le(y) interaction on leukocyte recruitment in inflammation. APPROACH AND RESULTS: The expression of Le(y) on the surface of human umbilical vein endothelial cells was increased by tumor necrosis factor-α stimulation. Direct binding of rTMD1 to Le(y) on the cell surface was observed. rTMD1 inhibited Le(y)-mediated leukocyte adhesion on the Le(y)-immobilized flow chamber and activated endothelium under a shear flow. The following leukocyte transmigration to endothelium was also reduced by rTMD1 through binding Le(y). In vivo, treatment of rTMD1 reduced leukocyte recruitment to the inflammatory sites in carotid ligation injury and thioglycollate-induced peritonitis. rTMD1 administration in apolipoprotein E-deficient mice effectively suppressed atherosclerotic plaque formation and macrophage infiltration in atherosclerotic lesions. Increased Le(y) expression, as well as administered rTMD1, was observed in inflamed vessels. CONCLUSIONS: rTMD1 suppresses vascular inflammation by inhibiting leukocyte recruitment to endothelium through attenuating Le(y)-mediated adhesion and further protects against atherosclerosis progression. The present study provides a mechanism showing that rTMD1 can inhibit inflammation by binding to its carbohydrate ligand Le(y).


Asunto(s)
Antiinflamatorios/administración & dosificación , Aterosclerosis/prevención & control , Quimiotaxis de Leucocito/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Neutrófilos/efectos de los fármacos , Trombomodulina/administración & dosificación , Vasculitis/prevención & control , Animales , Antiinflamatorios/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Placa Aterosclerótica , Estructura Terciaria de Proteína , Proteínas Recombinantes/administración & dosificación , Trombomodulina/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Vasculitis/inmunología , Vasculitis/metabolismo , Vasculitis/patología
12.
J Immunol ; 188(12): 6328-37, 2012 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-22573811

RESUMEN

Sepsis results from the host hyperinflammatory response to bacterial infection, causing multiple organ failure and high mortality. We previously demonstrated that LPS binds to monocytic membrane-bound thrombomodulin (TM), but the role of monocytic TM in LPS-induced inflammation remains unknown. In this study, we demonstrated that TM knockdown in human monocytic cells attenuated LPS-induced signaling pathways and cytokine production. Coimmunoprecipitation and immunofluorescence assays showed that monocytic TM interacted with the LPS receptors, CD14 and TLR4/myeloid differentiation factor-2 (MD-2) complex, indicating that it binds to LPS and triggers an LPS-induced inflammatory response by interacting with the CD14/TLR4/MD-2 complex. We also found that monocytic TM knockdown reduced cytokine production induced by gram-negative bacteria Klebsiella pneumoniae, suggesting that monocytic TM plays an important role in gram-negative bacteria-induced inflammation. To further investigate the function of monocytic TM in vivo, myeloid-specific TM-deficient mice were established and were found to display improved survival that resulted from the attenuation of septic syndrome, including reduced systemic inflammatory response and resistance to bacterial dissemination, after K. pneumoniae infection or cecal ligation and puncture surgery. The inhibition of bacterial dissemination in mice with a deficiency of myeloid TM may be caused by the early increase in neutrophil infiltration. Therefore, we conclude that monocytic TM is a novel component in the CD14/TLR4/MD-2 complex and participates in the LPS- and gram-negative bacteria-induced inflammatory response.


Asunto(s)
Inflamación/inmunología , Monocitos/inmunología , Sepsis/inmunología , Trombomodulina/inmunología , Animales , Línea Celular , Citocinas/biosíntesis , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Infecciones por Bacterias Gramnegativas/inmunología , Humanos , Inmunoprecipitación , Inflamación/metabolismo , Lipopolisacáridos/inmunología , Ratones , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis/metabolismo , Trombomodulina/metabolismo
13.
ScientificWorldJournal ; 2014: 902987, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24977233

RESUMEN

Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.


