Articles in PresS. Am J Physiol Heart Circ Physiol (December 5, 2014). doi:10.1152/ajpheart.00775.2014

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ELEVATED PRESSURE CAUSES ENDOTHELIAL DYSFUNCTION IN MOUSE CAROTID ARTERIES BY INCREASING LOCAL ANGIOTENSIN SIGNALING

Yingzi Zhao1,2, Sheila Flavahan1, Susan W. Leung2, Aimin Xu2, Paul M. Vanhoutte2, and Nicholas A. Flavahan1

1

Department of Anesthesiology and Critical Care Medicine Johns Hopkins University, Baltimore, MD 2 State Key Laboratory of Pharmaceutical Biotechnology and Department of Pharmacology and Pharmacy University of Hong Kong, Hong Kong, China

Running Title: Pressure-Induced Endothelial Dysfunction

Correspondence: Nicholas A. Flavahan, PhD. Department of Anesthesiology, Johns Hopkins University 720 Rutland Ave, Ross-1159 Baltimore, MD 21205 Phone: 410-502-6738 Fax: 410-502-7007 Email: [email protected]

Copyright © 2014 by the American Physiological Society.

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ABSTRACT Experiments were performed to determine whether or not acute exposure to elevated

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pressure would disrupt endothelium-dependent dilatation by increasing local angiotensin II

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(ANGII) signaling. Vasomotor responses of mouse isolated carotid arteries were analyzed in a

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pressure myograph at a control transmural pressure (PTM) of 80mmHg. Acetylcholine-induced

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dilatation was reduced by endothelial denudation or by inhibition of NO synthase (LNAME,

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100M). Transient exposure to elevated PTM (150mmHg, 180 minutes) inhibited dilatation to

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acetylcholine, but did not affect responses to the NO donor DEA-NONOate. Elevated PTM also

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increased endothelial reactive oxygen species (ROS), and the pressure-induced endothelial

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dysfunction was prevented by the direct antioxidant and NADPH oxidase inhibitor apocynin

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(100M). The increase in endothelial ROS in response to elevated PTM was reduced by the

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AT1 receptor (AT1R) antagonists losartan (3M) or valsartan (1M). Indeed, elevated PTM

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caused marked expression of angiotensinogen, the precursor of ANGII. Inhibition of ANGII

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signaling, by blocking angiotensin converting enzyme (ACE) (perindoprilat, 1M; captopril,

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10M) or blocking AT1Rs prevented the impaired response to acetylcholine in arteries exposed

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to 150mmHg, but did not affect dilatation to the muscarinic agonist in arteries maintained at

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80mmHg. After inhibiting ANGII, elevated pressure no longer impaired endothelial dilatation. In

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arteries treated with perindoprilat to inhibit endogenous formation of the peptide, exogenous

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ANGII (0.3M, 180 minutes) inhibited dilatation to acetylcholine. Therefore, elevated pressure

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rapidly impairs endothelium-dependent dilatation by causing ANG expression and enabling

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ANGII-dependent activation of AT1 receptors. These processes may contribute to the

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pathogenesis of hypertension-induced vascular dysfunction and organ injury.

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Keywords. angiotensin II, AT1 receptors, hypertension, endothelium

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INTRODUCTION In healthy human volunteers, short-term increases in arterial blood pressure cause long-

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lasting inhibition of endothelium-dependent dilatation (11,17,22). Likewise, in isolated arteries,

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acute exposure to pathological increases in transmural pressure (PTM) impairs endothelium-

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dependent relaxation (10,21,26). As occurs with chronic increases in pressure (hypertension),

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endothelial dysfunction caused by acute exposure to elevated PTM appears to be mediated by

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activation of NADPH oxidase and production of reactive oxygen species (ROS) causing

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disruption of endothelial NO-mediated dilatation (13,24,26). ANGII signaling plays a key role in

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the development and the pathological consequences of hypertension, including the occurrence

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of endothelial dysfunction (9,16,25). Indeed, ANGII is a powerful inducer of endothelial

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dysfunction, including diminution in endothelial NO dilator activity (7,14,24). ANGII activity in

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hypertension may reflect generation of the peptide by systemic and/or local ANG systems (2,19).

