Anti-inflammatory effects of angiotensin receptor blockers in the brain and the periphery

Abstract

In addition to regulating blood pressure, angiotensin II (Ang II) exerts powerful pro-inflammatory effects in hypertension through stimulation of its AT(1) receptors, most clearly demonstrated in peripheral arteries and in the cerebral vasculature. Administration of Ang II receptor blockers (ARBs) decreases hypertension-related vascular inflammation in peripheral organs. In rodent models of genetic hypertension, ARBs reverse the inflammation in the cerebral microcirculation. We hypothesized that ARBs could be effective in inflammatory conditions beyond hypertension. Our more recent studies, summarized here, indicate that this is indeed the case. We used the model of systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). LPS produces a robust initial inflammatory reaction, the innate immune response, in peripheral organs and in the brain. Pretreatment with the ARB candesartan significantly diminishes the response to LPS, including reduction of pro-inflammatory cytokine release to the general circulation and decreased production and release of the pro-inflammatory adrenal hormone aldosterone. In addition, the ARB very significantly decreased the LPS-induced gene expression of pro-inflammatory cytokines and microglia activation in the brain. Our results demonstrate that AT(1) receptor activity is essential for the unrestricted development of full-scale innate immune response in the periphery and in the brain. ARBs, due to their immune response-limiting properties, may be considered as therapeutically useful in a number of inflammatory diseases of the peripheral organs and the brain.

Fig. 1

Candesartan decreases LPS-induced pro-inflammatory cytokine release to the general circulation. Normal adult male Wistar Hanover rats have undetectable levels of the pro-inflammatory cytokines TNF-α and IL-6 in the circulation. Control rats received vehicle and/or sterile saline. LPS administration (50 μg/Kg, intraperitoneally) induces massive cytokine release to the general circulation, as determined by ELISA 3 h after endotoxin injection. Subcutaneous treatment with candesartan (1 mg/Kg/day for 3 days) does not affect cytokine release in control rats, but significantly reduces the pro-inflammatory cytokine release produced by LPS. Values are mean ± SEM for groups of 7–9 animals measured individually. * P < 0.05 compared with vehicle-saline treated group; ^# P < 0.05 compared with vehicle-LPS treated group; one-way ANOVA and post hoc Tukey’s test. ND, Non detectable. Modified from Sánchez-Lemus et al.

Fig. 2

Candesartan decreases LPS-induced aldosterone release to the general circulation, adrenal aldosterone content and adrenal aldosterone synthase mRNA and protein expression. a. Effect of candesartan on aldosterone release. To study hormone release, rats were cannulated in the tail artery and treated with vehicle or candesartan as described in Fig. 1. Blood samples were collected just before LPS injection and at the indicated times in conscious and undisturbed rats. Values represent mean ± SEM for groups of 5–10 animals measured individually. ^a P < 0.05 compared with correspondent vehicle-LPS treated group; two-way ANOVA and post hoc Bonferroni’s test. Areas under the curve were measured from 0 to 2 h, and from 2 to 4 h. Note that candesartan significantly decreased the enhanced aldosterone release produced by LPS only when the area under the curve was estimated between 0 and 2 h. Values are mean ± SEM for groups of 7–9 animals measured individually. * P < 0.05 compared with vehicle-saline treated group; ^# P < 0.05 compared with vehicle-LPS treated group; one-way ANOVA and post hoc Tukey’s test. b. Effect of candesartan on aldosterone synthesis. To study adrenal aldosterone content and aldosterone synthase mRNA and protein expression, rats were treated as described in Fig. 1. Candesartan treatment completely prevented the LPS-induced increase in adrenal aldosterone content and aldosterone synthase mRNA and protein expression. Data for aldosterone synthase mRNA and protein correspond to relative levels after normalization to 18S rRNA and β-actin, respectively. Note that candesartan treatment, in addition to its action to decrease LPS-induced effects on aldosterone formation and release, decreases the steady state expression of aldosterone synthase mRNA. Values are mean ± SEM for groups of 7–9 animals measured individually. * P < 0.05 compared with vehicle-saline treated group; ^# P < 0.05 compared with vehicle-LPS treated group; one-way ANOVA and post hoc Tukey’s test. Modified from Sánchez-Lemus et al.

Fig. 3

Candesartan significantly decreases LPS-induced TNFα, IL-1β, IκBα, iNOS, ICAM-1, and VCAM-1 mRNA expression in brain cortex. The rats were treated for 14 days with candesartan (1 mg/kg/day) or vehicle via subcutaneously implanted osmotic minipumps. At the end of the treatment the rats of each group were injected either with LPS (50 μg/kg i.p.) or saline and were sacrificed 3 h after the injection. The expression of pro-inflammatory genes was detected in whole cortex extracts by quantitative real-time PCR. Expression of all genes was normalized to the level of 18S rRNA and shown as a percent of Vehicle-Saline group. Data are expressed as means ± SEM (n = 5−8). * P < 0.05 compared with Vehicle-Saline; ^# P < 0.05 compared with Vehicle-LPS.; One-way ANOVA and Newman–Keuls post hoc test

Fig. 4

Candesartan suppresses LPS-induced microglia activation in the rat cingulate cortex. The representative pictures of microglia staining and quantitative analysis of microglia activation in rat cingulate cortex. The rats were treated for 14 days with candesartan (1 mg/kg/day) or vehicle via subcutaneously implanted osmotic minipumps. At the end of the treatment the rats of each group were injected either with LPS (50 μg/kg i.p.) or saline and were sacrificed 3 h after the injection. The formaldehyde-fixed brains were cut into 16 μm sections and microglia was stained with mouse monoclonal anti-rat Ox-42 antibody (CD11b/c, BD Pharmingen) and visualized with avidin-biotin enhanced horse radish peroxidase detection system. Microscopic images were acquired by AxioCam digital camera using AxioVision 4.6 software (Zeiss). For quantitative study, the images were subjected to color channel separation and particle analysis using Image J 1.37 software (NIH). Activated microglia was characterized by increased size, irregular shape and intensified Ox-42 staining. The Ox-42 positive area was used as a measure of microglia activation. Data are expressed as means ± SEM (n = 5−8). * P < 0.05 compared with Vehicle-Saline; ^# P < 0.05 compared with Vehicle-LPS. One-way ANOVA and Newman–Keuls post hoc test

Fig. 5

Angiotensin II and LPS—induced signaling pathways in endothelial cells in the control of the innate immune response. Activation of AT₁ receptor signaling cascade leads to ROS, PGE₂, and NO production. Prostaglandins are very important effectors of the Ang II–induced vascular permeability. Stimulation of endothelial cells either with Ang II or with bacterial endotoxin LPS activates several common downstream signaling pathways, including activation of mitogen-activated protein kinases, predominantly JNK, p38 and ERK, and transcription factors NF-κB and AP-1, resulting in enhanced expression of inflammatory cytokines, chemokines, inducible enzymes, and adhesion molecules mediating the interaction of endothelial cells with peripheral leukocytes. Pro-inflammatory cytokines activate microglia in brain parenchyma. Blockade of AT₁ receptors with candesartan eliminates Ang II—stimulated downstream signaling cascade, and suppresses activation of microglial cells. Another possible target in microglia regulation via candesartan could be AT₁—expressing neuronal cells