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(Circulation Research. 2000;86:1160.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Temporal Events Underlying Arterial Remodeling After Chronic Flow Reduction in Mice

Correlation of Structural Changes With a Deficit in Basal Nitric Oxide Synthesis

Radu Daniel Rudic, Mariarosaria Bucci, David Fulton, Steven S. Segal, William C. Sessa

From the Department of Pharmacology (R.D.R., M.B., D.F., W.C.S.), Yale University School of Medicine and Boyer Center for Molecular Medicine, and The John B. Pierce Laboratory and Department of Cellular and Molecular Physiology (S.S.S.), Yale University School of Medicine, New Haven, Conn.

Correspondence to William C. Sessa, PhD, Boyer Center for Molecular Medicine, Room 436D, Yale University School of Medicine, 295 Congress Ave, New Haven, CT 06536. E-mail william.sessa{at}yale.edu

	Abstract

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Abstract—To define the cellular events of vascular remodeling in mice, we measured blood flow and analyzed the morphology of remodeled vessels at defined points after a flow-reducing remodeling stimulus for 3, 7, 14, and 35 days. Acute ligation of the left external carotid artery reduced blood flow in the left common carotid artery (LC) compared with sham and contralateral right common carotid arteries (RCs). In morphometric analyses, the decrease in diameter in LCs was reversible by vasodilator perfusion 3 days after ligation, whereas ligation for 7 days or greater resulted in a permanent diameter reduction. Coincident with structural remodeling at day 7 was an increase in cell death in remodeled LCs. Functionally, rings from remodeled LCs contracted to prostaglandin F₂ and relaxed to acetylcholine in a manner identical to that of control arteries. However, remodeled LCs were hypersensitive to the nitrovasodilator sodium nitroprusside (at day 7) and exhibited a marked reduction in basal NO synthesis at 7 and 14 days after ligation. The impairment of endothelial NO synthase function was likely due to post-translational mechanisms, given that endothelial NO synthase mRNA and protein levels did not change in remodeled LCs. These data define the ontogeny of flow-triggered luminal remodeling in adult mice and suggest that endothelial dysfunction occurs during reorganization of the vessel wall.

Key Words: vascular • apoptosis • ß-actin • endothelial NO synthase • endothelium

	Introduction

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Vascular remodeling is the reorganization of blood vessel geometry in response to physiological alterations in blood flow or to pathological stimuli. For example, increases in blood flow triggered by a surgical anastomosis will increase lumen diameter and thus improve blood flow.¹ Conversely, dysregulated remodeling as found in severe atherosclerosis and vein grafts and after balloon angioplasty can result in luminal stenosis and potentially limit blood flow.^{2 3} These changes in blood vessel architecture are initiated by signal transduction events leading to cellular proliferation, migration, apoptosis, and/or survival of vascular cells. The integrity of the vascular endothelium⁴ is required for physiological remodeling of the vessel wall in response to flow changes, suggesting that abnormal remodeling in many disease states may be initiated and propagated by ongoing endothelial dysfunction.

One potential endothelium-derived mediator implicated in vascular remodeling is NO. Indeed, NO inhibits vascular smooth muscle cell proliferation^{5 6} and migration⁷ and stimulates endothelial cell migration and reorganization,^{8 9 10} phenotypes necessary for vascular remodeling. Accordingly, inhibition of NO synthase (NOS) using L-arginine analogues impairs high-flow remodeling of conduit arteries^{11 12} and triggers coronary artery remodeling and fibrosis.¹³ Genetic evidence in support of endothelium-derived NO as a major regulator of vascular architecture stems from the inability of endothelial NOS (eNOS) knockout mice to remodel their carotid arteries in response to a decrease in blood flow.¹⁴ Moreover, these animals displayed no change in lumen diameter and paradoxical medial thickening due to smooth muscle hyperplasia. eNOS knockout mice also exhibit an exaggerated hyperplastic response in response to vascular injury,¹⁵ suggesting that eNOS-derived NO regulates cellular events important for physiological and pathophysiological remodeling of the vessel wall.

The causal links between hemodynamic changes, signal transduction, cell turnover in the vessel wall, vasomotor function, and changes in vessel geometry during remodeling are not clearly defined. Thus, we undertook the present study to establish the molecular and cellular events underlying a remodeling response in a defined model of chronic flow reduction in mice. Here, we show that a decrease in basal, but not stimulated, NO production coincides with a loss of medial vascular smooth muscle cells and structural remodeling of the common carotid artery. These results suggest that a chronic reduction in blood flow per se can trigger endothelial dysfunction during a remodeling response.

