Interaction of CD44 and hyaluronic acid enhances biliary epithelial proliferation in cholestatic livers.

Biliary epithelia express high levels of CD44 in hepatobiliary diseases. The role of CD44-hyaluronic acid interaction in biliary pathology, however, is unclear. A rat model of hepatic cholestasis induced by bile duct ligation was employed for characterization of hepatic CD44 expression and extracellular hyaluronan distribution. Cell culture experiments were employed to determine whether hyaluronan can regulate cholangiocyte growth through interacting with adhesion molecule CD44. Biliary epithelial cells were found to express the highest level of CD44 mRNA among four major types of nonparenchymal liver cells, including Kupffer, hepatic stellate, and liver sinusoidal endothelial cells isolated from cholestatic livers. CD44-positive biliary epithelia lining the intrahepatic bile ducts were geographically associated with extracellular hyaluronan accumulated in the portal tracts of the livers, suggesting a role for CD44 and hyaluronan in the development of biliary proliferation. Cellular proliferation assays demonstrated that cholangiocyte propagation was accelerated by hyaluronan treatment and antagonized by small interfering RNA CD44 or anti-CD44 antibody. The study provides compelling evidence to suggest that proliferative biliary epithelia lining the intrahepatic bile ducts are a prime source of hepatic CD44. CD44-hyaluronan interaction, by enhancing biliary proliferation, may play a pathogenic role in the development of cholestatic liver diseases.

A method of isolation and culture of microvascular endothelial cells from mouse skin.

OBJECTIVES: The study of isolated microvascular endothelial cells from mice has long been impeded due to the many difficulties encountered in isolating and culturing these cells. We focused on developing a method to isolate microvascular endothelial cells from the skin fragments of newborn mice. We also aimed at establishing optimal culture conditions to sustain the growth of these cells. METHODS AND RESULTS: Isolation of murine dermal microvascular endothelial cells (mDMEC) from P3 newborn mice was based first on enzymatic separation of the skin epidermal layer from the dermis using dispase and then on disaggregating dermal cellular elements using collagenase. The cells obtained from the dermis were subjected to a continuous density gradient centrifugation. Cells situated between densities 1.033 and 1.047 were then cultured on collagen IV-coated culture flasks using optimized growth culture conditions. Cells were characterized by endothelial appearance and by the presence and genetic expression of endothelial markers like CD31, NOS3, VEGFR-2 and Tie-2. Uptake of acetylated low-density lipoprotein (Ac-LDL) was used as a functional assay. CONCLUSIONS: The methodology described herein for isolation and culture of murine microvascular endothelium offers a distinctive advantage for those using mouse models to study endothelial cell biology.

IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers.

Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [(3)H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml(-1). Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.

El papel de los contactos celulares en la recuperación de canales de membranas de células epiteliales / The role of cellular contacts in the recuperation of ion channels of epithelial cell membranes

Las corrientes catiónicas en células MDCK son casi exclusivamente debidas a canales de K+. El tratamiento con tripsina-EDTA destruye la mayoría de estos canales. Cuando son resembradas, los canales se recuperaron por medio de un proceso que requiere síntesis de proteínas y de mensajero. El presente trabajo demuestra que la recuperación requiere del contacto entre las células y concentraciones normales de Ca2+ (1.8 mM). Las células en monocapas confluentes en presencia de 1.8 mM de Ca2+ exhiben corrientes de K+ de 343 ñ 82 pA; las confluentes en un medio con bajo Ca2+ tienen sólo 90 ñ 12 pA (27 por ciento del control); y las que no establecen contactos celulares o que son incubadas a su confluencia pero en Ca2+ extracelular normal, tienen 104 ñ 21 pA (31 por ciento del control), con base en estos resultados concluimos que la expresión de canales de K+ depende de contactos celulares y presencia de Ca2+ normal. Es probable, entonces que esta expresión este mediada por uvomorulina