Angiotensin II Modulates BBB Permeability via Activation of the AT 1 Receptor in Brain Endothelial Cells

Abstract

Hypertensive encephalopathy occurs when acute changes in blood pressure cause breakdown of the blood—brain barrier (BBB). Angiotensin II (Ang II) plays a role in this pathophysiology. We determined whether Ang II directly regulates endothelial cell function at the BBB. In BBB microvessel endothelial cells (MECs), the Ang II (100 nmol/L; 0 to 6 h) effects on permeability to ¹²⁵I-albumin and transendothelial electrical resistance (TEER) were assessed. Angiotensin II (100 nmol/L) caused significant time-dependent changes in both ¹²⁵I-albumin permeability (25%) at 2 h and TEER (−8.87 Ω·cm²) at 6 h. Next, MECs were pretreated with the Ang II type 1 (AT₁) receptor blocker telmisartan (1 μmol/L) or the Ang II type 2 (AT₂) receptor blocker PD123,319 (1 μmol/L) followed by treatment with Ang II (100 nm). Telmisartan completely inhibited the Ang II-induced increase in ¹²⁵I-albumin permeability in MECs whereas PD123,319 had no effect. Using western blot analysis, we showed that MECs express AT₁ receptors but not AT₂ receptors. Treatment with Ang II (100 nmol/L; 0 to 6 h) also increased total protein kinase C activity. In contrast, Ang II had no effect on the expression of occludin, claudin 5, or actin. These results show that Ang II directly modulates transcytotic and paracellular permeability in BBB endothelial cells and could contribute to the pathophysiology of hypertensive encephalopathy.

Keywords

angiotensin II blood—brain barrier endothelial PKC

Introduction

In the United States, it is estimated that one in three persons have hypertension, making it one of the most common extensive medical conditions in the United States (AHA, 2008). Prolonged hypertension is a risk factor for cerebrovascular, cardiovascular, retinal, and end-stage renal diseases, whereas acute increases in blood pressure can be life threatening, leading immediately to organ dysfunction and permanent damage (AHA, 2008; Poulet et al, 2006). Severe acute changes in blood pressure are categorized as hypertensive emergencies when they are associated with end organ damage (cardiovascular, cerebrovascular, retinal, or renal). The most life-threatening event of a hypertensive emergency is the development of hypertensive encephalopathy, which results from an opening of the blood—brain barrier (BBB; Cherney and Straus, 2002; Gardner and Lee, 2007; Vaughan and Delanty, 2000).

The pathophysiology underlying hypertensive emergency is poorly understood. Because of its rapid onset, it is suggested that in most cases an initial trigger plus the higher blood pressure activates a positive feedback loop, which stimulates the release of vasoactive mediators (Haas and Marik, 2006; Kitiyakara and Guzman, 1998). Activation of this feedback loop begins a vicious cycle, which leads to continuous organ damage and continuous increases in blood pressure (Haas and Marik, 2006; Kitiyakara and Guzman, 1998). There are many factors, which contribute to the pathophysiology of hypertensive emergency. These include mechanical stress, endothelial dysfunction, oxidative stress and damage with potential release of cytokines, prostaglandins and nitric oxide. The renin—angiotensin system (RAS) in particular plays a critical role in the initiation and maintenance of hypertensive emergencies and in target organ damage (Haas and Marik, 2006; Kitiyakara and Guzman, 1998; Stefansson et al, 2000; Sunder-Plassmann et al, 2002).

Angiotensin II (Ang II), the major effector molecule of the RAS, plays an important role in maintaining blood pressure and fluid homeostasis by its actions in both peripheral tissues and the central nervous system (CNS; de Gasparo et al, 2000; Phillips and Sumners, 1998; Sumners et al, 2002). The physiologic effects of Ang II occur via the G-protein-coupled Ang II type 1 (AT₁) and Ang II type 2 (AT₂) receptors. The AT₁ receptor-mediated actions are the best characterized. In both peripheral tissues and the CNS, it is evident that the AT₁ receptor couples to a wide variety of signaling pathways (de Gasparo et al, 2000; Phillips and Sumners, 1998; Sumners et al, 2002). However, the classic AT₁ receptor-mediated signaling pathway in both the periphery and the CNS is activation of phospholipase C with subsequent increases in diacylglycerol and intracellular Ca²⁺, leading to activation of protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (de Gasparo et al, 2000; Phillips and Sumners, 1998; Sumners et al, 2002).

