Melissa W. Li

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.

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.

The effects of celiac ganglionectomy on sympathetic innervation to the splanchnic organs in the rat.

The sympathetic nervous system is important in blood pressure regulation, and regionally-specific increases in sympathetic nerve activity occur during the development of hypertension. Sympathetic neurons innervating the splanchnic organs may be especially critical, because sympathetic activity to the splanchnic region is increased early in hypertension development. The celiac ganglionic plexus contains the majority of the sympathetic neurons innervating the splanchnic organs and tissues. Celiac ganglionectomy (CGX) involves surgical removal of the celiac ganglionic plexus, and has been used to study the roles of the splanchnic sympathetic innervation in cardiovascular regulation. In the current study we characterized the short-term (two-week) and long-term (five-week and ten-week) effects of CGX in rats on splanchnic sympathetic nerve structure and function. In the short-term, norepinephrine concentrations in whole splanchnic organs and mesenteric arteries and veins were significantly decreased by CGX. Immunohistochemistry and glyoxylic acid staining showed an almost complete loss of the typical sympathetic innervation of mesenteric arteries and veins. Additionally, CGRP-containing sensory nerves largely disappeared. Constrictor responses of mesenteric arteries and veins to sympathetic nerve stimulation were abolished by CGX. However, the effects of CGX were time-dependent, since significant regeneration of sympathetic nerves in some organs was observed 5weeks after surgery. The inferior mesenteric ganglion had minimal impact on this reinnervation process. In vivo studies showed that CGX significantly lowers resting blood pressure in normal Sprague-Dawley rats. Therefore, CGX is an effective means to impair sympathetic input to the splanchnic organs, but the effect of the procedure is not permanent.

Chronic Activation of Endothelin B Receptors New Model of Experimental Hypertension

Endothelin (ET) exerts powerful pressor actions primarily through activation of the ETA receptor subtype. The ETB receptor (ETBR) subtype, on the other hand, is generally thought to initiate physiological actions that decrease arterial pressure. Such actions include clearing ET from the bloodstream, initiating endothelium-mediated vasodilation, and facilitating renal sodium and water excretion. The effect of long-term activation of the ETBR on arterial pressure, however, never has been directly tested. In this study we evaluated cardiovascular responses to chronic (5-day) activation of ETBR in male rats using continuous intravenous infusion of the selective agonist sarafotoxin 6c. Surprisingly, we found that sarafotoxin 6c caused a sustained increase in arterial pressure that rapidly reversed on termination of infusion. The hypertension was associated with increased renal excretion of sodium and water and decreased plasma volume. Alterations in daily sodium intake did not affect the magnitude of the hypertension. Hemodynamic studies revealed a decreased cardiac output and increased total peripheral resistance during sarafotoxin 6c infusion. Infusion of sarafotoxin 6c caused a small increase in plasma ET levels. Nevertheless, the hypertension was not affected by coadministration of a selective ETA receptor antagonist (atrasentan) but was completely prevented by treatment with a combined ETA receptor and ETBR antagonist (A186280). These experiments reveal for the first time that chronic activation of ETBR in rats causes sustained hypertension.

Increased superoxide levels in ganglia and sympathoexcitation are involved in sarafotoxin 6c-induced hypertension.

Endothelin (ET) type B receptors (ET(B)R) are expressed in multiple tissues and perform different functions depending on their location. ET(B)R mediate endothelium-dependent vasodilation, clearance of circulating ET, and diuretic effects; all of these should produce a fall in arterial blood pressure. However, we recently showed that chronic activation of ET(B)R in rats with the selective agonist sarafotoxin 6c (S6c) causes sustained hypertension. We have proposed that one mechanism of this effect is constriction of capacitance vessels. The current study was performed to determine whether S6c hypertension is caused by increased generation of reactive oxygen species (ROS) and/or activation of the sympathetic nervous system. The model used was continuous 5-day infusion of S6c into male Sprague-Dawley rats. No changes in superoxide anion levels in arteries and veins were found in hypertensive S6c-treated rats. However, superoxide levels were increased in sympathetic ganglia from S6c-treated rats. In addition, superoxide levels in ganglia increased progressively the longer the animals received S6c. Treatment with the antioxidant tempol impaired S6c-induced hypertension and decreased superoxide levels in ganglia. Acute ganglion blockade lowered blood pressure more in S6c-treated rats than in vehicle-treated rats. Although plasma norepinephrine levels were not increased in S6c hypertension, surgical ablation of the celiac ganglion plexus, which provides most of the sympathetic innervation to the splanchnic organs, significantly attenuated hypertension development. The results suggest that S6c-induced hypertension is partially mediated by sympathoexcitation to the splanchnic organs driven by increased oxidative stress in prevertebral sympathetic ganglia.

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.

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.