Talin Ebrahimian

Endothelial Nitric Oxide Synthase Uncoupling and Perivascular Adipose Oxidative Stress and Inflammation Contribute to Vascular Dysfunction in a Rodent Model of Metabolic Syndrome

The metabolic syndrome represents a constellation of cardiovascular risk factors that promote the development of cardiovascular disease. Oxidative stress is a mediator of endothelial dysfunction and vascular remodeling. We investigated vascular dysfunction in the metabolic syndrome and the oxidant mechanisms involved. New Zealand obese (NZO) mice with metabolic syndrome and New Zealand black control mice were studied. NZO mice showed insulin resistance and increased visceral fat and blood pressure compared with New Zealand black mice. Mesenteric resistance arteries from NZO mice exhibited increased media:lumen ratio and media cross-sectional area, demonstrating hypertrophic vascular remodeling. Endothelium-dependent relaxation to acetylcholine, assessed by pressurized myography, was impaired in NZO mice, not affected by NG-nitro-l-arginine methyl ester, inhibitor of endothelial NO synthase, and improved by the antioxidant Tempol, suggesting reduced NO bioavailability and increased oxidative stress. Dimer:monomer ratio of endothelial NO synthase was decreased in NZO mice compared with New Zealand black mice, suggesting endothelial NO synthase uncoupling. Furthermore, vascular superoxide and peroxynitrite production was increased, as well as adhesion molecule expression. Perivascular adipose tissue of NZO mice showed increased superoxide production and NADPH oxidase activity, as well as adipocyte hypertrophy, associated with inflammatory Mac-3–positive cell infiltration. Vasoconstriction to norepinephrine decreased in the presence of perivascular adipose tissue in New Zealand black mice but was unaffected by perivascular adipose tissue in NZO mice, suggesting loss of perivascular adipose tissue anticontractile properties. Our data suggest that this rodent model of metabolic syndrome is associated with perivascular adipose inflammation and oxidative stress, hypertrophic resistance artery remodeling, and endothelial dysfunction, the latter a result of decreased NO and enhanced superoxide generated by uncoupled endothelial NO synthase.

Angiotensin II Impairs Endothelial Progenitor Cell Number and Function In Vitro and In Vivo: Implications for Vascular Regeneration

Endothelial progenitor cells (EPCs) contribute to endothelial regeneration. Angiotensin II (Ang II) through Ang II type 1 receptor (AT1-R) activation plays an important role in vascular damage. The effect of Ang II on EPCs and the involved molecular mechanisms are incompletely understood. Stimulation with Ang II decreased the number of cultured human early outgrowth EPCs, which express both AT1-R and Ang II type 2 receptor, mediated through AT1-R activation and induction of oxidative stress. Ang II redox-dependently induced EPC apoptosis through increased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase phosphorylation; decreased Bcl-2 and increased Bax expression; and activation of caspase 3 but had no effect on the low cell proliferation. In addition, Ang II impaired colony-forming and migratory capacities of early outgrowth EPCs. Ang II infusion diminished numbers and functional capacities of EPCs in wild-type (WT) but not AT1a-R knockout mice (AT1a−/−). Reendothelialization after focal carotid endothelial injury was decreased during Ang II infusion. Salvage of reendothelialization by intravenous application of spleen-derived progenitor cells into Ang II-treated WT mice was pronounced with AT1a−/− cells compared with WT cells, and transfusion of Ang II–pretreated WT cells into WT mice without Ang II infusion was associated with less reendothelialization. Transplantation of AT1a−/− bone marrow reduced atherosclerosis development in cholesterol-fed apolipoprotein E–deficient mice compared with transplantation of apolipoprotein E–deficient or WT bone marrow. Randomized treatment of patients with stable coronary artery disease with the AT1-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT1-R activation, oxidative stress, and redox-sensitive apoptosis signal-regulating kinase 1–dependent proapoptotic pathways impairs EPCs in vitro and in vivo, resulting in diminished vascular regeneration.

