RESUMO
This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of 1.4(-0.6)(+0.9)x10(-6) cm/s and 1.7(-0.5)(+0.8)x10(-6) cm/s (geometric mean+/-95% CI), respectively, and 1.2+/-0.5 (mean+/-95% CI) focal leaks per mm. Exposure to low concentrations (3 microM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 microM) and/or the phosphodiesterase inhibitor Ro-20-1724 (20 microM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , AMP Cíclico/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Inibidores de Adenilil Ciclases , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Bucladesina/farmacologia , Caderinas/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , AMP Cíclico/análogos & derivados , Dextranos/metabolismo , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Homeodomínio/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Soroalbumina Bovina/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Engenharia Tecidual/métodos , Proteínas Supressoras de Tumor/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
This work describes the formation, perfusion, and maturation of three-dimensional microvascular tubes in vitro. These tubes consisted of confluent monolayers of human endothelial cells that lined open, cylindrical channels within collagen gels. Perivascular cells could be directly embedded within the gels or added after endothelial cells grew to confluence. The tubes spanned the entire 5-7 mm extent of the gels; their diameters initially ranged from 55 to 120 microm and increased to 75-150 microm after maturation. Endothelial tubes displayed a strong barrier function over 5 days, resisted adhesion of leukocytes, and reacted quickly to inflammatory stimuli by breakdown of the barrier and support of leukocyte adhesion. These tubes resembled venules and "giant" capillaries in both their cellular organization and function, and we believe that they will serve as useful in vitro models of inflammation under constant perfusion.
