Effect of Short-term Regression of Atherosclerosis on Reactivity of Carotid and Retinal Arteries
Background and Purpose This study tested the hypothesis that functional abnormalities of carotid and ocular arteries may improve after short-term regression of atherosclerosis, before regression of structural abnormalities.
Methods We examined effects of short-term dietary treatment of atherosclerosis on carotid and ocular vascular responses to serotonin and to platelet activation by collagen in vivo. Three groups of monkeys were studied: normal cynomolgus monkeys, monkeys fed an atherogenic diet for 34 months, and atherosclerotic monkeys that were fed a regression diet for 8.6±1.1 months (mean±SE). We measured changes in carotid blood flow (using a Doppler probe), retinal blood flow (using microspheres), and diameter of the internal carotid artery (using quantitative angiography). Endothelium-dependent relaxation to acetylcholine was studied in rings of internal carotid artery in vitro.
Results Carotid blood flow increased in response to both serotonin and collagen in normal monkeys, decreased in response to both agents in atherosclerotic monkeys, and was restored toward normal after regression. Serotonin had little effect on retinal blood flow in normal monkeys and produced a marked decrease in retinal blood flow in atherosclerotic monkeys; the vasoconstrictor response to serotonin was reduced after regression. Activation of platelets by collagen increased blood flow in normal monkeys, decreased blood flow in atherosclerotic monkeys, and had little effect after regression. Alterations in responses of the internal carotid artery were consistent with changes in carotid and ocular blood flow. Endothelium-dependent relaxation in vitro was impaired by atherosclerosis and was restored toward normal by regression. There was no reduction in intimal area of the atherosclerotic lesion in common carotid and ophthalmic arteries from regression monkeys, despite a marked reduction in cholesteryl ester.
Conclusions Within a few months of regression of atherosclerosis, endothelial function and hyperresponsiveness of carotid and ocular arteries to serotonin and platelet activation return toward normal. Functional improvement is associated with resorption of lipid from atherosclerotic lesions, but with little reduction in size of intimal lesions.
Atherosclerotic arteries undergo structural changes and pronounced functional abnormalities, including impaired endothelium-dependent relaxation and augmented vasoconstriction to platelet products.1 2 3 4 5 6 7 In atherosclerotic monkeys, structural and functional abnormalities can be restored toward normal by dietary treatment of hypercholesterolemia for 18 months or more.8 9 10 11
Because endothelium plays a critical role in modulation of vascular responses to activated platelets,12 changes in endothelial function during atherosclerosis and regression may have important implications. Reduction of plasma cholesterol appears to be beneficial in the treatment of coronary vascular dysfunction,13 14 15 16 17 but the contribution of hypercholesterolemia and atherosclerosis to clinical cerebrovascular events is less clear. We have suggested that in atherosclerosis, abnormal cerebral vascular responses to activated platelets may contribute to the pathophysiology of transient ischemic attacks18 and amaurosis fugax.19 Moreover, a recent study indicates that susceptibility to cerebrovascular events is decreased by treatment of hypercholesterolemia in patients with coronary heart disease.14 We found recently that in the monkey hindlimb, vascular function improves before structural improvement during regression of atherosclerosis, usually within a few months.20
In this study we tested the hypothesis that functional abnormalities of carotid and ocular arteries may improve after short-term regression of atherosclerosis, before significant reduction of structural abnormalities. Specifically, we determined whether exaggerated vasoconstrictor responses to serotonin and activation of platelets, and impairment of endothelium-dependent relaxation, in atherosclerotic monkeys return toward normal within approximately 9 months of normal (low-fat) diet.
Materials and Methods
Adult male cynomolgus monkeys (n=18; weight, 5 to 8 kg) were randomly assigned to receive either a normal or an atherogenic diet. Five monkeys (normal group) were fed commercial laboratory chow (Purina Monkey Chow). Thirteen monkeys (atherosclerotic group) were fed an atherogenic diet for 33.8±2.7 months. In seven of these atherosclerotic monkeys carotid vascular responses were examined in vivo, and the monkeys were then fed a normal diet to induce regression of atherosclerosis (regression group). The atherogenic diet consisted of 1 mg cholesterol per calorie (0.8% by weight) and 43% of total calories as fat. The protocol was approved by the University of Iowa Animal Care and Use Review Committee.
