© 1997 American Heart Association, Inc.
Articles |
Vascular Thrombin Receptor Regulation in Hypertensive Rats
From the Departments of Medicine, Divisions of Cardiology, Emory University School of Medicine (Q.C. IV, J.B.L., T.F., S.R., I.M., P.L., K.K.G., D.G.H., R.W.A., W.R.T.) and the Atlanta Veterans Administration Hospital (D.G.H., W.R.T.), Atlanta, Ga; the Department of Anesthesiology (B.A.F., W.R.B.), University of Alabama at Birmingham; the Department of Medicine (M.S.R.), Division of Cardiology, the University of Texas Medical Branch–Galveston; and Medical Department B (J.B.L.), Rigshospitalet, Copenhagen, Denmark.
Correspondence to W. Robert Taylor, MD, PhD, Woodruff Memorial Building, Room 308, Division of Cardiology, 1639 Pierce Dr, Emory University School of Medicine, Atlanta, GA 30322. E-mail wtaylor{at}emory.edu
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Abstract |
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Abstract Thrombin has been implicated as an important mediator of vascular lesion formation in atherosclerosis and restenosis. To investigate a potential role for thrombin signaling in the vascular response to hypertension, we have studied thrombin receptor (TR) expression and regulation in hypertensive rats. Aortic TR mRNA was upregulated by angiotensin II (Ang II)–induced hypertension (10.7±2.5 times control, P<.02), which correlated with a 4-fold increase in thrombin-induced constriction in isolated endothelium-denuded aortic rings. The AT1 receptor antagonist losartan normalized blood pressure and TR mRNA. Conversely, lowering blood pressure to the same degree with hydralazine did not abolish the upregulation of TR mRNA expression. When low-renin low–Ang II hypertension was induced in Dahl salt-sensitive rats, there was no detectable increase in the expression of aortic thrombin receptor mRNA. Finally, treatment with a chimeric heparin-binding form of the recombinant human Cu/Zn superoxide dismutase caused complete inhibition of TR mRNA upregulation, suggesting that an increased rate of superoxide anion production is an important signaling mechanism. Thus, increased TR expression via a redox-sensitive mechanism in the aortic smooth muscle of rats treated with Ang II represents a novel in vivo mechanism through which the hypertensive effects of Ang II are mediated.
Key Words: hypertension • aorta • vascular smooth muscle • angiotensin II • Dahl rat
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Introduction |
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Arterial hypertension has been identified as an important risk factor for cardiovascular mortality and morbidity. The use of antihypertensive drugs has resulted in a decrease in morbidity and mortality from stroke1 and renal failure.2 However, a similar reduction in ischemic heart disease–related deaths has not been achieved.3 Although several hypotheses have been proposed to explain this finding,4 it is clear that greater insight into the pathophysiology of hypertensive vascular disease is required to address this phenomenon. Recent studies have documented an increase in thrombin receptors in atherosclerotic lesions5 and in mechanically injured arteries,6 implying a thrombin receptor–mediated pathway of vascular lesion formation. The potential role of this receptor in the progression of hypertensive vascular disease has not previously been explored.
Thrombin is a serine protease that in addition to its well-defined role in the thrombosis cascade, appears to play a role in vascular adaptive responses to injury. Besides being a direct7 and indirect8 9 stimulus for vascular cell growth, thrombin can affect vascular tone by directly causing smooth muscle contraction10 or by inducing endothelial secretion of the potent vasoactive agents NO11 and endothelin-1.12 The pivotal role of thrombin in tissue inflammation and repair is apparent in its ability to induce inflammatory cell chemoattractant (MCP-1)13 and adhesion14 molecules and in its ability to stimulate collagen synthesis by fibroblasts.15 Finally, by regulating the procoagulant protein tissue factor16 and PAI-1,17 thrombin has an important influence on vascular wall thrombogenicity and fibrinolytic activity.