During a hypertensive emergency, there is an uncontrolled upregulation of the RAS. More Ang II is released, and Ang II leads to vasoconstriction, increased blood pressure, increased volume contraction, naturesis, and subsequent ischemia. These responses can result in hypoperfusion of the kidney, which in turn, will stimulate the release of renin to produce still more Ang II. Additionally, Ang II can stimulate the release of other vasoactive and hypertensive compounds such as vasopressin, aldosterone, and catecholamines as well as stimulate the production of proinflammatory cytokines. The latter can themselves mediate opening of the BBB (Haas and Marik, 2006; Kitiyakara and Guzman, 1998; Zhang et al, 2000).

The BBB forms a metabolic and physiologic regulatory interface between the peripheral circulation and the brain extracellular fluid. The BBB can be modulated by the crosstalk, which occurs between brain endothelial cells, astrocytes, pericytes, microglia, and neurons. This intercellular interaction has led to the concept of the neurovascular unit (NVU) where each cell type in the NVU contributes to BBB function (Hawkins and Davis, 2005). At the BBB, a lack of vesicular transport limits transcytosis (vesicular-mediated permeability across cells), whereas tight junctions (TJs) obliterate intercellular spaces and so eliminate paracellular permeability (passage between cells). The net result is a barrier with a high transendothelial electrical resistance (TEER; ∼2,000Ω·cm²), which primarily reflects paracellular permeability, and with little unrestricted leakage of proteins, which primarily reflects transcytosis (Hawkins and Davis, 2005).

Many studies have investigated the effect of hypertension on cerebral microvessels and have shown that hypertension causes changes in the cerebrovasculature, including alterations in BBB physiology. These effects of hypertension on the BBB have been attributed in part to the mechanical stresses that occur with increased pressure (Grubb and Raichle, 1981; Kaya et al, 2003; Kucuk et al, 2002; Oztas and Sandalci, 1984). However one study showed that inhibition of the AT₁ receptor blocks the hypertension-related increases in cell permeability and cerebral edema even in the absence of lowered blood pressure (Ito et al, 2000). This suggests a role for the RAS and Ang II in modulating BBB function. Early studies using bovine brain microvessel endothelial cells (MECs) showed that treatment with Ang II or an Ang II agonist stimulated an increase in fluid phase endocytosis (Guillot and Audus, 1990, 1991a; Guillot et al, 1990). Finally, it has been shown that Ang II itself undergoes AT₁ receptor-mediated transcytosis in BBB endothelial cells (Rose and Audus, 1998, 1999). This transcytosis is predominately from the apical (blood) to basolateral (brain) surface, thus allowing other cells of the NVU to be exposed to Ang II not only from the CNS but also the circulation.

However, the direct effects of the RAS, specifically Ang II, on BBB or brain MEC function have not been fully investigated. Such studies are vital if one is to differentiate the indirect effects of hypertension itself from the mediators of hypertension. As many of the actions of the humoral mediators likely evolved to be adaptive to events such as hypotensive crises and are pathologic only when activated inappropriately, it is a great advantage to be able to study their mechanisms of action in the absence of changes in blood pressure. In these studies, we investigate the direct effects of Ang II on cultured monolayers of BBB MECs and investigate possible cellular mechanisms underlying Ang II-mediated changes in BBB function.

Materials and methods

Preparation of Mouse Brain Microvessel Endothelial Cells

Brains were removed from male CD-1 mice (Charles River Laboratories, Wilmington, MA, USA) and cerebral MECs isolated using a modified method of Banks et al (2004). Briefly, microvessels were isolated from mouse cortical gray matter using a sequence of enzymatic digestions and differential centrifugations. Cells were suspended in culture medium (Dulbecco's Modified Eagle's media/F-12 nutrients, horse serum (20%), penicillin, streptomycin, bFGF (1 ng/mL), and puromycin (4 μg/mL)) and seeded on collagen/fibronectin-coated dishes. Three days after the initial plating, cells were then fed with culture medium without puromycin and fed every two days afterwards. Cells were passaged one time and seeded on either collagen/fibronectin-coated dishes or Transwell filters at 50,000 cells/cm². The confluent cells were used between days 7 to 10 after passage.

Preparation and Application of Angiotensin II, Peptides, and Drugs

The Ang II, pharmacologic agents and peptides were dissolved in appropriate solvents and then serially diluted in phosphate-buffered saline (PBS) to the appropriate concentrations for treatment. All the drugs, peptides, and control solutions (PBS) were added directly to the dishes of cells and incubated for various times.

