Effects of Endothelin Receptor Inhibition on Cerebral Arterioles in Hypertensive Rats
Abstract The purpose of this study was to examine the effects of endothelin receptor inhibition on cerebral arterioles in stroke-prone spontaneously hypertensive rats (SHRSP). Structure and mechanics of cerebral arterioles were examined in untreated Wistar-Kyoto rats (WKY) and SHRSP that were either untreated or treated for 3 months with bosentan, an inhibitor of endothelin receptors (100 mg/kg per day). We measured pressure, external diameter, and cross-sectional area of the vessel wall (histologically) in maximally dilated (EDTA) arterioles on the cerebrum. Bosentan reduced but did not normalize arteriolar mean pressure (103±3 and 81±5 mm Hg in untreated and treated SHRSP versus 51±4 mm Hg in WKY, P<.05; mean±SEM) and pulse pressure (40±2 and 33±2 mm Hg in untreated and treated SHRSP versus 25±3 mm Hg in WKY, P<.05) in SHRSP. Cross-sectional area of the vessel wall (CSA) was increased in untreated SHRSP (1627±173 μm2), and CSA in treated SHRSP (1287±78 μm2) was similar to that in WKY (1299±65 μm2). Bosentan had no effect on reductions in external diameter (remodeling) of cerebral arterioles (104±7 and 96±4 μm in untreated and treated SHRSP compared with 126±7 μm in WKY, P<.05). Stress-strain curves indicate that bosentan had no significant effect on distensibility of arterioles on the cerebrum in SHRSP. The results suggest that endothelin-1 may contribute to the development of hypertrophy, but not remodeling or changes in distensibility, of cerebral arterioles in SHRSP.
Determinants of vascular hypertrophy and remodeling are different. Possible determinants of cerebral vascular hypertrophy include increases in pressure,3 4 neurohumoral factors,5 6 7 8 9 10 11 and genetic factors.12 13 Possible determinants of vascular remodeling in cerebral arterioles of SHRSP include angiotensin II14 and genetic factors.2 Another determinant of vascular hypertrophy and remodeling during chronic hypertension may be ET-1.15 This suggestion is based on the finding that treatment with bosentan, a blocker of both ETA and ETB receptors, attenuates hypertrophy and remodeling in small mesenteric arteries of DOCA-salt hypertensive rats.15 The degree of attenuation of hypertrophy and remodeling was disproportionately greater than the modest reduction in arterial pressure during bosentan administration.
The effect of bosentan on the structure of small mesenteric arteries of DOCA-salt hypertensive rats offers a strong rationale for the possibility that ET-1 may contribute to vascular hypertrophy and remodeling during chronic hypertension. Alternative explanations are possible, however. First, we14 16 and others17 have provided evidence that pulse pressure may be more important than mean pressure in induction of vascular hypertrophy during chronic hypertension. Effects of treatment with bosentan on arterial pulse pressure are not known. It is possible that, if bosentan preferentially reduces pulse pressure, vascular hypertrophy may be attenuated by bosentan to a greater degree than predicted by reductions in mean pressure. Second, we have defined vascular remodeling during chronic hypertension as a reduction in external diameter that cannot be attributed to a decrease in distensibility.1 Because effects of bosentan on distensibility of vessels in hypertensive rats have not been examined, it is possible that attenuation of reductions in external diameter of vessels by bosentan15 may result from decreases in distensibility rather than remodeling.
The first goal of this study was to examine effects of treatment with bosentan on pulse pressure and hypertrophy of cerebral arterioles in SHRSP. Our hypothesis was that bosentan might attenuate hypertrophy of cerebral arterioles in proportion to reductions in arteriolar pulse pressure, but not mean pressure, in which case effects of bosentan could be attributed to effects on pulse pressure rather than a direct trophic action of ET-1. The second goal was to examine effects of bosentan on changes in distensibility of cerebral arterioles in SHRSP. Our third goal was to examine effects of bosentan on remodeling. We postulated that if attenuation of reduction in external diameter of vessels in hypertensive rats treated with bosentan15 is the result of changes in distensibility, treatment with bosentan may not attenuate remodeling of cerebral arterioles in SHRSP.