Angiotensin II/AT2 receptor-induced vasodilation in stroke-prone spontaneously hypertensive rats involves nitric oxide and cGMP-dependent protein kinase

Background Angiotensin II (Ang II) induces vasodilation, in part, through angiotensin type 2 receptor (AT2R)-induced actions in conditions associated with angiotensin type 1 receptor (AT1R) blockade and AT2R upregulation. Ang II/AT2R-induced vasodilation involves nitric oxide (NO)–cyclic guanosine monophosphate (cGMP)-dependent processes. We previously demonstrated that AT2R-mediated effects involve inhibition of the RhoA/Rho kinase pathway. However, molecular mechanisms underlying this phenomenon are unknown. Aims In the present in-vivo study we tested the hypothesis that AT2R-elicited vasodilation is associated with nitric oxide synthase (NOS) activation and NO production, and that a cGMP-dependent protein kinase (cGKI), which inactivates RhoA, is upregulated when stroke-prone spontaneously hypertensive rats (SHRSP) are treated with AT1R blockers. Methods SHRSP and Wistar–Kyoto (WKY) rats were treated with the AT1R blocker valsartan for 14 days. Dilatory responses to Ang II with or without the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) were performed in norepinephrine-precontracted vessels in the presence of valsartan. Expression of AT2R, endothelial NOS (eNOS) and cGKI was assessed by immunoblotting. NO bioavailability and NAD(P)H oxidase activity were evaluated by chemiluminescence. Results Ang II elicited vasodilation in valsartan-treated SHRSP. L-NAME inhibited this effect, indicating a role for NO. eNOS expression and NO concentration were increased twofold by valsartan, only in SHRSP. Expression of cGKI was reduced in SHRSP and restored after valsartan treatment. NAD(P)H oxidase activity was approximately threefold higher in SHRSP versus WKY (P < 0.05) and reduced by valsartan. Conclusions Ang II, via AT2R, facilitates vasodilation through NOS/NO-mediated pathways and downregulation of cGKI after chronic AT1R antagonism. These effects may contribute in part to beneficial actions of AT1R blockers in the treatment of hypertension.

Aldosterone-Induced Activation of Signaling Pathways Requires Activity of Angiotensin Type 1a Receptors

Rationale: Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)–mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT1R) and mineralocorticoid receptor (MR) are mostly unknown. Objectives: We tested whether the lack of Ang II type 1a receptor (AT1aR) or Ang II type 1b receptor (AT1bR) would decrease cellular effects induced by aldosterone. Methods and Results: We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT1aR, AT1bR, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-&kgr;B phosphorylation after a 20-minute stimulation. Small interference RNA for AT1aR downregulated phosphorylation of ERK1/2, JNK, and NF-&kgr;B after aldosterone stimulation compared to controls. Downregulation of AT1bR or MR only abolished the activation of NF-&kgr;B. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT1aR knockout mice. Conclusion: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT1aR and AT1bR. Our data suggest that aldosterone augments AT1R-dependent activation of ERK1/2, JNK, and NF-&kgr;B in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT1R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

Thioredoxin in vascular biology: role in hypertension

The thioredoxin (TRX) system consists of TRX, TRX reductase, and NAD(P)H, and is able to reduce reactive oxygen species (ROS) through interactions with the redox-active center of TRX, which in turn can be reduced by TRX reductase in the presence of NAD(P)H. Among the TRX superfamily is peroxiredoxin (PRX), a family of non-heme peroxidases that catalyzes the reduction of hydroperoxides into water and alcohol. The TRX system is active in the vessel wall and functions either as an important endogenous antioxidant or interacts directly with signaling molecules to influence cell growth, apoptosis, and inflammation. Recent evidence implicates TRX in cardiovascular disease associated with oxidative stress, such as cardiac failure, arrhythmia, ischemia reperfusion injury, and hypertension. Thioredoxin activity is influenced by many mechanisms, including transcription, protein-protein interaction, and post-translational modification. Regulation of TRX in hypertensive models seems to be related to oxidative stress and is tissue- and cell-specific. Depending on the models of hypertension, TRX system could be upregulated or downregulated. The present review focuses on the role of TRX in vascular biology, describing its redox activities and biological properties in the media and endothelium of the vessel wall. In addition, the pathopysiological role of TRX in hypertension and other cardiovascular diseases is addressed.