Iodination of ¹²⁵I-Albumin

Bovine serum albumin (5 μg; Sigma, St Louis, MO, USA) was radioactively labeled with ¹²⁵I (Perkin Elmer, Waltham, MA, USA) according to the chloramine T method. Labeled albumin was then purified on a column of G-10 Sephadex hydrated with 0.25 mol/L phosphate buffer containing 1% unlabeled bovine serum albumin. Phosphate buffer was used to elute the iodinated protein.

In Vitro Assay for ¹²⁵I-Albumin

Flux of ¹²⁵I-albumin across the MEC monolayer was performed to determine whether Ang II stimulated transcytosis. Briefly, MECs were seeded on 0.45 micron Transwells filters and allowed to grow for 7 days. After treatment, the growth media was replaced with assay buffer containing (in mmol/L) 141 NaCl, 4 KCl, 2.8 CaCl₂, 1 MgSO₄, 10 HEPES, 10 glucose, 1 K₂HPO₄, and 1% BSA for 30 mins at 37°C. ¹²⁵I-albumin (5 × 10⁶ c.p.m./mL) was added to the upper chamber (lumen). Immediately, 100 μL of solution was removed from the upper chamber to determine the concentration of ¹²⁵I-albumin in the donor chamber. Samples (500 μL) were removed from the lower chamber (abluminal) at times 15, 30, 60, and 90 mins and replaced with fresh assay buffer. The amount of radioactivity in each sample was determined and flux was calculated using the following equation:

F l u x = V \times C_{r} / C_{d}

where V is the volume in the receiver chamber (0.6 mL), C_d is the concentration of ¹²⁵I-albumin in the donor chamber at time 0, and C_r is the concentration of ¹²⁵I-albumin in the receiving chamber at time T, with the C_r for times 30 to 120 being corrected for the amount of radioactivity remaining in the chamber from the previous time point. Flux was then plotted versus time and the apparent permeability (PS_app) calculated using linear regression analysis. The permeability of the membrane (PS_membrane) without cells was also calculated. The permeability of the cells (PS_cells) was calculated using the following equation

1 / {P S}_{a p p} = 1 / {P S}_{m e m b r a n e} + 1 / {P S}_{c e l l s}

The permeability coefficient is calculated using the equation, PS_cells/SA, where SA is the surface area of the monolayer (0.33 cm²).

Transendothelial Electrical Resistance Measurements and Calculations

Transendothelial electrical resistance measurements were taken using an EVOM voltmeter and STX-2 electrode (World Precision Instruments, Sarasota, FL, USA). All TEERS are expressed as a change from pretreatment conditions.

Protein Kinase C Activity Assay

Total PKC activity in treated MECs was measured using the Non-radioactive Protein Kinase Assay kit from Calbiochem (San Diego, CA, USA). The protocol established by the company was used to measure PKC activity. Briefly, MECs were washed in ice-cold PBS and the cell extraction prepared using an extraction buffer containing (in mmol/L) 50 Tris-HCl, 5 EDTA, 10 EGTA, 50 β-mercaptoethanol, 10 benzamidine, and 1 PMSF, pH 7.5. Samples were sonicated and centrifuged according to the protocol. Protein concentration in the supernatant was determined using the Coomassie Plus—the better Bradford assay kit (Pierce, Rockford, IL, USA). Samples were then incubated with peptide pseudosubstrate followed by incubation with biotinylated antibody 2B9 and horseradish peroxidase-conjugated streptavidin. Aborbance was read at 492 nanometers. The absorbance is directly proportional to the amount of PKC activity and is corrected for the amount of protein in each sample.

Protein Analysis and Western Blot Analysis

Microvessel endothelial cells were washed in ice-cold PBS and protein was extracted using a RIPA buffer containing (in mmol/L) 150 NaCl, 50 Tris HCl, 1% NP-40, 0.5% Na deoxycholate, and 0.1% SDS. Protein concentrations were determined using the BCA Protein Assay kit (Pierce). SDS—polyacrylamide gel electrophoresis and western blot analysis were performed as described previously (Fleegal et al, 2005). Briefly, proteins were separated on 4% to 12% bis-tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose. The AT₁ and AT₂ receptors were detected by chemiluminescence (Pierce) using rabbit anti-AT₁ (1:500), rabbit anti-AT₂ (1:500), and HRP-conjugated anti-rabbit (1:10,000) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Occludin and claudin 5 were detected using mouse anti-occludin (1:2,000) and mouse anti-claudin 5 (1:2,000) antibodies (Invitrogen) and HRP-conjugated anti-mouse (1:10,000; Santa Cruz Biotechnology). Actin was run as a control to ensure that protein was loaded in equal amounts and that it was not degraded.