Experiments were performed on male WKY and male SHRSP. At 3 months of age, SHRSP were divided into 2 groups: a group that was treated with bosentan (100 mg/kg per day, n=8) and an untreated group (n=6). Bosentan was administered in powdered rat chow. Untreated WKY served as normotensive controls (n=7). Animals were allowed free access to food and tap water, housed at 25°C, and exposed to 12 hours of light each day. Arterioles on the cerebrum were examined when the animals reached approximately 6 months of age. Procedures followed in this study were in accordance with institutional guidelines for care and use of experimental animals at the University of Iowa.
Animals were weighed (WKY, 382±7 g; untreated SHRSP, 313±10 g; treated SHRSP, 313±4 g; mean±SEM), anesthetized with sodium pentobarbital (5 mg/100 g body wt IP), intubated, and mechanically ventilated with room air and supplemental O2. Paralysis of skeletal muscle was obtained with gallamine triethiodide (20 mg/kg IV). Because the animals were paralyzed, we evaluated them frequently for adequacy of anesthesia. Additional anesthesia (1.7 mg/100 g body wt IV) was administered when pressure to a paw evoked a change in blood pressure or heart rate.
A catheter was inserted into a femoral vein for injection of drugs and fluids. A catheter was inserted into a femoral artery to record systemic arterial pressure and to obtain blood samples for measurement of arterial blood gases, and a catheter was inserted into the other femoral artery to withdraw blood to produce hypotension.
Measurement of Arteriolar Pressure and Diameter
We measured pressure and diameter of first-order arterioles on the cerebrum18 through an open skull preparation.19 The head was placed in an adjustable head holder, and a 1-cm incision was made in the skin to expose the skull. The skin edges were retracted with sutures, and ports were placed for inflow and outflow of artificial CSF. A craniotomy was made over the left parietal cortex, and the dura was incised to expose cerebral vessels. The exposed brain was continuously suffused with artificial CSF, warmed to 37°C to 38°C, and equilibrated with a gas mixture of 5% CO2/95% N2. The composition of the CSF was (mmol/L) KCl 3.0, MgCl2 0.6, CaCl2 1.5, NaCl 131.9, NaHCO3 24.6, urea 6.7, and dextrose 3.7.19
Pressure in arterioles on the cerebrum was measured continuously with a micropipette connected to a servo-null pressure measuring device (model 5, Instrumentation for Physiology and Medicine, Inc). Pipettes were sharpened to a beveled tip of 3 to 5 μm in diameter, filled with 1.5 mol/L NaCl, and inserted into the lumen of an arteriole with a micromanipulator. The presence of the pipette tip in the vessel had no discernible effect on the diameter of arterioles on the cerebrum.
Arterioles were monitored through a Leitz microscope (×10 objective) connected to a closed-circuit video system with a final magnification of ×356. Images of arterioles were digitized with the use of a video frame grabber (Quick Image 24, MASS Microsystems) installed in a Macintosh computer (Quadra 900, Apple Computer). Arteriolar diameter was measured from the digitized images with the use of image analysis software (NIH Image, National Institutes of Health, Research Services Branch, NIMH). The precision of this system is 0.4 to 0.6 μm.
About 20 to 30 minutes after completion of surgery, arteriolar pressure and diameter were obtained under baseline conditions. Vascular smooth muscle was then deactivated by suffusion of arterioles with artificial CSF containing EDTA (67 mmol/L), which produces complete deactivation of smooth muscle in arterioles on the cerebrum.19 To examine pressure-diameter relationships in deactivated arterioles, hemorrhage was used to reduce arteriolar pressure in decrements of 10 mm Hg at pressures between 70 and 20 mm Hg and decrements of 5 mm Hg at pressures between 20 and 10 mm Hg. After each pressure step, arteriolar diameter achieved a steady state within 15 seconds. Inner diameter was measured approximately 30 seconds later.
After the last pressure step, blood was reinfused to restore arteriolar pressure to control levels. Suffusion of arterioles with artificial CSF containing EDTA was stopped, and the maximally dilated arterioles were fixed at physiological pressure in vivo by suffusion of vessels with glutaraldehyde fixative (2.25% glutaraldehyde in 0.10 mol/L cacodylate buffer) while maintaining arteriolar pressure at baseline levels. Arterioles were considered to be adequately fixed when blood flow through the arteriole had ceased.
