Quy N. Diep

Peroxisome Proliferator-Activated Receptors Vascular and Cardiac Effects in Hypertension

Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors acting as transcription factors on numerous target genes after heterodimerization with the retinoid X receptor. PPAR-alpha and PPAR-gamma may be activated by different agonists, although the endogenous ligands are unknown. Although PPAR-alpha is mainly involved in fatty acid oxidation and expressed in liver, kidney, and skeletal muscle, and PPAR-gamma is mainly involved in fat cell differentiation and insulin sensitivity, both are expressed in smooth muscle cells and myocardium, although PPAR-gamma are scarce in the latter. Activators of PPAR-alpha such as fatty acids and fibrates, and PPAR-gamma such as thiazolidinediones have been shown to exert antiproliferative effects, antagonize angiotensin II actions in vivo and in vitro, and exert antioxidant actions inhibiting generation of reactive oxygen species and activation of inflammatory mediators on blood vessels and the heart. These agents lowered blood pressure in several models of hypertension and corrected endothelial dysfunction. They exerted anti-inflammatory and antifibrotic actions on blood vessels and the heart. With the development of dual alpha/gamma-PPAR activators, these newer agents may become interesting therapeutic agents for prevention of vascular and cardiac complications of hypertension as well as for preventative therapy in other forms of cardiovascular disease.

Structure, Endothelial Function, Cell Growth, and Inflammation in Blood Vessels of Angiotensin II–Infused Rats Role of Peroxisome Proliferator–Activated Receptor-γ

Background—Pioglitazone and rosiglitazone, thiazolidinedione peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) activators, reduce blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that pioglitazone or rosiglitazone would prevent BP elevation and vascular dysfunction in angiotensin (Ang) II–infused rats by direct vascular effects. Methods and Results—Sprague-Dawley rats received Ang II (120 ng · kg−1 · min−1 SC) with or without pioglitazone (10 mg · kg−1 · d−1) or rosiglitazone (5 mg · kg−1 · d−1) for 7 days. Systolic BP, elevated in Ang II–infused rats (176±5 mm Hg) versus controls (109±2 mm Hg, P <0.01), was reduced by pioglitazone (134±2 mm Hg) or rosiglitazone (123±2 mm Hg). 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 the thiazolidinediones. In Ang II–infused rats, vascular DNA synthesis (by 3H-thymidine incorporation); expression of cell cycle proteins cyclin D1 and cdk4, angiotensin II type 1 receptors, vascular cell adhesion molecule-1, and platelet and endothelial cell adhesion molecule; and nuclear factor-&kgr;B activity were increased. These changes were abrogated by pioglitazone or rosiglitazone. Conclusions—Thiazolidinedione PPAR-&ggr; activators attenuated the development of hypertension, corrected structural abnormalities, normalized cell growth, and improved endothelial dysfunction induced by Ang II and prevented upregulation of angiotensin II type 1 receptors, cell cycle proteins, and proinflammatory mediators. Thiazolidinediones may be useful in the prevention and/or treatment of hypertension, particularly when it is associated with insulin resistance or diabetes mellitus.

PPARα 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 P P

Effect of Peroxisome Proliferator–Activated Receptor-α and -γ Activators on Vascular Remodeling in Endothelin-Dependent Hypertension

Objective—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. Methods and Results—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-&ggr; activator (rosiglitazone, 5 mg · kg−1 · d−1), or DOCA-salt+PPAR-&agr; 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. Conclusions—PPAR-&agr; and -&ggr; activators were able to modulate endogenous production of ET-1 and had beneficial vascular effects in endothelin-dependent hypertension.

Docosahexaenoic Acid, a Peroxisome Proliferator–Activated Receptor-α 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-&agr; (PPAR-&agr;) 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-&agr;, 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-&agr; 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-&agr; 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.

Peroxisome Proliferator-Activated Receptor-α and Receptor-γ Activators Prevent Cardiac Fibrosis in Mineralocorticoid-Dependent Hypertension

Abstract—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-&ggr; activator, 5 mg/kg per day), and DOCA-salt+fenofibrate (PPAR-&agr; 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.

Increased Expression of Peroxisome Proliferator-Activated Receptor-α and -γ in Blood Vessels of Spontaneously Hypertensive Rats

Abstract—Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors that include PPAR-&agr;, PPAR-&ggr;, and PPAR-&dgr;. We hypothesized that PPAR expression in blood vessels could be reduced in hypertension to result in increased vascular growth and reduced apoptosis. We investigated the abundance of PPAR-&agr; and PPAR-&ggr; in aorta and mesenteric arteries from young (6-week-old) and adult (16-week-old) spontaneously hypertensive rats (SHR) compared with age-matched control Wistar-Kyoto rats (WKY). mRNA levels of PPAR-&agr; and PPAR-&ggr; were determined by reverse transcription-polymerase chain reaction. Protein expression was evaluated by Western blot and by immunohistochemistry. PPAR-&ggr; was expressed in aortic and mesenteric vascular smooth muscle cells (VSMCs) from intact tissue and cultured cells. PPAR-&agr; was expressed in intact vascular tissue but was almost undetectable in cultured VSMCs. In mesenteric arteries from adult SHR, PPAR-&agr; and PPAR-&ggr; mRNA levels were significantly greater than in WKY (P <0.05). In aorta, PPAR-&agr; mRNA was significantly (P <0.05) more abundant in adult (but not in young) SHR than in WKY, whereas there was no difference in PPAR-&ggr; mRNA between WKY and SHR. PPAR-&agr; and PPAR-&ggr; mRNA were greater in mesenteric arteries (P <0.05) in young and adult SHR than in WKY. Expression of PPAR-&agr; and PPAR-&ggr; was similar in SHR and WKY in other tissues. In cultured mesenteric VSMCs, PPAR-&ggr; mRNA was 3-fold higher in SHR than in WKY. Immunohistochemistry demonstrated that PPAR-&ggr; resided constitutively in the cytoplasm in primary and low-passaged aortic and mesenteric VSMCs, whereas PPAR-&agr; was almost undetectable. Thus, aorta and mesenteric resistance arteries from SHR in the prehypertensive and the established phase of hypertension exhibit increased expression of both PPAR isoforms, whereas other tissues do not. Changes (increases) in PPAR expression may play a compensatory role in the remodeling of blood vessels in SHR.