Aldosterone induces arterial stiffness in absence of oxidative stress and endothelial dysfunction.

Aims Monocyte/macrophages participate in inflammatory responses that may play an important role in mineralocorticoid-induced vascular damage. We hypothesized that monocyte/macrophages modulate aldosterone effects on oxidative stress, endothelial function, and ultimately vascular stiffness. Methods Adult heterozygous osteopetrotic (Op/+) and wild-type mice were infused with aldosterone (600 μg/kg per day s.c. with Alzet osmotic minipumps) and received 1% NaCl in drinking water or were infused with vehicle for 14 days. Blood pressure was measured by the tail-cuff method. Endothelial function was determined in mesenteric arteries on a pressurized myograph by the response to acetylcholine following norepinephrine preconstriction. Extracellular matrix was quantified by immunohistochemistry, reactive oxygen species by image analysis of dihydroethidium staining, and reduced nicotinamide adenine dinucleotide phosphate oxidase activity by chemiluminescence. Results Body weight and blood pressure did not change following aldosterone treatment. Aldosterone induced stiffening of resistance arteries among all treated animals, as reflected by decreased sum of squares of strain from 2.07 ± 0.15 to 1.54 ± 0.29 in wild type, and from 2.68 ± 0.28 to 2.04 ± 0.15 in Op/+, and increased fibronectin-to-elastin ratio from 1.12 ± 0.40 to 4.52 ± 0.47 and 0.92 ± 0.47 to 5.26 ± 0.88, respectively. Endothelial function was impaired and reactive oxygen species increased only in aldosterone-treated wild-type mice. Reduced nicotinamide adenine dinucleotide phosphate oxidase activity was unaffected. Conclusion Monocyte/macrophage deficiency in Op/+ mice results in absence of aldosterone-induced oxidative stress and endothelial dysfunction, but does not play a role in aldosterone-induced arterial stiffness. Thus, although monocyte/macrophage-mediated inflammatory responses play a role in oxidative stress and endothelial dysfunction, vascular stiffening in response to aldosterone may be independent of inflammation.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension: Regulation of Oxidative Stress

Vascular oxidative stress and inflammation play an important role in angiotensin II–induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase–activated protein kinase 2 (MK2), a downstream target of p38 mitogen–activated protein kinase, is involved in angiotensin II–induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II–induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II–induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II–induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II–induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II–induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II–induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.

Differential regulation of thioredoxin and NAD(P)H oxidase by angiotensin II in male and female mice.

Objective We hypothesized that downregulation of the antioxidant thioredoxin system contributes to oxidative stress in angiotensin II-induced hypertension. As oestrogen may protect against oxidative stress, we also evaluated whether the thioredoxin system, particularly in the heart, is differentially regulated between females and males. Results C57Bl/6 male and intact or ovariectomized female mice were infused with angiotensin II (400 ng/kg per minute for 2 weeks). Systolic blood pressure (SBP) was increased by angiotensin II in both groups week 1 and increased further in males versus females in week 2. Angiotensin II increased SBP from 112 ± 6 to 143 ± 9 mmHg in ovariectomized mice. Basal cardiac thioredoxin expression and reductase activity were significantly higher (two to threefold) in females versus males. Angiotensin II increased thioredoxin expression (approximately threefold), thioredoxin reductase activity, nicotinamide adenine dinucleotide phosphate, reduced form (NAD(P)H) oxidase activity and plasma thiobarbituric acid-reducing substances in males but not in females. Angiotensin II increased thioredoxin expression and NAD(P)H oxidase activity in ovariectomized versus control mice. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE/Ref-1) activation, which interacts with thioredoxin to activate inflammatory transcription factors, was increased by angiotensin II only in males. Conclusion These results demonstrate sex dimorphism with respect to thioredoxin, oxidative stress and inflammation, and suggest the differential regulation of blood pressure, the cardiac thioredoxin system and NAD(P)H oxidase activity by angiotensin II in male and female mice. Whereas angiotensin II increases the activity of thioredoxin reductase and APE/Ref-1, enhances oxidative stress, and amplifies blood pressure elevation in males, it has little effect in females. Such differences may partly relate to the protective actions of oestrogens.

