Effects of angiotensin II on vascular endothelial cells: formation of receptor-mediated reactive nitrogen species.

Angiotensin II (ANG II) participates in many cardiovascular disease states, but the mechanisms involved are not completely defined. Doses of ANG II that do not affect blood pressure significantly can still cause early changes in vascular endothelial performance and cell-specific protein 3-nitrotyrosine formation (protein-3NT, marker of peroxynitrite formation) in vivo. Here, we have tested the hypothesis that ANG II induces endothelial cell peroxynitrite (ONOO-) formation in vitro, and investigated the mechanisms involved. Endothelial cells were incubated with ANG II (1nM-250 microM), and protein nitration was assessed by immunoblotting. ANG II caused concentration-dependent increases in protein-3NT above detectable basal control levels, at concentrations greater than 100nM. This response was inhibited significantly by co-incubation with losartan or diphenyleneiodonium chloride. Endothelial cell lysates incubated with nitrated protein standards demonstrated significant protein-3NT modification activity only in the presence of serum. However, endothelial cell lysates did not modify the free amino acid form of 3NT (free-3NT) in identical experimental conditions, assessed by capillary electrophoresis. Finally, free-3NT was cytotoxic to cultured endothelial cells (fitted LC(50)=98 microM). These data demonstrate that stimulation of angiotensin receptor subtype 1 by ANG II can cause increased endothelial cell protein nitration in vitro in the absence of other cell types or stimuli, at concentrations that are pathophysiologically relevant. Furthermore, endothelial cells selectively modified nitrated protein tyrosine residues only in the presence of a cofactor(s), and did not modify the free modified amino acid. Protein nitration may be a regulated endothelial signaling process, while free-3NT may be toxic to endothelial cells.

Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.