Regression or Progression
Dependency on Vascular Nitric Oxide
Abstract We have shown that chronic administration of the nitric oxide (NO) precursor l-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently, l-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area to 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration of l-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence that l-arginine therapy may be of potential clinical benefit.
- Received May 15, 1995.
- Accepted October 24, 1995.
Hypercholesterolemia induces a progressive loss of endothelium-dependent relaxation. This alteration in vascular function is seen before any observable morphological changes and is due to reduced bioactivity of endothelium-derived NO.1 2 3 In addition to being a potent vasodilator, NO is also known to inhibit platelet reactivity, vascular smooth muscle proliferation, and monocyte adherence, all of which are key contributors to the development of atherosclerosis.4 5 6 7 8 Moreover, NO reduces intracellular oxidative stress and thereby suppresses oxidant-responsive genes encoding adhesion molecules and chemokines involved in monocyte adhesion and accumulation.9 10 11 12 Because of the role that NO plays in modulating these events, we speculate that loss of NO activity promotes atherogenesis, whereas restoration of NO activity may inhibit atherogenesis. In support of our hypothesis, we have demonstrated that chronic administration of the NO precursor l-arginine to hypercholesterolemic rabbits restores vascular NO activity as assessed by bioassay and chemiluminescence.13 14 15 This restoration of vascular NO activity is associated with reduced endothelial adhesiveness for monocytes and diminished lesion formation in the aorta and coronary arteries. However, in these studies l-arginine was administered before the development of endothelial dysfunction and before the formation of lesions. To be of potential clinical benefit in patients with arterial disease, it must be determined whether supplemental l-arginine can slow the progression of lesion formation in vessels that manifest preexisting endothelial dysfunction and intimal lesions.
Accordingly, the aim of the present study was to determine whether oral administration of l-arginine to hypercholesterolemic rabbits would retard progression of preexisting intimal lesions.
Male New Zealand White rabbits (n=85) weighing 2.5 to 3.0 kg were entered into the study after a 1-week period of acclimation in the housing facilities of the Stanford Department of Comparative Medicine, during which time the animals were fed normal rabbit chow and received water ad libitum. All animals were inspected before the study by the Department of Comparative Medicine veterinarian and monitored daily by Department of Comparative Medicine technicians and the investigators. All experimental protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
Animals were then randomized into three dietary treatment groups. Rabbits in two of the groups were exposed to a high-cholesterol diet (0.5% cholesterol chow, Dyets) for 10 weeks. Subsequently, animals received vehicle (CHOL group) or l-arginine (2.25% wt/vol; ARG group) in the drinking water, and cholesterol chow was continued (for a total study duration of 23 weeks). The dose of l-arginine used in this study represents a sixfold increase in the daily arginine intake and in previous studies was associated with a doubling of the plasma arginine concentration.13 14 15
Animals from each of the three experimental groups were randomly selected to be killed at 10, 14, 18, or 23 weeks of dietary treatment (at which time animals in the ARG group had received 0, 4, 8, and 13 weeks of arginine supplementation, respectively). After an overdose of intravenous pentobarbital, blood was obtained for biochemical analysis and the thoracic aortae harvested for physiological and histological studies. Two segments (3 to 4 mm each) from the portion just distal to the subclavian artery were used for vascular reactivity studies, and the rest of the descending thoracic aorta was used for determination of plaque surface area.
Blood samples were obtained at the time of death from all animals for determination of total cholesterol, HDL, and plasma-free arginine. Serum cholesterol and HDL were analyzed with the use of a modification of the enzymatic method of Allain et al as developed by Sigma Diagnostics.13 16 Serum arginine was determined with the use of an automated amino acid analyzer, as described previously.13
The rings (3 to 4 mm each) of thoracic aorta were dissected free of connective tissue and immediately placed into oxygenated PSS that was composed of the following (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, and glucose 11.1. Pairs of rings from each animal were mounted horizontally on stainless steel wires placed through the lumen and connected to force transducers. The vascular rings were suspended in the organ chambers filled with oxygenated PSS at 37°C. Over a period of 60 minutes, rings were progressively stretched to the optimal point of their length-tension relation (determined previously to be 4 g).13 Subsequently, the EC50 of norepinephrine was determined by exposing the tissues to increasing concentrations of norepinephrine (in half-log increments from 10−9 to 10−4 mol/L). Once a maximal response was obtained, the rings were washed repeatedly with fresh PSS for 60 minutes until the tension returned to the previous baseline value. Response to vasodilating agents (nitroglycerin, acetylcholine) was studied after the rings were precontracted with the EC50 concentration of norepinephrine. After a stable contraction was obtained, the rings were exposed to increasing doses of the vasodilator.
