RESUMEN
Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity-dependent process, was decreased to a greater extent by general TG inhibitors vs. Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, and Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4, and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2, and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature and that TG1 and TG4 are expressed in vascular tissues.
Asunto(s)
Aorta Torácica/enzimología , Proteínas de Unión al GTP/biosíntesis , Miocitos del Músculo Liso/enzimología , Transglutaminasas/biosíntesis , Animales , Aorta Torácica/citología , Western Blotting , Cadaverina/análogos & derivados , Cadaverina/antagonistas & inhibidores , Reactivos de Enlaces Cruzados , Inhibidores Enzimáticos/farmacología , Factor XIII/biosíntesis , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Proteínas de Unión al GTP/antagonistas & inhibidores , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Ratones Noqueados , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transglutaminasas/antagonistas & inhibidores , Vena Cava Superior/citología , Vena Cava Superior/enzimologíaRESUMEN
Vascular polarity is a fundamental feature of angiogenesis and left-right asymmetry of the vascular network. Contrary to this importance, the molecular basis of vascular polarity is completely unknown. In this report, we show that the combinatorial function of angiopoietin-1 and the orphan receptor TIE1 is critical specifically for the development of the right-hand side venous system but is dispensable for the left-hand side venous system. Furthermore, our current finding reveals the existence of a distinct genetic program for the establishment of the right-hand side and left-hand side vascular networks well before the network asymmetry becomes morphologically discernible.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Vena Cava Inferior/embriología , Vena Cava Superior/embriología , Angiopoyetina 1 , Animales , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Fenotipo , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor TIE-1 , Receptores de Superficie Celular/genética , Receptores TIE , Vena Cava Inferior/enzimología , Vena Cava Superior/enzimologíaRESUMEN
PURPOSE: The aim of this study was to measure the distribution of endogenous plasminogen activators during thrombolysis with an endothelial-conserving model of laminated thrombosis. METHODS: Thrombi were raised in the inferior vena cava of rats with thrombin and flow reduction. The thrombi, adjacent vein wall, and distant veins (the superior vena cava) were removed at intervals from 1 hour to 21 days from formation and then cryohomogenized and assayed with specific bioimmunoassays for tissue-type (t-PA) and urokinase-type plasminogen activators (u-PA). RESULTS: The measured t-PA activity of the vein wall around the thrombus was reduced compared with the control inferior vena cava at 4 days. Both the u-PA and t-PA content of the thrombus increased progressively during thrombolysis. The t-PA activity increased significantly in the distant vein walls in the animals with thrombi. Immunocytochemistry and in situ hybridization localized the t-PA to a mononuclear cell infiltrate and showed up-regulation of mRNA for rat t-PA in these monocytes. CONCLUSIONS: The local plasminogen activator response was predominantly within the thrombus itself. Increased t-PA activity was additionally found in distant veins but was reduced in the vessel wall adjacent to the thrombus. This is the first report to show that u-PA activity is increased within organizing thrombus in vivo and that most of the t-PA activity is localized to a monocyte infiltrate.