B cell-specific knockout of AID protects against atherosclerosis.

Antigen-naive IgM-producing B cells are atheroprotective, whereas mature B cells producing class-switched antibodies promote atherosclerosis. Activation-induced cytidine deaminase (AID), which mediates class switch recombination (CSR), would thus be expected to foster atherosclerosis. Yet, AID also plays a major role in the establishment of B cell tolerance. We sought to define whether AID affects atherosclerotic plaque formation. We generated Ldlr-/- chimeras transplanted with bone marrow from Aicda-/- or wild-type (WT) mice, fed a HFD for 14 weeks. Decreased B cell maturation in Ldlr-/-Aicda-/- mice was demonstrated by 50% reduction in splenic and aortic BAFFR expression, a key signaling component of B2 cell maturation. This was associated with increased plasma IgM in Ldlr-/-Aicda-/- compared with Ldlr-/-WT animals. Importantly, Ldlr-/-Aicda-/- mice had reduced atherosclerotic lesion area (0.20 ± 0.03mm2) compared with Ldlr-/-WT (0.30 ± 0.04mm2, P < 0.05), although no differences in plaque composition were noted between groups. In addition, immunofluorescence analysis revealed increased splenic B and T cell areas independent of cell number. AID depletion directly inhibits atherosclerotic plaque formation.

Design and development of Branched Poly(ß-aminoester) nanoparticles for Interleukin-10 gene delivery in a mouse model of atherosclerosis.

Atherosclerosis progression is a result of chronic and non-resolving inflammation, effective treatments for which still remain to be developed. We designed and developed branched poly(ß-amino ester) nanoparticles (NPs) containing plasmid DNA encoding IL-10, a potent anti-inflammatory cytokine to atherosclerosis. The NPs (NP-VHPK) are functionalized with a targeting peptide (VHPK) specific for VCAM-1, which is overexpressed by endothelial cells at sites of atherosclerotic plaque. The anionic coating affords NP-VHPK with significantly lower toxicity than uncoated NPs in both endothelial cells and red blood cells (RBCs). Following injection of NP-VHPK in ApoE-/- mice, Cy5-labelled IL-10 significantly accumulates in both whole aortas and aortic sinus sections containing plaque compared to injection with a non-targeted control. Furthermore, IL-10 gene delivery results in an attenuation of inflammation locally at the plaque site. NP-VHPK may thus have the potential to reduce the inflammatory component of atherosclerosis in a safe and effective manner. STATEMENT OF SIGNIFICANCE: Atherosclerosis is a chronic inflammatory disease that results in the formation of lipid-laden plaques within vascular walls. Although treatments using drugs and antibodies are now beginning to address the inflammation in atherosclerosis, neither is sufficient for long-term therapy. In this paper, we introduce a strategy to deliver genes encoding the anti-inflammatory protein interleukin-10 (IL-10) in vivo. We showed that Branched Poly(ß-aminoester) carrying the IL-10 gene are able to localize specifically at the plaque via surface-functionalized targeting moieties against inflamed VCAM-1 and/or ICAM-1 and to facilitate gene transcription by ECs to increase the local concentration of the IL-10 within the plaque. To date, there is no report involving non-viral nanotechnology to provide gene-based therapies for atherosclerosis.

MNK2 governs the macrophage antiinflammatory phenotype.

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.

FHL2 Is Essential for Spleen T Cell-Dependent B Cell Activation and Antibody Response.

Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2-/- mouse spleens displayed enlarged follicles with more B cells. When a T cell-dependent immune response was elicited using SRBC, FHL2-/- germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2-/- mice, and circulating plasma cells were also reduced in FHL2-/- This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2-/- B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell-deficient µMT mice transplanted with WT or FHL2-/- bone marrow, suggesting that the defects observed in FHL2-/- mice were not B cell intrinsic. However, spleen lysates from FHL2-/- mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell-dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.

Endothelial nitric oxide synthase overexpressing human early outgrowth cells inhibit coronary artery smooth muscle cell migration through paracrine functions.

