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To determine the reactivity and accompanying structural changes in thoracic aorta and carotid artery from nitric oxide (NO)-deficient hypertensive and spontaneously hypertensive rats (SHR).
For the functional study, isolated rat arterial rings were precontracted with a submaximal concentration of phenylephrine (1 μM) and relaxant responses to cumulative concentrations of acetylcholine were obtained. For the morphological study, arteries were processed by a standard method for electron microscopy. The geometry of the arteries – the inner diameter and the wall thickness (tunica intima plus tunica media) – was evaluated by light microscopy.
Increased systolic blood pressure was accompanied by increased heart weight to body weight ratio in both NO-deficient and SHR compared with normotensive controls, indicating cardiac hypertrophy. Morphometry of the thoracic aorta and carotid artery in both models of hypertension showed increased wall thickness, cross-sectional area and wall to diameter ratio. The inner diameter increased in aorta but not in carotid artery. In isolated arteries from normotensive rats, the addition of acetylcholine to precontracted vessels resulted in dose-dependent relaxation. The relaxing effect was more prominent in thoracic aorta than in carotid artery. Endothelium-dependent relaxation of arteries from NO-deficient hypertensive rats was markedly reduced. On the other hand, in aorta and carotid artery from SHR, the endothelium-dependent relaxation in response to acetylcholine was not significantly attenuated. The relaxation of arteries from SHRs, as well as the residual relaxation of arteries from NO-deficient hypertensive rats, was abolished by addition of NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase, to the incubation medium.
These results suggest that increased systolic blood pressure and accompanying structural changes are not primarily responsible for impairment of endothelium-dependent relaxation in experimental hypertension.
It has been shown that acetylcholine-induced relaxation is impaired in arteries of various models of experimental hypertension including renovascular (1), coarctation-induced (2), nitric oxide (NO)-deficient (3), deoxycorticosterone acetate-salt (4) and genetic spontaneous hypertension (5–7). Impaired endothelial function, as evaluated by the vasodilator response to acetylcholine, has also been detected in essential and secondary forms of human hypertension (8,9). Conflicting results indicate that relaxation of arteries from hypertensive animals is not impaired under all circumstances (10,11). Impaired relaxation may also be related to increased blood pressure (in coarctation-induced hypertension) because reversal of hypertension caused a renewed endothelium-dependent response (2).
Experimental models of NO-deficient and spontaneously hypertensive rats (SHR) have provided controversial data concerning cardiac and vascular wall hypertrophy (12–15). Long term administration of the NO synthase inhibitor resulted in a sustained increase in systolic pressure that is accompanied by a remarkable increase in arterial wall thickness. In SHR the increase in arterial thickness is already present before blood pressure increases (16,17). The question arose whether blood pressure is the main cause of pathological alterations in the cardiovascular system.
The aim of the study was to compare endothelium-dependent relaxation of isolated conduit arteries from normotensive rats, NO-deficient hypertensive rats and SHR; and to assess the structural changes in isolated conduit arteries (thoracic aorta and carotid artery) from NO-deficient hypertensive rats compared with those of SHR.
The procedures followed the guidelines presented in the Guide for the Use of Laboratory Animals (Ethics Committee for Experimental Work, Slovak Academy of Sciences, 1995).
The experiments were performed on the thoracic aorta and carotid artery from Wistar rats and SHR. Ten-week-old rats were divided into three groups: normotensive control rats; rats in which hypertension was induced by administration of NG-nitro-l-arginine-methyl ester (L-NAME, 50 mg/kg/day) in drinking water for six weeks; and age-matched SHR. Systolic blood pressure and heart rate were measured weekly by the indirect tail-cuff technique.