Differential activation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen activated-protein kinase by AT1 receptors in vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats.

Objectives The present study investigates effects of angiotensin II on activation of extracellular signal-regulated protein kinase (ERK) 1/2, p38 mitogen activated-protein kinase (p38MAPK) and c-Jun amino terminal kinase (JNK) in vascular smooth muscle cells from spontaneously hypertensive rats (SHR). Methods Vascular smooth muscle cells (VSMC) from mesenteric arteries of Wistar–Kyoto (WKY) rats and SHR were studied. Angiotensin II-induced phosphorylation of ERK1/2, JNK and p38MAPK were assessed by Western blot analysis. c-fos mRNA expression by angiotensin II was determined by reverse transcriptase-polymerase chain reaction in the absence and presence of PD98059, selective inhibitor of ERK1/2-dependent pathways and SB202190, selective p38MAPK inhibitor. Results Angiotensin II increased phosphorylation of ERK1/2 and p38MAPK, but not JNK. Responses were significantly increased in SHR compared with WKY. Irbesartan, AT1 receptor antagonist, but not PD123319, AT2 receptor blocker, abolished angiotensin II-induced effects. PP2, selective Src inhibitor, decreased angiotensin II-mediated activation of MAP kinases. Angiotensin II increased c-fos mRNA expression in SHR and had a small stimulatory effect in WKY. These actions were inhibited by PD98059, whereas SB202190 had no effect. Conclusions Angiotensin II-induced activation of vascular ERK1/2 and p38MAPK is increased in SHR. These effects are mediated via AT1 receptors, which activate Src-dependent pathways. Overexpression of c-fos mRNA in SHR is due to ERK1/2-dependent, p38MAPK-independent pathways. Our results suggest that angiotensin II activates numerous MAP kinases in VSMCs and that differential activation of these kinases may be important in altered growth signaling in VSMCs from SHR.

Endothelin-1 Attenuates ω3 Fatty Acid–Induced Apoptosis by Inhibition of Caspase 3

Endothelin-1 (ET-1) may be involved in the induction of vascular hypertrophy in hypertension. ET-1 may also modulate vascular growth through the exertion of antiapoptotic effects. The omega3 fatty acids (omega3 FAs), which have antiproliferative effects in various cell types, may have a beneficial role in hypertension. We tested the hypothesis that ET-1 could act as a survival factor against omega3 FA-induced apoptosis and attempted to elucidate possible molecular mechanisms underlying the protective action of ET-1 on docosahexaenoic acid (DHA)-induced apoptosis. Mesenteric vascular smooth muscle cells were stimulated with DHA, a representative omega3 FA. Dose-response curves of DHA at different apoptotic stages were assessed with the use of flow cytometry: (1) very early: plasma membrane phosphatidylserine (PS) translocation; (2) early: change in mitochondrial transmembrane potential (DeltaPsim); and (3) late: cell cycle analysis. Expression of the proapoptotic protein bax and the antiapoptotic protein bcl-2 was determined with Western blot assay. The activity and the expression of caspase 3, which is a critical proteolytic enzyme involved in the death-signaling pathway, were evaluated with a fluorometric immunosorbent enzyme assay and Western blot analysis, respectively. Apoptosis, which was detected with PS translocation, DeltaPsim disruption, and cell cycle analysis, was increased dose dependently by DHA. DHA-induced apoptosis was attenuated through exposure to ET-1 for 1 hour before DHA in cell cycle analysis. The interference of ET-1 with DHA-induced apoptosis, as detected with cell cycle analysis, was not apparent at the membrane (PS translocation) or the mitochondrial (DeltaPsim) level. The increase in bax/bcl-2 ratio in DHA-stimulated cells was not affected by ET-1. However, DHA increased both caspase 3 activity and the active forms of caspase 3 (20 and 17 kDa), resulting in enhanced DNA fragmentation as shown through Hoechst staining and fluorescence microscopy, which were attenuated by ET-1 pretreatment. In conclusion, DHA, an omega3 FA, induced apoptosis in vascular smooth muscle cells in a dose-dependent manner. ET-1 exerted important protective effects through the attenuation of DHA-induced caspase 3 activation and subsequent DNA fragmentation in the late stages of apoptosis.

Expression of Cell Cycle Proteins in Blood Vessels of Angiotensin II–Infused Rats Role of AT1 Receptors

Angiotensin II is an important modulator of cell growth through AT1 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 AT1-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 3H-thymidine incorporation. The expression of cyclin D1, cdk4, p21, and p27, which play critical roles during the G1-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 3H-thymidine incorporation (cpm/100 &mgr;g 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 AT1 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 AT1 receptor stimulation.