On completion of the vascular reactivity studies, the aortic rings were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with an elastic van Gieson stain for light microscopy and histomorphometric measurements. Measurements of intimal and medial cross-sectional areas (expressed in square millimeters) were made with the aid of a computerized image analyzer (Image Analyst, Automatix). At least six sections from each aortic ring were examined, and the values from each were averaged to derive a value for each ring.
Lesion Surface Area
The segment of descending thoracic aorta not used for vascular reactivity studies was fixed in formalin immediately after dissection. Subsequently, the aorta was incised longitudinally, opened, and placed flat under a glass slide for photography. The photographs were projected and the lesion surface and total surface areas quantified by planimetry by an observer blinded to the treatment groups.
Measurement of Nitrogen Oxides and Superoxide Anion
A separate group of rabbits (n=6) was used for determination of vascular NO and superoxide anion elaboration. Two groups of three rabbits each were fed 0.5% cholesterol for 10 weeks. At week 10, one group (ARG) received 2.25% l-arginine in addition to the cholesterol diet, whereas the other group continued on the cholesterol diet only (CHOL). The animals were killed at 12 weeks (at which time the ARG animals had received l-arginine for a total of 2 weeks).
Before they were killed, the animals were lightly sedated with pentobarbital (200 to 250 mg) and the central ear artery cannulated for measurement of intra-arterial blood pressure. Subsequently, an overdose of intravenous pentobarbital was administered and the thoracic aortae harvested.
For measurement of nitrogen oxides, rings (15 mm) from the arch of the thoracic aorta were removed and placed in ice-cold, oxygenated PSS. After the adventitia was removed, the segment was then carefully opened longitudinally and incubated in 2 mL of Hanks’ balanced salt solution (Irvine Scientific), with the endothelial surface exposed to the medium. The medium contained calcium ionophore (1 μmol/L) and l-arginine (100 μmol/L) at 37°C. At selected time points (0, 30, 60, 120 minutes), the medium was collected for measurement of nitrogen oxide and replaced with 2 mL of fresh media. After incubation the aortic segment was blotted dry and weighed. Nitrogen oxide in the incubation medium was measured with a commercially available chemiluminescence apparatus (model 2108, Dasibi Corp), as previously described.15 The samples (100 μL) were injected into boiling acidic vanadium (III). The reaction uses acidic vanadium (III) at 98°C to reduce both NO2− and NO3− to NO, which is then quantified by the chemiluminescence detector after the reaction with ozone. Signals from the detector were analyzed by a computerized integrator and recorded as areas under the curve. Standard curves for NO2/NO3 were linear over the range of 50 pmol to 10 nmol.
For determination of vascular superoxide anion, rings of the suprarenal abdominal aorta 5 mm in length were dissected free of adventitia and transferred to glass vials containing 0.25 mmol lucigenin per 1 mL of phosphate-buffered saline. After a delay of 30 seconds, superoxide release was measured by chemiluminescence at 1-minute intervals at room temperature with the use of a commercially available luminometer (Turner TD-20e Luminometer). Vials containing all components with the exception of aortic rings were counted and these blank values subtracted from the chemiluminescence signals obtained from aortic rings.17
All solutions were prepared in distilled water made fresh the day of the experiment and stored on ice. Norepinephrine bitartrate, acetylcholine chloride, and l-arginine were purchased from Sigma Chemical Co, nitroglycerin from DuPont Chemicals, and calcium ionophore (A23187) from Calbiochem.
