Asia Rehman

T Regulatory Lymphocytes Prevent Aldosterone-Induced Vascular Injury

Aldosterone mediates actions of the renin-angiotensin-aldosterone system inducing hypertension, oxidative stress, and vascular inflammation. Recently, we showed that angiotensin II–induced hypertension and vascular damage are mediated at least in part by macrophages and T-helper effector lymphocytes. Adoptive transfer of suppressor T-regulatory lymphocytes (Tregs) prevented angiotensin II action. We hypothesized that Treg adoptive transfer would blunt aldosterone-induced hypertension and vascular damage. Thirteen to 15-week–old male C57BL/6 mice were injected intravenously at 1-week intervals with 3×105 CD4+CD25+ cells (representing Treg) or control CD4+CD25− cells and then infused or not for 14 days with aldosterone (600 &mgr;g/kg per day, SC) while receiving 1% saline to drink. Aldosterone induced a small but sustained increase in blood pressure (P<0.001), decreased vasodilatory responses to acetylcholine by 66% (P<0.001), increased both media:lumen ratio (P<0.001) and media cross-sectional area of resistance arteries by 60% (P<0.05), and increased NADPH oxidase activity 2-fold in aorta (P<0.001), kidney and heart (P<0.05), and aortic superoxide production. As well, aldosterone enhanced aortic and renal cortex macrophage infiltration and aortic T-cell infiltration (all P<0.05), and tended to decrease Treg in the renal cortex. Treg adoptive transfer prevented all of the vascular and renal effects induced by aldosterone. Adoptive transfer of CD4+CD25− cells exacerbated aldosterone effects except endothelial dysfunction and increases in media:lumen ratio of resistance arteries. Thus, Tregs suppress aldosterone-mediated vascular injury, in part through effects on innate and adaptive immunity, suggesting that aldosterone-induced vascular damage could be prevented by an immunomodulatory approach.

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.

Vascular Effects of Antihypertensive Drug Therapy

Hypertension is associated with structural and functional alterations in the vasculature that lead to hemodynamic disturbances and target organ damage. The benefit of reducing blood pressure on risk reduction is well established. Antihypertensive drugs partially correct hypertensive vascular changes by a number of mechanisms, but their influence may vary in different vascular beds. Recently, combinations of drugs with complementary or synergistic effects have shown favorable effects on the vasculature; these combinations may contribute to risk reduction and improve outcomes in the future. Clinical trial evidence has shown an improvement in morbidity and mortality indicators that could be related to vascular effects of antihypertensive drugs, but this effect needs to be proven in future long-term prospective studies involving simultaneous evaluation of small-artery and large-artery properties.

Inducible Human Endothelin-1 Overexpression in Endothelium Raises Blood Pressure via Endothelin Type A Receptors

The mechanisms of blood pressure regulation by endothelin-1 produced by endothelial cells are complex and still unclear. Transgenic mice with endothelium-restricted human endothelin-1 (EDN1) overexpression presented vascular damage but no significant change in blood pressure, which could be because of adaptation to life-long exposure to elevated endothelin-1 levels. We now generated a tamoxifen-inducible endothelium-restricted EDN1 overexpressing transgenic mouse (ieET-1) using Cre/loxP technology. Sixteen days after tamoxifen treatment, ieET-1 mice presented ≥10-fold increase in plasma endothelin-1 (P<0.01) and ≥20 mm Hg elevation in systolic blood pressure (P<0.01), which could be reversed by atrasentan (P<0.05). Endothelin-1 overexpression did not cause vascular or kidney injury or changes in kidney perfusion or function. However, endothelin type A and B receptor expression was differentially regulated in the mesenteric arteries and the kidney. Our results demonstrate using this ieET-1 mouse model that 21 days of induction of endothelin-1 overexpression caused endothelin-1–dependent elevated blood pressure mediated by endothelin type A receptors.

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.

MPS 18-02 ENDOTHELIN-1 OVEREXPRESSION PRESERVES ENDOTHELIAL FUNCTION IN MICE WITH VASCULAR SMOOTH MUSCLE CELL-SPECIFIC DELETION OF PPAR-GAMMA