	Materials and Methods

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Vascular Casting
All procedures and protocols were approved by the Yale Animal Care and Use Committee. The left external carotid artery (EC) was ligated in C57BL/6J male mice for 2 weeks.¹⁴ After exsanguination, mice were perfused with a casting media consisting of a 2:1 mix of methyl methacrylate and cranioplastic powder.

Measurement of Carotid Arterial Blood Flow
Mice were anesthetized with a ketamine/xylazine mix and maintained at 37°C with a heating pad. Right or left common carotid artery (RC or LC, respectively) blood flow was measured at the vessel midpoint using an ultrasonic flow probe (0.5 mm V-series probe, Transonic Systems Inc).

Time Course of Vascular Remodeling
EC ligations were performed in mice for 3, 7, 14, and 35 days, as previously described.¹⁴ In addition, parallel sections of carotid artery were excised, cut longitudinally, and mounted en face with the endothelium facing upward. Propidium iodide (PI)–positive cells were observed using a Bio-Rad 600 confocal microscope.

Functional Studies in Isolated Carotid Arteries
Control or ligated mice were lightly anesthetized with methoxyflurane (Mallinckrodt Veterinary, Inc). RCs and LCs were carefully excised and placed into Krebs-Henseleit bicarbonate buffer solution. Adventitial fat was carefully removed and carotid arteries cut into rings (2 mm thickness). The rings were suspended by 2 tungsten wires (25 µm diameter) and mounted in a vessel myograph system (5 mL chamber size, Kent Scientific).

Competitive Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) for Quantification of eNOS mRNA Levels
RT-PCR was performed as described.¹⁶

Western Blotting
Four individual carotid arteries from the groups were pooled to permit detection of specific proteins, pulverized on dry ice, and then immersed into protein lysis buffer.

Statistics
Data are mean±SEM, with n referring to number of mice per group. Significant differences were analyzed using a Student t test or ANOVA followed by a Dunnett multiple-comparison post hoc test. Differences were considered statistically significant with P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Previously, we have shown that ligation of the left EC in mice stimulates a decrease in lumen diameter in the LC, a response associated with a decrease in the number of vascular smooth muscle cells.¹⁴ To delineate the perfusion field of the carotid circulation in mice after EC ligation, we assessed the anatomy of this circulation by vascular casting. Figure 1A illustrates the arterial circulation of the neck and head of a mouse with ligation of the left EC for 2 weeks. The right EC (clearly absent on the left side) advances ventrally and divides into its branches depicted by the unlabeled arrows (seen in Figure 1B). The left internal carotid artery is visible as a result of the absence of the left EC occluded by the ligation. Asymmetry due to EC ligation between the right and left side is more readily apparent under higher magnification (Figure 1B). Also seen is the reduction in LC diameter relative to RC. Control vascular casts showed no obvious anatomic differences in the circulation or between left and right common carotid arteries (not shown).

View larger version (69K): [in this window] [in a new window]   Figure 1. Vascular cast from a mouse with left EC ligation. A, Low-magnification image of the casted arterial circulation. A indicates aorta; Br, brachiocephalic artery; IC, internal carotid artery; O, ophthalmic artery; Ba, basilar artery; and w, circle of Willis. Bar=4 mm. B, Higher magnification of the vascular cast. Bar=0.5 mm.

To establish that EC ligation triggered a decrease in LC blood flow, we directly measured flow using an ultrasonic probe. As seen in the experimental trace (Figure 2A), acute ligation of the left EC (shown by the arrow) resulted in a rapid decrease in LC blood flow followed by an increase in flow to a stable baseline that was lower than the original flow rate. Acute ligation of EC decreased peak blood flow by 30% (quantified in Figure 2B) without changing blood flow in the contralateral RC. Because arterial remodeling is a process that occurs over days to weeks,¹⁷ blood flow was quantified in mice that underwent left EC ligation for 2 weeks. Figure 2B (right) shows that ligated mice exhibited a decrease in LC peak blood flow (by 50%) compared with sham-operated mice.

View larger version (33K): [in this window] [in a new window]   Figure 2. Measurement of carotid arterial blood flow after left EC ligation. A, Tracing of blood flow measurement in LC after acute left EC ligation. A loose ligature was placed around left EC and tightened (arrow) while blood flow in LC was continuously recorded. B, Peak blood flow changes after acute or chronic (2 weeks) ligation of EC. Values are mean±SEM. *P<0.05 vs baseline LC; P<0.05 vs control LC. n=5 mice per group.

