Opposing effects of bim and bcl-2 on lung endothelial cell migration

Abstract

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

Aberrant production of extracellular matrix proteins and dysfunction in kidney endothelial cells with a short duration of diabetes

“…Cells were progressively passed to larger plates, maintained, and propagated in 1% gelatin-coated 60-mm dishes. Kidney endothelial cells were positive for B4-lectin (a mouse endothelial cell-specific lectin) and expressed PECAM-1 and vascular endothelial (VE)-cadherin as previously described (7,13). The experiments described here were performed with three separate isolations of cells with similar results.…”

“…Immortomice expressing a temperature-sensitive SV40 large T antigen were obtained from Charles River Laboratories (Wilmington, MA). Akita/ϩ mice (Jackson Laboratory, Bar Harbor, ME) were crossed with the Immortomouse and screened as previously described (7,23). To isolate kidney endothelial cells, kidneys from three 8-wk-old wild-type and Akita/ϩ Immortomice were dissected out aseptically and placed in serum-free DMEM (no.…”

“…Plates were then rinsed with PBS and fed with growth medium containing 100 ng/ml of 5-FU to rule out the potential contribution of differences in cell proliferation. The wounds were observed and photographed up to 72 h. The distance migrated was determined as the percentage of total distance for quantitative assessments as described previously (7). These experiments were repeated at least twice with two different isolations with similar results.…”

“…Following a 40-min incubation at 33°C, fresh endothelial cell medium was placed on the cells, and the incubation continued for 20 min. The wells were washed with TBS, the cells in each well were resuspended in 100 l of TBS, and the absorbance was read at 495/515 nm using a fluorescence plate reader (7). These experiments were performed in triplicate and repeated twice with similar results.…”

“…7C. Previous studies from our laboratories indicated that optimal endothelial cell migration is essential for capillary morphogenesis in vitro (4,7,14,29). Next, we determined whether decreasing eNOS expression restored capillary morphogenesis.…”