Many of the cellular effects of thrombin are mediated through a recently cloned receptor,18 19 which is similar to other G protein–coupled receptors in that it has seven transmembrane regions. This receptor, however, differs from most receptors in its novel mode of activation. Thrombin cleaves the extracellular amino-terminal end of its receptor, revealing a new amino terminus that acts as a tethered ligand, folding into a transmembrane pocket to activate the receptor.18 This novel mechanism of receptor activation allows one molecule of thrombin to activate multiple receptors, in contrast to most cellular receptor–mediated events, in which receptor occupancy by the ligand is necessary to maintain an activated state. Thus, the magnitude of thrombin-mediated events is strongly influenced by receptor concentration.
The signaling pathways responsible for the regulation of thrombin receptor expression have not been well characterized. Recent evidence from our laboratory suggests that reactive oxygen species can upregulate thrombin receptor mRNA expression in vitro.20 This is of particular relevance given the recent demonstration of increased superoxide production by aortic tissues from rats made hypertensive by Ang II infusion.21 22 These data provide a mechanistic basis for implicating thrombin receptor expression in the vascular pathology of Ang II–induced hypertension.
Considering the emerging concept that thrombin plays an important role in vascular lesion formation, the potential role of reactive oxygen species in upregulating thrombin receptor mRNA expression, and the accepted but incompletely understood influence of hypertension on atherosclerosis, we hypothesized that thrombin signaling plays a role in the pathogenesis of hypertensive vascular disease. Here, we present our finding that the vascular thrombin receptor is upregulated in experimental hypertension via a redox-sensitive mechanism, implying a mechanistic link between hypertension and atherosclerosis and suggesting a novel paradigm for hypertensive vascular disease.
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Materials and Methods |
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Reagents and Materials
Losartan was a gift from Dr R.D. Smith (Dupont de Nemours Co, Wilmington, Del), and purified
-thrombin was provided by
Dr S. Krishnaswamy (Emory University, Atlanta, Ga). TRI reagent was
purchased from Molecular Research Center. Alzet osmotic minipumps were
from Alza Corp, [32P]dCTP was purchased from DuPont NEN,
and all rats and chows were from Harlan Sprague-Dawley, Inc
(Indianapolis, Ind). Magna NT nylon membranes were from Micron
Separation, Inc. Prime-it II probe labeling kits were purchased from
Stratagene, and Biospin P30 columns were from Bio-Rad. All other
chemicals and drugs were purchased from Sigma Chemical Co.
Experimental Models of Hypertension and Antihypertensive
Therapy
To mimic high-renin hypertension, 250- to 300-g male
Sprague-Dawley rats received Ang II infusions from subcutaneously
implanted osmotic minipumps for 1, 3, and 5 days (Alzet, model 2001) or
for 8 and 14 days (Alzet, model 2002). Rats were anesthetized
with 10 mg/kg xylazine and 80 mg/kg ketamine hydrochloride
injected intraperitoneally. Using sterile
techniques, a 1-cm horizontal interscapular incision was made, and a
subcutaneous pocket was opened over the right shoulder region by blunt
dissection. An osmotic pump containing Ang II dissolved in a solution
of 0.15 mol/L NaCl and 0.01N acetic acid at a concentration calculated
to deliver 
0.75 mg/kg per day of the drug was placed in the
subcutaneous space. In addition, some animals received HB-SOD (700 U/d)
infused via the internal jugular vein. Control animals were either
sham-operated with no pump implanted or implanted with pumps containing
vehicle only. The skin was closed with surgical staples.
To mimic low-renin hypertension, 11-week-old male Dahl salt-sensitive rats (JJ/ss) were fed either low-salt (0.4%) or high-salt (8%) chow for 3 weeks with water provided ad libitum. This inbred strain of rats reproducibly responds to high-salt feeding with a gradual sustained elevation of blood pressure. Of particular importance to this study is the presence of very low plasma renin and renal renin activity in these animals.23 For these experiments, the same strain of rats maintained on a low-salt diet served as controls.