Animals were sedated with ketamine hydrochloride (10 mg/kg IM initially and supplemental doses as needed) and anesthetized with pentobarbital (20 mg/kg IV). Studies were performed under sterile conditions in an animal catheterization laboratory. A polyethylene catheter with multiple side holes and a 60° directional Doppler ultrasound transducer was inserted via an arteriotomy into the right axillary artery. The catheter was passed retrogradely under fluoroscopic visualization to the origin of the right subclavian artery, ie, to the bifurcation of the right brachiocephalic artery into the right common carotid and subclavian arteries. Mean and phasic arterial pressure and Doppler frequency were recorded continuously. Cineangiograms of the right internal carotid artery were obtained in a projection that was 45° to the anteroposterior plane. Power injections of nonionic contrast (iohexol, Sanofi-Winthrop Pharmaceuticals) were made at a rate of 15 mL/s through the catheter in the brachiocephalic artery.
To avoid injection of excessive angiographic contrast medium, only four angiograms were obtained in each study: at baseline, after infusion of 30 μg/min serotonin (IA) for 10 minutes, at baseline, and after 150 μg/min collagen (IA) for 10 minutes. Arterial pressure and velocity of blood flow were also recorded. Changes in carotid blood flow are expressed as percent change in Doppler flow velocity. Blood gases were maintained within normal levels.
Arteriotomy sites were sutured, and the animals received an injection of penicillin. A lethal dose of KCl was given intravenously to normal and atherosclerotic animals while the monkeys were anesthetized approximately 2 weeks after angiography. Regression animals underwent angiograms after 34±3.6 months of atherogenic diet. They were then placed on a normal diet, and angiography was repeated in 6 of the 7 monkeys after 7.2±0.2 months. After 7 months of regression, 1 monkey was not studied because of a systemic reaction to injection of contrast medium. Two regression monkeys were found to have no reduction in hyperresponsiveness to serotonin after 7 months on normal diet. Therefore, 3 of 7 regression monkeys were maintained on normal diet, and angiograms were repeated after 12±0.1 months of normal diet.
Angiographic data were analyzed quantitatively with computerized arterial lumen edge detection software. This process is described in detail elsewhere.21 Briefly, a cine frame was selected for image focus and for uniform, complete opacification of the right internal carotid artery. The image was digitized and magnified, and a segment of the artery was selected for analysis. Criteria for selection of segments were that the section of artery was straight and without major branches, overlying structures, or crossing vessels. An approximate centerline was defined by the operator. Edge detection and smoothing were performed by the software package, with correction by the operator only for obvious deviation that was usually a result of vessel branching. Mean arterial diameter was measured over the length of the right internal carotid artery, and a focal diameter was taken as a minimum caliber of six to eight computer-defined subsegments of the same segment of artery. Diameter of the brachiocephalic artery (artery of origin) was measured with a similar approach. Arterial diameter at the point of measurement of blood flow velocity was stable during infusion of drugs. Total blood flow in the right common carotid artery was calculated from Doppler frequency shift and brachiocephalic artery mean diameter.
Measurement of Retinal Blood Flow
In terminal studies, catheters were inserted into the left atrial appendage for injection of microspheres and infusion of serotonin (40 μg/kg per minute). Catheters were also placed in both brachial arteries for withdrawal of reference blood samples during infusion of microspheres.
Collagen (150 μg/min), which was used to activate platelets, was infused directly into the right common carotid artery to minimize systemic platelet aggregation. Intracarotid infusion of collagen for 10 minutes decreased the blood platelet count in the ipsilateral internal jugular vein by approximately 30% in all groups of monkeys (normal, −35±3%, n=5; atherosclerotic, −28±3%, n=13; regression, −28±4%, n=7; P>.05 between groups).