Lincoplex Analysis of Cytokine Release

Cytokine release into the media by MECs was assayed using the mouse cytokine/chemokine multiplex technology of the LINCOplex Biomarker Immunoassay kit (Millipore, St Charles, MO, USA). The protocol established by the company was used. Briefly, after treatment, media were collected from the cells and centrifuged to remove cell debris. Cytokine controls, standards, and samples were incubated with antibody-immobilized beads for the cytokines/chemokines overnight at 4°C followed by incubation with cytokine/chemokine detection antibodies at room temperature for 1 h. Cytokines were detected using streptavidin phycoerythrin and quantified using the Luminex200. Regression analysis was used to determine the concentration of cytokines in the media. This kit was used to assay for the following 22 cytokines: IL1α, IL1β, IL2, IL4, IL6, IL10, IL12, GM-CSF, IFNγ, TNFα, IL5, IL7, IL9, IL13, IL15, IL17, GCSF, IP-10, KC, MCP1, MIP-1α, and RANTES.

Results

Angiotensin II Alters Blood—Brain Barrier Endothelial Cell Permeability

In the first series of experiments, we determined the effects of Ang II on TEER, a measurement of paracellular permeability, and ¹²⁵I-albumin transport, a measure of transcytosis, in isolated BBB endothelial cells. After treatment with Ang II (100 nmol/L; 0 to 6 h), there was a time dependent increase in ¹²⁵I-albumin transport (25%) with the maximum increase seen at 2 h (Figure 1A). Additionally, there was a time dependent decrease in TEER with the maximum seen 6 h after Ang II treatment (Figure 1B). These results suggest that Ang II-mediated changes in ¹²⁵I-albumin transport and TEER may involve different regulatory mechanisms. In the remaining experiments, we focused on investigating the mechanisms involved in the effects of Ang II on ¹²⁵I-albumin transport at 2 h and TEER at 6 h. However it is important to note that when we looked at the 2 h time point only, Ang II also caused a significant decrease in TEER as well as the significant increase in ¹²⁵I-albumin transport (Figure 2).

Figure 1

Ang II treatment increases ¹²⁵I-albumin permeability and decreases TEER in a time-dependent manner in cultured BBB MECs. Confluent monolayers of cultured MECs were exposed to either PBS (0) or Ang II (100 nmol/L) for 0.5 to 6 h and permeability to ¹²⁵I-albumin (A) and TEER (B) were assessed. TEER was measured pre-Ang II treatment and after Ang II treatment and change in TEER was calculated. (A) Bar graph showing the mean±s.e.m. ¹²⁵I-albumin permeability (expressed as % of control). (B) Bar graph showing the mean±s.e.m. change in TEER (expressed as a change in TEER from pre-Ang II treatment). ∗P<0.05 compared with control (0) (n=5 to 9).

Figure 2

Ang II alters BBB permeability in cultured endothelial cells. Cultured brain MECs were exposed to either PBS (0) or Ang II (100 nmol/L) for 2 h and change in ¹²⁵I-albumin permeability (A) and TEER (B) were measured. All data is expressed as the mean±s.e.m. change ∗P<.05 compared with control (0) (n=16 to 18).

Angiotensin II-Mediated Changes in ¹²⁵I-Albumin Transport Involve the Angiotensin II type 1 Receptor

We next determined whether the changes in ¹²⁵I-albumin transport and TEER involved the AT₁ or the AT₂ receptor. For this set of experiments, cultured MECs were pretreated with either the AT₁ receptor blocker, telmisartan (1 μmol/L; 15 mins) or the AT₂ receptor blocker, PD123,319 (1 μmol/L; 15 mins) followed by Ang II (100 nmol/L) for 2 h. Treatment with telmisartan significantly attenuated the Ang II stimulated increase in ¹²⁵I-albumin transport whereas the AT₂ receptor blocker PD123,319 had no effect on ¹²⁵I-albumin transport (Figure 3). Inhibition of the AT₁ receptor reduced the change in TEER at 6 h but this effect was not statistically significant (data not shown).

