Background-The remodeled vessel wall in many vascular diseases such as restenosis after injury is characterized by proliferative and apoptosis-resistant vascular smooth muscle cells. There is evidence that proproliferative and antiapoptotic states are characterized by a metabolic (glycolytic phenotype and hyperpolarized mitochondria) and electric (downregulation and inhibition of plasmalemmal K ϩ channels) remodeling that involves activation of the Akt pathway. Dehydroepiandrosterone (DHEA) is a naturally occurring and clinically used steroid known to inhibit the Akt axis in cancer. We hypothesized that DHEA will prevent and reverse the remodeling that follows vascular injury. Methods and Results-We used cultured human carotid vascular smooth muscle cell and saphenous vein grafts in tissue culture, stimulated by platelet-derived growth factor to induce proliferation in vitro and the rat carotid injury model in vivo. DHEA decreased proliferation and increased vascular smooth muscle cell apoptosis in vitro and in vivo, reducing vascular remodeling while sparing healthy tissues after oral intake. Using pharmacological (agonists and antagonists of Akt and its downstream target glycogen-synthase-kinase-3␤ [GSK-3␤]) and molecular (forced expression of constitutively active Akt1) approaches, we showed that the effects of DHEA were mediated by inhibition of Akt and subsequent activation of GSK-3␤, leading to mitochondrial depolarization, increased reactive oxygen species, activation of redox-sensitive plasmalemmal voltage-gated K ϩ channels, and decreased [Ca 2ϩ ] i . These functional changes were accompanied by sustained molecular effects toward the same direction; by decreasing [Ca 2ϩ ] i and inhibiting GSK-3␤, DHEA inhibited the nuclear factor of activated T cells transcription factor, thus increasing expression of Kv channels (Kv1.5) and contributing to sustained mitochondrial depolarization. These results were independent of any steroidrelated effects because they were not altered by androgen and estrogen inhibitors but involved a membrane G protein-coupled receptor. Conclusions-We suggest that the orally available DHEA might be an attractive candidate for the treatment of systemic vascular remodeling, including restenosis, and we propose a novel mechanism of action for this important hormone and

In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Srcmediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.Apoptosis is a cell suicide program that plays a crucial role in the maintenance of cellular integrity (77). Two classical pathways for induction of apoptosis exist in mammalian cells, the intrinsic, or mitochondrial, and extrinsic, or death receptor, pathways, and both involve the activation of caspases, a family of cysteine proteases with aspartate specificity (3,4,28,39). This self-amplifying caspase cascade culminates in the proteolytic inactivation of critical components of survival pathways and activation of proapoptotic functions, which altogether lead to the disassembly of the cell. Despite the general role of caspases in apoptotic processes, several modes of caspaseindependent induction of cell death also exist, but the mechanisms involved are poorly understood. In general, the cytosolic hallmarks of apoptosis predominate (e.g., rounder and shrunken morphology, deformations of the plasma membrane, and membrane blebs) and are associated with DNA condensation but not with classical DNA degradation. This programmed cell death response has been termed type II apoptosis. Notably, such suicide programs can be driven by the growth suppressor PML (63), the c-myc-interacting protein Bin1 (17), the Fas-binding protein Daxx (7), and the adenoviral death factor E4orf4 (42). Caspase-...

We propose that HIF-1 is centrally involved in carotid artery remodelling in response to arterial injury and that localized inhibition of HIF-1 may be a novel therapeutic strategy to prevent carotid stenosis.