Sofiane Ouerd

γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular InjuryClinical Perspective

Background: Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the &ggr;&dgr; T cell receptor (TCR) rather than the &agr;&bgr; TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in &ggr;&dgr; T cells; deficiency in &ggr;&dgr; T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and &ggr;&dgr; T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcr&dgr;−/− mice, which are devoid of &ggr;&dgr; T cells, or wild-type mice injected IP with control isotype IgG or &ggr;&dgr; T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR &ggr; constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between &ggr;&dgr; T cells and SBP. Results: Seven- and 14-day Ang II infusion increased &ggr;&dgr; T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcr&dgr;−/− mice (P<0.01). Anti-TCR&ggr;&dgr; antibody-induced &ggr;&dgr; T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcr&dgr;−/− mice (P<0.01). In humans, the association between SBP and &ggr;&dgr; T cells was demonstrated by a multiple linear regression model integrating whole blood TCR &ggr; constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). Conclusions: &ggr;&dgr; T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. &ggr;&dgr; T cells might contribute to the development of hypertension in humans.

Gamma Delta T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury

Background: Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the &ggr;&dgr; T cell receptor (TCR) rather than the &agr;&bgr; TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in &ggr;&dgr; T cells; deficiency in &ggr;&dgr; T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and &ggr;&dgr; T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcr&dgr;−/− mice, which are devoid of &ggr;&dgr; T cells, or wild-type mice injected IP with control isotype IgG or &ggr;&dgr; T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR &ggr; constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between &ggr;&dgr; T cells and SBP. Results: Seven- and 14-day Ang II infusion increased &ggr;&dgr; T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcr&dgr;−/− mice (P<0.01). Anti-TCR&ggr;&dgr; antibody-induced &ggr;&dgr; T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcr&dgr;−/− mice (P<0.01). In humans, the association between SBP and &ggr;&dgr; T cells was demonstrated by a multiple linear regression model integrating whole blood TCR &ggr; constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). Conclusions: &ggr;&dgr; T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. &ggr;&dgr; T cells might contribute to the development of hypertension in humans.

Aldosterone-Induced Vascular Remodeling and Endothelial Dysfunction Require Functional Angiotensin Type 1a Receptors

We investigated the role of angiotensin type 1a receptors (AGTR1a) in vascular injury induced by aldosterone activation of mineralocorticoid receptors in Agtr1a−/− and wild-type (WT) mice infused with aldosterone for 14 days while receiving 1% NaCl in drinking water. Aldosterone increased systolic blood pressure (BP) by ≈30 mm Hg in WT mice and ≈50 mm Hg in Agtr1a−/− mice. Aldosterone induced aortic and small artery remodeling, impaired endothelium-dependent relaxation in WT mice, and enhanced fibronectin and collagen deposition and vascular inflammation. None of these vascular effects were observed in Agtr1a−/− mice. Aldosterone effects were prevented by the AGTR1 antagonist losartan in WT mice. In contrast to aldosterone, norepinephrine caused similar BP increase and mesenteric artery remodeling in WT and Agtr1a−/− mice. Agtr1a−/− mice infused with aldosterone did not increase sodium excretion in response to a sodium chloride challenge, suggesting that sodium retention could contribute to the exaggerated BP rise induced by aldosterone. Agtr1a−/− mice had decreased mesenteric artery expression of the calcium-activated potassium channel Kcnmb1, which may enhance myogenic tone and together with sodium retention, exacerbate BP responses to aldosterone/salt in Agtr1a−/− mice. We conclude that although aldosterone activation of mineralocorticoid receptors raises BP more in Agtr1a−/− mice, AGTR1a is required for mineralocorticoid receptor stimulation to induce vascular remodeling and inflammation and endothelial dysfunction.

Endothelin-1 Overexpression Exaggerates Diabetes-Induced Endothelial Dysfunction by Altering Oxidative Stress.