Systolic blood pressure was measured at baseline and before tissue harvesting in the Sprague-Dawley rats and twice weekly in the Dahl rats by the previously described tail-cuff sphygmomanometer method.24 Measurements were performed in triplicate, and a mean value was obtained.
Additional Ang II–infused rats were treated with antihypertensive drugs. Losartan, a nonpeptide competitive antagonist of the Ang II AT1 receptor (25 mg/kg per day), and the nonspecific vascular smooth muscle relaxant hydralazine (15 mg/kg per day) were given orally by supplementation of the drinking water. Antihypertensive drug treatment was initiated 24 hours before pump implantation.
Tissue Harvesting and RNA Preparation
Sprague-Dawley rats were studied 1, 3, 5, 8, or 14 days
after pump implantation, and Dahl rats were studied 3 weeks after the
initiation of low- or high-salt feeding. Briefly, CO2
narcosis was induced by CO2 inhalation, after which
intraperitoneal xylazine (40 mg) was administered.
A midline ventral incision was then made. With the heart still beating,
an intracardiac injection of 5000 U heparin was given. The aorta was
then dissected from the renal arteries to the ascending arch, removed
en bloc, and placed in ice-cold PBS (pH 7.4). Extravascular tissue was
removed rapidly with forceps, and the vessel lumen was flushed
sequentially with heparin and PBS. The specimen was placed in a
precooled microcentrifuge tube, immediately submerged in liquid
nitrogen, and transferred to a -70°C freezer for storage. Aortas
were homogenized in TRI reagent using a Polytron
homogenizer (Brinkman). The TRI reagent method of RNA
extraction was then followed, as described previously.25
Ten micrograms of total RNA per specimen was loaded onto a
formaldehyde-containing 1% agarose gel.
Northern Blotting and Hybridization
After size fractionation on a denaturing agarose-formaldehyde
gel, total RNA was transferred to a nylon membrane (MSI, Inc) and
exposed to ultraviolet light to cross-link the RNA. Membranes were
prehybridized for 2 hours at 42°C with prehybridization buffer (50%
[vol/vol] formamide, 1 mol/L NaCl, 5x Denhardt's solution, 0.5%
SDS, 50 mmol/L Tris buffer, pH 7.4, and 100 µg/mL denatured
salmon testes DNA). Subsequently, membranes were hybridized overnight
with a random-primed [32P]dCTP-labeled Pst I
fragment of rat vascular smooth muscle thrombin receptor cDNA.
Membranes were then washed two to four times for 15 minutes in 0.5x
SSC and 0.1% SDS at 55°C and exposed to x-ray film at -70°C for
24 to 48 hours. Autoradiographic bands were quantified via
densitometry and normalized to 28S ribosomal RNA.
Thrombin-Induced NO Release
Isolated aortic rings from Ang II–infused hypertensive and
control rats were compared with respect to their basal and
thrombin-stimulated NO release. Each ring was incubated in
Krebs-Henseleit buffer for 30 minutes at 37°C, assayed for basal NO
secretion, and then incubated with 1 U/mL thrombin; some rings were
also stimulated with 40 µmol/L TRAP
(NH2-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-COOH).
To ensure an intact NO-secreting capacity of the rings, each ring was
treated with acetylcholine (0.1 mmol/L) as well as the calcium
ionophore A23187 (10-5 mol/L), a
receptor-independent activator of NO synthase. A 30-minute
incubation in agonist-free buffer preceded each agonist treatment. The
eluant from the rings was analyzed via chemiluminescence for
nitrite and nitrate, the stable degradation products of NO. This
technique has been previously used to accurately analyze NO
release.26 NO measurements were indexed to the dry weight
of the rings in milligrams and are reported as picomoles per
milligram.
