Induction of hypertension and peripheral inflammation by reduction of extracellular superoxide dismutase in the central nervous system

Abstract

The circumventricular organs (CVOs) lack a well-formed blood-brain barrier and produce superoxide in response to angiotensin II and other hypertensive stimuli. This increase in central superoxide has been implicated in the regulation of blood pressure. The extracellular superoxide dismutase (SOD3) is highly expressed in cells associated with CVOs and particularly with tanycytes lining this region. To understand the role of SOD3 in the CVOs in blood pressure regulation, we performed intracerebroventricular injection an adenovirus encoding Cre-recombinase (5x10(8) particles per milliliter) in mice with loxP sites flanking the SOD3 coding region (SOD3(loxp/loxp) mice). An adenovirus encoding red-fluorescent protein was injected as a control. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low-dose angiotensin II (140 ng/kg per day), whereas intracerebroventricular injection of adenovirus encoding red-fluorescent protein had minimal effects on these parameters. Adenovirus encoding Cre-recombinase-treated mice exhibited increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production, and T-cell activation as characterized by increased circulating CD69(+)/CD3(+) cells. Deletion of CVO SOD3 also markedly increased vascular T-cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in the regulation of blood pressure, and its loss promotes T-cell activation and vascular inflammation, in part by modulating sympathetic outflow. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli, such as angiotensin II.

Figure 1

Effect of ICV AdCre and AdRFP on SOD3 protein in various organs. Panels A–C show immunostaining for SOD3 (brown staining) in the subfornical organ (Panel A) the kidneys (Panel B) and the aortas (Panel C) of mice injected with either AdRFP or AdCre. Panels D and E show Western blots for SOD3 in kidney and aortas with b-actin as a loading control. Adjacent to each Western blot are mean densitometric analyses presented as a bar graph (n = 4 for each, n.s. = not significant).

Figure 2

Effect of SOD3 deletion in the circumventricular structure on hemodynamics. Mice received ICV injections of either AdCre or AdRFP and blood pressure was measured by tail cuff (Panel A) or radiotelemetry (Panel B). Heart rate (Panel C) and systolic blood pressure (Panel D) variability was analyzed using Hemolab software package (LF/HF = Low Frequency/High Frequency).

Figure 3

Indices of oxidative stress as detected by nitrotyrosine staining and superoxide detection using electron spin resonance. Mice underwent ICV injections of either AdCre or AdRFP and subsequent infusion of angiotensin II as in figure 2. Panel A shows immunostaining for nitrotyrosine in the subfornical organ (SFO) and adjacent structures in the 3rd ventricle (3V). Panel B shows quantification of pixel intensities from experiments depicted in A (n = 4–5). Panel C shows quantification of vascular superoxide production as detected by ESR and the spin probe CAT1-H (n = 6).

Figure 4

Role of circumventricular SOD3 in T cell activation and vascular infiltration of inflammatory cells. Mice were treated as in figure 2 and 14 days following either buffer or angiotensin II infusion (140 ng/kg/min) FACS was employed for analysis of peripheral blood and vascular cells. Panel A shows the percent of total peripheral blood mononuclear cells for circulating CD3+ cells or percent of CD3+ that are CD4+. Panel B shows the percent of CD4+ cells expressing the early activation marker CD69. Panel C shows the percent of total T cells (CD3+) that are double negative for both CD4 and CD8. Panel D shows FACS analysis of single cell suspensions of aortic homogenates. Panel E shows the total number of CD4+/CD69+ cells per aorta. Panel F shows immunostaining for CD3+ cells in aortas and perivascular fat mice treated with either ICV AdRFP or angiotensin II or AdCre and angiotensin II for two weeks (PBMC: Peripheral Blood Mononuclear Cells, n.s. = not significant).