Role of NO in Flow-Induced Remodeling of the Rabbit Common Carotid Artery
Flow-induced changes in vessel caliber tend to restore baseline wall shear stress (WSS) and have been reported to be endothelium-dependent. To investigate the role of endothelium-derived nitric oxide (NO) in the adaptive increase in artery diameter in response to a chronic increase in blood flow, an arteriovenous fistula was constructed between the left common carotid artery (CCA) and the external jugular vein in 22 New Zealand White rabbits, and NO synthesis was inhibited in 14 animals by long-term administration of NG-nitro-l-arginine-methyl ester (L-NAME) in drinking water given for 4 weeks. The remaining 8 animals served as controls. Mean arterial blood pressure was not significantly altered by L-NAME treatment (91±2 in control versus 98±3 mm Hg in L-NAME–treated rabbits). Blood flow significantly increased in the left CCA in both groups but was lower in L-NAME–treated than control animals (106.1±10.7 versus 196.2±32.3 mL/min, P<.003). The diameter of the flow-loaded left CCA also increased significantly in both groups compared with the right CCA (2.15±0.12 and 2.54±0.1 mm, respectively, P<.02), but the increase was less in the L-NAME–treated than the control group (3.24±0.09 and 4.64±0.17 mm, respectively, P<.0001). The diameter of the anastomosed veins was also increased but to a much lesser degree in L-NAME–treated animals than in controls (4.14±0.29 versus 7.94±0.51 mm, P<.0001). As a result of artery enlargement, WSS was normalized in the flow-loaded left CCA of the control group (8.87±0.77 dynes/cm2) regardless of blood flow values. In L-NAME–treated animals, however, WSS was only partially regulated, the mean value being significantly increased (18.7±2.2 dynes/cm2, P<.006). Moreover, a highly significant positive correlation between WSS and blood flow was obtained in L-NAME–treated animals (r=.84, P<.0001). We also found remodeling of the artery wall, with a larger increase in the medial cross-sectional area associated with an increased number of smooth muscle cells, in the control group compared with the L-NAME–treated group (0.75±0.09 versus 0.49±0.04 mm2 and 4504±722 versus 2717±282 cells/mm2, P<.03). We conclude that NO plays a role in the increase of vessel caliber in response to chronic increase in blood flow. As yet unidentified additional metabolic processes appear to be necessary for a complete regulatory response.
- Received October 17, 1995.
- Revision received April 2, 1996.
Clinical findings indicate that chronic changes in blood flow rates through large arteries induce corresponding adjustments of artery diameter. Vessels exposed to elevated flow, such as arteries feeding an AV fistula1 2 or collateral vessels after arterial occlusion, tend to enlarge.3 4 Moreover, adaptive enlargement and remodeling are known to accompany early human coronary atherosclerosis.5 Experimental studies have demonstrated that blood flow regulates the diameter of arteries both acutely6 7 8 9 and chronically in association with the reorganization of vascular wall cellular and extracellular components.10 An endothelial reaction appears to mediate this response.8 11 12 13 Thus, the endothelium functions as a mechanically sensitive signal-transduction interface between the blood and the artery wall. In vitro and in vivo studies in large and small arteries6 7 9 13 14 indicate that acute flow-induced dilation is mediated by the endothelial release of NO, but the messenger(s) responsible for the chronic local adaptation to blood flow conditions have not been identified.
The aim of the present study was to test whether NO participates in eliciting chronic arterial and venous enlargement in response to increased WSS and stimulates the corresponding vascular mural tissue adaptation. To that end, we produced an AV shunt between the CCA and the external jugular vein in rabbits to induce increased blood flow, vessel enlargement, and vessel wall remodeling and examined the effects of inhibiting NO synthesis by means of long-term oral administration of L-NAME.
The study was performed in 22 male New Zealand White rabbits (8 weeks old, weighing 2.2 to 2.5 kg). The animals were anesthetized with sodium pentobarbital (0.5 mg/kg body wt IV) followed by injections of ketamine hydrochloride (0.5 mg/kg body wt IV) every 15 minutes. Each rabbit received heparin (500 U IV) at the beginning of the dissection.