Antiproliferative effect of estrogen in vascular smooth muscle cells is mediated by Kruppel-like factor-4 and manganese superoxide dismutase

The mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) and the zinc finger transcription factor Kruppel-like factor-4 (KLF4) are involved in the regulation of redox homeostasis, apoptosis and cell proliferation. We have shown that estrogen exerts antioxidative actions via induction of MnSOD in cultured rat aortic vascular smooth muscle cells (VSMC). The purpose of the present study was to investigate whether estrogen inhibits VSMC proliferation via alteration of KLF4 and MnSOD expression. In cultured rat aortic VSMC, estrogen binding to estrogen receptor-alpha led to rapid increase in KLF4 expression and reduction of cell proliferation by 50%. Protein separation revealed that KLF4 was shifted to the nucleus when VSMC were treated with estrogen. Estrogen-mediated induction of KLF4 and the antiproliferative effect involved activation of PI-3 kinase, Akt phosphorylation and induction of NO synthase activity. Experiments in freshly isolated denuded aortic segments revealed an increase in KLF4 abundance after estrogen treatment and demonstrated that eNOS is expressed in the media at low levels. Transfection experiments showed that estrogen-induced overexpression of MnSOD required KLF4 and that both KLF4 and MnSOD were indispensable for the observed antiproliferative effect of estrogen in VSMC. To confirm these data in vivo, we investigated neointima formation after carotid artery injury in wild-type (WT) and MnSOD+/− mice. Estrogen deficiency led to enhanced neointima formation and higher numbers of Ki67-positive proliferating cells in the neointima of ovariectomized WT and MnSOD+/− mice. Moreover, MnSOD+/− mice showed more extensive neointima formation and Ki67 immunostaining. Interestingly, estrogen replacement prevented neointima formation in WT mice but failed to completely inhibit neointima formation in MnSOD+/− mice. Cultured VSMC derived from MnSOD+/− mice showed enhanced proliferation as compared to WT VSMC, and estrogen treatment failed to inhibit proliferation in MnSOD+/− VSMC. In conclusion, these data demonstrate the importance of MnSOD and KLF4 for proliferation control in VSMC. Our results provide novel insights into how proliferation of VSMC is regulated by estrogen and may help to identify novel targets for the treatment of vascular diseases such as restenosis.

Inactivation of endothelial proprotein convertase 5/6 decreases collagen deposition in the cardiovascular system: role of fibroblast autophagy

Proprotein convertase (PC) 5/6 belongs to a family of secretory proteases involved in proprotein proteolysis. Several studies suggest a role for PC5/6 in cardiovascular disease. Because lethality at birth of mice lacking PC5/6 precluded elucidation of its function in the adult, we generated mice in which the gene of PC5/6 (pcsk5) is specifically inactivated in endothelial cells (ecKO), which are viable and do not exhibit overt abnormalities. In order to uncover the function of PC5/6 in the cardiovascular system, the effect of ecKO was studied in aging mice. In 16 to 18-month-old ecKO mice, the left ventricle (LV) mass, media cross-sectional area of aorta and coronary arteries, and media-to-lumen ratio of mesenteric arteries were decreased. The LV presented decreased diastolic function, and mesenteric arteries showed decreased stiffness. Collagen was decreased in the LV myocardial interstitium and perivascularly in coronary arteries and aorta. Cardiovascular hypotrophy likely develops with aging, since no significant changes were observed in 2-month-old ecKO mice. Fibroblasts, as a source of collagen in myocardium and vasculature, may play a role in the decrease in collagen deposition. Fibroblasts co-cultured with ecKO endothelial cells showed decreased collagen production, decreased insulin-like growth factor (IGF)-1/Akt/mTOR signaling, and enhanced autophagic activation. PC5/6 inactivation in endothelial cells results in cardiovascular hypotrophy associated with decreased collagen deposition, decreased LV diastolic function, and vascular stiffness, suggesting a trophic role of endothelial PC5/6 in the cardiovascular system, likely mediated by IGF-1/Akt/mTOR signaling and control of autophagy.