Carotid artery and thoracic aorta were removed, cleaned of connective tissue and cut into rings, as described previously (18). The rings were set up for isometric tension recording in 20 mL organ baths containing modified Krebs bicarbonate solution at 37ºC and bubbled continuously with a 95% O2 and 5% CO2 gas mixture to maintain the pH at 7.3 to 7.4. The modified Krebs bicarbonate solution used had the following composition (in mM): NaCl 118, NaHCO3 25, KCl 5, MgSO4.7H2O 1.2, CaCl2 2.5, glucose 11, CaNa2EDTA 0.03 and ascorbic acid 1.1. The rings were mounted on stainless steel hooks, and one side of the tissue was connected by a thread to a force-displacement transducer (Sanborn FT 10, USA) to measure changes in isometric contraction, which were recorded with a TZ 4200 polygraph (Labora, Czech Republic). A resting tension of 10 mN was applied to the tissue and was readjusted every 15 min during a 60 to 90 min equilibration period.
Arterial endothelium-dependent relaxation was measured after active tension had been elicited with phenylephrine (1 μM/L). When the contractile response had reached a plateau, acetylcholine was added to the organ bath in a cumulative manner. All preparations were precontracted with indomethacin (10 μM/L) to avoid the possible participation of prostaglandins in endothelium-dependent relaxation. To assess the mechanisms of relaxation further, some arteries were treated with L-NAME (10 μM/L) before the concentration response curves to acetylcholine were determined.
Morphometrical analysis of the thoracic aorta and carotid artery was performed by a technique previously described (19). In brief, the cardiovascular system of rat was perfused through the left ventricle under a pressure of 120 mmHg by a glutaraldehyde fixative. The thoracic aorta and carotid artery were excised and processed by a standard method for electron microscopy. The sections were cut perpendicularly to the longitudinal axis of the arteries. The geometry of the arteries – the inner diameter and the wall thickness (tunica intima plus tunica media) – was evaluated by light microscopy. The cross-sectional area (tunica intima plus tunica media) was calculated.
Results are presented as mean ± SEM. The statistical difference in mean values between the control and experimental groups was evaluated by two-way analysis of variance, and Bonferroni test for unpaired variables was used. Results were considered significantly different at P<0.05.
Table 1 gives the characteristics of the control and experimental groups. The mean systolic blood pressure measured after six weeks of L-NAME administration was higher in the experimental groups than in the control. The ratio of heart weight to body weight increased in both experimental groups, suggesting the occurrence of cardiac hypertrophy.
In the thoracic aorta from control rats, acetylcholine (1 nM to 10 μM) relaxed phenylephrine-precontracted artery in a concentration-dependent manner (Figure 1). Acetylcholine-induced endothelium-dependent relaxation was significantly attenuated in NO-deficient hypertensive rats. The maximum relaxation due to acetylcholine was 89.3±2.1% (n=16) in the control rats and 40.3±3.0% (P<0.01, n=16) in NO-deficient hypertensive rats. In contrast, in SHR acetylcholine produced dose-related relaxation of aorta similar in magnitude to that observed in controls.
Figure 2 illustrates that the relaxation of thoracic aorta from SHR, as well as the residual relaxation of aorta from NO-deficient hypertensive rats, was abolished by administration of L-NAME (0.1 mM) to the bath medium.
A similar pattern of relaxation in control and experimental groups was seen in carotid artery (Figure 3). In control rats this artery has a relatively small relaxant capacity to acetylcholine (and to other agonists) but the pattern of the dose-response curve is similar to that of thoracic aorta.
Acute addition of L-NAME (0.1 mM) to the incubating medium abolished the residual relaxation of carotid artery in NO-deficient hypertensive rats and in SHR (Figure 4).
The wall thickness of the thoracic aorta in control rats was 60.41±2.37 μm whereas it increased in NO-deficient hypertensive rats to 78.35±1.42 μm and in SHR to 80.79±2.26 μm (P<0.01) (Figure 5, left). There was no significant difference between NO-deficient rats and SHR.
Because wall thickness may be influenced by the perfusion pressure during fixation, the cross-sectional area of the arterial wall was also evaluated. The calculated cross-sectional area in control aortas was 331,600±11,600 μm2, and it increased significantly both in NO-deficient rats (458,000±9640 μm2) and in SHR (478,900±1969 μm2) (Figure 5, right).