Blood flow to the retina of the right eye, which was ipsilateral to infusion of collagen, was measured after injection into the left atrium of radioactive microspheres 15 μm in diameter as described in detail for monkeys.19 22
Morphological Studies and Analysis of Arterial Lipids
After the monkeys were killed, right common carotid and right ophthalmic arteries were removed and fixed in formalin. Specimens were embedded in paraffin and stained with hematoxylin and eosin and Verhoeff-van Gieson’s stain. Intimal and medial areas were measured as described previously.23
Loose adventitia was removed from other segments of right common carotid artery, and the vessel was blotted dry, weighed, and homogenized. Arterial contents of free cholesterol and cholesteryl ester were then measured as described in detail previously.20
Studies of Arterial Rings In Vitro
Segments (3 to 5 mm long) of internal carotid artery were cleaned of loose connective tissue. Vascular rings were mounted in organ baths at 2 g resting tension as described previously.24 After a 60-minute equilibration period, the vascular rings were contracted twice with 80 mmol/L KCl and rinsed three times after each contraction.
When vascular tone had returned to basal level, the rings were again contracted to a steady level with prostaglandin F2α (1 to 10 μmol/L; approximately 70% of maximum prostaglandin F2α contraction), and acetylcholine or sodium nitroprusside was added in cumulatively increasing concentrations. The order of application of acetylcholine and sodium nitroprusside was randomized.
Results are expressed as mean±SEM. Mean values between groups were compared by an ANOVA with Bonferroni’s correction. A logarithmic transformation was used in some analyses when variance exceeded that of the normal group. Statistical significance was assumed at P<.05.
Total plasma cholesterol was 115±10 mg/dL in the normal group and 560±64 mg/dL in the atherosclerotic group. Plasma cholesterol in regression monkeys was 564±34 mg/dL while on the atherogenic diet and 108±12 mg/dL after 7 to 12 months of normal diet.
Carotid Blood Flow and Angiograms
Serotonin produced an increase in carotid blood flow (Fig 1⇓) and a decrease in mean diameter of the internal carotid artery (Fig 2⇓) in normal monkeys. In contrast, in atherosclerotic monkeys serotonin produced a decrease in carotid blood flow (P<.05 versus normal, Fig 1⇓) and a larger decrease in mean diameter of the internal carotid artery (P<.05 versus normal, Fig 2⇓).
Serotonin had little effect on flow in the regression group (Fig 1⇑; P<.05 versus atherosclerotic group), and the reduction in diameter of the internal carotid artery in response to serotonin was normal (Fig 2⇑). Values for the regression group were taken from the final angiographic study for each animal, obtained 8.6±1.1 months (range, 7 to 14 months) after starting a regression (normal) diet.
Collagen produced an increase in carotid blood flow in normal monkeys but a decrease in blood flow in atherosclerotic monkeys (Fig 1⇑). In regression monkeys, the response to collagen returned toward normal (Fig 1⇑; P<.05 versus atherosclerotic). Collagen produced little change in internal carotid artery diameter in the three groups (normal, +8±4%; atherosclerotic, −2±4%; regression, −5±4%; P>.05 between groups). Baseline diameter of the internal carotid artery was similar in all groups (normal, 1.80±0.10 mm; atherosclerotic, 1.82±0.10 mm; regression, 1.80±0.10 mm; P>.05 between groups). Arterial pressure was not significantly different in the three groups (normal, 97±4 mm Hg; atherosclerotic, 88±5 mm Hg; regression, 88±4 mm Hg; P>.05 between all groups) and did not change significantly during administration of serotonin and collagen.
Retinal Blood Flow
Serotonin had no significant effect on blood flow to the retina in normal monkeys but produced a profound reduction in blood flow in atherosclerotic monkeys (Fig 3⇓; P<.05 atherosclerotic versus normal). After regression of atherosclerosis, the response to serotonin was less than that in the atherosclerotic monkeys (P<.05) and was not significantly different from the normal group (Fig 3⇓).
Collagen produced an increase in blood flow to the retina in normal monkeys (Fig 3⇑). In contrast, collagen decreased retinal blood flow in atherosclerotic monkeys (Fig 3⇑; P<.05 versus normal). Regression of atherosclerosis restored retinal blood flow response to collagen toward normal (Fig 3⇑; P>.05 versus normal).