Figure 3

Inhibition of the AT₁ receptor and not the AT₂ receptor attenuates the Ang II-induced changes in ¹²⁵I-albumin permeability. Confluent monolayers of cultured MECs were pretreated with either PBS, telmisartan (Tel; 1 μmol/L) or PD123,319 (PD; 1 μmol/L) for 15 mins and then exposed to Ang II (100 nmol/L) for 2 h. After the treatment paradigm, permeability to ¹²⁵I-albumin was assessed. Bar graph shows the mean±s.e.m. ¹²⁵I-albumin permeability (expressed as % of control). ∗P<0.05 compared with control; ^†P<.05 compared with Ang II (n=8 to 18).

As there is conflicting evidence on whether both the AT₁ and AT₂ receptors are expressed in BBB endothelial cells (Zhou et al, 2006), the next set of experiments was performed to determine which of these receptors are present on cultured BBB MECs. Protein was isolated from cultured brain MECs and from whole mouse brain, a control known to express both the AT₁ and the AT₂ receptors. Western blot analysis showed that whole mouse brain expressed both Ang II receptors (Figure 4). On the contrary, cultured MECs expressed only the AT₁ receptor and not the AT₂ receptor (Figure 4). This suggests that all actions of Ang II in BBB endothelial cells occur via the AT₁ receptor.

Figure 4

Cultured mouse brain microvessel endothelial cells express the AT₁ receptor but not the AT₂ receptor. Protein expression was evaluated using western blot analysis in whole brain (B) and mouse brain microvessel endothelial cells (EC).

Angiotensin II Stimulates Protein Kinase C Activity in Cultured Microvessel Endothelial Cells

In the next series of experiments, the intracellular mechanisms altered by Ang II in BBB endothelial cells were investigated. As discussed previously, many effects mediated by Ang II and the AT₁ receptor occur via activation of the PKC pathway (Sumners et al, 2002). We next determined whether Ang II stimulates PKC activity in cultured brain MECs. In BBB endothelial cells treated with Ang II (100 nmol/L) for 0 to 6 h, there was a significant increase in PKC activity seen as early as 1 h, which remained elevated at both 2 and 6 h (Figure 5).

Figure 5

Ang II stimulates PKC activity in mouse BBB endothelial cells. Confluent monolayers of cultured MECs were exposed to either PBS (0) or Ang II (100 nmol/L) for 1 to 6 h and total PKC activity was measured as described in the methods. Bar graph shows the mean±s.e.m. PKC activity. ∗P<0.05 compared with control (0) (n=5 to 8).

As studies have shown that PKC can regulate paracellular permeability and several studies have suggested that PKC regulates the TJs (Clarke et al, 2000; Fleegal et al, 2005; Wosik et al, 2007), we also looked at whether Ang II altered the expression of the TJ proteins occludin and claudin 5. There was no change in the expression of these two proteins or the cytoskeletal protein, actin (Figure 6; Table 1). Finally, we looked at the effects of Ang II on the release of 22 different cytokines. There was no significant change in the release of these cytokines at either the 2 or 6 h time points (data not shown).

Figure 6

Ang II does not alter expression of claudin 5, occludin or actin. MECs were treated with PBS (0) or Ang II (100 nmol/L) for 0.25 to 6 h and protein expression was evaluated using western blot analysis. Representative western blots showing the expression of claudin 5, occludin, and actin.

Table 1

Protein expression of claudin 5, occludin, and actin is not regulated by Ang II in vitro

	Control	Ang II 0.25 h	Ang II 0.5 h	Ang II 1 h	Ang II 1.5 h	Ang II 2 h	Ang II 4 h	Ang II 6 h
Claudin 5	100±0.00	95.05±5.85	96.92±1.95	97.11±1.67	96.84±2.63	95.07±4.52	97.51±5.96	96.98±4.35
Occludin	100±0.00	102.55±4.75	101.11±4.68	97.95±4.68	101.80±2.09	95.36±3.45	98.58±15.45	101.98±1.39
Actin	100±0.00	96.98±2.81	91.33±3.01	86.30±1.95	90.94±3.10	89.82±2.67	93.79±2.92	90.49±3.59

Confluent monolayers of cultured MECs were exposed to Ang II either PBS (0) or Ang II (100 nmol/L) for 0.25 to 6 h and protein was collected (n=3). Western blot analysis was used to determine the relative expression of claudin 5, occludin, and actin. Data is expressed as mean±s.e.m. of percent of control.