Data are expressed as mean±SEM. Concentration-effect curves to norepinephrine are expressed as contractions in grams above the resting tension. The concentration-effect curves were characterized by determining the maximal response and the EC50. The concentration-effect curves to acetylcholine or nitroglycerin are expressed as the percentage of tension attained by precontraction with norepinephrine. Comparisons between the three experimental groups were made by ANOVA. A value of P<.05 was considered significant.
Biochemical and Physiological Measurements
At the time the animals were killed, there were no significant differences in body weight between the three experimental groups at any time point (Table 1⇓). There was no difference in mean arterial pressure between the ARG (74±1.6 mm Hg) and CHOL (76±1.5 mm Hg) groups 2 weeks after initiation of l-arginine supplementation.
At all time points, the plasma cholesterol level was markedly elevated in each of the two experimental groups receiving the atherogenic diet compared with the animals receiving normal chow (Table 1⇑). The addition of dietary l-arginine supplementation did not have a significant effect on the plasma cholesterol level. There was no difference in plasma HDL levels between the three experimental groups. Plasma arginine levels were increased by 20% to 65% in the ARG group compared with the CHOL group (Table 2⇓).
The response to norepinephrine was not different in the three experimental groups at any time point, with potency and maximal tensions that were similar (data not shown).
Endothelium-independent vasorelaxation was also not different between the experimental groups, with potency and maximal relaxations that were similar (Table 3⇓).
NO-dependent vasodilation to acetylcholine was significantly impaired in the CHOL group at all time points compared with the animals receiving normal chow (Fig 1⇓). In the CHOL group, endothelium-dependent vasodilation (as expressed by the maximal response to acetylcholine) became progressively attenuated over the course of the study (from 53.4% at week 10 to 17.4% at week 23; Fig 1⇓). By contrast, at 14 and 18 weeks animals in the ARG group (hypercholesterolemic animals that had received l-arginine supplementation for 4 and 8 weeks, respectively) exhibited significantly improved responses compared with the CHOL animals at the same time points (Fig 1⇓, top right and bottom left panels). This beneficial effect was lost by week 23 in 4 of the 7 animals (“nonresponders”) at this time point (Fig 1⇓, bottom right panel). However, despite 23 weeks of persistent hypercholesterolemia in 3 of the 7 ARG animals, endothelium-dependent relaxation remained normal (as judged by a maximal response to acetylcholine that was 2 SDs above the mean value for the CHOL group at this time point [dashed line in Fig 2⇓, top panel]).
Lesion Surface Area
Planimetry of the lesion within the thoracic aorta was also performed to determine the amount of surface area involved by lesions. After 10 weeks on the atherogenic diet, each of the two groups receiving high-cholesterol chow had a similar percentage of surface area involved by lesion (30.3% versus 27.3%, ARG versus CHOL; P=NS; Fig 2⇑, bottom panel). At week 14, the CHOL animals manifested an increase in lesion surface area (to 36.5%), whereas ARG animals exhibited a reduction in lesion surface area (to 20.9%). This trend was maintained at week 18, with CHOL animals manifesting a progression in lesion surface area (to 43.7%), whereas ARG animals exhibited a significant reduction in lesion surface area (to 18.3%). By week 23, the animals in the CHOL group exhibited a further increase in lesion surface area (to 56.5%). At this time point there was a loss of the antiatherogenic effect of arginine in the four nonresponders that coincided with the loss of the ability of arginine to restore NO activity in these animals (Fig 2⇑, top panel). However, in 3 of the 7 ARG animals, NO activity remained persistently normal despite 23 weeks on the high-cholesterol diet (dashed line in Fig 2⇑, top panel). In these 3 responders, lesion surface area was only 5.4% (dotted line in Fig 2⇑, bottom panel), a remarkable finding in view of the persistent hypercholesterolemia.
The differences in lesion surface between the responders and nonresponders could not be accounted for by differences in lipid values (Table 4⇓).