Relaxation of Internal Carotid Artery In Vitro
Acetylcholine produced relaxation of precontracted internal carotid arteries from normal monkeys (Fig 4⇓). Acetylcholine had little or no relaxant effect in carotid arteries from atherosclerotic monkeys (Fig 4⇓; maximum response, P<.05 versus normal). After regression of atherosclerosis for 8.6±1.1 months, responses of the internal carotid artery were similar to those of the normal group (Fig 4⇓; P>.05).
Sodium nitroprusside, an endothelium-independent vasodilator, produced similar relaxation of internal carotid arteries from normal, atherosclerotic, and regression monkeys (Fig 4⇑).
Arterial Wall Lipids
Free cholesterol content of the common carotid artery was higher in atherosclerotic than in normal monkeys (P<.05 versus normal) and was normal in the regression group (Fig 5⇓). Similarly, there was marked accumulation of cholesteryl ester in the carotid arterial wall of atherosclerotic monkeys (Fig 5⇓; P<.05 versus normal). Cholesteryl ester content in regression animals was normal (Fig 5⇓; P>.05 versus normal).
There was a substantial increase in intimal area of the common carotid (Figs 6⇓ and 7⇓) and ophthalmic (Figs 6⇓ and 7⇓) arteries in atherosclerotic monkeys, with no significant reduction in regression monkeys (common carotid, P<.05 for normal versus atherosclerotic and regression; ophthalmic artery, P=.07 for normal versus atherosclerotic and P<.05 for normal versus regression). There was no effect of atherosclerosis or regression on medial area in either artery (quantitated morphometric data not shown).
The major findings of this study are as follows. First, dietary treatment for 7 to 14 months abrogates the marked augmentation of constrictor responses that occurs in carotid and ocular arteries of atherosclerotic primates in response to serotonin or platelet activation. Second, infusion of collagen and serotonin in vivo produces more pronounced changes in blood flow than in diameter of large conductance arteries in which lesions are more prevalent. Third, endothelial function in carotid arteries is markedly improved after 7 to 14 months of regression of atherosclerosis. Improvement in endothelial function may contribute to normalization of responses to collagen and serotonin after dietary treatment of atherosclerosis.
Constrictor responses to serotonin are exaggerated in atherosclerotic arteries.1 11 25 In addition, vasodilatation in response to vasoactive products that are released by activated platelets is attenuated or reversed to vasoconstriction by atherosclerosis.26 Serotonin is released by platelets during activation,27 and augmented vasoconstrictor responses to activated platelets may be mediated in part by serotonin.
Dietary treatment of experimental atherosclerosis for at least 18 months results in both a significant reduction in intimal area8 11 and improvement of hyperresponsiveness to serotonin and impaired responses to ADP in vivo.2 11 18 28 Improvement in structure, with consequent improvement in maximal vasodilator responses, varies in the leg and cerebral vessels after 18 months of regression of atherosclerosis.23 Recently we reported that after 4 to 12 months of regression of atherosclerosis, functional improvement occurs in vessels in the leg before detectable reduction in mass of the atherosclerotic lesion.20 We now demonstrate that the function of both carotid and ocular arteries is also improved by short-term dietary treatment of atherosclerosis.
Regression of Atherosclerosis and Reduction of Cerebrovascular Events
Many transient ischemic attacks are thought to be produced by platelet adhesion, aggregation, and embolization from plaques in large extracranial arteries.29 Release of serotonin during aggregation of platelets, coupled with augmented constrictor responses to serotonin in atherosclerotic arteries, may produce pronounced vasoconstriction and perhaps contribute to cerebral ischemia.22
Treatment of hypercholesterolemia restores endothelial function in coronary arteries of patients after 6 to 12 months.15 16 17 Hypercholesterolemia is a major risk factor for coronary vascular events, but plasma cholesterol is thought to have only a small effect on susceptibility to cerebrovascular events. The present and previous24 findings indicate that regression of atherosclerosis improves endothelium-dependent relaxation of monkey common carotid arteries and responses of cerebral and retinal vessels to activation of platelets. Of interest is the finding that in patients with coronary heart disease who received a cholesterol-lowering agent for approximately 5 years, reduction of cholesterol reduced cerebral vascular events by one third.14 The present findings are consistent with a possible therapeutic effect of reduction of cholesterol on cerebrovascular dysfunction. Improvement of endothelial function during regression of atherosclerosis may contribute to reduction of cerebral vascular events.