Discussion

The results presented here show that Ang II modulates permeability of BBB endothelial cells. These effects are presumed to involve the AT₁ receptor but not the AT₂ receptor for two reasons. (1) The AT₁ receptor blocker telmisartan inhibited the Ang II stimulated increase in ¹²⁵I-albumin transport whereas the AT₂ receptor blocker had no effect on the Ang II-mediated increase in ¹²⁵I-albumin transport. (2) Western blot analysis confirmed the presence of the AT₁ receptor but not the AT₂ receptor in cultured brain MECs. These studies have also begun to investigate the intracellular signaling mechanisms involved in the Ang II-modulated changes in both transcellular and paracellular permeability. These studies have shown the activation of PKC in cultured brain MECs before, and sustained throughout, the Ang II-mediated effects on cellular permeability.

Although these studies are not the first to suggest that Ang II plays a role in modulation of the BBB, they are the first to directly link Ang II to changes in BBB endothelial cell permeability. Early in vivo studies have suggested that Ang II does modulate changes in the cerebrovasculature. For example, monkeys showed an increase in ¹⁵O-water in the brain after intracerebroventricular injection of Ang II, indicating an increase in cerebrovasculature permeability (Grubb and Raichle, 1981). Additionally, studies in rat showed that intravenous injections of Ang II, which cause acute hypertension, caused a reversible increase in BBB permeability to Evans blue albumin (Oztas and Sandalci, 1984). However, it is difficult to determine if these were direct effects of Ang II or indirect effects because of mechanical stresses from increased blood pressure and flow. Therefore, it is a great advantage to be able to study the mechanisms of action of Ang II in the absence of changes in blood pressure. The studies presented here are in agreement with early studies that used bovine brain MECs to show that treatment with Ang II, an Ang II agonist or the PKC activator, phorbol myristate acetate stimulates an increase in fluid phase endocytosis (Guillot and Audus, 1990, 1991a; Guillot et al, 1990). However, it is important to note that a recent study has shown that Ang II released from astrocytes contributes to establishing and maintaining BBB properties in vitro (Wosik et al, 2007). The main reason there could be a difference in results between this recently published study and our current study is the treatment paradigm. We were focused on studying the effects of increased Ang II in an already established cultured monolayer whereas the paper by Wosik et al (2007) was looking at the effects of Ang II on a monolayer of endothelial cells as it was growing and developing its BBB properties.

In addition to showing a role for Ang II in modulating BBB function, these studies also suggest that in our model system, Ang II mediates its physiologic effects through the AT₁ receptor. Other studies have shown the presence of Ang II binding sites and the presence of both AT₁- and AT₂-receptor mRNA and protein in brain MECs (Guillot and Audus, 1991b; Ibaragi and Niwa, 1989; Zhou et al, 2006). However, only the AT₁ receptor has been shown to colocalize with the endothelial cell marker Glut1 (Zhou et al, 2006). Our results confirm the presence of the AT₁ but not the AT₂ receptor in cultured BBB endothelial cells. Our results along with the literature suggest that the AT₂ receptor is not expressed by the BBB endothelial cell but is expressed by other cells of the NVU (Gallinat et al, 2000; Zhou et al, 2006). These results would then suggest that any effects of Ang II on cultured BBB endothelial cells involve the AT₁ receptor.

Finally, our studies have begun to investigate the intracellular signaling mechanisms regulated by Ang II. As stated earlier, classically, Ang II-mediated physiologic responses involve activation of the AT₁ receptor, which leads to activation of PKC (Sumners et al, 2002). The studies presented here show that Ang II also increases PKC activity in cultured brain MECs. Whether Ang II stimulates activity of similar isoforms of PKC in endothelial cells as compared with other cells of the CNS and periphery remains to be determined. However, a prime candidate to be investigated is the PKCδ isoform. Inhibition of the PKCδ isoform has been shown to prevent BBB breakdown and protect against hypertensive encephalopathy (Qi et al, 2008).

Protein kinase C is not the only intracellular signaling mechanism capable of modulating the physiologic effects of Ang II. A recent study showed that Ang II via the AT₁ receptor stimulates adhesion of platelets and leukocytes in the cerebral microvasculature (Ishikawa et al, 2007). Studies have also shown that antagonism of the AT₁ receptor will reverse macrophage infiltration of the microvessels and decrease expression of IL1β and TNFα in SHR (Takemori et al, 2000; Zhou et al, 2005, 2004). Other studies have shown alterations in the distribution of the TJs in microvessels of the SPSHR (Ito et al, 2000; Lippoldt et al, 2000). We also looked at the effects of Ang II on changes in both TJ protein expression and release of cytokines in cultured MEC monolayers. We showed no change in TJ protein expression and the release of 22 cytokines, which included but was not limited to IL1β and TNFα. Further studies need to investigate in greater detail the cellular mechanisms used by Ang II to alter both transcellular (¹²⁵I-albumin transport) and paracellular (TEER) permeability in BBB endothelial cells.

