Daniel A. Kasal

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.

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, Aldosterone, and Anti-Inflammatory Lymphocytes: Interplay and Therapeutic Opportunities.

Inflammation is recognized as an important factor in the pathophysiology of hypertension, with the renin-angiotensin-aldosterone system (RAAS) playing a key role in the disease. Initially described because of its contribution to extracellular fluid and electrolyte homeostasis, the RAAS has been implicated in endothelial dysfunction, vascular remodeling, oxidative stress, proinflammatory cytokine production, and adhesion molecule synthesis by the vascular wall. Both angiotensin II and aldosterone are involved in these systemic effects, activating innate and adaptive immune responses. This paper highlights some aspects connecting RAAS to the hypertensive phenotype, based on experimental and clinical studies, with emphasis on new findings regarding the contribution of an increasingly studied population of T lymphocytes: the T-regulatory lymphocytes. These cells can suppress inflammation and may exert beneficial vascular effects in animal models of hypertension.

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.

Vascular Dysfunction as Target Organ Damage in Animal Models of Hypertension

Endothelial dysfunction is one of the main characteristics of chronic hypertension and it is characterized by impaired nitric oxide (NO) bioactivity determined by increased levels of reactive oxygen species. Endothelial function is usually evaluated by measuring the vasodilation induced by the local NO production stimulated by external mechanical or pharmacological agent. These vascular reactivity tests may be carried out in different models of experimental hypertension such as NO-deficient rats, spontaneously hypertensive rats, salt-sensitive rats, and many others. Wire myograph and pressurized myograph are the principal methods used for vascular studies. Usually, increasing concentrations of the vasodilator acetylcholine are added in cumulative manner to perform endothelium-dependent concentration-response curves. Analysis of vascular mechanics is relevant to identify arterial stiffness. Both endothelial dysfunction and vascular stiffness have been shown to be associated with increased cardiovascular risk.

Research proposal: inflammation and oxidative stress in coronary artery bypass surgery graft: comparison between diabetic and non-diabetic patients

BackgroundDiabetes mellitus patients (DM) have more severe progression of atherosclerotic disease than non-diabetic (NDM) individuals. In situ inflammation and oxidative stress are key points in the pathophysiology of atherosclerosis, a concept largely based on animal model research. There are few studies comparing inflammation and oxidative stress parameters in medium-sized arteries between DM and NDM patients. A fragment of the internal mammary artery used in coronary artery bypass grafting (CABG) will be employed for this purposeObjectiveTo assess the expression of inflammatory markers tumor necrosis factor-α, transforming growth factor-β1, nuclear factor kappa B, the enzymes superoxide dismutase, and catalase in the vascular wall of the arterial graft used in CABG, comparing DM and NDM patientsResultsThe present study will add information to the vascular degenerative processes occurring in diabetic patients.

16 ENDOTHELIN-1-INDUCED OXIDATIVE STRESS AND INFLAMMATORY CELL INFILTRATION ARE ASSOCIATED WITH DEVELOPMENT OF ANEURYSMS IN HIGH-FAT DIET-FED APOLIPOPROTEIN E KNOCKOUT MICE

Background: Endothelin (ET)-1 has been implicated in the pathogenesis of atherosclerosis. Plasma and tissue ET-1 are increased in human and animal with atherosclerosis. ET-1 overexpression exacerbates high-fat diet (HFD)-induced atherosclerosis in apolipoprotein E knockout (apoE-/-) mouse. Abdominal aorta aneurysms (AAA) occur in association with atherosclerosis. We hypothesized that ET-1-induced ROS and inflammation would increase the occurrence of AAA in HFD fed apoE-/- mice. Design and methods: Eight-week-old male transgenic mice overexpressing preproET-1 in the endothelium (eET-1), apoE-/-, eET-1/apoE-/- and wild type mice were fed a HFD for 8 weeks. Suprarenal aortic perimeter was determined using Oil Red O stained-sections. ROS production using dihydroethidium staining and monocyte/macrophage and T cell infiltration using immunofluorescence with MOMA-2 and anti-CD4 antibodies, respectively, were determined in perivascular fat and media in suprarenal aorta sections. Results: Aneurysms were observed at a suprarenal level in 6 of 15 eET-1/apoE-/- compared to none of 15 apoE-/- (P<0.05). The aortic perimeter was increased 2.5-fold in eET-1/apoE-/- with AAA compared to apoE-/- (P<0.01). ROS production was increased 2.8- and 3.8-fold in perivascular fat and media of eET-1/apoE-/- compared to apoE-/-, respectively (P<0.05). Monocyte/macrophage infiltration was increased 2.6-fold in perivascular fat of eET-1/apoE-/- compared to apoE-/- (P<0.01). CD4+ T cell infiltration was observed in perivascular fat and plaque of of 6 eET-1/apoE-/- compared to none of 6 apoE-/-, respectively (P<0.05). Conclusions: The results suggest that ET-1 plays an important role in development of AAA by increasing oxidative stress and monocyte/macrophage and T cell infiltration in the aorta.