Cells mobilized from the bone marrow can contribute to endothelial regeneration and repair. Nevertheless, cardiovascular diseases are associated with diminished numbers and function of these cells, attenuating their healing potential. Gene transfer of endothelial nitric oxide synthase (eNOS) can restore the activity of circulating cells. Furthermore, estrogen accelerates the reendothelialization capacity of early outgrowth cells (EOCs). We hypothesized that overexpressing eNOS alone or in combination with estrogen stimulation in EOCs would potentiate the beneficial effects of these cells in regulating smooth muscle cell (SMC) function. Native human EOCs did not have any effect on human coronary artery SMC (hCASMC) proliferation or migration. Transfecting EOCs with a human eNOS plasmid and/or stimulating with 17ß-estradiol (E2) increased NO production 3-fold and enhanced EOC survival. Moreover, in co-culture studies, eNOS overexpressing or E2-stimulated EOCs reduced hCASMC migration (by 23% and 56% respectively), vs. control EOCs. These effects do not implicate ERK1/2 or focal adhesion kinases. Nevertheless, NOS-EOCs had no effect on hCASMC proliferation. These results suggest that overexpressing or activating eNOS in EOCs increases their survival and enhances their capacity to regulate SMC migration through paracrine effects. These data elucidate how eNOS overexpression or activation in EOCs can prevent vascular remodeling.

Matrix metalloproteinase-2 knockout prevents angiotensin II-induced vascular injury.

AIMS: Matrix metalloproteinases (MMPs) have been implicated in the development of hypertension in animal models and humans. Mmp2 deletion did not change Ang II-induced blood pressure (BP) rise. However, whether Mmp2 knockout affects angiotensin (Ang) II-induced vascular injury has not been tested. We sought to determine whether Mmp2 knockout will prevent Ang II-induced vascular injury. METHODS AND RESULTS: A fourteen-day Ang II infusion (1000 ng/kg/min, SC) increased systolic BP, decreased vasodilatory responses to acetylcholine, induced mesenteric artery (MA) hypertrophic remodelling, and enhanced MA stiffness in wild-type (WT) mice. Ang II enhanced aortic media and perivascular reactive oxygen species generation, aortic vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 expression, perivascular monocyte/macrophage and T cell infiltration, and the fraction of spleen activated CD4+CD69+ and CD8+CD69+ T cells, and Ly-6Chi monocytes. Study of intracellular signalling showed that Ang II increased phosphorylation of epidermal growth factor receptor and extracellular-signal-regulated kinase 1/2 in vascular smooth muscle cells isolated from WT mice. All these effects were reduced or prevented by Mmp2 knockout, except for systolic BP elevation. Ang II increased Mmp2 expression in immune cells infiltrating the aorta and perivascular fat. Bone marrow (BM) transplantation experiments revealed that in absence of MMP2 in immune cells, Ang II-induced BP elevation was decreased, and that when MMP2 was deficient in either immune or vascular cells, Ang II-induced endothelial dysfunction was blunted. CONCLUSIONS: Mmp2 knockout impaired Ang II-induced vascular injury but not BP elevation. BM transplantation revealed a role for immune cells in Ang II-induced BP elevation, and for both vascular and immune cell MMP2 in Ang II-induced endothelial dysfunction.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

OBJECTIVE: Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. APPROACH AND RESULTS: Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6-/- mice contain less inflammatory (CCR2hiCX3CR1lo) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2hiCX3CR1lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). CONCLUSIONS: This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2hiCX3CR1lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis.

Absence of Four-and-a-Half LIM Domain Protein 2 Decreases Atherosclerosis in ApoE-/- Mice.