Thrombin-Induced Smooth Muscle Constriction
Some aortas were carefully dissected, rinsed, and
submerged in indomethacin-supplemented-Krebs' buffer
(mmol/L: NaCl 118.3, KCl 4.7, MgSO4 1.2,
KH2PO4 1.2, CaCl2 2.5,
NaHCO3 25, disodium EDTA 0.026, and glucose 5.5), pH 7.4,
and placed on ice. Three to four 5-mm segments were cut from each
aorta, and segments were denuded of endothelium by
rubbing with cotton-tipped swabs. Successful denudation was confirmed
by demonstrating a preserved constrictor response to KCl in rings with
an absent vasodilation to acetylcholine. Rings were suspended between a
fixed base and strain gauge for measurement of isometric force. The
rings were placed in an organ bath containing Krebs' buffer with
10-5 mol/L indomethacin at
37°C. The length of the smooth muscle was increased stepwise for 90
minutes to adjust basal tension to 2 to 3 g. Rings were treated
sequentially with 5 and 10 U/mL purified -thrombin. Results are
shown as the percent of constriction elicited by 80 mmol/L
KCl.
Statistical Analysis
All data are given as mean±SEM. Statistical significance was
determined using Student's t test or two-way ANOVA, and
Duncan's multiple-range test was used for post hoc analysis.
Means were considered significantly different at values of
P<.05.
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Results |
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Aortic Thrombin Receptor mRNA Regulation by Ang II–Induced Hypertension
Hypertension was achieved in the Ang II–infused animals by day 3 after pump implantation and persisted for 14 days (Figs 1
and 2A). For most experiments, rats
were studied on day 5, when the mean systolic blood pressure
was 190±10 mm Hg in the Ang II group versus 113±2 mm Hg
in the control group (P<.0001). In the control aortas,
thrombin receptor mRNA was nearly undetectable. There was a marked
10-fold increase in thrombin receptor mRNA by 5 days of Ang II
infusion (P=.019) (Fig 2B). Obvious increases were detected
after 1 day, with a peak occurring at 5 days. A significant increase
persisted through 14 days of Ang II infusion (data not shown).
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Effect of Antihypertensive Drugs on Systolic Hypertension
and Thrombin Receptor mRNA
Both hydralazine and losartan prevented an
elevation of systolic blood pressure during the Ang II infusion
(Fig 2A
). When systolic hypertension was prevented by blocking
AT1 receptor activation with losartan, thrombin
receptor mRNA remained at basal levels (Fig 2B). In contrast, with
hydralazine treatment, thrombin receptor mRNA levels remained
significantly elevated over control levels (6.0±3.2 times control,
P=.02) and not significantly different from levels seen in
animals treated with Ang II alone (P=.26).
Thrombin Responsiveness of Aortic Rings From Control and Ang
II–Infused Rats
Rings from control and Ang II–infused rats displayed
similar basal NO release that increased after treatment with
acetylcholine (0.1 mmol/L) or the receptor-independent stimulator
of NO synthase, A23187 (10-5 mol/L). Purified
-thrombin (1 U/mL) also induced an increase above basal NO
secretion, as did 40 µmol/L of TRAP (Fig 3). No
significant differences in thrombin- or TRAP-induced NO secretion were
detected between segments from Ang II–infused hypertensive rats and
sham-operated control rats (P=NS for each agonist
treatment).
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To determine if there was an increase in thrombin receptor–mediated
smooth muscle constriction, the thrombin reactivity of isolated
endothelium-denuded aortic segments was examined. We
found that aortic rings from Ang II–infused animals displayed a 4-fold
increase in thrombin-induced constriction (44±7% versus 9±6%
maximal KCl constriction) (Fig 4).
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Modulation of Systolic Blood Pressure and Aortic Thrombin
Receptor mRNA in Dahl Salt-Sensitive Rats
High-salt feeding induced an increase in blood pressure in the
Dahl salt-sensitive rats (210±5 mm Hg, P<.001),
whereas no significant blood pressure change was detected in the rats
fed low-salt chow (131±6 mm Hg). The peak systolic blood
pressure in the hypertensive Dahl rats was similar to that achieved in
the rats infused with Ang II (214±4 versus 210±5 mm Hg,
P=.56). Thrombin receptor mRNA was barely detectable in the
aortas from normotensive Dahl salt-sensitive rats and was not altered
by salt-induced hypertension (105±12% of control, P=.79,
Fig 5).