In conclusion, these studies show that Ang II directly modulates BBB endothelial cell function by altering both transcellular and paracellular permeability. Furthermore, these studies confirm the presence of the AT₁ receptor and not the AT₂ receptor in cultured brain MECs. This leads us to suggest that the effects of Ang II in this model system occur via activation of the AT₁ receptor. The cellular mechanisms involved in the Ang II-mediated changes in BBB endothelial cell function are not quite clear. However, they may involve PKC because Ang II stimulates PKC activity in these cells. On the contrary, Ang II does not appear to regulate the expression of the TJ proteins nor does it seem to regulate the release of cytokines. These studies indicate that Ang II may directly regulate BBB function by altering transcellular and paracellular permeability and by stimulating PKC activity.

References

AHA (2008) Diseases and Conditions: High Blood Pressure—High Blood Pressure Statistics. www.americanheart.org

Banks

Coon

Robinson

Moinuddin

Shultz

Nakaoke

Morley

(2004) Triglycerides induce leptin resistance at the blood-brain barrier. Diabetes 53:1253–60

Cherney

Straus

(2002) Management of patients with hypertensive urgencies and emergencies: a systematic review of the literature. J Gen Intern Med 17:937–45

Clarke

Marano

Peralta Soler

Mullin

(2000) Modification of tight junction function by protein kinase C isoforms. Adv Drug Deliv Rev 41:283–301

de Gasparo

Catt

Inagami

Wright

Unger

(2000) International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52:415–72

Fleegal

Hom

Borg

Davis

(2005) Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation. Am J Physiol Heart Circ Physiol 289:H2012–9

Gallinat

Busche

Raizada

Sumners

(2000) The angiotensin II type 2 receptor: an enigma with multiple variations. Am J Physiol Endocrinol Metab 278:E357–74

Gardner

Lee

(2007) Hyperperfusion syndromes: insight into the pathophysiology and treatment of hypertensive encephalopathy. CNS Spectr 12:35–42

Grubb

Jr Raichle

(1981) Intraventricular angiotensin II increases brain vascular permeability. Brain Res 210:426–30

10.

Guillot

Audus

(1990) Angiotensin peptide regulation of fluid-phase endocytosis in brain microvessel endothelial cell monolayers. J Cereb Blood Flow Metab 10:827–34

11.

Guillot

Audus

(1991a) Angiotensin peptide regulation of bovine brain microvessel endothelial cell monolayer permeability. J Cardiovasc Pharmacol 18:212–8

12.

Guillot

Audus

(1991b) Some characteristics of specific angiotensin II binding sites on bovine brain microvessel endothelial cell monolayers. Peptides 12:535–40

13.

Guillot

Audus

Raub

(1990) Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers. Microvasc Res 39:1–14

14.

Haas

Marik

(2006) Current diagnosis and management of hypertensive emergency. Semin Dial 19:502–12

15.

Hawkins

Davis

(2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173–85

16.

Ibaragi

Niwa

(1989) Atrial natriuretic peptide and angiotensin II binding sites in cerebral capillaries of spontaneously hypertensive rats. Cell Mol Neurobiol 9:221–31

17.

Ishikawa

Sekizuka

Yamaguchi

Nakadate

Terao

Granger

Minamitani

(2007) Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature. Am J Physiol Heart Circ Physiol 292:H2306–15

18.

Ito

Takemori

Kawai

Suzuki

(2000) AT1 receptor antagonist prevents brain edema without lowering blood pressure. Acta Neurochir Suppl 76:141–5

19.

Kaya

Kalayci

Kucuk

Arican

Elmas

Kudat

Korkut

(2003) Effect of losartan on the blood-brain barrier permeability in diabetic hypertensive rats. Life Sci 73:3235–44

20.

Kitiyakara

Guzman

(1998) Malignant hypertension and hypertensive emergencies. J Am Soc Nephrol 9: 133–42

21.

Kucuk

Kaya

Kalayci

Cimen

Kudat

Arican

Elmas

Korkut

(2002) Effects of losartan on the blood-brain barrier permeability in long-term nitric oxide blockade-induced hypertensive rats. Life Sci 71:937–46

22.

Lippoldt

Kniesel

Liebner

Kalbacher

Kirsch

Wolburg

Haller

(2000) Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells. Brain Res 885:251–61

23.