Objective: Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents. Ppar&ggr; inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular damage. Transgenic mice overexpressing endothelin (ET)-1 in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (smPpar&ggr;−/−) will exaggerate ET-1-induced vascular damage. Design and Method: Eleven week-old male control, eET-1, smPpar&ggr;−/− and eET-1/smPpar&ggr;−/− mice were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 4 weeks later. BP was measured by telemetry. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining, monocyte/macrophage infiltration by immunofluorescence and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA) or perivascular fat (PVAT). Spleen T cell and monocyte profiles were assessed by flow cytometry. Results: Systolic BP was 20 mmHg higher in eET-1 and unaffected by Ppar&ggr; inactivation. MA vasorelaxation to acetylcholine was impaired 37% only in smPpar&ggr;−/−. Likewise, ET-1-induced contractions were enhanced only in smPpar&ggr;−/−. ROS levels were increased 1.7-fold in smPpar&ggr;−/− and 2.5-fold in eET-1/smPpar&ggr;−/−. Monocyte/macrophage infiltration in PVAT was 2-fold higher in eET-1 and smPpar&ggr;−/−, which was further increased 2-fold in eET-1/smPpar&ggr;−/−. The percentage of CD11b+ cells was increased 2.3-fold in smPpar&ggr;−/− and further increased 1.5-fold in eET-1/smPpar&ggr;−/−. The percentage of Ly-6Chi monocytes was increased ∼1.8-fold in eET-1 and smPpar&ggr;−/− but not eET-1/smPpar&ggr;−/−. The percentage of T regulatory cells was increased 1.5-fold in smPpar&ggr;−/− and decreased by 26% in eET-1, which was further decreased by 38% in eET-1/smPpar&ggr;−/−. Conclusions: These results suggest that increased ET-1 paradoxically preserves endothelial function in mice with inactivated VSMC Ppar&ggr; despite enhanced oxidative stress. Flow cytometry data indicate that infiltrating monocyte/macrophages in these mice might be anti-inflammatory.

OS 15-02 MATRIX METALLOPROTEINASE 2 KNOCKOUT PROTECTS FROM ANGIOTENSIN II-INDUCED VASCULAR INJURY IN PART VIA INHIBITION OF EGFR AND ERK1/2 ACTIVATION.

Objective: Matrix metalloproteinase 2 (MMP2) is involved in cardiovascular disease. Whether MMP2 plays a role in hypertension and vascular damage is unknown. We hypothesized that Mmp2 knockout will prevent angiotensin (Ang) II-induced hypertension and vascular injury. Design and Method: Mmp2 knockout (Mmp2−/−) and wild-type (WT) mice were infused with Ang II (1000 ng/kg/min, SC) for 14 days. Systolic blood pressure (SBP) was measured by telemetry, and mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography. Reactive oxygen species (ROS) generation using dihydroethidium staining, and vascular cell adhesion protein 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) expression and monocyte/macrophage infiltration by immunofluorescence were determined in the aortic wall or perivascular fat (PVAT). Spleen T cell and monocyte profiles were assessed by flow cytometry. Vascular smooth muscle cells (VSMCs) were isolated from MA of WT and Mmp2−/− mice, stimulated 5 min with Ang II (100 nM) and epidermal growth factor receptor (EGFR) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation measured by Western blot. Results: Ang II increased SBP by 50 mmHg (P < 0.01), decreased by 50% MA vasodilator responses to acetylcholine (P < 0.01) and increased 1.7-fold MA media-to-lumen ratio (P < 0.01), 1.4-fold media cross-sectional area (P < 0.05), and stiffness (P < 0.01), as shown by a leftward shift of the stress/strain relationship, in WT. Furthermore, Ang II enhanced 12-fold aortic ROS generation (P < 0.01), 3.4-fold aortic VCAM-1 (P < 0.01), 6.5-fold MCP-1 expression (P < 0.01), 8-fold PVAT monocyte/macrophage infiltration (P < 0.05), and ∼2-fold spleen activated CD4+CD69+ and CD8+CD69+ T cells, and pro-inflammatory Ly-6Chi monocytes (P < 0.05) in WT. Ang II increased phosphorylation of EGFR 1.9-fold and ERK1/2 1.4-fold in VSMCs (P < 0.05). Mmp2 knockout prevented or reduced all of the above (P < 0.05) except SBP elevation. Conclusions: MMP2 plays an important role in Ang II-induced vascular injury, which could be mediated at least in part through EFGR and ERK1/2 activation in VSMCs.

Endothelin-1-induced oxidative stress and inflammatory cell infiltration contribute to high-fat diet induced-atherosclerosis and aneurysm formation in apolipoprotein E knockout mice

monocyte/macrophage infiltration by 160% (P b 0.001), and raised plasma ET-1 by 130% (P b 0.05). EPO had no effect on wild-type mice. Exercise training prevented all of the above effects of EPO as well as ET-1 (P b 0.05). EPO-induced SBP rise and adverse vascular effects are dependent on the pre-existing level of ET-1 expression. Exercise training prevented EPO-induced BP rise and adverse vascular effects in part by inhibiting ET-1 overexpression-induced oxidative stress, inflammation and immune activation.