Asunto(s)
Anoicis , Receptores ErbB/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Serina Proteasas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Serina Endopeptidasas
14.
J Mol Cell Cardiol ; 63: 79-88, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23880609

RESUMEN

Kringle 1-5 (K1-5), an endogenous proteolytic fragment of human plasminogen (Plg), is an angiostatin-related protein that inhibits angiogenesis. Many angiostatin-related proteins have been identified, but the detailed molecular mechanisms underlying their antiangiogenic effects remain unclear. Thrombomodulin (TM) is a transmembrane glycoprotein that plays a major role in the anticoagulation process in endothelial cells. Previously, we demonstrated that recombinant TM could interact with Plg to enhance Plg activation. In the present study, we investigated the interaction between TM and K1-5, and their functions in endothelial cells. We found that K1-5 colocalized with TM and directly interacted with TM through the TM lectin-like domain. After K1-5 interacted with TM, it induced TM internalization and degradation. In addition, the K1-5-induced TM internalization and degradation in proteasomes after ubiquitin modification were dependent on protein kinase A (PKA). Moreover, a PKA-specific inhibitor reversed the effects of K1-5 on cell migration and tube formation. Consistent with these findings, TM overexpression resulted in increased cell migration; moreover, K1-5 inhibited the increase of TM-mediated cell migration in a PKA-dependent manner. We determined that TM acts as a K1-5 receptor and that K1-5 induces TM internalization, ubiquitination, and degradation through the PKA pathway, by which K1-5 may inhibit endothelial cell migration and tube formation.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Plasminógeno/farmacología , Trombomodulina/metabolismo , Animales , Bovinos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Células Endoteliales/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Plasminógeno/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Trombomodulina/química , Ubiquitinación/efectos de los fármacos
15.
J Biol Chem ; 287(19): 15739-48, 2012 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-22416129

RESUMEN

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.


Asunto(s)
Daboia/metabolismo , Coagulación Intravascular Diseminada/metabolismo , Heparina/farmacología , Inhibidor de Proteína C/farmacología , Proteína C/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Coagulación Intravascular Diseminada/inducido químicamente , Sinergismo Farmacológico , Humanos , Cinética , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Unión Proteica , Proteína C/metabolismo , Inhibidor de Proteína C/genética , Inhibidor de Proteína C/metabolismo , Daboia/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Venenos de Víboras/genética , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacología , Venenos de Víboras/envenenamiento
16.
Ann Surg ; 258(6): 1103-10, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23295319

RESUMEN

OBJECTIVE: To investigate whether recombinant thrombomodulin containing all the extracellular domains (rTMD123) has therapeutic potential against aneurysm development. SUMMARY BACKGROUND DATA: The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and proteolytic degradation of extracellular matrix. Thrombomodulin, a transmembrane glycoprotein, exerts anti-inflammatory activities such as inhibition of cytokine production and sequestration of proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that may sustain inflammation and tissue damage. METHODS: The in vivo effects of treatment and posttreatment with rTMD123 on aortic dilatation were measured using the CaCl2-induced AAA model in mice. RESULTS: Characterization of the CaCl2-induced model revealed that HMGB1 and RAGE, both localized mainly to macrophages, were persistently upregulated during a 28-day period of AAA development. In vitro, rTMD123-HMGB1 interaction prevented HMGB1 binding to macrophages, thereby prohibiting activation of HMGB1-RAGE signaling in macrophages. In vivo, short-term treatment with rTMD123 upon AAA induction suppressed the levels of proinflammatory cytokines, HMGB1, and RAGE in the aortic tissue; reduced the infiltrating macrophage number; and finally attenuated matrix metalloproteinase production, extracellular matrix destruction, and AAA formation without disturbing vascular calcification. Consistently, posttreatment with rTMD123 seven days after AAA induction alleviated vascular inflammation and retarded AAA progression. CONCLUSIONS: These data suggest that rTMD123 confers protection against AAA development. The mechanism of action may be associated with reduction of proinflammatory mediators, blockade of macrophage recruitment, and suppression of HMGB1-RAGE signaling involved in aneurysm formation and downstream macrophage activation.


Asunto(s)
Aneurisma de la Aorta Abdominal/prevención & control , Trombomodulina/uso terapéutico , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Cloruro de Calcio/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/fisiología , Proteínas Recombinantes/uso terapéutico
17.
Microcirculation ; 20(2): 190-201, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23140507