Under sterile conditions, a longitudinal cervical incision was made, and the left jugular vein and CCA were dissected free and flooded with topical papaverine to prevent vessel spasm during dissection. Flow through the CCA was interrupted by means of microvascular clamps, and the vein was clamped proximally. The jugular vein was transected distally, and an end-to-side anastomosis was performed between vein and artery by using continuous 7/0 polypropylene suture under ×2.5 magnification. The size of the anastomotic openings was the same for each animal (5 mm). Flow was then reestablished through the fistula by releasing the carotid artery clamps. The incisions were closed in layers, and the animals were allowed to recover. Animals were housed individually and cared for in accordance with the European Community Standards on the Care and Use of Laboratory Animals (No. 00577).
Eight animals were given normal drinking water for 1 month; these constituted the control group. Ten rabbits were given drinking water containing 0.5 g/L L-NAME, an l-arginine analogue that inhibits NO synthesis,15 for 1 month beginning the day of surgery. To obviate the effect of dosage, it was elected to expose 4 rabbits to a higher dose of L-NAME (1.5 g/L for 1 month). Since no differences in any of the parameters under study were evident between animals treated with the different doses of L-NAME, results were pooled.
After 1 month, rabbits were anesthetized by using the procedure described above and again received heparin 500 U IV. The carotid arteries and jugular veins of both sides were exposed. The right CCA and vein served as nonoperated internal controls. Left and right arterial and venous diameters were measured in situ by using a focusing eyepiece and a bow compass.
Arterial pressure was measured by using a catheter introduced via the femoral artery and connected to a Statham model P23ID pressure transducer (Gould).
Blood flow velocity was measured by using a perivascular ultrasonic flow velocity transducer placed around the vessel wall. The perivascular transducer selected in each instance was always of a diameter (2, 3, 4, or 5 mm) slightly smaller than that of the vessel itself, so that the value of the cross-sectional area for use in the calculation of the blood flow was that of the appropriate selected transducer and was not estimated from the vessel diameter measured by using the focusing eyepiece. The ultrasound probe was connected to a range-gated Doppler velocimeter (Alvar, 8 MHz). Saline was used to couple the ultrasound probe to the artery, and the probe position and pulsed Doppler range gate were adjusted to give the maximum velocity signal. Pulsatile and mean Doppler shifts were recorded on a Gould polygraph recorder. Velocities were calculated from the measured Doppler shifts by using the conventional Doppler equation,16 and volume flow was obtained by multiplying the mean velocity by the cross-sectional area of the perivascular transducer that best fitted the vessel. Blood flow was measured in the left CCA both proximal and distal to the AV fistula and in the right CCA.
To assess the immediate effect of the AV fistula on blood flow, the blood flow rate of both CCAs was measured in seven animals immediately after carotid and vein dissection but before the anastomosis was performed and again at the end of the carotid and jugular anastomosis 15 minutes after flow was reestablished. Blood flow was also measured at 1 month when the animals were killed.
Calculation of WSS
WSS, the frictional force exerted by flowing blood on the vascular lumen surface, was calculated for right and left CCAs. Assuming laminar flow, WSS in dynes per square centimeter was calculated by using the Poiseuille formula: , where μ is the blood viscosity (taken to be 0.035 poise), Q is the blood flow (in milliliters per second), and r is the radius (in centimeters).
The AV fistula site and the right CCA were exposed in nine L-NAME–treated rabbits and all untreated animals. Both carotid arteries were cannulated at the thoracic aorta and ligated. The jugular veins were dissected free, and the end of the vein close to the carotid-jugular anastomosis was ligated. Animals were then killed by an overdose of sodium pentobarbital. The CCAs were fixed in situ as follows. Blood was washed out by perfusion with normal saline solution followed by a fixing solution of 5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 mL/L cacodylate buffer, pH 7.4, for 15 minutes. A constant pressure of 100 cm H2O was maintained throughout the fixing procedure. The CCAs were then excised and stored in a 10% buffered formaldehyde solution. For histological studies, samples were embedded in paraffin. Transverse cross sections were cut at 5-μm intervals, stained with hematoxylin-eosin, orcein, Weigert–van Gieson's, or Masson trichrome stains, and assessed by using an automatic image-analysis processor (Microvision 15000). Medial cross-sectional areas were measured by planimetry at six axial locations with respect to the AV fistula (three in the proximal CCA and three in the distal portion) and at three locations in the right CCA. Because there were no significant differences among the three sections in each location, the values were pooled. Measurements were averaged for each arterial segment. The number of nuclei stained with hematoxylin was counted automatically for each cross section.