When all l-arginine-treated hypercholesterolemic animals (from weeks 14, 18, and 23) were treated as one group (n=20), their maximal relaxation to acetylcholine was significantly greater and intimal lesion surface area significantly less than all hypercholesterolemic animals not treated with arginine (from weeks 14, 18, and 23; Fig 3⇓).
The cross-sectional area of the media was not different between the two hypercholesterolemic groups at any time point (Table 5⇓).
There was no measurable intima in vessels from normocholesterolemic animals. There was a progressive time-dependent increase in aortic intimal area in the CHOL group. In contrast, in the ARG group intimal lesion area progressed at a slower rate and was significantly less than that of the CHOL group at the 18-week time point (Table 5⇑). The analysis of intima-media ratios demonstrated a similar pattern, with ARG animals manifesting a significant reduction in intima-media ratio at 18 weeks.
Vascular NO and Superoxide Anion
At 12 weeks, vascular elaboration of nitrogen oxides was significantly increased in ARG animals compared with CHOL animals (11.6±3.0 versus 8.5±0.7 pmol/mg tissue per 100 μL buffer; P<.05).
Aortas from ARG animals also manifested a significant reduction in superoxide anion elaboration compared with CHOL animals at the same time point (2.7±0.4 versus 6.7±2.4 U/mg tissue; P<.05). The ratio of NO generation to superoxide anion release was significantly increased in the ARG animals at this time point (4.3±1.0 versus 1.5±0.4, ARG versus CHOL; P<.05).
The salient findings of this study are as follows: (1) Administration of 0.5% cholesterol chow induces a progressive endothelial vasodilator dysfunction associated with intimal lesion formation. (2) Addition of supplemental l-arginine to the high-cholesterol diet at week 10 does not alter the lipoprotein profile yet partially restores endothelium-derived NO activity by weeks 12 through 18. (3) This improvement in vascular NO activity is associated with a reduction in superoxide anion generation by the vessel wall and regression of intimal lesions. (4) After longer courses of a high-cholesterol diet (23 weeks), the salutary effect of l-arginine on endothelium-dependent relaxation is lost in half of the animals; the failure to restore vascular NO activity at this time point is associated with progression of disease. (5) In half of the animals, endothelium-dependent relaxation remains enhanced by l-arginine treatment even after 23 weeks of cholesterol chow; in animals with persistent normalization of NO activity, intimal lesion surface area is reduced by an order of magnitude compared with vehicle-treated animals at the same time point.
This is the first demonstration that restoration of vascular NO activity is associated with regression of preexisting lesions. This finding is of scientific interest and potential clinical relevance.
Numerous studies have focused on regression of atherosclerotic lesions in various animal species, including rabbits, pigs, and nonhuman primates. One of the first such studies was by Armstrong and colleagues,18 who induced atherosclerosis in monkeys using a diet high in cholesterol. A reduction in lesion size was seen after the animals were returned to a normal diet. In subsequent studies investigators observed that regression occurred parallel with a restoration of endothelium-dependent relaxation.19 20 21 22 In hypercholesterolemic rabbits, previous studies have documented that angiotensin-converting enzyme inhibitors, calcium antagonists, fish oil, and lipid-lowering agents may slow the progression of disease and even induce regression of preexisting lesions.20 21 22 23 24 Regression was observed only in those studies in which the animals had been returned to a normal chow diet.
What is remarkable about the current findings is that l-arginine administration was associated with a reduction in intimal lesions despite continued intake of the high-cholesterol diet and persistent hypercholesterolemia. The observed regression in intimal lesions at 14 and 18 weeks was not correlated with a change in total cholesterol, as levels remained elevated by 20- and 18-fold, respectively. The three responders at 23 weeks did have a slightly (but not significantly) lower cholesterol level, which was still 16-fold elevated compared with the values in the control animals.
The loss of the effect of l-arginine in the nonresponders at 23 weeks is not explained by either a decreased intake or increased metabolism of l-arginine, since there is no significant difference in plasma arginine levels between the responders and nonresponders. The loss of the antiatherogenic effect of arginine in the nonresponders may be due to an inability to restore NO activity in this group or may even reflect an adverse effect of arginine at later time points.