RESUMEN

OBJECTIVE: This study examines the effect of Dextromethorphan (d-3-methoxy-17-methylmorphinan; DXM), a commonly used cough-suppressing drug, on the expression of VCAM-1 and ICAM-1 in human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS). METHODS: The effect of DXM on expression of cell adhesion molecules induced by LPS was evaluated by monocyte bindings in vitro and ex vivo and transmigration assays. The signaling pathways involved in the inflammation inhibitory effect of DXM were analyzed by Western blot and immunofluorescent stain. RESULTS: Pretreatment of HUVECs with DXM inhibited LPS-induced adhesion of THP-1 cells in vitro and ex vivo, and reduced transendothelial migration of these cells. Furthermore, treatment of HUVECs with DXM can significantly decrease LPS-induced expression of ICAM-1 and VCAM-1. DXM abrogated LPS-induced phosphorylation of ERK and Akt. The translocation of early growth response gene-1 (Egr-1), a downstream transcription factor involved in the mitogen-activated kinase (MEK)-ERK signaling pathway, was suppressed by DXM treatment. Furthermore, DXM inhibited LPS-induced IκBα degradation and nuclear translocation of p65. CONCLUSIONS: Dextromethorphan inhibits the adhesive capacity of HUVECs by reducing the LPS-induced ICAM-1 and VCAM-1 expression via the suppression of the ERK, Akt, and NF-κB signaling pathways. Thus, DXM is a potential anti-inflammatory therapeutic that may modulate atherogenesis.


Asunto(s)
Dextrometorfano/farmacología , Células Endoteliales/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Antiinflamatorios/farmacología , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Interacciones Farmacológicas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
18.
Hepatology ; 56(5): 1913-23, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22505209

RESUMEN

UNLABELLED: The liver architecture plays an important role in maintaining hemodynamic balance, but the mechanisms that underlie this role are not fully understood. Hepsin, a type II transmembrane serine protease, is predominantly expressed in the liver, but has no known physiological functions. Here, we report that hemodynamic balance in the liver is regulated through hepsin. Deletion of hepsin (hepsin(-/-) ) in mice resulted in enlarged hepatocytes and narrowed liver sinusoids. Using fluorescent microbeads and antihepsin treatment, we demonstrated that metastatic cancer cells preferentially colonized the hepsin(-/-) mouse liver as a result of the retention of tumor cells because of narrower sinusoids. The enlarged hepatocytes expressed increased levels of connexin, which resulted from defective prohepatocyte growth factor (pro-HGF) processing and decreased c-Met phosphorylation in the livers of hepsin(-/-) mice. Treatment of hepsin(-/-) mice with recombinant HGF rescued these phenotypes, and treatment of wild-type mice with an HGF antagonist recapitulated the phenotypes observed in hepsin(-/-) mice. CONCLUSION: Our findings show that the maintenance of hepatic structural homeostasis occurs through HGF/c-Met/connexin signaling by hepsin, and hepsin-mediated changes in liver architecture significantly enhance tumor metastasis to the liver.


Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Hepatocitos/patología , Neoplasias Hepáticas/secundario , Hígado/metabolismo , Hígado/patología , Metástasis de la Neoplasia/patología , Serina Endopeptidasas/metabolismo , Animales , Conexinas/metabolismo , Hemodinámica , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Fosforilación , Proteínas Proto-Oncogénicas c-met/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal
19.
FASEB J ; 26(8): 3440-52, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22593542

RESUMEN

Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound healing. Thrombomodulin (TM) is an integral membrane protein that participates in cell-cell adhesion through its extracellular lectin-like domain. However, the molecular basis of TM-mediated cell-cell adhesion is poorly understood. Here, we demonstrate that TM is linked to the actin cytoskeleton via ezrin. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified (522)RKK(524) as important ezrin-binding residues. In epidermal epithelial A431 cells, TM colocalized with ezrin and actin filaments at cell-cell contacts. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, stimulated the interaction between ezrin and TM. In skin wound healing of mice, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient melanoma A2058 cells promoted collective cell migration. In summary, TM, which associates with ezrin and actin filaments, maintains epithelial morphology and promotes collective cell migration.


Asunto(s)
Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Trombomodulina/fisiología , Actinas/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Línea Celular , Células Epidérmicas , Factor de Crecimiento Epidérmico , Humanos , Ratones , Estructura Terciaria de Proteína , Trombomodulina/genética , Cicatrización de Heridas/fisiología
20.
Clin Sci (Lond) ; 123(3): 147-59, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22339730

RESUMEN

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.


Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Tetrazoles/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cilostazol , Colágeno/farmacología , Ensayo de Unidades Formadoras de Colonias , Combinación de Medicamentos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Isquemia/patología , Laminina/farmacología , Ratones , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/efectos de los fármacos , Proteoglicanos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Regulación hacia Arriba/efectos de los fármacos
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