Vascular Reactivity Studies
To ensure that L-NAME successfully blocked NO synthesis, endothelium-dependent relaxation to Ach was examined in aortic rings from L-NAME–treated rabbits. Ring segments of aorta 3 mm long were dissected free of adventitial fat and connective tissue and mounted between two stainless steel wires in a 30-mL organ bath containing physiological salt solution of the following composition (in mmol/L): NaCl 135.0, NaHCO3 15.0, KCl 4.6, CaCl2 1.5, MgSO4 1.2, glucose 11.0, and HEPES 5.0. The pH was adjusted to 7.4, and the solution was bubbled with 95% O2 and 5% CO2. One wire was attached to a fixed support; the second was connected to a movable holder supporting a tension transducer (Grass FT.03) so that isometric force measurements could be recorded on a physiograph (Grass SD90). The artery segments were allowed to recover for 30 minutes, during which time the physiological salt solution was replaced at 15-minute intervals. Following this recovery period, a 2-g preload was applied to the aortic segments, which were then allowed to equilibrate for an additional 90 minutes. Responses to Ach (10−8 to 10−4 mol/L) were tested after precontraction of the aorta with phenylephrine (10−6 mol/L). Data are expressed as percent dilation of phenylephrine-induced preconstriction.
Results are expressed as mean±SEM. Comparisons between the control and L-NAME groups were performed by using an unpaired Student's t test, and intragroup comparisons between left and right CCAs were done by using a paired Student's t test. Linear regression analysis was used to evaluate the relationship between measured blood flow and calculated WSS values. Two-factor ANOVA was used to compare the concentration-response curves to Ach. Differences were considered significant at a value of P<.05.
Characteristics of L-NAME–Treated Rabbits
Animal weight at the beginning of the study was 2.18±0.04 kg. At the end of the 1-month period of treatment, the body weights of the L-NAME–treated rabbits were significantly lower than those of the control group (Table 1⇓). Long-term oral administration of L-NAME did not significantly affect arterial BP (Table 1⇓).
L-NAME treatment did not alter the contractions of aortic rings to phenylephrine, but relaxations caused by Ach (10−8 to 10−4 mol/L) were inhibited in aortic rings from rabbits treated with L-NAME (0.5 or 1.5 g/L) in drinking water (Fig 1⇓).
Blood flow in the left CCA before the construction of the AV fistula was 22.2±2.9 mL/min. It increased significantly to 51.8±2.9 mL/min 15 minutes after the AV fistula was created (n=7, P<.0003). Subsequent flow measurements at the time of death revealed that the AV fistula had resulted in a large increase in proximal carotid blood flow (Table 2⇓). In the flow-loaded left CCA, blood flow was increased 3.2-fold in the L-NAME group and 6.6-fold in untreated animals compared with right CCA flow. Left CCA blood flow ranged from 41 to 138 mL/min in the L-NAME–treated group and from 82 to 350 mL/min in the untreated group (P<.003). Mean blood flow in the right CCA was similar for both groups.
In contrast, blood flow in the left CCA distal to the AV fistula was 15.1±2.7 mL/min in the L-NAME–treated group and 20.3±3.9 mL/min in the untreated group. Although blood flow was retrograde distal to the AV fistula, the magnitude of flow was not statistically different for both groups.
The diameter of the left CCA increased significantly from 1.84±0.12 mm before construction of the fistula to 2.03±0.15 mm 15 minutes after release of the clamps (P<.003).