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It is currently unknown whether or not local ANG signaling contributes to the endothelial

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dysfunction caused by acute increases in pressure. Prolonged organ culture of rabbit aortae at

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high PTM (3 days, 150mmHg) causes an ANGII-dependent increase in fibronectin expression (3),

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indicating that pressure can induce local ANGII signaling in the blood vessel wall. Furthermore,

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AT1 receptors (AT1Rs) can be activated directly by mechanical stretch, independently of ANGII

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(1,23,28). The goal of the present experiments was to determine whether or not an acute,

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transient increase in PTM causes endothelial dysfunction in carotid arteries via ANGII-dependent

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or ANGII-independent activation of AT1Rs.

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MATERIALS AND METHODS

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Animals: Male C57BL6 mice (10-12 weeks old) were obtained from Jackson Laboratories (Bar

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Harbor, ME), and killed by CO2 asphyxiation. Animal use was approved by the Institutional

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Animal Care and Use Committee and complied with the NIH Guide for the Care and Use of

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Laboratory Animals.

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Blood Vessel Analysis: Carotid arteries were isolated and placed in cold Krebs-Ringer

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bicarbonate solution (control solution)(20). They were cannulated with glass micropipettes,

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secured within a microvascular chamber, and maintained at a control PTM of 80mmHg in the

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absence of flow (Living Systems, Burlington, VT). The chamber was superfused with control

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solution (37°C, pH 7.4, 16% O2-5% CO2-balance N2) and placed on the stage of an inverted

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microscope (20). The artery image was captured by a video camera and the internal diameter

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continuously determined by a video dimension analyzer and recorded using a data acquisition

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system (BIOPAC, Santa Barbara, CA)(20).

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To optimize the role of NO, dilatation was assessed in arteries constricted with 34mM KCl

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(in equimolar replacement of NaCl) after inhibition of cyclooxygenase with indomethacin (10M).

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Concentration-effect curves to dilator agonists were generated by increasing their concentration

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in half-log increments once the response to the preceding concentration had stabilized.

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Acetylcholine (1M) caused 69.2 + 2.9% dilatation of KCl-constricted arteries under control

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conditions and 7.6 + 1.5% dilatation after inhibition of NO synthase (LNAME, 100M)(P < 0.001,

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n = 3). Endothelial denudation by wire abrasion (70m)(20) abolished acetylcholine-induced

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dilatation. Following washout (with control solution) and return of the arterial diameter to basal

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levels, PTM was maintained at 80mmHg or increased to 150mmHg for up to three hours (12,13).

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PTM was then returned to 80mmHg, the arteries were constricted again with KCl (34mM) and

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responses to dilator agonists reassessed. There was no significant time-dependent change in

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dilator responses in arteries maintained under control conditions at 80mmHg. When assessing

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inhibitors, arteries were incubated for 30 minutes with the drugs before and during exposure to

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acetylcholine and during exposure of the arteries to elevated PTM (inhibitors were reintroduced

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into the superfusate if required, following washout and recovery of the arteries). The inhibitors

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did not significantly affect constriction to KCl. The effect of exogenous ANGII (0.3 M, 180

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minutes) was studied in arteries treated with perindoprilat (1M, to inhibit endogenous

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production of the peptide) and exposed to increased pressure (150mmHg).

