Tlili Barhoumi

T Regulatory Lymphocytes Prevent Angiotensin II–Induced Hypertension and Vascular Injury

Angiotensin (Ang) II induces hypertension by mechanisms mediated in part by adaptive immunity and T effector lymphocytes. T regulatory lymphocytes (Tregs) suppress T effector lymphocytes. We questioned whether Treg adoptive transfer would blunt Ang II–induced hypertension and vascular injury. Ten- to 12-week–old male C57BL/6 mice were injected IV with 3×105 Treg (CD4+CD25+) or T effector (CD4+CD25−) cells, 3 times at 2-week intervals, and then infused or not with Ang II (1 &mgr;g/kg per minute, SC) for 14 days. Ang II increased systolic blood pressure by 43 mm Hg (P<0.05), NADPH oxidase activity 1.5-fold in aorta and 1.8-fold in the heart (P<0.05), impaired acetylcholine vasodilatory responses by 70% compared with control (P<0.05), and increased vascular stiffness (P<0.001), mesenteric artery vascular cell adhesion molecule expression (2-fold; P<0.05), and aortic macrophage and T-cell infiltration (P<0.001). All of the above were prevented by Treg but not T effector adoptive transfer. Ang II caused a 43% decrease in Foxp3+ cells in the renal cortex, whereas Treg adoptive transfer increased Foxp3+ cells 2-fold compared with control. Thus, Tregs suppress Ang II–mediated vascular injury in part through anti-inflammatory actions. Immune mechanisms modulate Ang II–induced blood pressure elevation, vascular oxidative stress, inflammation, and endothelial dysfunction.

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.

Endothelin-1 Overexpression Exacerbates Atherosclerosis and Induces Aortic Aneurysms in Apolipoprotein E Knockout Mice

Objective—Endothelin (ET)-1 plays a role in vascular reactive oxygen species production and inflammation. ET-1 has been implicated in human atherosclerosis and abdominal aortic aneurysm (AAA) development. ET-1 overexpression exacerbates high-fat diet–induced atherosclerosis in apolipoprotein E−/− (Apoe−/−) mice. ET-1–induced reactive oxygen species and inflammation may contribute to atherosclerosis progression and AAA development. Approach and Results—Eight-week-old male wild-type mice, transgenic mice overexpressing ET-1 selectively in endothelium (eET-1), Apoe−/− mice, and eET-1/Apoe−/− mice were fed high-fat diet for 8 weeks. eET-1/Apoe−/− had a 45% reduction in plasma high-density lipoprotein (P<0.05) and presented ≥2-fold more aortic atherosclerotic lesions compared with Apoe−/− (P<0.01). AAAs were detected only in eET-1/Apoe−/− (8/21; P<0.05). Reactive oxygen species production was increased ≥2-fold in perivascular fat, media, or atherosclerotic lesions in the ascending aorta and AAAs of eET-1/Apoe−/− compared with Apoe−/− (P<0.05). Monocyte/macrophage infiltration was enhanced ≥2.5-fold in perivascular fat of ascending aorta and AAAs in eET-1/Apoe−/− compared with Apoe−/− (P<0.05). CD4+ T cells were detected almost exclusively in perivascular fat (3/6) and atherosclerotic lesions (5/6) in ascending aorta of eET-1/Apoe−/− (P<0.05). The percentage of spleen proinflammatory Ly-6Chi monocytes was enhanced 26% by ET-1 overexpression in Apoe−/− (P<0.05), and matrix metalloproteinase-2 was increased 2-fold in plaques of eET-1/Apoe−/− (P<0.05) compared with Apoe−/−. Conclusions—ET-1 plays a role in progression of atherosclerosis and AAA formation by decreasing high-density lipoprotein, and increasing oxidative stress, inflammatory cell infiltration, and matrix metalloproteinase-2 in perivascular fat, vascular wall, and atherosclerotic lesions.

Deficiency of T-regulatory cells exaggerates angiotensin II-induced microvascular injury by enhancing immune responses.