BACKGROUND Increased endothelin (ET)-1 expression causes endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we hypothesized that overexpression of ET-1 in the endothelium would exaggerate diabetes-induced endothelial dysfunction. METHODS Diabetes was induced by streptozotocin treatment (55mg/kg/day, i.p.) for 5 days in 6-week-old male wild type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription/quantitative PCR were determined. RESULTS Endothelium-dependent vasodilatory responses to acetylcholine of MA were reduced 24% by diabetes in WT ( P < 0.05), and further decreased by 12% in eET-1 ( P < 0.05). Diabetes decreased MA media/lumen in WT and eET-1 ( P < 0.05), whereas ET-1 overexpression increased MA media/lumen similarly in diabetic and nondiabetic WT mice ( P < 0.05). Vascular ROS production was increased 2-fold by diabetes in WT ( P < 0.05) and further augmented 1.7-fold in eET-1 ( P < 0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3 ) expression in eET-1 by 31% ( P < 0.05) but not in WT. Induction of diabetes caused a 52% ( P < 0.05) increase in superoxide dismutase 1 ( Sod1 ) and a 32% ( P < 0.05) increase in Sod2 expression in WT but not in eET-1. CONCLUSIONS Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by decrease in eNOS expression, increase in vascular oxidative stress, and decrease in antioxidant capacity.

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.

Three-Month Endothelial Human Endothelin-1 Overexpression Causes Blood Pressure Elevation and Vascular and Kidney InjuryNovelty and Significance

Endothelium-derived endothelin (ET)-1 has been implicated in the development of hypertension and end-organ damage, but its exact role remains unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited blood pressure rise after a 3-week induction in an ET type A (ETA) receptor-dependent manner, in absence of vascular and renal injury. It is unknown whether long-term ET-1 overexpression results in sustained blood pressure elevation and vascular and renal injury. Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were induced with tamoxifen and 2.5 months later, were treated with or without the ETA receptor blocker atrasentan for 2 weeks. Three-month induction of endothelial human ET-1 overexpression increased blood pressure (P<0.01), reduced renal artery flow (P<0.001), and caused mesenteric small artery stiffening (P<0.05) and endothelial dysfunction (P<0.01). These changes were accompanied by enhanced mesenteric small artery Col1A1 and Col3A1 expression, and perivascular adipose tissue oxidative stress (P<0.05) and monocyte/macrophage infiltration (P<0.05). Early renal injury was demonstrated by increased kidney injury molecule-1 expression in renal cortex tubules (P<0.05), with, however, undetectable lesions using histochemistry staining and unchanged urinary albumin. There was associated increased myeloid (CD11b+) and myeloid-derived suppressive cell (CD11b+Gr-1+) renal infiltration (P<0.01) and greater frequency of myeloid and renal cells expressing the proinflammatory marker CD36 (P<0.05). Atrasentan reversed or reduced all of the above changes (P<0.05) except the endothelial dysfunction and collagen expression and reduced renal artery flow. These results demonstrate that long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation and vascular and renal injury via ETA receptors.

OS 21-01 NOX1 OR NOX4 DELETION PREVENTS TYPE 1 DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION.

Objective : The prognosis of type-1 diabetes remains poor and is primarily related to the increased risk of vascular complications. Overproduction of reactive oxygen species by NADPH oxidase (NOX) is believed to play an important role in diabetes-related vascular injury. NOX1 may play a role in the macrovascular disease, whereas NOX4 may have protective actions. Nevertheless, their role in diabetic microangiopathy is less well understood. We hypothesized that deletion of Nox1 would prevent diabetes-induced endothelial dysfunction and vascular remodeling of small arteries whereas Nox4 would exaggerate vascular injury. Design and Method : Diabetes-related vascular injury were studied in atherosclerosis-prone apolipoprotein knockout (Apoe-/-) mice. Six-week-old male Apoe-/- mice, Apoe-/- mice deficient in Nox1 (Apoe-/-/Nox1y/-) and Nox4 (Apoe-/-/Nox4-/-) were rendered diabetic by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days. Mice were studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Results: Apoe-/- mice presented a maximal endothelium-dependent vasodilatory response to acetylcholine of only 46%, which was further decreased by ∼50% by diabetes. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe-/-/Nox1y/- and Apoe-/-/Nox4-/- mice compared to vehicle-treated Apoe-/- mice. Endothelium-independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in all groups. Diabetes decreased MA stiffness in Apoe-/- mice, as indicated by a rightward displacement of the stress-strain curves, which was blunted by Nox1 or Nox4 knockout. MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe-/- mice. Conclusions: These results suggest that NOX1 and NOX4 play a pathophysiological role in diabetes-induced endothelial dysfunction and contribute to potentially maladaptive changes in vascular stiffness. NOX4 also seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.

