Chronic Inhibition of Nitric Oxide Synthesis Causes Coronary Microvascular Remodeling in Rats
Abstract The aim of the present study was to investigate the effects of long-term blockade of nitric oxide synthesis with the l-arginine analogue Nω−nitro-l-arginine methyl ester (L-NAME) for 8 weeks on coronary vascular and myocardial structural changes. Four groups of Wistar-Kyoto rats were studied: those with no treatment, those treated with L-NAME 1 g/L (3.7 mmol/L in drinking water), those treated with L-NAME 0.1 g/L (0.37 mmol/L in drinking water), and those treated with L-NAME 1.0 g/L and hydralazine 120 mg/L (0.6 mmol/L in drinking water). After 8 weeks, the heart was excised, and the degrees of structural changes in coronary arteries (wall-to-lumen ratio and perivascular fibrosis), myocardial fibrosis, and myocyte size were quantified by an image analyzer. Chronic inhibition of nitric oxide synthesis increased arterial pressure compared with control animals. Chronic inhibition of nitric oxide synthesis caused significant microvascular remodeling (increased wall-to-lumen ratio and perivascular fibrosis). Cardiac hypertrophy was also observed after chronic inhibition of nitric oxide synthesis. Coadministration of hydralazine prevented arterial hypertension but did not affect microvascular remodeling and cardiac hypertrophy induced by the chronic inhibition of nitric oxide synthesis. In addition, chronic inhibition of nitric oxide synthesis caused scattered lesions of myocardial fibrosis, which was significantly attenuated by cotreatment with hydralazine. These results suggest that long-term blockade of nitric oxide synthesis caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo by a mechanism other than arterial hypertension. In contrast, arterial hypertension contributed to the development of myocardial fibrosis induced by long-term blockade of nitric oxide synthesis.
- nitric oxide
- endothelium-derived relaxing factor
- coronary circulation
- cardiac hypertrophy
It is now clear that the vascular endothelium plays an important role in the regulation of vascular tone, platelet aggregation, thrombus formation, and proliferation/remodeling of the blood vessel wall by releasing endothelium-derived relaxing factors such as nitric oxide (NO).1 2 3 4 5 NO is synthesized from l-arginine through a metabolic pathway mediated by NO synthase, which is inhibited by l-arginine analogues.4 5
Recent studies in humans have shown that the presence of coronary risk factors such as arterial hypertension and hypercholesterolemia is associated with endothelial dysfunction of coronary arteries,6 7 8 9 which may alter coronary blood flow regulation and thus contribute to myocardial ischemia.10 11 12 Extensive evidence suggests that NO is involved importantly in atherosclerotic changes of large arteries.1 2 3 10 11 12 13 14 However, it is not known whether defective NO synthesis causes structural changes in coronary microvessels and myocardium in vivo. Coronary vascular and myocardial structural changes that might result from the chronic inhibition of NO synthesis are poorly understood. Recent studies in rats15 16 17 18 19 20 21 have shown that chronic administration of l-arginine analogues such as Nω-nitro-l-arginine methyl ester (L-NAME) caused systemic arterial hypertension, decreased intracellular cGMP levels (the second messenger of NO) in vascular smooth muscle, and induced structural changes of the renal microvessels. However, it is not known whether inhibition of NO synthesis per se contributed to the development of vascular changes in those rats, because they were markedly hypertensive.
The aim of this study was to examine the effects of chronic inhibition of NO synthesis with L-NAME on structural changes in the coronary arteries and myocardium in rats. We also examined the relative contribution of arterial hypertension and inhibition of NO synthesis in L-NAME–induced structural changes.
The protocol of the present study was approved by the Institutional Committee on Animal Care and Use of Laboratory Animals of Kyushu University. This study was reviewed by the Committee on the Ethics of Animal Experiments of the Faculty of Medicine, Kyushu University, and carried out under the control of the Guidelines for Animal Experimentation of the Faculty of Medicine, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government.