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Western Blotting: Isolated carotid arteries (maintained at 80mmHg or exposed to 150mmHg for

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180 minutes) were frozen in liquid nitrogen. Because of their small size, three carotid arteries

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were pooled to obtain one sample (‘n’ = 1). Arteries were disrupted in cold RIPA Lysis and

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Extraction Buffer (Thermo Fisher, Rockford, IL) containing Protease Inhibitor Cocktail (Thermo

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Fisher) and homogenized (5 minutes) using Tissuelyser LT (QIAGEN, Valencia, CA). The

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lysate was then cleared by centrifugation (14,000g, 5 minutes, 4oC). Western blot detection of

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angiotensinogen (ANGogen) was performed under denaturing conditions using a primary goat

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polyclonal antibody (1:200, sc-7419, recognizing ANGogen, ANGI and ANGII, Santa Cruz

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Biotechnology, Dallas, TX), and quantified using ImageQuant TL (GE Healthcare) within

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consistent specified Regions of Interest.

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ROS activity: Isolated carotid arteries that had been maintained at 80mmHg or exposed to

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150mmHg for 15 or 180 minutes were rapidly removed from the chamber and incubated under

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non-pressurized conditions with the ROS-sensitive fluorescent probe 5-(and 6)-chloromethyl-

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29,79-dichlorodihydro-fluorescein diacetate (DCDHF, 5 g/mL)(Life Technologies, Grand Island,

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NY) and Draq5 (5M, nuclear stain)(Biostatus, Leicestershire, UK) for 20 minutes (37°C, control

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solution)(20). They were then placed in control solution (4oC) and the endothelial surface

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viewed using a Leica SP5 laser-scanning microscope (20X air objective, 0.7NA). Images

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(1024x1024 pixels) were obtained using sequential acquisition, a pinhole of 1 Airy unit, scan

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speed of 400 Hz, 6 line averaging and an optical zoom of 3.0. For DCDHF, excitation was

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488nm and emission captured from 492-541nm; and for DRAQ, excitation was 633nm and

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emission captured from 650-750nm. Arteries were processed at the same time, using the same

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approach and instrument settings. Fluorescence intensity is therefore expressed as detector

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units. The entire endothelial surface of each carotid artery was scanned (~7 images/artery),

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with the average fluorescent intensity representing the arterial fluorescence (n = 1).

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Drugs: Acetylcholine, bradykinin, indomethacin, L-NAME and ANGII were from Sigma-Aldrich

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(St. Louis, MO), apocynin, losartan, valsartan and captopril from Tocris Biosciences (Bristol,

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UK), DEA-NONOate from Enzo Life Sciences (Farmingdale, NY) and perindoprilat from Santa

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Cruz Biotechnology.

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Data Analysis: Vasomotor responses were expressed as a percent change in baseline

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diameter. Agonist concentrations causing 30% dilatation of the KCl constriction (EC30) were

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calculated by regression analysis and compared as -log EC30. Data are expressed as means +

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S.E.M., where ‘n’ equals the number of animals from which blood vessels were studied (except

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as stated for Western blotting). Statistical evaluation of the data was performed by Student's t-

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test for paired or unpaired observations. When more than two means were compared, analysis

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of variance was used. If a significant F value was found, then Student-Newman-Keuls or

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Dunnett tests for multiple comparisons were employed to identify differences among groups.

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Values were considered to be statistically different when P was less than 0.05.

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RESULTS Increased Pressure Inhibits Endothelium-Dependent Dilatation to Acetylcholine:

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Acetylcholine-induced dilatation was significantly inhibited following transient exposure to

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elevated PTM (150mmHg) (Fig 1). After 60 minutes, elevated PTM caused a significant rightward

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shift in the concentration-effect curve to acetylcholine (log shift of 0.20 + 0.08, 1.6-fold, n = 7, P

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< 0.05) with no significant change in maximal dilatation. After 180 minutes, elevated PTM

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exerted more profound depression, causing a larger rightward shift in the concentration-effect

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curve and a significant depression in the maximal dilatation (Fig 1). Elevated PTM (150mmHg,

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180 minutes) did not significantly affect dilatation to the NO donor NONOate (data not shown).

