RESUMEN
WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Neovascularización Patológica/inducido químicamente , Proteína wnt2/metabolismo , Proteína wnt2/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Microambiente Tumoral/fisiologíaRESUMEN
OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.
Asunto(s)
Proteínas CCN de Señalización Intercelular/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Proteínas Proto-Oncogénicas/metabolismo , Proteína wnt2/metabolismo , Animales , Proteínas CCN de Señalización Intercelular/deficiencia , Proteínas CCN de Señalización Intercelular/genética , Proteínas CCN de Señalización Intercelular/farmacología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Fenotipo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Interferencia de ARN , Proteínas Recombinantes/farmacología , Factores de Transcripción TCF/metabolismo , Transfección , Vía de Señalización Wnt , Proteína wnt2/deficiencia , Proteína wnt2/genética , Proteína wnt2/farmacología , beta Catenina/metabolismoRESUMEN
BACKGROUND: No substantial work has been conducted to date in context to cationic proteins with antilithiatic activity. We explored the antilithiatic cationic proteins present in human calcium oxalate (CaOx) stones and also examined their molecular interactions with calcium oxalate crystals in silico. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to cation exchange chromatography followed by molecular-sieve chromatography. The effect of these purified cationic proteins was tested against CaOx nucleation and growth and on oxalate injured MDCK cells for their activity. Proteins were identified by MALDI-TOF MS. Molecular interaction studies with COM crystals in silico were also investigated. RESULTS: Three antilithiatic cationic proteins were identified as histone-lysine N-methyltransferase, inward rectifier K channel and protein Wnt-2 (MW~53, ~44, and ~42 kDa respectively) by MALDI-TOF MS based on database search with MASCOT server. Further molecular modeling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSION: We identified histone-lysine N-methyltransferase, inward rectifier K channel and protein Wnt-2 as novel antilithiatic proteins which play a vital role in the kidney function and have been associated with various kidney diseases.
Asunto(s)
Oxalato de Calcio/química , N-Metiltransferasa de Histona-Lisina/química , Cálculos Renales/química , Canales de Potasio de Rectificación Interna/química , Proteína wnt2/química , Adulto , Animales , Sitios de Unión , Cationes , Supervivencia Celular/efectos de los fármacos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cristalización , Citoprotección , Perros , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , N-Metiltransferasa de Histona-Lisina/farmacología , Humanos , Células de Riñón Canino Madin Darby , Simulación del Acoplamiento Molecular , Canales de Potasio de Rectificación Interna/aislamiento & purificación , Canales de Potasio de Rectificación Interna/farmacología , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteína wnt2/aislamiento & purificación , Proteína wnt2/farmacologíaRESUMEN
Recently, we have shown that Wnt2 is an autocrine growth and differentiation factor for hepatic sinusoidal endothelial cells. As Wnt signaling has become increasingly important in vascular development and cancer, we analyzed Wnt signaling in non-sinusoidal endothelial cells of different vascular origin (HUVEC, HUAEC, HMVEC-LLy). Upon screening the multiple components of the Wnt pathway, we demonstrated lack of Wnt2 expression, but presence of Frizzled-4, one of its receptors, in cultured non-sinusoidal endothelial cells. Treatment of these cells by exogenous Wnt2 induced endothelial proliferation and sprouting angiogenesis in vitro. Upon analysis of Wnt2 tissue expression as a basis for paracrine Wnt2 effects on non-sinusoidal endothelial cells in vivo, Wnt2 was found to be expressed in densely vascularized murine malignant tumors and in wound healing tissues in close proximity to CD31+ endothelial cells. By gene profiling, stanniocalcin-1 (STC1), a known regulator of angiogenesis, was identified as a target gene of Wnt2 signaling in HUVEC down-regulated by Wnt2 treatment. Tumor-conditioned media counter-acted Wnt2 and up-regulated STC1 expression in HUVEC. In conclusion, we provide evidence that Wnt2 acts as an angiogenic factor for non-sinusoidal endothelium in vitro and in vivo whose target genes undergo complex regulation by the tissue microenvironment.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Glicoproteínas/genética , Neovascularización Fisiológica/efectos de los fármacos , Proteína wnt2/farmacología , Inductores de la Angiogénesis/farmacología , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Glicoproteínas/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica/genética , Ratas , Ratas Sprague-Dawley , Proteína wnt2/fisiologíaRESUMEN
Blood vessel growth is critical for embryonic development and contributes to pathologies including cancer and diabetic retinopathy. A growing body of evidence suggests that signaling via the Wnt/beta-catenin pathway contributes to angiogenesis, and that paracrine Wnt signaling might alter endothelial cell function. To test the hypothesis that Wnt signaling promotes endothelial cell proliferation and vessel growth, we treated bovine aortic endothelial cells with Wnt1, Wnt2 and Wnt5a derived from coculture with Wnt-expressing fibroblasts. Endothelial cells cultured in the presence of Wnt1 displayed increased Wnt/beta-catenin signaling, proliferation and capillary stability in vitro. Wnt5a, which primarily signals via an alternate Wnt pathway, the Wnt/Ca(++) pathway, decreased both cell number and capillary length. Wnt2, which in other cell types activates the Wnt/beta-catenin pathway, did not activate signaling, affect cell number or increase capillary length. These results suggest that Wnt/beta-catenin and Wnt/Ca(++) signals might have opposing effects on angiogenesis.