Eight-week-old male Wistar-Kyoto rats were obtained from an established colony at the Animal Research Institute of Kyushu University Faculty of Medicine. Four groups of rats were studied. Ten control rats (C group) received no treatment. Fifteen rats (L1 group) received L-NAME 1g/L (3.7 mmol/L in drinking water). At this concentration, the daily intake of L-NAME was ≈100 mg (0.37 mmol)/d. Six rats (L2 group) received L-NAME 0.1g/L (0.37 mmol/L in drinking water). At this concentration, the daily intake of L-NAME was ≈10 mg (0.037 mmol)/d. Ten rats (L+H group) received L-NAME 1 g/L (3.7 mmol/L in drinking water) and hydralazine 120 mg/L (0.6 mmol/L in drinking water). All rats of either group were housed in a viral antigen–free facility and were fed with normal rat chow for 8 weeks. Systolic blood pressure and heart rate were measured every week by the tail-cuff method. At 8 weeks of treatment, all rats were anesthetized with an intraperitoneal injection of thiopentobarbital, and a carotid artery was cannulated with tubing for determination of arterial pressure. Arterial pressure was measured by a pressure transducer (Nihon-Kohden Inc) connected to the tubing, and then the rats were killed by exsanguination.
Excised hearts were perfused with physiological saline solution containing adenosine 10 μg/kg and nitroglycerin 10 μg/kg for 10 minutes and then with 6% formaldehyde solution for 30 minutes via retrograde infusion into the ascending aorta at a pressure of 90 mm Hg. The left ventricle was separated from the right ventricle, the atria, and the great vessels. The left ventricle was weighed and cut into five pieces perpendicular to the long axis. The tissue was fixed in 6% formaldehyde for a few days and dehydrated with graded concentrations of alcohol for embedding in paraffin.
Paraffin slices from each heart were stained with hematoxylin-eosin and Masson’s trichrome stains. All histopathological sections of each animal were carefully scanned with a Nikon light microscope equipped with a Sony two-dimensional analysis system (Sony Inc), and histopathological findings of the myocardium and coronary arteries were examined.
To assess thickening of the coronary arterial wall and perivascular fibrosis, the transsectional images of the small arterioles with internal diameters ≤100 μm, small coronary arteries with diameters 100 to 300 μm, and large epicardial conduit arteries with diameters ≥300 μm were studied. The inner border of the lumen and the outer border of the tunica media were traced in each arterial image with hematoxylin-eosin staining at ×100 to ×200 magnification, and the areas encircled by the tracings were calculated. In quantification, nonround vessels resulting from oblique transsection or branching were excluded, and only round vessels were studied. The wall-to-lumen ratio (the area of the vessel wall divided by the area of the total blood vessel lumen) was determined. The area of fibrosis immediately surrounding blood vessels was calculated, and perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total area of the vessel. In each heart, ≈20 arterioles, 10 small coronary arteries, and 5 large epicardial coronary arteries were studied, and averaged values in each size vessel were used for analysis.
To assess the area of myocardial fibrosis, the area of pathological collagen deposition (stained with aniline blue) was measured in the microscopic field (×10 to ×40) of each Masson’s trichrome–stained section. The areas of the myocardium that normally contain collagen, such as the perivascular space, were excluded. The ratio (in percent) of the total area of fibrosis within the left ventricular myocardium to the total area of the left ventricular myocardium in each heart was calculated and was used for analysis.
Morphometry of left ventricular myocytes was performed to measure the myocyte cross-sectional area. In Masson’s trichrome–stained sections of the lateral mid free wall of the left ventricle, the myocyte cross-sectional area was measured in myocytes that were cut transversely and had a visible nucleus and an unbroken cellular membrane. The outer borders of the myocytes were traced, and the myocyte areas were calculated. Approximately 100 cells per heart were counted, and the averaged value was used for analysis.
Data are expressed as mean±SEM. Paired data were compared by Student’s t tests. Comparisons of myocardial fibrosis and hemodynamic parameters such as arterial pressure and heart rate were performed by one-way ANOVA followed by Bonferroni’s multiple-comparison t tests. Comparisons of the vascular wall-to-lumen ratio and perivascular fibrosis were performed by two-way ANOVA followed by the multiple-comparison tests. A probability of <.05 was considered statistically significant.
Five of the 15 rats of the L1 group died spontaneously between day 5 and week 8 of treatment. The myocardium of 3 rats that died before 4 weeks of treatment showed no significant structural changes, and the myocardium of 2 rats that died between 6 and 8 weeks of treatment had structural changes comparable to those in rats that survived for 8 weeks. No rats in the C group, L2 group, or L+H group died during the period of treatment.