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Mechanisms Contributing to Pressure-Induced Disruption of Endothelial Dilatation

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ROS: Elevated PTM is known to increase arterial NADPH oxidase activity and ROS production,

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thereby reducing endothelial NO-mediated dilatation (13,24,26). Indeed, elevated PTM (80 to

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150mmHg) increased endothelial ROS activity (Fig 2). Although the direct antioxidant and

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NADPH oxidase inhibitor, apocynin (100M) did not significantly affect dilatation to acetylcholine

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in arteries maintained at 80mmHg, it amplified responses in arteries exposed to 150mmHg (180

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minutes), significantly increasing the maximal dilatation and causing a significant leftward shift

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of the concentration-effect curve (Fig 1). After incubation with apocynin, increased pressure no

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longer significantly inhibited dilatation to acetylcholine (Fig 1).

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ANG SIGNALING: The AT1R antagonist losartan (3M) did not influence the immediate

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increase in endothelial ROS activity in response to elevated PTM (150mmHg, 15 minutes), but

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significantly reduced the ROS increase occurring after more prolonged exposure to elevated

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PTM (150 mmHg, 180 minutes) (Fig 2). Similar results were obtained with the AT1R antagonist

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valsartan (1M) (data not shown).

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ANG Expression: Immunoblot analysis of arterial lysates selectively identified molecular

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species consistent with glycosylated forms of ANGogen in blood vessels exposed to elevated

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PTM (150mmHg, 180 minutes) (Fig 2). The exposure to elevated PTM caused significant

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induction of this ANGogen-like activity in mouse carotid arteries (Fig 2).

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ACE Inhibition: Perindoprilat (1M) or captopril (10M) did not significantly affect dilatation to

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acetylcholine in arteries maintained at 80mmHg, but amplified responses to the agonist in

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arteries exposed to 150mmHg (180 minutes), significantly increasing the maximal dilatation and

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causing significant leftward shifts in the concentration-effect curves (Fig 3). After ACE

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inhibition, increased pressure no longer significantly inhibited dilatation to acetylcholine (Fig 3).

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ACE inhibition could potentially amplify endothelial dilatation by preventing the breakdown of

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endogenous kinins, including bradykinin. However, bradykinin (0.1 to 100nM) had no significant

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effects on arterial diameter. During KCl-induced constriction, diameter was 69.2 + 2.6% of

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baseline before and 68.1 + 3.2% five minutes after administration of bradykinin 100nM (n = 5,

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NS). Bradykinin also did not significantly affect arterial diameter after ACE inhibition

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(perindoprilat) or after the transient increase in PTM (150 mmHg, 180 minutes) (n = 5, data not

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shown). Furthermore, bradykinin did not amplify the response to acetylcholine: after the

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transient elevation in PTM (150 mmHg, 180 minutes) the maximal response to acetycholine was

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57.1 + 5.0% of the constriction to KCl under control conditions and 59.2 + 5.0% after

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administration of bradykinin (100nM, five minutes before acetylcholine) (n = 5, NS).

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AT1R Antagonism: Losartan (3M) or valsartan (1M) did not significantly affect dilatation to

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acetylcholine in arteries maintained at 80mmHg, but amplified responses to the agonist in

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arteries exposed to 150mmHg (180 minutes), significantly increasing the maximal dilatation and

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causing significant leftward shifts in the concentration-effect curves (Fig 4). After AT1R

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antagonism, increased pressure no longer significantly inhibited dilatation to acetylcholine (Fig

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4).

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Exogenous ANGII: ANGII (0.3 M, 180 minutes) depressed acetylcholine-induced dilatation,

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causing significant inhibition of the maximal response (from 97.3 + 1.0% to 93.7 + 0.3%

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dilatation of KCl constriction, n = 7, P < 0.05) and a significant rightward shift in the

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concentration-effect curve (log shift of 0.25 + 0.03, 1.8-fold, n = 7, P < 0.001). ANGII did not

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cause constriction of the arteries and did not significantly affect constriction to KCl (data not

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shown).