OBJECTIVE: Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial cells, vascular smooth muscle cells, and leukocytes. It regulates cell survival, migration, and inflammatory response, but its role in atherogenesis is unknown. APPROACH AND RESULTS: To investigate the role of FHL2 in atherosclerosis, FHL2-deficient mice were crossed with ApoE-deficient mice, to generate ApoE/FHL2-/- mice. After high-fat diet, ApoE/FHL2-/- mice had significantly smaller atherosclerotic plaques than ApoE-/- mice in the aortic sinus, the brachiocephalic artery, and the aorta. This was associated with enhanced collagen and smooth muscle cell contents and a 2-fold reduction in macrophage content within the plaques of ApoE/FHL-2-/- versus ApoE-/- mice. This could be explained, in part, by the reduction in aortic ICAM-1 (intracellular adhesion molecule) mRNA and VCAM-1 (vascular cell adhesion molecule) protein expression in the plaque. Aortic gene expression of the chemokines CX3CL1 and CCL5 was increased in ApoE/FHL2-/- versus ApoE-/- mice. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of proinflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- versus ApoE-/- mice. Furthermore, mRNA levels of CX3CR1 were 2-fold higher in monocytes from ApoE/FHL2-/- versus ApoE-/- mice. Finally, we investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. After being irradiated, ApoE-/- or ApoE/FHL2-/- mice were transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After high-fat diet, both chimeric groups developed smaller plaques than ApoE-/- transplanted with ApoE-/- bone marrow. CONCLUSIONS: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting proinflammatory chemokine production, adhesion molecule expression, and proinflammatory monocyte recruitment.

Inhibition of four-and-a-half LIM domain protein-2 increases survival, migratory capacity, and paracrine function of human early outgrowth cells through activation of the sphingosine kinase-1 pathway: implications for endothelial regeneration.

RATIONALE: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. OBJECTIVE: We investigated the role of FHL2 in the regulation of EOCs. METHODS AND RESULTS: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell-derived factor-1-induced EOC migration through upregulation of αv/ß3, αv/ß5, and ß2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2(-/-) mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2(-/-) versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2(-/-) versus WT EOCs. CONCLUSIONS: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.

Protective role of vascular smooth muscle cell PPARγ in angiotensin II-induced vascular disease.

AIMS: Vascular peroxisome proliferator-activated receptor γ (PPARγ) activation improves vascular remodelling and endothelial function in hypertensive rodents. The goal of this study was to determine that vascular smooth muscle cell (VSMC) PPARγ exerts a vascular protective role beyond its metabolic effects. METHODS AND RESULTS: We generated a model of adult inducible VSMC-specific Pparγ inactivation to test the hypothesis that PPARγ counteracts angiotensin (Ang) II-induced vascular remodelling and endothelial dysfunction. Inducible VSMC Pparγ knockout mice were generated by crossing Pparγ floxed mice with mice expressing a tamoxifen-inducible Cre recombinase Smooth muscle (Sm) myosin heavy chain promoter control. Eight-to-ten-week-old SmPparγ(-/-) and control mice were infused with a nonpressor dose of Ang II for 7 days. Blood pressure was unaffected. Mesenteric arteries showed eutrophic remodelling in Ang II-infused control mice and hypertrophic remodelling in Ang II-infused SmPparγ(-/-) mice. Endothelium-dependent relaxation to acetylcholine was reduced in SmPparγ(-/-) mice and further impaired by Ang II infusion, and was unaffected by an inhibitor of NO synthase, suggesting a defect of NO-mediated relaxation. SmPparγ deletion increased the sensitivity to Ang II-induced contraction. SmPparγ(-/-) mice exhibited enhanced Ang II-induced vascular NADPH oxidase activity and adhesion molecule ICAM-1 and chemokine monocyte chemotactic protein-1 expression. The antioxidant Superoxide dismutase 3 expression was decreased by SmPparγ deletion. Ang II infusion increased the expression of CD3 T-cell co-receptor chain δ and decreased Adiponectin in perivascular adipose tissue of SmPparγ(-/-) mice. CONCLUSION: Inducible Pparγ inactivation in VSMCs exacerbated Ang II-induced vascular remodelling and endothelial dysfunction via enhanced vascular oxidative stress and inflammation, revealing the protective role of VSMC PPARγ in angiotensin II-induced vascular injury.