The mechanism(s) by which restoration of vascular NO activity may induce regression was not elucidated by this study, but recent observations from our laboratory and others provide some clues. Using an ex vivo functional binding assay, we have previously shown that supplemental l-arginine suppresses endothelial adhesiveness for monocytes in the thoracic aorta of hypercholesterolemic rabbits.15 By contrast, when vascular NO synthesis is inhibited (by chronic administration of an NO synthase antagonist), endothelial adhesiveness is markedly increased and lesion formation accelerated.15 25 26
One of the major chemokines mediating monocyte-endothelial cell interaction is MCP-1.27 28 Preliminary studies from our laboratory indicate that the expression of MCP-1 in the thoracic aorta of hypercholesterolemic rabbits is reduced by treatment (2 weeks) with l-arginine. By contrast, treatment of normocholesterolemic animals with the NO synthase antagonist nitro-arginine induces the expression of MCP-1 in the vessel wall (P.T. Tsao and J.P. Cooke, unpublished data, 1995). The mechanism by which vascular NO modulates MCP-1 expression is not fully understood but does appear to involve transcriptional regulation.29
The expression of chemokines and adhesion molecules mediating endothelial-monocyte interaction appears to be regulated by redox-sensitive transcriptional pathways.10 11 12 30 Hypercholesterolemia may activate these transcriptional pathways by increasing endothelial oxidative stress.17 NO may interfere with these transcriptional pathways by virtue of its capability to scavenge superoxide anion, disrupt the autocatalytic chain of lipid peroxidation initiated by oxygen-derived free radicals, or inhibit oxidative enzyme activity.31 32 33 Alternatively, arginine itself may reduce the generation of superoxide anion. LDL cholesterol induces a dysregulation of NO synthase in cultured endothelial cells such that the enzyme begins to generate superoxide anion; this can be reversed by increasing arginine availability to the enzyme.34 The present study reveals that l-arginine treatment in vivo enhances vascular NO activity and reduces generation of superoxide anion by the aorta of the hypercholesterolemic rabbit.
The inhibition by NO of monocyte adherence may fully account for the regression of lesions observed in this study if the accumulation of monocytes depends on a balance between influx and efflux of cells. Lipid-laden foam cells may exit the vessel wall through the endothelium, reenter the circulation, and eventually become enmeshed in the reticuloendothelial system.35 If enhanced vascular NO activity were to inhibit MCP-1 expression, the dissipation of a chemotactic gradient might promote macrophage efflux as well as reduce monocyte adherence.
Programmed cell death, or apoptosis, might be another mechanism by which vascular NO induces regression. NO donors are known to induce apoptosis in macrophages and vascular smooth muscle in vitro.36 37 Therefore, it is possible that the enhanced elaboration of vascular NO could induce apoptosis of cells in the lesion to slow or even reverse lesion growth.
To conclude, this study demonstrates for the first time that enhancement of vascular NO activity can induce regression of preexisting lesions in the thoracic aorta of hypercholesterolemic rabbits. This investigation provides additional support for our hypothesis that endothelium-derived NO is an endogenous antiatherogenic molecule and justifies future clinical studies to determine whether enhancement of endogenous NO activity is a viable therapeutic strategy for individuals afflicted by atherosclerotic vascular disease.
Selected Abbreviations and Acronyms
|ARG group||=||animals that received l-arginine (2.25%) plus a high-cholesterol diet|
|CHOL group||=||animals that received vehicle plus a high-cholesterol diet|
|EC50||=||concentration of drug inducing a half-maximal response|
|MCP-1||=||monocyte chemotactic protein-1|
|PSS||=||physiological saline solution|
This study was supported by a grant from the National Institutes of Health (R01-HL-48638) and was performed during the tenure of a Grant-in-Aid Award from the American Heart Association and Sanofi Winthrop. Drs Candipan and Tsao are recipients of National Service Research Awards (1F32-HL-08779). Dr Wang is the recipient of a Ho Tim-Stanley Ho-Li Shing Award from the Stanford University-Asia Medical Fund. Dr Cooke is a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute (1K07-HC-02660).
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