Arterial Pressure, Heart Rate, and Body Weight
In the L1 and L2 groups, systolic arterial pressure increased progressively and plateaued after 4 weeks of L-NAME treatment (Fig 1⇓, Table⇓). No significant increase in systolic arterial pressure was observed in either the C or the L+H group. At 8 weeks, systolic arterial pressure was higher in the L1 and L2 groups than in the C and L+H groups and was comparable between the C and L+H groups. Heart rate was comparable among the four groups, and no interval change was noted. Animals in the C group gained body weight, whereas those in the L1 group lost body weight during the course of this study. Body weight did not change in animals in the L2 and L+H groups.
Wall-to-Lumen Ratio and Perivascular Fibrosis
There was no significant difference among arterioles, small coronary arteries, and large arteries with regard to the wall-to-lumen ratio and perivascular fibrosis in the C group. The degrees of wall-to-lumen ratios and degrees of group (Figs 2 through 4⇓⇓⇓). The wall-to-lumen ratios in the arterioles and small coronary arteries were significantly greater in the L1, L2, and L+H groups than in the C group, whereas the ratios in large epicardial coronary arteries did not differ significantly among the four groups. The degrees of perivascular fibrosis in the arterioles were significantly greater in the L1, L2, and L+H groups than in the C group, whereas the perivascular fibrosis in small coronary arteries and large epicardial coronary arteries did not differ significantly among the groups. The wall-to-lumen ratios and degrees of perivascular fibrosis did not differ between the L1, L2, and L+H groups.
In addition to the microvascular remodeling, there is an impressive accumulation of collagen (stained with blue in Fig 2⇑) within the media of microvessels in the L1, L2, and L+H groups.
Compared with the C group, the L1, L2, and L+H groups (Figs 5⇓ and 6⇓) showed scattered areas of myocardial fibrosis associated with myocyte necrosis. The ratio of total area of myocardial fibrosis to total left ventricular area was significantly greater in the L1 group than in the L2 group. Myocardial fibrosis in the L+H group was significantly less than in the L1 and L2 groups. In the L1, L2, and L+H groups, microvascular luminal occlusion was sometimes seen in the area of myocardial fibrosis.
The absolute left ventricular weight did not differ significantly among the four groups. However, a significant increase in the relative left ventricular weight was observed in the L1, L2, and L+H groups compared with the C group (Table⇑). There was no significant difference among the L1, L2, and L+H groups in relative left ventricular weight.
The myocyte cross-sectional areas were greater (P<.01) in the L1, L2, and L+H groups than in the C group. The myocyte cross-sectional areas did not differ significantly among the L1, L2, and L+H groups.
This study confirmed that chronic administration of L-NAME caused systemic arterial hypertension in rats in vivo.15 16 17 18 19 20 21 It is reasonable to assume that the arterial hypertension observed in these rats was due to the decreased NO synthesis, because previous studies in rats showed that (1) long-term administration of L-NAME caused a marked decrease in the arterial wall content of cGMP (the second messenger of NO)15 16 and a decrease in urinary secretion of the stable products of NO (NO2− and NO3−),21 and (2) the L-NAME–induced hypertension was reversed or prevented by short-term administration of excess l-arginine but not by d-arginine.20
Microvascular Remodeling Induced by Chronic Inhibition of NO Synthesis
The major novel finding of the present study is that chronic inhibition of NO synthesis with L-NAME for 8 weeks was associated with structural changes of coronary microvessels (thickening of the media and increased perivascular fibrosis) but not with those in large coronary arteries. In animals with L-NAME treatment, an impressive accumulation of collagen also was observed in the media of microvessels. The presence of connective tissue in media and adventitia may contribute to altered vasomotor reactivity and coronary vasodilator reserve and lead to myocardial ischemia. It is unlikely that the observed microvascular changes were an artifact caused by vasoconstriction, because histopathological specimens were fixed under pressure perfusion after maximum vasodilation. The pathogenesis of microvascular remodeling in our model involves at least two possibilities: (1) adaptive responses to arterial hypertension22 23 and (2) increased production of mitogen- or growth-promoting factors due to decreased NO synthesis.1 2 3 10 11 12 13 14 Our results support the latter possibility, because the microvascular remodeling did not appear until week 4 despite the presence of arterial hypertension at week 2 and because the effective antihypertensive treatment with hydralazine did not affect the L-NAME–induced microvascular remodeling. It is unlikely that our results suffered from the lack of an animal group that was treated only with hydralazine, because treatment with hydralazine does not affect coronary vascular and myocardial structure in normotensive normal rats (see References 2222 and 2424 ). These results suggest that arterial hypertension may not be prerequisite to the occurrence of L-NAME–induced microvascular remodeling. The development of vascular remodeling in microvessels and not in large vessels may be related to altered receptor expression of the coronary microcirculation. Further studies are needed to elucidate why chronic administration of L-NAME caused structural changes in coronary microvessels but not in large coronary arteries.
