Docosahexaenoic acid, a peroxisome proliferator-activated receptor- ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase

Omega-3 fatty acids (n-3 FAs) have been shown to exert a blood pressure-lowering effect in hypertension, possibly in part by influencing vascular structure. We previously demonstrated that n-3 FAs induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on the structure of blood vessels. In the present study, we investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured mesenteric VSMCs from Sprague-Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FAs. Morphological changes in apoptosis and DNA fragmentation were examined with phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, we evaluated the expression of phosphorylated p38 mitogen-activated protein kinases, bax, bcl-2, cytochrome c, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) with Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing, and apoptotic bodies in VSMCs. DHA time-dependently activated p38 mitogen-activated protein kinases, bax, PPAR-alpha, and cytochrome c, with maximal effects obtained after 5 and 30 minutes and 1 and 3 hours, respectively. SB-203580 and SB-202190, selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPAR-alpha but had no effect on the expression of bax or cytochrome c. The present results indicate that DHA induces apoptosis in VSMCs through >/=2 distinct mechanisms: (1) a p38-dependent pathway that regulates PPAR-alpha and (2) a p38-independent pathway via dissipation of mitochondrial membrane potential and cytochrome c release. The death-signaling pathway stimulated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA may play a pathophysiological role in vascular remodeling in cardiovascular disease

Expression of cell cycle proteins in blood vessels of angiotensin II-inused rats: role of AT(1) receptors

Angiotensin II is an important modulator of cell growth through AT(1) receptors, as demonstrated both in vivo and in vitro. We investigated the role of proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase inhibitors p21 and p27 in blood vessels of angiotensin II-infused rats and the effect therein of the AT(1)-receptor antagonist losartan. Male Sprague-Dawley rats were infused for 7 days with angiotensin II (120 ng/kg per minute SC) and/or treated with losartan (10 mg/kg per day orally). DNA synthesis in mesenteric arteries was evaluated by radiolabeled (3)H-thymidine incorporation. The expression of cyclin D1, cdk4, p21, and p27, which play critical roles during the G(1)-phase of the cell cycle process, was examined by Western blot analysis. Tail-cuff systolic blood pressure (mm Hg) was elevated (P<0.01, n=9) in angiotensin II-infused rats (161.3+/-8.2) versus control rats (110.1+/-5.3) and normalized by losartan (104.4+/-3.2). Radiolabeled (3)H-thymidine incorporation (cpm/100 microgram DNA) showed that angiotensin II infusion significantly increased DNA synthesis (152+/-5% versus 102+/-6% of control rats, P<0.05). Expression of cyclin D1 and cdk4 was significantly increased in the angiotensin II group to 213.7+/-8% and 263.6+/-37% of control animals, respectively, whereas expression of p21 and p27 was significantly decreased in the angiotensin II group to 23.2+/-10.4% and 10.3+/-5.3% of control animals, respectively. These effects induced by angiotensin II were normalized in the presence of losartan. Thus, when AT(1) receptors are stimulated in vivo, DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT(1) receptor stimulation

SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension

We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85α and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85α and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85α-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR

Effect of peroxisome proliferator-activated receptor-α and -γ activators on vascular remodeling in endothelin-dependent hypertension

<b>Objective—</b> Peroxisome proliferator–activated receptors (PPARs) may modulate in vitro the vascular production of vasoactive peptides such as endothelin-1 (ET-1). Thus, we investigated in vivo the interaction between PPARs and ET-1 in deoxycorticosterone acetate (DOCA)–salt rats that overexpress vascular ET-1.<p></p> <b>Methods and Results—</b> Unilaterally nephrectomized 16-week-old Sprague-Dawley rats (Uni-Nx) were divided into 4 groups (n=6 each): control group, DOCA-salt group, DOCA-salt+PPAR-γ activator (rosiglitazone, 5 mg · kg−1 · d−1), or DOCA-salt+PPAR-α activator (fenofibrate, 100 mg · kg−1 · d−1). Systolic blood pressure was significantly increased in the DOCA-salt group (240±11 vs 121±2 mm Hg in Uni-Nx, P<0.01). Progression of hypertension was partially prevented by coadministration of rosiglitazone (172±3 mm Hg vs DOCA-salt, P<0.05) but not by fenofibrate. Both PPAR activators abrogated the increase in prepro-ET-1 mRNA content in the mesenteric vasculature of DOCA-salt rats. The media-to-lumen ratio was increased in DOCA-salt rats (10.3±0.9% vs 4.9±0.5% in Uni-Nx rats, P<0.01). Rosiglitazone and fenofibrate prevented the hypertrophic remodeling observed in DOCA-salt rats without affecting vascular stiffness. Rosiglitazone but not fenofibrate prevented endothelial dysfunction in pressurized mesenteric arteries. Finally, both rosiglitazone and fenofibrate prevented the vascular increase in superoxide anion production induced in DOCA-salt animals.<p></p> <b>Conclusions—</b> PPAR-α and -γ activators were able to modulate endogenous production of ET-1 and had beneficial vascular effects in endothelin-dependent hypertension