Values for right and left CCA diameters in L-NAME–treated and untreated rabbits 1 month after surgery are given in Table 3⇓. L-NAME animals had smaller right CCA diameters than untreated animals (P<.02). The diameter of the left CCA proximal to the AV fistula was significantly increased by 83% in the untreated group but by only 51% in the L-NAME group compared with the contralateral right CCA (P<.0001). Left CCA diameters in L-NAME animals were significantly smaller than in untreated rabbits (P<.0001). Fig 2⇓ shows diameter–blood flow relationships for both groups. Diameter values in the L-NAME group were lower than those in untreated animals, even at similar blood flow values.
Diameters of the left CCA distal to the AV fistula were reduced by 27% in the L-NAME group (P<.0003) and by 18% in the untreated group (P<.02) compared with the contralateral right CCA.
The vein diameter before anastomosis to the CCA was 2.03±0.15 mm; this diameter increased to 2.33±0.24 mm 15 minutes after flow was established through the AV fistula (n=7, P<.004). After 1 month, the caliber of the anastomosed vein averaged 4.14±0.29 mm in L-NAME–treated rabbits and 7.94±0.51 mm in untreated animals (P<.0001). This vein enlargement was not due to increased BP in the anastomosed vein, for pressure measured in the vein just proximal to the fistula remained low (between 5 and 7 mm Hg).
In untreated animals, WSS values calculated for the flow-loaded left and contralateral right CCAs were not significantly different (Table 4⇓). In the L-NAME group, WSS was significantly higher in both right and left CCAs.
To estimate the compensatory effect of the adaptive diameter changes to increased blood flow, WSS values were plotted against blood flow values (Fig 3⇓). In the untreated group the data points were clustered close to a theoretical horizontal line representing complete regulation. All but one of the calculated WSS values were between 6.8 and 11.4 dynes/cm2, indicating that the regulation of WSS had been nearly completed in 1 month. Only in one rabbit, in which the greatest flow load was observed (350 mL/min), the WSS, at 20.1 dyne/cm2, remained twofold larger than the control value, suggesting an incomplete regulatory response in this animal. Conversely, in the L-NAME group, WSS values varied between 7.1 and 37.5 dynes/cm2, and a highly significant positive correlation with a regression slope of 0.16±0.03 dynes·min·cm−5 was obtained between blood flow and WSS values (r=.84, P<.0001, n=14). However, based on the mean value of the right CCA diameter (2.15±0.12 mm), the regression slope should be 0.6±0.1 dynes·min·cm−5 if there were no regulation. This value was significantly different from that found in the L-NAME group (P<.001), suggesting that a partial regulation of WSS occurred after long-term inhibition of NO synthesis.
Cross-sectional area of the media of the contralateral right CCA was significantly (P<.005) lower in L-NAME–treated than untreated animals (Table 5⇓). The medial cross-sectional area of the left CCA proximal to the AV fistula was increased compared with the contralateral right CCA in both groups (Table 5⇓) but was significantly lower in the L-NAME than in the untreated group (P<.01). In the left CCA distal to the AV fistula, the mean medial cross-sectional area remained unchanged compared with the right CCA in both groups.
The SMC density of the media measured in transverse sections obtained from right or left CCAs in L-NAME–treated or untreated animals (Table 6⇓) was not significantly changed, except that this value was decreased in the flow-loaded left CCA of L-NAME–treated animals compared with its contralateral CCA.
Using cell density and medial cross-sectional area values, the number of SMCs per medial cross-sectional area was unchanged in the right CCAs of L-NAME–treated and untreated animals (Table 7⇓). However, the number of SMCs in the media in the left CCA proximal to the AV fistula was increased more than twofold in control animals compared with the right CCA (P<.03), whereas it was increased by only 53% in L-NAME rabbits (P<.02). Increased cell number in the left CCAs was significantly lower in L-NAME–treated than untreated animals (P<.03). No difference was observed in the left CCAs distal to the AV fistula in either group.