After the animal was killed by an injection of KCl, the arteriolar segment used for pressure-diameter measurements was removed with a microsurgical knife. Fixed arterioles were processed, embedded in Spurr’s low viscosity resin while cross-sectional orientation was maintained, and sectioned using an ultramicrotome. Cross-sectional area of the vessel wall was determined with the use of a light microscope interfaced with the video image analyzing system described above. Luminal and total (lumen plus vessel wall) cross-sectional areas of the arteriole were measured by tracing the inner and outer edges of the vessel wall. Cross-sectional area of the vessel wall was calculated by subtraction of luminal cross-sectional area from total cross-sectional area.
Calculation of Mechanical Characteristics
Circumferential stress (ς) was calculated from mean pressure (AP), inner diameter (ADi), and wall thickness (WT) of arterioles on the cerebrum: ς=(AP·ADi)/(2WT). Arteriolar pressure was converted from mm Hg to newtons per meter squared (1 mm Hg=1.334×102 N/m2). Because the volume of the vessel wall does not change during changes in intravascular pressure,20 21 we assumed that cross-sectional area of the vessel wall remains constant with changes in arteriolar diameter. Thus, wall thickness can be calculated from cross-sectional area of the vessel wall (CSA) and inner arteriolar diameter: WT=[(4CSA/π+ADi2)1/2−ADi]/2. External diameter of arterioles on the cerebrum (ADe) was calculated as: ADe=ADi+2WT. Histological determinations of cross-sectional area were used in all calculations of wall thickness and circumferential stress.
Circumferential strain (ε) was calculated as ε=(ADi−ADo)/ADo, where ADo is original diameter. Original diameter has been defined as the diameter at very low or 0 mm Hg pressure with the vessel extended to in situ length.21 22 Blood flow through arterioles on the cerebrum at 10 mm Hg of arteriolar pressure was adequate to maintain an intact red blood cell column. Because reduction of pressure to 0 mm Hg stops blood flow, and because passive vascular collapse is likely at 0 mm Hg, it was not possible to obtain reliable measurements of inner diameter of arterioles on the cerebrum at 0 mm Hg. Therefore, we calculated strain using diameter at 10 mm Hg as the original diameter, as in previous studies.16 19
Measurements of systemic mean pressure, arteriolar pressure, diameter, and cross-sectional area of the vessel wall were compared using ANOVA (JMP for the Macintosh, SAS Institute Inc). Probability values were calculated using a Student’s t test.
Systemic arterial mean pressure and systolic, diastolic, pulse, and mean pressures in arterioles on the cerebrum in SHRSP treated with bosentan were significantly less than in untreated SHRSP but were significantly higher than in WKY (Table⇓).
Cross-sectional area of the vessel wall was significantly greater in untreated SHRSP than in WKY (Table⇑). Bosentan reduced the cross-sectional area of the vessel wall in SHRSP to levels not significantly different from those in WKY (Table⇑). Thus, treatment with bosentan prevented hypertrophy of cerebral arterioles in SHRSP without normalizing arteriolar pressures.
Diameters of arterioles on the cerebrum both before and after deactivation with EDTA were significantly less in untreated SHRSP than in WKY. Bosentan had no significant effect on external or internal diameters of arterioles in SHRSP (Table⇑). Thus, treatment with bosentan did not attenuate remodeling of cerebral arterioles in SHRSP.
Internal and external diameters at comparable pressures were smaller in untreated SHRSP than in WKY (Fig 1⇓). Treatment with bosentan did not significantly alter pressure-diameter relationships for either internal or external diameter in SHRSP (Fig 1⇓).
The stress-strain curve in untreated SHRSP was shifted to the right of the curve in WKY (Fig 2⇓). Treatment with bosentan in SHRSP did not affect stress-strain curves (Fig 2⇓). This finding suggests that bosentan had no effect on distensibility of cerebral arterioles in SHRSP, despite prevention of hypertrophy.
There were three major findings in this study. First, treatment with bosentan, an inhibitor of both ETA and ETB receptors, prevented hypertrophy of arterioles on the cerebrum in SHRSP, even though it did not normalize arteriolar pulse pressure. This finding suggests that ET-1 may contribute directly to hypertrophy of cerebral arterioles during chronic hypertension in SHRSP. Second, in contrast to treatment with an angiotensin-converting enzyme inhibitor14 or carotid clipping,16 which prevent both hypertrophy and increases in distensibility of cerebral arterioles in SHRSP, inhibition of ET receptors in SHRSP had no effect on distensibility of cerebral arterioles, despite preventing hypertrophy. Third, inhibition of ET receptors did not prevent reductions of external diameter of arterioles on the cerebrum in SHRSP. This finding suggests that ET-1 does not contribute to remodeling of cerebral arterioles in SHRSP.