OS 21-06 THREE-MONTH EXPOSURE TO ENDOTHELIN-1 OVEREXPRESSION INCREASES BLOOD PRESSURE AND CAUSES SMALL ARTERY INJURY.

Objective: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited a BP increase after three weeks of induction in an ET type A receptor-dependent manner, in absence of evident vascular injury. It is unknown whether long-term exposure to ET-1 overexpression results in persistent BP elevation and vascular injury. Design and Method: Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. BP by telemetry, mesenteric artery (MA) endothelial function and vascular remodeling using pressurized myography and reactive oxygen species (ROS) generation using dihydroethidium staining and immune cell infiltration by immunofluorescence in MA or perivascular fat (PVAT) were determined at the end of the study. Results: Systolic BP was increased by 27 mmHg in ieET-1 compared with ieCre mice (P < 0.001). Endothelium-dependent relaxation responses to acetylcholine were decreased by 50% in ieET-1 compared to ieCre mice (P < 0.01), whereas endothelium-independent relaxation to sodium nitroprusside were unchanged. MA media/lumen and media cross-sectional area were similar in both groups, but stiffness was increased in ieET-1 compared to ieCre mice, as indicated by leftward displacement of the stress-strain curves (14% decrease in strain at 140 mmHg, P < 0.05). ROS generation was enhanced 1.5-fold in PVAT of ieET-1 compared to ieCre mice (P < 0.05). Monocyte/macrophage infiltration was similar whereas CD3+ cell infiltration was 1.4-fold higher in MA PVAT of ieET-1. Conclusions: The results demonstrate that long-term exposure to endothelial ET-1 overexpression caused sustained BP elevation, endothelial dysfunction and vascular stiffening, oxidative stress and CD3+ cell infiltration.

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.

PS 16-31 ENDOTHELIN-1 EXAGGERATES TYPE-1 DIABETES-ACCELERATED ATHEROSCLEROSIS THROUGH NADPH OXIDASES 1 AND 4

Objective: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe−/−) mice exaggerated high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We hypothesized that ET-1 overexpression in the endothelium would exaggerate diabetes-accelerated atherosclerosis through a mechanism involving NOX1 but not NOX4. Design and method: Six-week-old male Apoe−/− mice, eET-1/Apoe−/− and eET-1/Apoe−/− mice deficient in Nox1 (eET-1/Apoe−/−/Nox1y/−) or Nox4 (eET-1/Apoe−/−/Nox4−/−) were rendered diabetic with 55 mg/kg/day streptozotocin (STZ) IP for 5 days and studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Aortic atherosclerotic lesions were quantified using Oil Red O staining. Results: Diabetic Apoe−/− mice presented an impaired maximal endothelium-dependent vasodilatory response to acetylcholine (21%), which was not observed in diabetic eET-1/Apoe−/−, eET-1/Apoe−/−/Nox1y/− or eET-1/Apoe−/−/Nox4−/− mice (99%). Endothelium-independent relaxation to the nitric oxide (NO) donor sodium nitroprusside was similar between groups. Blockade of NO synthase with L-NAME completely blunted endothelium-dependent relaxation to acetylcholine in diabetic eET-1/Apoe−/− mice, which was prevented by Nox1 but not by Nox4 knockout. ET-1 overexpression caused a 1.8-fold increase in MA media/lumen of diabetic Apoe−/− mice, which was further exaggerated 1.2-fold by Nox4 but not Nox1 knockout. ET-1 overexpression exaggerated > 2-fold the atherosclerotic lesion area in the aortic sinus in diabetic Apoe−/− mice, which was reduced ∼40% by Nox1 and Nox4 knockout. Conclusions: Increased levels of ET-1 exaggerate diabetes-accelerated atherosclerosis through NOX1 and NOX4, despite paradoxically improving endothelium-dependent relaxation in small arteries.