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Potential Role of Superoxide in the Upregulation of Thrombin
Receptor mRNA Expression
Previous studies have demonstrated that superoxide
production is increased in Ang II–induced
hypertension.21 22 Thus, we sought to determine the
potential contribution of superoxide to the upregulation of thrombin
receptor expression in Ang II–induced hypertension by administering
HB-SOD intravenously to rats also receiving Ang II. This
supplemental superoxide dismutase completely inhibited Ang II–induced
thrombin receptor expression (114±30% of control, P=.79).
A representative Northern blot and mean data are shown
in Fig 6.
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Discussion |
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In the present study, we show a dramatic increase in aortic thrombin receptor mRNA expression in animals with Ang II–induced hypertension via a superoxide-dependent mechanism. We have also demonstrated an increase in thrombin-mediated constriction of aortic segments from these animals, suggesting that the primary anatomic location of thrombin receptor upregulation is the vascular smooth muscle cells. Based on our experiments with the antihypertensive drugs hydralazine and losartan, the critical stimulus for thrombin receptor regulation in this model appears to be activation of the Ang II AT1 receptor. These findings may have important implications regarding the mechanisms of Ang II–induced vascular pathology.
The finding that activation of the AT1 receptor is more important than hypertension per se in terms of increasing thrombin receptor mRNA is based on two lines of evidence. First, blood pressure reduction with hydralazine was not sufficient to normalize thrombin receptor mRNA during the Ang II infusion. In contrast, losartan, which reduced blood pressure to the same degree, prevented the thrombin receptor mRNA induction. Finally, although we cannot rule out the possibility that the rate of development of hypertension could be a stimulus regulating the thrombin receptor expression, the peak systolic blood pressures in the low-renin low–Ang II hypertensive Dahl rats were nearly identical to those induced by Ang II infusion, yet there was no induction of thrombin receptor mRNA in the former group. Thus, in these experiments, the effects of Ang II on vascular gene expression can be separated from its effects on vascular tone. These findings imply that protection from hypertension-induced vascular pathology requires more than nonspecific blood pressure reduction.
Ideally, correlative measurements of thrombin receptor protein would have been desirable; however, there are currently no reproducible methods available for the accurate detection and quantification of rat vascular thrombin receptor protein in whole tissues. Furthermore, because of the unique fashion in which thrombin interacts with its receptor, ligand binding studies are not possible. Therefore, we used measurements of NO production and thrombin-induced constriction as an approach to gain insight into the presence of functional thrombin receptors in the endothelium and vascular smooth muscle, respectively. Although we cannot rule out the possibility of alterations in receptor coupling masking increases in thrombin receptor protein, the finding that thrombin receptor activation leads to identical amounts of vascular NO release from control and Ang II–treated rats is consistent with the conclusion that endothelial thrombin receptors were not functionally altered in this model. In contrast, thrombin-induced constriction in endothelium-denuded aortic rings from Ang II–infused rats was 4-fold higher than that observed in sham-operated control rats. This finding suggests that thrombin receptors were functionally increased in the vascular smooth muscle layer and implies a role for thrombin in modulating aortic tone and stiffness in hypertensive states.
Given the myriad effects of thrombin on smooth muscle cells,8 10 13 16 27 thrombin receptor activation in the vascular smooth muscle layer may contribute to the characteristic changes of hypertensive vascular disease: medial hypertrophy at the expense of the lumen,28 impaired vasodilatory capacity,29 and inflammatory cell infiltration of the vessel wall.30 Many studies have concentrated on the importance of the endothelium in orchestrating vascular pathophysiology in hypertension, specifically smooth muscle growth and tone.31 However, thrombin receptors appear to be selectively increased in the AT1 receptor–rich vascular smooth muscle layer of Ang II–infused hypertensive rats, and this increase proceeds via an AT1 activation pathway. Thus, our data imply that the medial layer in hypertension can be directly activated to induce vascular growth and inflammation via paracrine and autocrine mechanisms and imply a direct role for vascular smooth muscle cells in promoting vascular pathology.