Oztas

Sandalci

(1984) Reversibility of blood-brain barrier dysfunction in acute hypertension induced by angiotensin. Exp Neurol 84:666–70

24.

Phillips

Sumners

(1998) Angiotensin II in central nervous system physiology. Regul Pept 78:1–11

25.

Poulet

Gentile

Vecchione

Distaso

Aretini

Fratta

Russo

Echart

Maffei

De Simoni

Lembo

(2006) Acute hypertension induces oxidative stress in brain tissues. J Cereb Blood Flow Metab 26:253–62

26.

Inagaki

Sobel

Mochly-Rosen

(2008) Sustained pharmacological inhibition of deltaPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats. J Clin Invest 118:173–82

27.

Rose

Audus

(1998) Receptor-mediated angiotensin II transcytosis by brain microvessel endothelial cells. Peptides 19:1023–30

28.

Rose

Audus

(1999) AT1 receptors mediate angiotensin II uptake and transport by bovine brain microvessel endothelial cells in primary culture. J Cardiovasc Pharmacol 33:30–5

29.

Stefansson

Ricksten

Rymo

Aurell

Herlitz

(2000) Angiotensin-converting enzyme gene I/D polymorphism in malignant hypertension. Blood Press 9:104–9

30.

Sumners

Fleegal

Zhu

(2002) Angiotensin AT1 receptor signalling pathways in neurons. Clin Exp Pharmacol Physiol 29:483–90

31.

Sunder-Plassmann

Kittler

Eberle

Hirschl

Woisetschlager

Derhaschnig

Laggner

Horl

Fodinger

(2002) Angiotensin converting enzyme DD genotype is associated with hypertensive crisis. Crit Care Med 30:2236–41

32.

Takemori

Ito

Suzuki

(2000) Effects of the AT1 receptor antagonist on adhesion molecule expression in leukocytes and brain microvessels of stroke-prone spontaneously hypertensive rats. Am J Hypertens 13:1233–41

33.

Vaughan

Delanty

(2000) Hypertensive emergencies. Lancet 356:411–7

34.

Wosik

Cayrol

Dodelet-Devillers

Berthelet

Bernard

Moumdjian

Bouthillier

Reudelhuber

Prat

(2007) Angiotensin II controls occludin function and is required for blood brain barrier maintenance: relevance to multiple sclerosis. J Neurosci 27:9032–42

35.

Zhang

Smith

Howlett

Stanimirovic

(2000) Inflammatory activation of human brain endothelial cells by hypoxic astrocytes in vitro is mediated by IL-1beta. J Cereb Blood Flow Metab 20:967–78

36.

Zhou

Ando

Macova

Dou

Saavedra

(2005) Angiotensin II AT1 receptor blockade abolishes brain microvascular inflammation and heat shock protein responses in hypertensive rats. J Cereb Blood Flow Metab 25:878–86

37.

Zhou

Jezova

Elkahloun

Saavedra

(2004) Angiotensin II AT1 receptor antagonism downregulates stress-related gene expression in brain microvessels from spontaneously hypertensive and normotensive rats. Stroke 37:1271–6

38.

Zhou

Pavel

Macova

Imboden

Nishioku

Dou

Delgiacco

Saavedra

(2006) AT1 receptor blockade regulates the local angiotensin II system in cerebral microvessels from spontaneously hypertensive rats. Stroke37:1271–1276

Angiotensin II Modulates BBB Permeability via Activation of the AT 1 Receptor in Brain Endothelial Cells

Abstract

Keywords

Introduction

Materials and methods

Preparation of Mouse Brain Microvessel Endothelial Cells

Preparation and Application of Angiotensin II, Peptides, and Drugs

Iodination of 125I-Albumin

In Vitro Assay for 125I-Albumin

Transendothelial Electrical Resistance Measurements and Calculations

Protein Kinase C Activity Assay

Protein Analysis and Western Blot Analysis

Lincoplex Analysis of Cytokine Release

Results

Angiotensin II Alters Blood—Brain Barrier Endothelial Cell Permeability

Angiotensin II-Mediated Changes in 125I-Albumin Transport Involve the Angiotensin II type 1 Receptor

Angiotensin II Stimulates Protein Kinase C Activity in Cultured Microvessel Endothelial Cells

Discussion

References

Iodination of ¹²⁵I-Albumin

In Vitro Assay for ¹²⁵I-Albumin

Angiotensin II-Mediated Changes in ¹²⁵I-Albumin Transport Involve the Angiotensin II type 1 Receptor