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

Diabetic nephropathy is the most common cause of end-stage renal disease and is a major risk factor for cardiovascular disease. In the United States, microvascular complications during diabetic nephropathy contribute to high morbidity and mortality rates. However, the cell-autonomous impact of diabetes on kidney endothelial cell function requires further investigation. Male Akita/+ [autosomal dominant mutation in the insulin II gene (Ins2)] mice reproducibly develop diabetes by 4 wk of age. Here, we examined the impact a short duration of diabetes had on kidney endothelial cell function. Kidney endothelial cells were prepared from nondiabetic and diabetic mice (4 wk of diabetes) to delineate the early changes in endothelial cell function. Kidney endothelial cells from Akita/+ mice following 4 wk of diabetes demonstrated aberrant expression of extracellular matrix proteins including decreased osteopontin and increased fibronectin expression which correlated with increased α5-integrin expression. These changes were associated with the attenuation of migration and capillary morphogenesis. Kidney endothelial cells from Akita/+ mice had decreased VEGF levels but increased levels of endothelial nitric oxide synthase(eNOS) and NO, suggesting uncoupling of VEGF-mediated NO production. Knocking down eNOS expression in Akita/+ kidney endothelial cells increased VEGF expression, endothelial cell migration, and capillary morphogenesis. Furthermore, attenuation of sprouting angiogenesis of aortas from Akita/+ mice with 8 wk of diabetes was restored in the presence of the antioxidant N-acetylcysteine. These studies demonstrate that aberrant endothelial cell function with a short duration of diabetes may set the stage for vascular dysfunction and rarefaction at later stages of diabetes.

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

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.

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.

Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium- and temperature-dependent autolysis. The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.