After establishing the fundamental basis of this model, luminal remodeling was quantified at defined time points after ligation. Mice undergoing ligation were perfusion-fixed with a vasodilator (VD) cocktail or with PBS alone (VD-free) to examine the temporal relationship between arterial constriction (a reversible, VD-sensitive process) versus structural adaptation (a permanent VD-insensitive process) during arterial remodeling. Mice underwent left EC ligation for 3, 7, 14, and 35 days followed by morphometric analysis of RC and LC lumen diameters. RC and LC diameters were identical in the absence or presence of VD in the perfusate (see Table online at http://www.circresaha.org). However, after a 3-day remodeling stimulus, LC diameter decreased relative to the contralateral RC, an effect that was completely reversible by VD, suggesting that vasoconstriction accounted for the decreased diameter. By day 7 after ligation through day 35, a permanent LC lumen diameter reduction occurred, consistent with structural adaptation of the vessel wall. These effects are more clearly discernable when the absolute data are expressed as a change in lumen diameter (calculated by subtracting RC from LC lumen diameters in each mouse at different time points after a remodeling stimulus; see Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Time course of luminal remodeling in carotid arteries after EC ligation. Mice undergoing left EC ligation for 3, 7, 14, and 35 days were perfusion-fixed in the absence or presence of a VD cocktail (0.1 mmol/L adenosine, 0.3 mmol/L papaverine, or VD-free) in the perfusate, and then lumen diameter was analyzed by quantitative morphometry. Data are presented as mean±SEM differences in lumen diameters between LCs and RCs. See Table online (http://www.circresaha.org) for number of mice per group. *P<0.05 vs control or day 0; P<0.05 comparing VD-free vs VD at day 3.

Previously, we have shown that the decreased lumen diameter after a 2-week ligation in mice was accompanied by a loss of vascular smooth muscle cell nuclei consistent with an apoptotic event.¹⁴ Thus, we injected mice with PI to label dying cells throughout the time course of remodeling followed by en face, confocal imaging. Dramatic PI incorporation was only observed in remodeled LCs after 7 days of a remodeling stimulus. (depicted in Figure 4A, right, and quantified in Figure 4C). The PI staining was most prominent in smooth muscle cells, as indicated by their orientation perpendicular to the vector of flow (Figure 4B). At all other time points, little or no PI staining was detected in RCs or LCs. Identical results demonstrating enhanced PI labeling at 7 days after ligation were seen in an additional time course study (data not shown). Interestingly, the permanent diameter reduction at day 7 (Figure 3) was temporally coincident with increases in cell death. For this reason, the 7-day time point was used for further experimentation.

View larger version (82K): [in this window] [in a new window]   Figure 4. Increased cell death in remodeled carotid arteries after 7 days of EC ligation. Mice were intravenously injected with PI 15 minutes before euthanization. RCs and LCs were isolated after exsanguination and perfusion fixation, cut lengthwise and pinned en face for fluorescence imaging, and quantified as described in Materials and Methods. A, Remodeled LC (7 days) shows dramatic patches of PI staining, whereas negligible fluorescence is visible in an identical section of the contralateral RC. Arrow indicates direction of blood flow. Magnification, x200. B, Higher magnification (x400) of another 7-day remodeled LC. C, Quantitative data demonstrating an increase in PI-positive vascular smooth muscle cells in remodeled LC compared with RC. Values are mean±SEM. *P<0.05, n=5 mice per group.

To correlate structural features of remodeling with vascular function, isometric tension development and vascular relaxations were studied in isolated carotid arteries. As seen in Figure 5, prostaglandin F₂ (PGF₂) increased isometric tension to the same extent in RCs and LCs at 0 (control), 7, and 14 days after ligation. In addition, endothelium-dependent relaxations in response to acetylcholine (Ach) were identical in RCs and LCs from the 3 groups of mice. Endothelium-dependent responses to Ach in all 3 groups were attenuated (70% to 80%) by the NOS inhibitor nitro-L-arginine methyl ester (L-NAME 100 µmol/L, data not shown), demonstrating that NO is the major relaxing factor in carotid arteries, as previously described.¹⁸ Interestingly, endothelium-independent relaxations to sodium nitroprusside (SNP) were identical in RCs versus LCs at days 0 and 14 but were enhanced at day 7 only in LCs, suggesting an increase in sensitivity to the nitrovasodilator.