Aims: T-regulatory lymphocyte (Treg) adoptive transfer prevented angiotensin (Ang) II-induced hypertension and microvascular injury. Scurfy mice are deficient in Treg because of a mutation in the transcription factor forkhead box P3 (Foxp3) gene. Enhanced Ang II effects in the absence of Treg would unambiguously demonstrate their vascular protective role. We hypothesized that adoptive transfer of Scurfy vs. wild-type T cells will exacerbate Ang II-induced microvascular damage in T and B-cell-deficient recombination-activating gene 1 (Rag1) knockout mice. Methods and results: Rag1 knockout mice were injected with vehicle, 107 T cells from wild-type or Scurfy mice or 106 wild-type Treg alone or in combination with Scurfy T cells, and then infused or not with Ang II (490 ng/kg per min, subcutaneous) for 14 days. Ang II increased SBP in all the groups, but DBP only in wild-type and Scurfy T-cell groups. Ang II-induced endothelial dysfunction and oxidative stress in perivascular adipose tissue (PVAT) of mesenteric arteries of the wild-type T-cell group, whereas these were exaggerated in the Scurfy T-cell group. Ang II enhanced microvascular remodeling and stiffness in vehicle and Scurfy T-cell groups. Ang II increased monocyte chemotactic protein-1 expression in the vascular wall and PVAT, monocyte/macrophage infiltration and proinflammatory polarization in PVAT and the renal cortex, and T-cell infiltration in the renal cortex only in the Scurfy T-cell group. Treg coinjection in the vehicle and Scurfy T-cell groups prevented or reduced the effects of Ang II. Conclusion: FOXP3+ Treg deficiency exaggerates Ang II-induced microvascular injury by modulating innate and adaptive immune responses.

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.

Erythropoietin-induced hypertension and vascular injury in mice overexpressing human endothelin-1: exercise attenuated hypertension, oxidative stress, inflammation and immune response.

Objective: Erythropoietin used to correct anaemia in chronic kidney disease (CKD) has been shown to increase blood pressure (BP) in CKD patients and experimental animals. Endothelin (ET)-1 expression is increased in CKD animals and patients, and enhanced by erythropoietin. Erythropoietin-induced BP rise was blunted by ETA receptor blockers. This study was designed to determine whether preexisting endothelin (ET)-1 overexpression is required for erythropoietin to cause adverse vascular effects and whether this could be prevented by exercise training. Methods: Eight to 10-week old male wild-type mice and mice with endothelial-specific ET-1 overexpression (eET-1) were treated or not with EPO (100 IU/kg, SC, 3 times/week). eET-1 was subjected or not to swimming exercise training (1 h/day, 6 days/week) for 8 weeks. SBP, mesenteric artery endothelial function and remodelling, NADPH oxidase activity, reactive oxygen species (ROS) generation, vascular cell adhesion protein (VCAM)-1, monocyte/macrophage infiltration, T regulatory cells (Tregs) and tissue ET-1 and plasma endothelin were determined. Results: Erythropoietin increased SBP by 24 mmHg (P < 0.05) and decreased by 25% vasodilatory responses to acetylcholine (P < 0.01) in eET-1 mice. Erythropoietin enhanced ET-1 induced increase in resistance artery media/lumen ratio (31%, P < 0.05), aortic NADPH oxidase activity (50%, P < 0.05), ROS generation (93%, P < 0.001), VCAM-1 (80%, P < 0.01) and monocyte/macrophage infiltration (159%, P < 0.001), and raised plasma and aortic ET-1 levels (≥130%, P < 0.05). EPO had no effect in wild-type mice. Exercise training prevented all of the above (P < 0.05). Conclusion: Erythropoietin-induced adverse vascular effects are dependent on preexisting elevated ET-1 expression. Exercise training prevented erythropoietin-induced adverse vascular effects in part by inhibiting ET-1 overexpression-induced oxidative stress, inflammation and immune activation.

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.