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DISCUSSION

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In the present study, an acute transient increase in PTM impaired endothelium-dependent,

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NO-mediated dilatation to acetylcholine but did not influence dilatation to a NO-donor. This

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pressure-induced endothelial dysfunction was associated with an increase in endothelial ROS

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and was prevented by the direct antioxidant and NADPH oxidase inhibitor apocynin, consistent

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with previous reports (13,24,26). AT1Rs are considered mechanosensitive and can be

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activated directly by mechanical stretch in an ANGII-independent manner (1,18,28). Although

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valsartan is a strong inverse agonist and can effectively inhibit such ANGII-independent AT1R

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activity, losartan is considered a poor inverse agonist and is ineffective (1,18). However, both

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agents are powerful AT1R antagonists and inhibit responses to ANGII. The immediate

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pressure-induced increase in endothelial ROS (15 minutes) was not affected by these AT1R

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inhibitors, indicating that the initial increase in ROS is not dependent on AT1R activation.

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However, the increase in ROS activity following a more prolonged elevation in pressure (180

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minutes) was reduced by either agent, suggesting that the role of AT1Rs involves ANGII-

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dependent activation rather than direct mechanosensitive stimulation. Indeed, prolonged

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exposure to elevated pressure caused a dramatic induction of ANGogen expression. Moreover,

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the pressure-induced disruption of endothelial dilatation was prevented by antagonizing AT1Rs

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with losartan or valsartan and by preventing ANGII formation with the ACE inhibitors captopril or

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perindoprilat. The protective effects of ACE inhibition on endothelial dilatation are unlikely to be

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mediated by production of endogenous kinins because bradykinin did not initiate endothelium-

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dependent dilatation and did not prevent the pressure-induced depression of responses to

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acetylcholine. Although inhibition of AT1Rs or ACE markedly increased the depressed

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responses to acetylcholine in arteries exposed to elevated pressure, these interventions had no

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effect in preparations maintained at a control pressure. Therefore, pathological local ANGII

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signaling is present only at elevated pressure, consistent with the lack of angiotensinogen

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expression under control conditions. A previous report suggested that elevated pressure in

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isolated porcine coronary arteries caused endothelial dysfunction by direct mechanical

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activation of AT1Rs (15). However, this was based solely on the effects of losartan (15), which

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would be expected to inhibit AT1R responses initiated by ANGII but not direct mechanical

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activation (1,18). Thus, the present study indicates that acute pressure-induced endothelial

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dysfunction is mediated by local induction of ANGII signaling and ANGII-dependent activation of

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AT1Rs.

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The concept that elevated pressure increases local vascular ANGII signaling is consistent

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with previous studies. Hypertension induced by aortic banding was associated with increased

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ANGogen expression in coronary arteries (24). Likewise, prolonged culture of rabbit aortae at

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elevated PTM (150mmHg, three days) caused an ANGII-dependent increase in fibronectin

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production (3). Furthermore, cyclic stretch of cultured endothelium increased ANG expression

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and caused an ANGII-dependent decrease in NO activity (6). The present study demonstrates

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that an acute elevation in pressure causes endothelial dysfunction by rapidly increasing local

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ANGII signaling. A key positive regulator of ANGogen expression is NF-κB (4). In mouse

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carotid arteries, elevated PTM (150mmHg) caused a peak increase in NF-κB activity within 15

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minutes, which appears to be mediated by the pressure-induced generation of ROS (12,13).

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Pressure-induced modulation of endothelial dilatation appears to be developmentally-regulated.