The results of the present study demonstrate that the regulation of WSS in flow-loaded arteries is at least in part NO dependent. Long-term oral administration of L-NAME did not raise BP in rabbits, as has been reported in rats.17 Our findings do agree with other studies in rabbits,18 19 but the reasons for this discrepancy between species have yet to be elucidated. Thus, the effect of L-NAME treatment in our experiments can be considered to have resulted directly from its inhibition of NO synthesis and was not the result of a pressure effect. The inhibition of NO synthesis by L-NAME was attested, too, by the inhibition of the endothelium-dependent relaxation to Ach of aortic rings from L-NAME–treated animals. Although alkyl esters of l-arginine have been reported to block muscarinic receptors in vitro,20 such an effect in chronic administration of L-NAME in vivo is unlikely since L-NAME is rapidly (t1/2=2 minutes) metabolized into the pure NO synthesis inhibitor Nω-nitro-l-arginine with a slow rate of elimination.21 L-NAME–treated animals gained significantly less weight than the controls. Similar weight loss caused by chronic L-NAME treatment has been reported in rabbits,18 rats,17 and mice,22 probably due to the inhibitory effect of L-NAME on relaxation in the digestive tract and to smaller food intake resulting from neurotoxic effects of methanol, the second metabolite of L-NAME in vivo.21
Our findings support the concept that arteries enlarge in response to time-averaged blood flow rates and that artery enlargement tends to restore baseline mean shear stress. After effective long-term blockade of NO synthesis, the WSS in the flow-loaded CCA varied directly with blood flow (Fig 3⇑), indicating that eliminating the NO pathway significantly prevents the vascular adaptation to increased blood flow.
A number of in vivo studies suggest that blood flow conditions directly affect vascular growth. Guyton and Hartley16 have shown that flow restriction of one carotid artery in juvenile rats inhibits growth of arterial diameter. Langille and O'Donnell11 report that 2 weeks after left external carotid artery ligation in the rabbit (resulting in carotid artery blood flow reduction), the left CCA was 21% smaller in diameter than the right CCA. This chronically reduced flow leads to permanent reduction in arterial diameter since the smooth muscle relaxant papaverine did not attenuate the response. Conversely, Kamiya and Togawa,23 who studied the adaptation of the dog carotid artery to increased flow produced by carotid-to-jugular anastomosis, showed that the flow-loaded artery adapted completely by enlargement to reduce the WSS to physiological baseline values of 15 dynes/cm2. Moreover, Zarins et al24 used the monkey iliac artery to show that when flow was increased 10-fold, the artery adapted to restore WSS to a baseline value (between 15 and 16 dynes/cm2) after 6 months. Our present findings are in agreement with these experiments. Although we obtained a wide range of increased flow values, the diameter of the left CCA increased in each untreated animal in such a way that the WSS tended to be reduced to 8.9 dynes/cm2, which did not differ significantly from the value in the right CCA (10.7 dynes/cm2). This corresponds closely to values of 11 to 12 dynes/cm2 reported by Walpola et al25 in the rabbit CCA.
Only in one animal, in which the left CCA was exposed to a very high shunt flow (more than 10-fold higher than the control), was a complete regulation of WSS not observed (Fig 3⇑). The adaptive response to such great stresses might require longer than 1 month. It is also likely that abnormal infrastructural changes might be induced by such extreme stresses and that these may disturb the physiological adaptation, as shown by Tohda et al,26 who used a model of rat carotid-jugular AV fistula.
The presence of the endothelium is required for vascular response to chronic flow alterations. Langille and O'Donnell,11 using a rabbit carotid stenosis model, report that deendothelialization eliminates the diameter decrease that otherwise would result from flow reduction. Tohda et al26 have shown that deendothelialized segments of flow-loaded rat CCAs fail to dilate. The capacity of the endothelium to sense shear stress is therefore an important determinant of lumen diameter and overall vessel structure remodeling. However, the mechanisms by which blood flow changes are transduced or the second messengers that elicited the responses of the media to chronically altered blood flow have not been elucidated, even though a large quantity of data is available concerning the endothelial metabolic and structural responses to shear.27
The present study also revealed that long-term administration of L-NAME partially blocked carotid artery enlargement and the corresponding wall tissue growth in response to increased blood flow, yielding to incomplete regulation of WSS. This finding suggests that endothelium-derived NO plays a significant direct role in blood flow–induced vascular remodeling and is the likely mediator, at least in part, of the long-term structural adaptation to altered blood flow. Other lines of evidence support a probable role for NO as a modulator of the adaptive modeling of flow-loaded arteries.