The finding that the thrombin receptor increase appears to be localized to the muscular layer led us to question how thrombin accesses these receptors. There are at least three possibilities: First, there is evidence that the coagulation cascade, and thus thrombin formation, may occur underneath an intact endothelium. Membrane-bound tissue factor, an important mediator of thrombin formation,32 is known to be present in subendothelial layers of the vascular wall33 and is regulated in vascular smooth muscle cells by Ang II.34 Clotting factors VII, X, and prothrombin may gain access to the media in hypertension through an abnormally permeable endothelial barrier (a characteristic of hypertensive arteries35 ) and thus supply the subendothelial compartment with the necessary components for tissue factor–mediated thrombin formation. Alternatively, recent studies have documented the existence of a basal intravascular pool of thrombin36 that could access medial thrombin receptors via transendothelial migration.37
There is an additional consideration regarding the long-term functional consequences of upregulated arterial thrombin receptor expression in hypertension. Since the thrombin receptor has been associated with atherosclerotic lesions, it is possible that a preexisting increase in thrombin receptor number may exaggerate the thrombogenic and proliferative responses of the vessel wall to injury. In this way, Ang II–induced hypertension may "prime" the arterial wall for atherosclerosis.
The finding that thrombin receptors are increased in a model of hypertension and the implication that a coagulation-promoting enzyme may play a role in hypertensive vascular disease are novel. To date, several lines of evidence have suggested an association between hypertensive vascular disease and thrombosis: platelets from hypertensive animals and humans have an increased tendency to degranulate and display increased adhesiveness in vitro38 ; clinical studies have shown increased circulating levels of the prothrombotic proteins PAI-1, von Willebrand factor, and fibrinogen in hypertensive subjects39 ; and finally, Ang II, a mediator of arterial hypertension, has procoagulant effects in vitro40 and in vivo.41 By showing an enhanced responsiveness to thrombin in the vasculature of hypertensive rats, the present study provides evidence implicating the vascular wall in the proposed link between the renin-angiotensin system, hypertension, and thrombosis.
Finally, our demonstration of an inhibition of thrombin receptor upregulation by superoxide dismutase provides evidence for redox-sensitive thrombin receptor regulation. We have recently shown that exposure to exogenously generated superoxide anion increases thrombin receptor expression in cultured vascular smooth muscle cells.20 Furthermore, recent studies have demonstrated that Ang II–induced hypertension is perhaps uniquely associated with an increase in vascular superoxide production22 by a membrane-associated NADH/NADPH oxidase.21 22 Chronic administration of HB-SOD to Ang II–infused animals abolishes the hypertensive response.21 These data are then consistent with the hypothesis that in vivo Ang II induces superoxide production via the membrane-associated NADH/NADPH oxidase and that the subsequent upregulation of the thrombin receptor occurs via this redox-sensitive mechanism.
In conclusion, vascular thrombin receptor expression is increased in Ang II–induced experimental hypertension; the increase appears to be AT1-mediated and is localized to the smooth muscle layer. As outlined earlier, thrombin receptor activation in vascular smooth muscle is not only a potent contractile and growth stimulus but stimulates synthesis of a variety of proinflammatory proteins. Our findings provide a basis for the hypothesis that thrombin receptor activation represents one link between hypertension and atherosclerosis and introduces a new paradigm for the pathogenesis of hypertensive vascular disease that focuses attention on the medial layer as an initiator of, and not simply a passive target of, aberrant growth stimuli.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was partially supported by National Institutes of Health grants HL-48667 and HL-09185 and a Merit Review Board grant from the Veterans Administration. Jørn Bech Laursen and Toshiki Fukui both contributed equally to the scientific content of this manuscript.
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Footnotes |
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This work was presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.
Received December 11, 1996; accepted February 25, 1997.
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