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 (AT(1)-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 AT(1)-R and Ang II type 2 receptor, mediated through AT(1)-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 AT(1)a-R knockout mice (AT(1)a(-/-)). 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 AT(1)a(-/-) 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 AT(1)a(-/-) 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 AT(1)-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT(1)-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.

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.

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.

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.

Countervailing vascular effects of rosiglitazone in high cardiovascular risk mice: role of oxidative stress and PRMT-1.

In the present study, we tested the hypothesis that the PPARgamma (peroxisome-proliferator-activated receptor gamma) activator rosiglitazone improves vascular structure and function in aged hyperhomocysteinaemic MTHFR (methylene tetrahydrofolate reductase) gene heterozygous knockout (mthfr+/-) mice fed a HCD (high-cholesterol diet), a model of high cardiovascular risk. One-year-old mthfr+/- mice were fed or not HCD (6 mg x kg-1 of body weight x day-1) and treated or not with rosiglitazone (20 mg x kg-1 of body weight x day-1) for 90 days and compared with wild-type mice. Endothelium-dependent relaxation of carotid arteries was significantly impaired (-40%) only in rosiglitazone-treated HCD-fed mthfr+/- mice. Carotid M/L (media-to-lumen ratio) and CSA (cross-sectional area) were increased (2-fold) in mthfr+/- mice fed or not HCD compared with wild-type mice (P<0.05). Rosiglitazone reduced M/L and CSA only in mthfr+/- mice fed a normal diet. Superoxide production was increased in mthfr+/- mice fed HCD treated or not with rosiglitazone, whereas plasma nitrite was decreased by rosiglitazone in mice fed or not HCD. PRMT-1 (protein arginine methyltransferase-1), involved in synthesis of the NO (nitric oxide) synthase inhibitor ADMA (asymmetric omega-NG,NG-dimethylarginine), and ADMA were increased only in rosiglitazone-treated HCD-fed mthfr+/- mice. Rosiglitazone had both beneficial and deleterious vascular effects in this animal model of high cardiovascular risk: it prevented carotid remodelling, but impaired endothelial function in part through enhanced oxidative stress and increased ADMA production in mice at high cardiovascular risk.

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 N(G)-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.

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 (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown. OBJECTIVES: We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) 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 AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. 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 AT(1a)R knockout mice. CONCLUSION: We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

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 microg/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.

Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause.

Oxidative stress is implicated in menopause-associated hypertension and cardiovascular disease. The role of antioxidants in this process is unclear. We questioned whether the downregulation of thioredoxin (TRX) is associated with oxidative stress and the development of hypertension and target-organ damage (cardiac hypertrophy) in a menopause model. TRX is an endogenous antioxidant that also interacts with signaling molecules, such as apoptosis signal-regulated kinase 1 (ASK-1), independently of its antioxidant function. Aged female wild-type (WT) and follitropin receptor knockout (FORKO) mice (20-24 wk), with hormonal imbalances, were studied. Mice were infused with ANG II (400 ng x kg(-1) x min(-1); 14 days). Systolic blood pressure was increased by ANG II in WT (166+/-8 vs. 121+/-5 mmHg) and FORKO (176+/-7 vs. 115+/-5 mmHg; P<0.0001; n=9/group) mice. In ANG II-infused FORKO mice, cardiac mass was increased by 42% (P<0.001). This was associated with increased collagen content and augmented ERK1/2 phosphorylation (2-fold). Cardiac TRX expression and activity were decreased by ANG II in FORKO but not in WT (P<0.01) mice. ASK-1 expression, cleaved caspase III content, and Bax/Bcl-2 content were increased in ANG II-infused FORKO (P<0.05). ANG II had no effect on cardiac NAD(P)H oxidase activity or on O(2)(*-) levels in WT or FORKO. Cardiac ANG II type 1 receptor expression was similar in FORKO and WT. These findings indicate that in female FORKO, ANG II-induced cardiac hypertrophy and fibrosis are associated with the TRX downregulation and upregulation of ASK-1/caspase signaling. Our data suggest that in a model of menopause, protective actions of TRX may be blunted, which could contribute to cardiac remodeling independently of oxidative stress and hypertension.

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.