The inner diameter of the thoracic aorta in control rats was 1683±39.70 μm, and it increased in NO-deficient rats to 1793±25.48 μm (P<0.01) and in SHR to 1805±44.62 μm (P<0.01) (Figure 6, left). No significant differences were observed between hypertensive groups.
The ratio of wall thickness to inner diameter (3.56±0.19×10−2 in control rats) increased to 4.39±0.11×10−2 (P<0.01) in NO-deficient rats and to 4.49±0.16×10−2 (P<0.01) in SHR (Figure 6, right). No significance differences were observed between experimental groups.
The wall thickness of carotid artery was 25.01±1.27 μm in control rats, and it increased significantly in NO-deficient rats (42.94±1.43 μm, P<0.01) and SHR (48.20±1.52 μm, P<0.01) (Figure 7, left). No significant differences were observed between NO-deficient rats and SHR.
The cross-sectional area in control rats was 67,152±3340 μm2, in NO-deficient rats it increased to 112,037±10,000 μm2 (P<0.01), and in SHR it was 145,253±6120 μm2 (P<0.01) (Figure 7, right). No significant differences were observed between NO-deficient rats and SHR.
The inner diameter did not differ significantly among the groups (Figure 8, left). In control rats it was 833±31.53 μm, in NO-deficient rats it was 802±22.19 μm, and in SHR it was 883±18.45 μm.
On the other hand, the wall to diameter ratio in the control group (3.06±0.24×10−2) was significantly increased in NO-deficient rats (5.43±0.31×10−2) and in SHR (5.49±0.23×10−2, P<0.01) (Figure 8, right). No significant difference was observed between experimental groups.
In the present study increased systolic blood pressure was accompanied by an increase in heart weight to body weight ratio in both NO-deficient hypertensive rats and SHR, thus indicating cardiac hypertrophy.
Our findings are consistent with similar studies using the same concentration of L-NAME and approximately the same period of administration (13,20–22). Cardiac hypertrophy in NO-deficient hypertensive rats was shown to be linked to increased myocardial fibrosis and elevated concentrations of metabolic proteins and nucleic acids (23,24). It seems that increased blood pressure lasting six weeks in rats with experimental hypertension contributes to morphological changes in the cardiovascular system similar to other models of experimental hypertension (25). Cardiac hypertrophy is considered to be a universal mechanism for adapting the heart to long lasting increased blood pressure.
Functional studies showed that there are differences in arterial responses to acetylcholine between the preparations from NO-deficient hypertensive rats and those from control normotensive rats. The reduced maximum relaxation to acetylcholine in rats with NO deficiency cannot be explained solely by structural alterations such as vascular hypertrophy.
The relaxation response to acetylcholine in arteries was thought to be mediated mainly by NO because it was blocked by removal of the endothelium or by acute or chronic application of L-NAME. The observation that inhibited residual relaxation of arteries from NO-deficient hypertensive rats can be partly restored by administration of exogenous l-arginine supports this suggestion (22).
There was no difference between the arteries from control normotensive rats and those from SHR in the magnitude of the relaxation response produced by acetylcholine. On the other hand, arteries from NO-deficient hypertensive rats were less sensitive to the relaxant effect of acetylcholine. This difference does not seem to be caused by decreased sensitivity of the vascular smooth muscle to NO because relaxation elicited by nitroprusside was not significantly different between either SHR or NO-deficient hypertensive rats and control normotensive rats (6,22).
The relaxation response to acetylcholine is not diminished in SHR with similar vascular hypertrophy and with even higher systolic blood pressure, suggesting heterogeneity in the alteration of endothelial function in different models of hypertension. No generalization can be made as to the relative roles played by NO in experimental hypertensive and normotensive animals (25). In NO-deficient hypertension as well as in stroke-prone SHR, mineralocorticoid hypertension and renal hypertension, the NO system is depressed but it may be overactive in SHR (26–28). Also in our experiments, marked relaxation of arteries from SHR probably reflects increased release of NO compared with normotensive rats (Figures 1,,3).3). Relaxant action of vascular NO counteracts the contradictory pressor effect of hypertrophied arterial wall in SHR. These results are in good agreement with the interpretation of Arnal et al (21), who found that cyclic GMP content in the aortic wall was not significantly different under control conditions between normotensive and SHR. They concluded that basal release of NO does not appear to be impaired in SHR, at least in conduit arteries, but is a major counter-regulatory mechanism for the increased vascular resistance in the genetic model of arterial hypertension. Long term administration of l-arginine did not influence the geometry of conduit arteries in SHR (19).