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In contrast to adult carotid arteries, elevated PTM increased endothelium-dependent NO-

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mediated dilatation to acetylcholine in carotid arteries of newborn mice (8). Despite differences

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in baseline pressure at these distinct developmental stages, the elevation in P TM (20-50mmHg in

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newborns; 80-150mmHg in adults) caused similar increases in internal diameter (31.0 + 2.4% in

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newborns, 29.3 + 1.9% in adults, n = 15; NS). The pressure-induced mechanical stimulus to the

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endothelium would therefore be of similar magnitude. Thus, increased pressure can exert

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protective effects on endothelium-dependent dilatation, but in adult arteries this mechanism is

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suppressed by pressure-induced pathological expression of ANGII signaling.

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The present study did not identify the cellular source of ANGogen or the mechanism(s)

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responsible for the initial processing of this ANG precursor. Numerous cell types located in the

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arterial wall can express ANGogen and contribute to vascular ANGII signaling including

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endothelial cells, smooth muscle cells and perivascular adipocytes (5,6,19). Conversion of

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ANGogen to ANGI, the substrate for ACE can be accomplished by renin expressed within the

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blood vessel wall, by (pro)renin captured from the circulation by (pro)renin receptors, or by

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renin-independent mechanisms (2,19). Experiments confirmed that, in arteries treated with an

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ACE inhibitor, endothelial dysfunction could be restored by exogenous ANGII (7). The

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pathological effects of ANGII on the endothelium have been extensively studied, including

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demonstrating a role of increased NADPH oxidase and ROS production in causing endothelial

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dilator dysfunction (7,14,24). Interestingly, exogenous ANGII did not cause constriction or

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change the response to KCl in mouse carotid arteries confirming a minimal role for AT1Rs in the

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smooth muscle of this blood vessel (27).

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In conclusion, the results of the present study demonstrate that transient exposure to

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elevated pressure impairs endothelium-dependent NO-mediated dilatation by increasing local

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ANGII signaling and causing ANGII-dependent activation of AT1Rs. Pressure-induced local

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ANGII signaling is likely a key mediator in the pathological effects of increased arterial blood

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pressure on the arterial wall.

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GRANTS

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NIH award to NAF (HL102715)

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DISCLOSURES

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None

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FIGURE CAPTIONS

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Figure 1: Effects of a transient increase in PTM on acetylcholine-induced dilatation in mouse

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isolated carotid arteries. Dilatation was analyzed at a PTM of 80mmHg during KCl (34mM)

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constriction in arteries that had been maintained at 80mmHg (P80) or exposed to a transient

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increase in PTM (150mmHg, 180 minutes)(P150). The upper panel presents the effects of

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elevated PTM in untreated arteries, whereas the middle and lower panel presents the influence

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of the direct antioxidant and NADPH oxidase inhibitor apocynin (100M) in arteries exposed to

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150 mmHg or maintained at 80 mmHg, respectively. Data are expressed relative to baseline

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diameter and presented as means + SEM (n = 6). B, baseline; C, KCl constriction. Vertical

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arrows indicate statistical comparisons between maximal responses, whereas horizontal arrows

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indicate comparisons between -log EC30 values (***, P < 0.001; ** P < 0.01; NS, not significant).

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Figure 2: Effects of elevated PTM on endothelial ROS activity and the expression of ANGogen in

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mouse carotid arteries. A: ROS activity was assessed using DCDHF fluorescence in isolated

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arteries maintained at 80mmHg or exposed to 150mmHg for 15 minutes (P15015) or 180

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minutes (P150180). Effects of elevated PTM were assessed under control conditions and after

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AT1R antagonism by losartan (3M). Representative images (green, DCDHF; blue, DRAQ) and

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combined data with means + SEM (n = 3 to 4) are presented. Fluorescence is expressed as

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detector units. B: ANGogen expression was assessed in arteries maintained at a PTM of 80

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mmHg or exposed to elevated PTM (150mmHg, 180 minutes). A representative immunoblot

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and combined density data (relative to -actin expression) with means + SEM (n = 6) are

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presented. The mean at 150mmHg was set as 100%, and the intensity of all measurements

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expressed relative to that value. For A and B, ***, P