Cerebral arterioles undergo hypertrophy in several models of experimental hypertension. Determinants that may contribute to cerebral vascular hypertrophy during chronic hypertension include increases in pressure,3 4 neurohumoral factors,5 6 7 8 9 10 11 and genetic factors.12 13 ET-1 also may contribute to vascular hypertrophy in some forms of chronic hypertension. Treatment of DOCA-salt hypertensive rats with bosentan prevents hypertrophy of small mesenteric arteries.15 Furthermore, DOCA-salt hypertension in SHR augments vascular hypertrophy and ET-1 mRNA in small mesenteric arteries.23
In a previous study, hypertrophy in small mesenteric, coronary, renal, and femoral arteries was not significantly different in SHR treated with bosentan than in untreated SHR.24 In contrast, we found in this study that treatment with bosentan prevented hypertrophy in arterioles on the cerebrum of SHRSP. Several factors may account for the different findings. First, ET-1 may contribute to the hypertrophy of cerebral vessels but not mesenteric, coronary, renal, or femoral vessels. Second, the contribution of ET-1 to vascular hypertrophy may vary with vessel size. Third, ET-1 may contribute to hypertrophy only when arterial pressure increases above a critical threshold. This possibility is suggested by several findings. Arterial pressure is somewhat higher in SHRSP than in SHR.25 26 Furthermore, as shown in this study, bosentan lowers arterial pressure in SHRSP, whereas in a previous study it was found to have no effect on blood pressure in SHR.24 Finally, treatment of SHR with DOCA salt increases systolic arterial pressure and, at the same time, results in increases of ET-1 mRNA and hypertrophy in small mesenteric arteries.23
Several studies suggest that pulse pressure may play an important role in the development of vascular hypertrophy during chronic hypertension. Both hydralazine and cilazapril, an angiotensin-I–converting enzyme inhibitor, prevent hypertrophy of cerebral arterioles in SHRSP and are equally effective in reducing pulse pressure in cerebral arterioles of SHRSP, even though hydralazine is less effective in reducing mean arterial pressure.14 Effectiveness of different treatments in reducing media-to-lumen ratio of small mesenteric arteries in SHR correlates to a greater degree with arterial pulse pressure than with arterial mean or systolic pressure.17 Furthermore, there is a strong correlation between cross-sectional area of the vessel wall and pulse pressure, but not systolic pressure or mean pressure, in cerebral arterioles of WKY and SHRSP.16 An implication of these findings is that, even if effects of bosentan on arterial pulse pressure contribute to the prevention of hypertrophy in cerebral arterioles in SHRSP, it is likely that the modest reduction in arteriolar pressure that is produced by bosentan accounts for only a portion of the reduction in the cross-sectional area of the vessel wall that was found in this study. Another implication of the findings is that ET-1 may contribute directly to hypertrophy of cerebral arterioles during chronic hypertension in SHRSP.
Distensibility of fully relaxed cerebral arterioles is increased paradoxically in SHRSP, SHR, and rats with one-kidney, one clip renal hypertension, despite hypertrophy of the arteriolar wall.2 19 Furthermore, prevention of hypertrophy in cerebral arterioles of SHRSP by treatment with an inhibitor of the angiotensin-converting enzyme14 or carotid clipping16 normalizes arteriolar distensibility. We were surprised therefore by the finding in this study that treatment with bosentan had no effect on increases in distensibility of arterioles on the cerebrum in SHRSP, even though it prevented hypertrophy.
We have proposed previously that increases in passive distensibility that accompany hypertrophy of cerebral arterioles may be due to a reduction in the proportion of stiff (collagen and basement membrane) to compliant (smooth muscle, elastin, and endothelium) components of the arteriolar wall in cerebral arterioles.2 6 16 27 Therefore, a possible explanation for the finding in this study that treatment with bosentan did not prevent increases in cerebral arteriolar distensibility in SHRSP, despite preventing hypertrophy, is that bosentan did not normalize proportional composition of the arteriolar wall.