The role of blood pressure levels in endothelium-dependent relaxation in hypertensive animals is not completely clarified. Our results suggest that the influence of blood pressure is of minor, if any, importance. The increase in arterial pressure, per se, most likely does not directly cause abnormal relaxation because the arteries in SHR and NO-deficient hypertensive rats were exposed to increased systemic blood pressures, yet the impaired relaxation was seen only in arteries from NO-deficient hypertensive rats.
Morphometrical studies have indicated that thoracic aorta and carotid artery undergo structural changes in both groups of hypertensive rats. The increase of heart weight and vascular hypertrophy occurring during chronic hypertension contributes to hemodynamic changes in the organism. Folkow (29) proposed that hypertrophy is responsible for both enhanced vasoconstriction and impaired vasodilation in chronic hypertension.
We have found that structural alterations in the arterial wall of the thoracic aorta and carotid artery in the SHR are present at the established phase of hypertension. The increase in the media of the thoracic aorta and carotid artery in SHR was probably due to hypertrophy of the smooth muscle cells, as has also been shown by other authors (30,31).
Long term administration of L-NAME resulted in a remarkable increase in arterial wall thickness of both examined arteries. These observations are in agreement with previous findings on conduit arteries (13,32–34). Examination of the volume density of the cellular and extracellular components in conduit arteries showed that the main contribution to the wall thickness results from accumulated extracellular matrix in the tunica media (33).
In other experimental models of hypertension, thickening of the vessel wall of conduit arteries was also found, but specific changes were different, depending on the model and the duration of hypertension (35,36).
Structural changes of arteries (increase in wall thickness or cross-sectional area) may act as an amplifier that increases the effect of any vasoconstricting stimulus in hypertension (37) and in the maintenance of high blood pressure. Thickening of the vessel wall seems to be secondary adaptive change, at least in NO-deficient hypertensive rats. Lee (38) in three animal models of hypertension showed that structural alterations in vessel wall (hypertrophy of the smooth muscle cells) are secondary adaptive changes because these changes occur after hypertension develops. Quantitative differences in vascular structure may be explained partly by differences in the severity of experimental hypertension.
Vascular remodelling (rearrangement of existing material around a diameter) seems to play an important part in the altered structure of small arteries (39–41). In NO-deficient hypertensive rats the structural changes in conduit arteries reflected reduced NO release. A model of NO-deficient hypertension is characterized by decreased NO synthase activity in the rat aorta. Enhanced DNA synthesis and proteosynthesis of the aorta also were consequences of NO deficiency (34). These findings suggest that altered NO availability to smooth muscle may also influence cellular and extracellular components in the vessel wall and affect vascular growth.
In both groups of hypertensive rats, the inner diameter in the carotid artery was not significantly altered. On the other hand, the inner diameter was increased slightly in the thoracic aorta. These differences might be explained by the differences in biomechanical properties of these arteries and by the relation between the passive and active components of their vascular wall.
The NO system has been firmly established as an important physiological regulator of arterial pressure. Reports indicating that NO production in hypertension is reduced suggest that increased systolic pressure reflects the deficit in this vasodilator system. Excessive NO production in SHR is interpreted as indicating that NO is a compensatory vasodilator system responding to hypertension. Marked inhibition of acetylcholine-induced relaxation in SHR by acute addition of L-NAME to the incubating medium (Figures 2, ,4)4) strongly supports this statement.
In conclusion, our data support the hypothesis that endothelial dysfunction is independent of the etiology of hypertension and of the degree of accompanying vascular structural alterations.
The research reported in this article was supported by grant VEGA No 2/7240/20 and by Slovakofarma, Joint Stock Company, Hlohovec, Slovak Republic.