It is now clear, for example, that endothelial NO release mediates the acute vasodilation response to increased flow observed in vivo.6 9 Increased shear stress in rabbit iliac artery activates a flow-sensitive potassium channel that induces hyperpolarization and promotes calcium influx leading to NO release,28 and bovine aortic endothelial cells cultured under flow conditions show increased NO synthase expression29 and increased NO release in response to high shear stress.30 Others31 have shown that chronic increases in blood flow in dog femoral arteries resulting from an AV fistula enhance the tonic and receptor-stimulated production of NO and that the arterial wall upstream of a chronic aortocaval fistula has increased cGMP content.32 Although NO-mediated vasodilation may participate in the carotid enlargement we observed in response to increased blood flow, this process is unlikely to be sufficient to account for the almost twofold increase in carotid diameter seen in untreated animals. In our experiments, the diameter of the flow-loaded carotid artery increased slightly (by 10%) but significantly 15 minutes after opening the AV fistula.
In addition to its relaxing effect on SMCs, endothelium-derived NO may favor artery enlargement through other mechanisms. Recent findings indicate that fluid flow stimulates metalloproteinase production by endothelial cells.33 Other data suggest that NO activates metalloproteinase, as has been shown in articular cartilage.34 If this is also the case in endothelial cells, NO-stimulated protease activity by endothelial cells could produce local erosions and interruptions of the internal elastic lamina that would compromise tensile support and favor artery enlargement. Tears in the internal elastic lamina have been reported by Greenhill and Stehbens35 in AV fistula models. We observed internal elastic fragmentation in the present work (Fig 4⇓), but the precise temporal relationship of this injury to the enlargement phenomenon remains to be established.
In general, the enlargement of arteries in response to increased blood flow is accompanied by tissue proliferation. In flow-loaded iliac arteries of cynomolgus monkeys, Zarins et al24 found an 84% increase in medial cross-sectional area after 6 months. This change parallels our findings of a 108% increase in flow-loaded left CCAs of the untreated rabbits after 1 month. This increase in medial mass was due to cellular hyperplasia, in agreement with results reported by Lehman et al36 in flow-loaded rabbit basilar arteries. Wall tissue growth was, however, prevented in the L-NAME–treated rabbits. The increase in medial mass may be a biosynthetic and proliferative reaction to the increased wall tension associated with the artery enlargement.37 The growth adaptation to the changes associated with increased blood flow could also be attributable to increased production or activity of endothelium-derived growth factors stimulated by NO release. Shear stress increases platelet-derived growth factor (PDGF) gene expression in cultured endothelial cells, and Hsieh et al38 have proposed that “PDGF may be involved in the adaptation of blood vessels to flow mediated by the endothelium.” Moreover, it is noteworthy that while a steady laminar shear stress of 15 dynes/cm2 induces only a mild and transient increase in basic fibroblast growth factor mRNA expression in cultured bovine aortic endothelial cells, exposure to a shear stress of 36 dynes/cm2 for 6 hours induces an almost fivefold increase, which remained elevated about threefold at 9 hours.39 On the other hand, NO, which has been found to inhibit vascular SMC proliferation,40 amplifies the mitogenic activity of basic fibroblast growth factor in primary culture of rat aortic SMCs.41
In conclusion, the present study demonstrates, we believe for the first time, that NO synthesis blockade in vivo inhibits adaptive WSS regulation in vessels subjected to chronic increased blood flow. The effect is partial, however, indicating that other factors are probably involved. The pathway(s) by which the action of NO is mediated may include effects other than its direct influence on SMC relaxation. These include metalloproteinase activation and induction of growth factor mitogenic activity. The possible role of NO as an initiating or amplifying factor could explain our finding that long-term blockade of NO synthesis did not totally abolish the adaptive responses to blood flow increase. What may prove to be yet another finely tuned regulatory cascade merits further detailed investigation.
Selected Abbreviations and Acronyms
|CCA||=||common carotid artery|
|SMC||=||smooth muscle cell|
|WSS||=||wall shear stress|
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