In addition to hypertrophy, cerebral arterioles in SHRSP and SHR undergo remodeling of the vessel wall with a reduction in external diameter.1 2 Angiotensin II and genetic factors, but not arterial pressure per se, may be determinants of remodeling of cerebral arterioles in SHRSP.2 14 It has been proposed recently that another determinant of remodeling in small mesenteric arteries in DOCA-salt hypertension may be ET-1.15 Our finding in this study, however, that external diameter of arterioles on the cerebrum was no different in SHRSP treated with bosentan than in untreated SHRSP indicates that ET-1 probably does not contribute to remodeling of these vessels in SHRSP.
There are at least two possible reasons for the apparent discrepancy that treatment with bosentan attenuates reductions in external diameter in small mesenteric arteries of DOCA-salt hypertensive rats15 but not in arterioles on the cerebrum of SHRSP. First, determinants of remodeling may vary in different vascular beds. Second, and perhaps more likely, DOCA-salt hypertension may reduce the external diameter of small mesenteric arteries in rats as a consequence of reduced distensibility and not as a consequence of remodeling. We define remodeling as a reduction in external diameter that cannot be attributed to a decrease in distensibility of the vessel wall.1 Our reason for emphasizing the role of distensibility in the definition of remodeling is exemplified by the finding that distensibility is reduced in large branches and increased in small branches,28 whereas external diameter is reduced in both large and small branches of the posterior cerebral artery in SHRSP. Furthermore, reductions in external diameter of large branches of posterior cerebral artery in SHRSP can be attributed entirely to reductions in distensibility. Thus, we speculate that attenuation of reductions in external diameter of small resistance arteries caused by bosentan in DOCA-salt hypertensive rats may have resulted from the effects of bosentan on distensibility of these arteries and not from an effect on remodeling.
Our findings also suggest that remodeling, with reduction in external diameter, may play a larger role than hypertrophy in impairment of maximal dilator capacity of cerebral arterioles in SHRSP. This statement is based on the finding that internal diameter of cerebral arterioles during maximal dilatation with EDTA was reduced to the same extent in SHRSP treated with bosentan as in untreated SHRSP, even though the cross-sectional area of the vessel wall was substantially less in the treated group. If hypertrophy was an important factor in impaired maximal dilatation, we would have expected prevention of hypertrophy by bosentan to restore at least a portion of the maximal dilator capacity that is lost in cerebral arterioles of SHRSP compared with WKY.
Speculations and Implications
We conclude that treatment of SHRSP with an antagonist of ETA and ETB receptors prevents hypertrophy of cerebral arterioles. Furthermore, the effect of ET receptor blockade on cross-sectional area of the vessel wall in cerebral arterioles of SHRSP is disproportionate to its modest effect on cerebral arteriolar pressure. Finally, in contrast to its effect on hypertrophy, treatment with an ET receptor blocker has no effect on distensibility or remodeling of cerebral arterioles in SHRSP.
An implication of these findings relates to the possibility that trophic effects of pulse pressure on cerebral arterioles may be mediated by endothelial factors, such as ET-1. This possibility is suggested by the following observations. First, there is a strong correlation between pulse pressure and cross-sectional area of the vessel wall in cerebral arterioles of WKY and SHRSP.16 Second, ET-1 stimulates growth of vascular smooth muscle in tissue culture.29 30 Finally, cyclical strain enhances the production of ET-1 by endothelium in tissue culture.31 32 From these findings, one can construct a sequence of events in which pulse pressure exerts an effect on growth of cerebral arterioles through cerebral vascular endothelium by regulating the production of endothelium-derived growth factors, such as ET-1.
Selected Abbreviations and Acronyms
|SHR||=||spontaneously hypertensive rats|
This work was supported by NIH grants HL-22149, NS-24621, HL-16066, and HL-14388 and by funds from the Iowa Affiliate of the American Heart Association. Jean-Marc Chillon is the recipient of a Fellowship Award from the Iowa Affiliate of the American Heart Association. Gary L. Baumbach is the recipient of an Established Investigator Award from the American Heart Association. We thank Hoffmann–La Roche Ltd and Dr Martine Clozel for their support and the gift of bosentan. We also thank Shams Ghoneim and Ronald McElmurry for technical assistance and Dr William Haynes for critical review of the manuscript.
Reprint requests to Jean-Marc Chillon, PhD, Department of Pathology, 146 Medical Laboratories, University of Iowa College of Medicine, Iowa City, IA 52242.
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