Circulating angiotensin II gains access to the hypothalamus and brain stem during hypertension via breakdown of the blood-brain barrier

Abstract

Angiotensin II-mediated vascular brain inflammation emerged as a novel pathophysiological mechanism in neurogenic hypertension. However, the precise underlying mechanisms and functional consequences in relation to blood-brain barrier (BBB) integrity and central angiotensin II actions mediating neurohumoral activation in hypertension are poorly understood. Here, we aimed to determine whether BBB permeability within critical hypothalamic and brain stem regions involved in neurohumoral regulation was altered during hypertension. Using digital imaging quantification after intravascularly injected fluorescent dyes and immunohistochemistry, we found increased BBB permeability, along with altered key BBB protein constituents, in spontaneously hypertensive rats within the hypothalamic paraventricular nucleus, the nucleus of the solitary tract, and the rostral ventrolateral medulla, all critical brain regions known to contribute to neurohumoral activation during hypertension. BBB disruption, including increased permeability and downregulation of constituent proteins, was prevented in spontaneously hypertensive rats treated with the AT1 receptor antagonist losartan, but not with hydralazine, a direct vasodilator. Importantly, we found circulating angiotensin II to extravasate into these brain regions, colocalizing with neurons and microglial cells. Taken together, our studies reveal a novel angiotensin II-mediated feed-forward mechanism during hypertension, by which circulating angiotensin II evokes increased BBB permeability, facilitating in turn its access to critical brain regions known to participate in blood pressure regulation.

Keywords: angiotensin II; blood–brain barrier; brain stem; hypertension; hypothalamus; receptor, angiotensin, type 1.

Figure 1. BBB permeability quantification

Samples of PVN (A), NTS (B), and RVLM (C) sections (TOTO counterstaining). D, RHO70 (red) and FITC10 (green) staining in the PVN of an SHR. For quantification, pixels containing both signals colocalized are isolated (E, white color) and processed as a binary image (F). Concurrently, a binary image containing only the FITC10 signal is obtained (G). To determine extravasation, image (F) is subtracted from image (G), resulting in a new image containing only the extravascular FITC10 signal (H) from which the density of extravasated FITC10 is calculated. Scale bar: 50µm. 3V, 4V: third and fourth ventricles; NA: nucleus ambiguous.

Figure 2. Increased BBB permeability in SHRs is prevented by an AT1 receptor blocker

Staining for intravascularly-delivered RHO70 (red) and FITC10 (green) within the PVN, NTS and RVLM of WKY (A1,B1,C1) and SHRs (A2,B2,C2). The corresponding extravasated FITC10 are shown in respective panels 3 and 4. A5, B5 and C5: mean extravasated FITC10 (FITC10_EV) in WKY, SHR, SHR treated with Losartan (SHR-los) and SHR treated with hydralazine (SHR-hyd). C6, high magnification image of the RVLM (SHR), showing intravascular FITC10 (white arrows) and extravasated FITC10 in neuronal-like profiles (red arrows).***P<0.001 vs WKY; †††P<0.001 vs SHR; n=8 SHR/WKY; n=4 SHR-los/SHR-Hyd. Scale bars: 50µm. 3V: third ventricle.

Figure 3. Circulating AngII leaks through the disrupted BBB in SHRs

A–F, Intravascularly-delivered AngII_fluo within the PVN, NTS and RVLM of WKYs (A,B,C) and SHRs (D,E,F). Insets show respective squared areas at higher magnification. G, Mean extravasated AngII_fluo. *P<0.05 vs WKY, n=4/group. Scale bar: 50µm; insets: 25µm. 3V: third ventricle.

Figure 4. Extravasated AngII in SHRs is localized in neurons and microglia

A–D, intravascularly-delivered AngII_fluo (A) co-localized with the neuronal marker NeuN (B) (C, merged; D, higher magnification) within the RVLM. E–H, RHO70 (E), AngII_fluo (F) and NeuN (G) showing extravasation of the AngII_fluo but not RHO70 and its colocalization with neurons (H) within the PVN. I–L, Leaked AngII_fluo (I) co-localized with the microglial marker CD11b (J) (K, merged; L, higher magnification). M–P, Lack of co-localization between leaked AngII_fluo (M), RHO70 (N) and the astrocytic marker GFAP (O), merged in P. Solid and empty arrows: intravascular and leaked AngII_fluo. Scale bars: 50µm.

Figure 5. AngII-dependent diminished endothelial barrier antigen (EBA) expression in SHRs

A–F, EBA staining within the PVN, NTS and RVLM of WKYs (A–C; n=5) and SHRs (D–E; n=5). G and H, EBA immunostainning (red) and isolectin IB4 (H) within the PVN of a WKYs. I, merged figure showing co-localization (yellow). J, mean EBA immunoreactivity density in WKY, SHR and SHR-LOS (n=3).***P<0.0001 vs. WKY and ⁺⁺⁺P<0.0001 vs. SHR. Scale bar: 50µm. 3V: third ventricle.