PPARalpha activator effects on Ang II-induced vascular oxidative stress and inflammation

Docosahexaenoic acid (DHA), a peroxisome proliferator–activated receptor-α (PPARα) activator, reduces blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that DHA would prevent BP elevation and improve vascular dysfunction in angiotensin (Ang) II–infused rats by modulating of NADPH oxidase activity and inflammation in vascular wall. Sprague-Dawley rats received Ang II (120 ng/kg per minute SC) with or without DHA (2.5 mL of oil containing 40% DHA/d PO) for 7 days. Systolic BP (mm Hg), elevated in Ang II–infused rats (172±3) versus controls (108±2, P<0.01), was reduced by DHA (112±4). In mesenteric small arteries studied in a pressurized myograph, media/lumen ratio was increased (P<0.05) and acetylcholine-induced relaxation impaired in Ang II–infused rats (P<0.05); both were normalized by DHA. In blood vessels of Ang II–infused rats, NADPH oxidase activity measured by chemiluminescence and expression of adhesion molecules intercellular adhesion molecule and vascular cell adhesion molecule-1 were significantly increased. These changes were abrogated by DHA. PPARα activator DHA attenuated the development of hypertension, corrected structural abnormalities, and improved endothelial dysfunction induced by Ang II. These effects are associated with decreased oxidative stress and inflammation in the vascular wall

Peroxisome proliferator-activated receptor-α and receptor-γ activators prevent cardiac fibrosis in mineralocorticoid-dependent hypertension

Peroxisome proliferator-activated receptor (PPAR) activation may prevent cardiac hypertrophy and inhibit production of endothelin-1 (ET-1), a hypertrophic agent. The aim of this in vivo study was to investigate the effects of PPAR activators on cardiac remodeling in DOCA-salt rats, a model overexpressing ET-1. Unilaterally nephrectomized 16-week-old Sprague-Dawley rats (Uni-Nx) were randomly divided into 4 groups: control rats, DOCA-salt, DOCA-salt+rosiglitazone (PPAR-γ activator, 5 mg/kg per day), and DOCA-salt+fenofibrate (PPAR-α activator, 100 mg/kg per day). After 3 weeks of treatment, mean arterial blood pressure was significantly increased in DOCA-salt by 36 mm Hg. Mean arterial blood pressure was normalized by coadministration of rosiglitazone but not by fenofibrate. Both PPAR activators prevented cardiac fibrosis and abrogated the increase in prepro–ET-1 mRNA content in the left ventricle of DOCA-salt rats. Coadministration of rosiglitazone or fenofibrate failed to prevent thickening of left ventricle (LV) walls as measured by echocardiography and the increase in atrial natriuretic peptide mRNA levels. However, rosiglitazone and fenofibrate prevented the decrease in LV internal diameter and thus concentric remodeling of the LV found in DOCA-salt rats. Taken together, these data indicate a modulatory role of PPAR activators on cardiac remodeling in mineralocorticoid-induced hypertension, in part associated with decreased ET-1 production

Fat intake and cardiovascular response

High dietary fat intake is a major risk factor for the development of obesity, which is frequently associated with diseases such as hypertension and diabetes and thus accelerated atherosclerosis. Angiotensin II and endothelin-1 are powerful growth factors and vasoconstrictors implicated in regulating vascular tone, vascular structure, and inflammation. Reduced bioactivity of nitric oxide and increased formation of reactive oxygen species (ROS) have been associated with obesity and high dietary fat intake. This article reviews the effects of high-fat diet on vascular functional changes in rodents and humans. Changes include alterations in vasoconstrictor function and receptor expression, and modulators of endothelium-dependent vascular tone (eg, nitric oxide- or endothelium-dependent contracting factor-mediated responses). Novel vasodilator effects of ROS and the anatomic heterogeneity of vascular responses are discussed. The beneficial effects of vasoactive mediators on vascular function could play a role for susceptibility to obesity-dependent hypertension, which is present in many, but not all, obese patients