RESUMEN
A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay--a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.
Asunto(s)
Linfangiogénesis/fisiología , Tejido Linfoide/metabolismo , Modelos Biológicos , Técnicas de Cultivo de Tejidos/métodos , Animales , Simulación por Computador , Femenino , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfangiogénesis/efectos de los fármacos , Tejido Linfoide/citología , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/enzimología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Serpina E2 , Serpinas/genética , Serpinas/metabolismoRESUMEN
Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.