The marked decrease in blood flow to the retina in response to serotonin in atherosclerotic monkeys is associated with reversible impairment of retinal responses to light.19 Thus, reductions in blood flow to the eye in response to products of platelet aggregation appear to be important functionally. We19 and others30 31 have suggested that vasoconstriction may contribute to amaurosis fugax. In this study we have confirmed previous observations19 26 32 that the decrease in blood flow to the retina in response to collagen-induced platelet aggregation resembles the response to serotonin in atherosclerotic monkeys. Moreover, we now demonstrate that hyperresponsiveness to serotonin and platelet aggregation may be abrogated by short-term regression of atherosclerosis. The data are consistent with a previous finding that amaurosis fugax and transient ischemic attacks did not recur in patients with carotid atherosclerosis who were treated for hypercholesterolemia.33
Despite the absence of morphological evidence of disease in small vessels, vascular abnormalities of atherosclerosis extend to the microcirculation.13 34 35 36 37 In this study we found that the effects of atherosclerosis and regression on changes in carotid and ocular blood flow are more dramatic than responses of large cerebral arteries in vivo. Although large cerebral arteries contribute significantly to total cerebral vascular resistance38 and endothelial function in these vessels is impaired by atherosclerosis and normalized by regression, alterations in microvascular function may be more critical than alterations of large arteries for determining cerebral perfusion during atherosclerosis and regression.
Increases in carotid and retinal blood flow induced by collagen are probably due largely to endothelium-dependent vasodilatation in response to ADP,12 39 a major product of platelet activation. Serotonin also releases endothelium-derived relaxing factor in some vessels, which either produces a dilator response to serotonin or attenuates the constrictor response, depending on the vascular bed. Previous studies in vitro demonstrated that abnormal endothelium-dependent relaxation in atherosclerosis is improved after 18 months of regression.10 Hyperresponsiveness to serotonin in the leg improves during much shorter periods of regression, associated with reabsorption of lipids from the vessel wall, without reduction in overall mass of the lesion.20 Using rings of internal carotid artery, the present experiments demonstrate that endothelium-dependent vascular relaxation is also restored by short-term dietary treatment of atherosclerosis. Thus, impaired release or activity of endothelium-derived relaxing factor during atherosclerosis, and normalization during regression, could contribute to the functional alterations in vascular responses to serotonin or activated platelets.
It is likely that carotid and retinal vascular function improved after regression of atherosclerosis as the result of improvement within the vascular wall and not simply as a response to reduction of plasma cholesterol. In an earlier study, we found that elevation of plasma cholesterol before development of atherosclerotic lesions does not alter vascular function and that reduction of plasma cholesterol in atherosclerotic monkeys before resorption of vascular cholesterol does not normalize vascular function.20
Thus, functional abnormalities in carotid and ocular arteries during atherosclerosis can be markedly improved within a few months of dietary treatment. We speculate that treatment of hypercholesterolemia and atherosclerosis may be beneficial for carotid and ocular vascular function, even before reduction in size of intimal lesions.
These studies were supported by National Institutes of Health grants HL-14388, NS-24621, HL-16066, AG-10269, and HL-38901; by Research Funds from the Veterans Administration; and by a Grant-in-Aid from the American Heart Association (95014510). Dr Faraci is an Established Investigator of the American Heart Association. Dr Sobey is the recipient of a C.J. Martin Fellowship from the National Health and Medical Research Council of Australia and a Michael J. Brody Fellowship in Basic Cardiovascular Research from the University of Iowa. We thank Robert M. Brooks II and Pam Tompkins for technical assistance, Dr Sohan Hayreh for expert advice, and Arlinda LaRose for secretarial assistance.
- Received October 24, 1995.
- Revision received January 26, 1996.
- Accepted February 13, 1996.
- Copyright © 1996 by American Heart Association
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