Abstract-CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO-1, and Prx I protein levels. Because PPAR-␥ can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.
Cellular aging is often associated with an increase in protein carbonyl groups arising from oxidation- and glycation-related phenomena and suppressed proteasome activity. These "aged" polypeptides may either be degraded by 20S proteasomes or cross-link to form structures intractable to proteolysis and inhibitory to proteasome activity. Carnosine (beta-alanyl-l-histidine) is present at surprisingly high levels (up to 20 mM) in muscle and nervous tissues in many animals, especially long-lived species. Carnosine can delay senescence in cultured human fibroblasts and reverse the senescent phenotype, restoring a more juvenile appearance. As better antioxidants/free-radical scavengers than carnosine do not demonstrate these antisenescent effects, additional properties of carnosine must contribute to its antisenescent activity. Having shown that carnosine can react with protein carbonyls, thereby generating "carnosinylated" polypeptides using model systems, we propose that similar adducts are generated in senescent cells exposed to carnosine. Polypeptide-carnosine adducts have been recently detected in beef products that are relatively rich in carnosine, and carnosine's reaction with carbonyl functions generated during amino acid deamidation has also been described. Growth of cultured human fibroblasts with carnosine stimulated proteolysis of long-labeled proteins as the cells approached their "Hayflick limit," consistent with the idea that carnosine ameliorates the senescence-associated proteolytic decline. We also find that carnosine suppresses induction of heme-oxygenase-1 activity following exposure of human endothelial cells to a glycated protein. The antisenescent activity of the spin-trap agent alpha-phenyl-N-t-butylnitrone (PBN) towards cultured human fibroblasts resides in N-t-butyl-hydroxylamine, its hydrolysis product. As hydroxylamines are reactive towards aldehydes and ketones, the antisenescent activity of N-t-butyl-hydroxylamine and other hydroxylamines may be mediated, at least in part, by reactivity towards macromolecular carbonyls, analogous to that proposed for carnosine.
Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic and antirestenotic effects on type 2 diabetes, in part due to an induction of vascular smooth muscle cell (VSMC) apoptosis. We aimed to study the role of transforming growth factor (TGF)- in rat aortic VSMC. Pioglitazone at 100 mol/l increased apoptosis without affecting DNA synthesis, and this effect was reversed by an anti-TGF-1 antibody. Extracellular TGF-1 levels were rapidly increased after treatment with pioglitazone in a peroxisome proliferator-activated receptor (PPAR)-␥-dependent mechanism because this secretion was blocked by the PPAR-␥ inhibitor GW9662. Pioglitazone subsequently increased the nuclear recruitment of phospho-Smad2, without any effect on protein expression. According to our results, we propose that the apoptotic effect of pioglitazone on VSMC depends on the following sequence: PPAR-␥ activation, TGF-1 release, and selective phospho-Smad2 nuclear recruitment. Management of Smad signaling on VSMC might provide future clinical benefits in vascular diseases. Diabetes 54: [811][812][813][814][815][816][817] 2005 T ype 2 diabetes is a major risk factor for atherosclerosis, the leading cause of morbidity and mortality in developed countries (1). In addition, diabetic patients show an increased risk for suffering postangioplasty restenosis (2). Vascular wall size depends on a relative balance between proliferation and apoptosis (3). When the ratio of proliferation to apoptosis increases, an early atherosclerotic plaque or a postangioplasty restenosis is faced.Thiazolidinediones (TZDs) are an emerging class of antidiabetic drugs that enhance insulin sensitivity in a peroxisome proliferator-activated receptor (PPAR)-␥-dependent fashion (4). However, they have also been demonstrated to exert some direct effects in decreasing neointimal growth after balloon injury (5) and angioplasty (6), two situations with an increased ratio for vascular smooth muscle cell (VSMC) proliferation to apoptosis. Many of the studies concerning the cellular effects of TZD have focused on cell cycle and DNA synthesis regulation. Surprisingly, these effects seem to be PPAR-␥ independent (7). On the other hand, at higher doses, TZDs have been proven to induce apoptosis in VSMCs by involving PPAR-␥ (8).Transforming growth factor (TGF)-1 is an essential cytokine involved in the control of the balance between proliferation and apoptosis in VSMCs. TZDs and TGF-1 share the induction of apoptosis by similar mechanisms, such as GADD45 (9,10). In addition, some crosstalk between TGF-1 and PPAR-␥ has been described; thus, in the long term, TGF-1 inhibits PPAR-␥ expression (11), whereas PPAR-␥ agonists and PPAR-␥ itself interact with the TGF-1 signaling pathway by inhibiting Smad3 (12). Therefore, Smad2 appears as a likely target for TGF- in TZD-treated VSMCs.In the present study, we show that the apoptotic effect of the TZD pioglitazone in VSMC depends on the following sequence: PPAR-␥ agonism, rapid TGF-1 autocrine secretion, and select...
An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation. Diabetes 55: [1243][1244][1245][1246][1247][1248][1249][1250][1251] 2006
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