The mechanisms of L-NAME–induced microvascular remodeling were not explored in the present study. We consider the following three possibilities that may be related to the mechanisms. First, extensive evidence suggests that NO may inhibit vascular smooth muscle proliferation in vitro and in vivo.1 2 3 10 11 12 13 14 Inhibition of NO synthesis upregulates the synthesis of peptide growth factors such as platelet-derived growth factor.21 It is likely, therefore, that the effects of L-NAME on microvascular remodeling were due to its inhibition of the antiproliferating action of NO. Second, it was reported in this rat model of hypertension that plasma renin activity decreased slightly at 4 weeks of L-NAME administration15 19 but increased significantly at 6 or 8 weeks of L-NAME administration.17 18 It is possible that this secondary rise in plasma renin activity, which probably results from upregulation of renin synthesis due to nephroangiosclerosis,16 17 might result in an increase in the synthesis of angiotensin II. It is also possible that defective NO synthesis might directly upregulate the local elaboration of trophic factors such as angiotensin II and endothelin.25 Angiotensin II is also related to expression of transforming growth factor-β1 and to elaboration of endothelins.2 3 Third, chronic administration of L-NAME might increase sympathetic nerve activity, which may contribute to vascular remodeling. Sakuma et al26 showed that renal sympathetic nerve activity increased after administration of L-NAME.
One third of the rats in the L1 group died before 8 weeks of L-NAME treatment, and rats of the L1 group that survived for 8 weeks lost weight during the course of this study, suggesting the possibility of malabsorption and/or toxicity induced by oral administration of L-NAME. However, all rats that received a lower dose of L-NAME (the L2 group) survived 8 weeks and did not lose weight during the course of this study. It is unlikely that malabsorption and/or toxicity accounted for coronary vascular and myocardial remodeling in our animal model, because chronic subcutaneous infusion of L-NAME by osmotic infusion pump caused similar structural changes in coronary arteries and myocardium in rats without weight loss.27
Myocardial Fibrosis and Cardiac Hypertrophy Induced by Chronic Inhibition of NO Synthesis
Another novel finding of the present study is that a significant myocardial fibrosis was noted in animals with chronic administration of L-NAME (the L1 and L2 groups). Weber et al28 suggested that the differentiation between reparative fibrosis that follows myocyte necrosis and reactive fibrosis that represents perivascular and interstitial processes may be important in pathophysiological states. In contrast to the lack of its effect on microvascular remodeling, the antihypertensive treatment with hydralazine markedly attenuated L-NAME–induced myocardial fibrosis, which suggests that arterial hypertension contributed to the development of L-NAME–induced myocardial fibrosis in our rats. The presence of myocyte necrosis associated with microvascular luminal occlusion suggests that L-NAME–induced myocardial fibrosis might have resulted from reparative/replacement fibrosis. It is possible that increased myocardial metabolic demand due to arterial hypertension in the presence of microvascular luminal narrowing, hypertension, and cardiac hypertrophy facilitated myocardial ischemia and caused myocardial necrosis. Also, a possible local elaboration of angiotensin II, endothelins, or catecholamines related to arterial hypertension could have led to myocardial necrosis and subsequent fibrosis.
For interpretation of the increased relative weight of the left ventricle in rats that received L-NAME, the body weight loss and the loss of myocardial myocytes (replaced by fibrosis) need to be considered. The increase in the myocyte cross-sectional area in the L1 and L2 groups suggests that myocyte hypertrophy actually occurred. This study demonstrated that despite prevention of arterial hypertension, hydralazine did not affect the increase in the relative left ventricular weight or myocyte hypertrophy induced by chronic L-NAME administration. This suggests that arterial hypertension was not the sole factor responsible for the development of cardiac hypertrophy in our model.
In summary, our results demonstrated that chronic inhibition of NO synthesis with L-NAME caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo by mechanisms other than arterial hypertension. In contrast, arterial hypertension contributed to the development of the L-NAME–induced myocardial fibrosis.
This study was supported by Grants-in-Aid for Scientific Research 06670725 and 06404034 from the Ministry of Education, Science, and Culture, Tokyo, and a research grant from the Uehara Memorial Foundation, Tokyo, Japan.
- Received February 24, 1995.
- Revision received April 3, 1995.
- Accepted August 22, 1995.
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