View larger version (34K): [in this window] [in a new window]   Figure 5. Isometric tension development and relaxation in remodeled carotid arteries. PGF2, Ach, and SNP concentration response curves were performed on isolated RC () and LC () from mice undergoing 7-day and 14-day EC ligation were compared with vessels from age-matched control mice. *P<0.05, n=7 rings from at least 4 mice per group (1 to 2 rings per RC or LC from each mouse).

To assess whether the increased sensitivity to SNP could be related to a deficit in basal NO production, we examined the ability of L-NAME to further increase isometric tension in PGF₂-precontracted RCs and LCs (Figure 6A). L-NAME enhanced isometric tension development in RCs and LCs from control mice to the same extent. However, at day 7 after ligation, L-NAME–induced increases in tension were markedly depressed in the remodeled LCs (right trace in A) compared with contralateral RCs (left trace in A). By day 14, the impairment in the responsiveness to L-NAME in LCs was beginning to normalize to that observed in RCs (Figure 6B).

View larger version (23K): [in this window] [in a new window]   Figure 6. L-NAME–induced increases in isometric tension are reduced in remodeled LC. A, Typical bioassay trace of carotid arterial rings from a mouse undergoing 7 days of a remodeling stimulus. Left, RC ring was preconstricted with PGF2 and Ach added to document the presence of endothelium. Drugs were washed out (w), and PGF2 was added again to induce a stable contraction. The addition of L-NAME (100 µmol/L) further increased isometric tension development indicative of removal of basal NO tone on the vessel. Right, Same experiment from a remodeled contralateral LC from the same mouse. L-NAME–induced increases in isometric tension were reduced in remodeled LC. B, Summary of L-NAME–induced isometric tension in carotid arterial rings of control mice and mice after 7 and 14 days of EC ligation. Values are mean±SEM. *P<0.05; n=6 rings from 4 mice per group.

Next, we performed experiments to determine whether the observed impairment of eNOS function could be attributed to changes in eNOS mRNA or protein levels. Utilizing a competitive (quantitative) RT-PCR strategy (Figure 7), eNOS mRNA levels were quantified from individual RCs and LCs from controls and 7 days after ligation. Figure 7A demonstrates that increasing concentration of mouse eNOS cDNA competes with the eNOS competitor cDNA generated as an internal control for reverse transcription and amplification. Identical results were obtained using increasing concentrations of competitor versus a fixed amount of cDNA. As seen in Figure 7B and summarized in Figure 7C, eNOS mRNA levels were not different in RCs and LCs isolated from control or ligated mice. Similarly, immunoblotting showed no changes in eNOS levels relative to platelet-endothelial cell adhesion molecule (PECAM) (a protein expressed exclusively in the endothelium of blood vessels), Hsp90, and Akt in RCs and LCs from control or ligated mice (Figure 7D). However, the expression of ß-actin was markedly increased in remodeled LCs at 7 and 14 days after ligation, consistent with migration and reorganization of vascular cells during arterial remodeling.

View larger version (39K): [in this window] [in a new window]   Figure 7. Carotid arterial eNOS mRNA and protein levels do not change in response to vascular remodeling. A, Ethidium-stained gel demonstrating that increasing amounts of eNOS cDNA linearly competes with the eNOS-C cDNA in an RT-PCR reaction. B, Typical experiment comparing eNOS mRNA levels in single RC and LC isolated from control and ligated mice (7-day ligation). C, Densitometric analysis of the eNOS levels from individual carotid arteries compared with a standard curve demonstrate no differences in eNOS mRNA levels in control RC and LC nor in RC and 7-day remodeled LC. n=5 individual carotid arteries (RC and LC) from 5 mice per group. D, Western blot analysis was performed on tissue homogenates (60 µg) from 4 pooled RC and LC with the indicated antisera. Changes in vascular architecture during LC remodeling are associated with enhanced levels of ß-actin but no difference in eNOS, PECAM, Hsp90, or Akt protein levels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study demonstrates the temporal relationship between a change in blood flow, cell death, vascular remodeling, and vascular function in carotid arteries of mice. Here we show that chronic flow reduction triggers a VD-sensitive decrease in lumen diameter followed by a more permanent, VD-insensitive change in vascular architecture. The permanent luminal remodeling occurs contemporaneously with an increase in cell death. Concomitant with the permanent architectural changes is a decrease in basal, but not stimulated, NO release, suggesting an interrelationship between the structural and functional changes in the vessel wall during low-flow remodeling.

The major goal of the present study was to fully characterize low-flow stimulated vascular remodeling in a genetically tractable species such as the mouse. Ligation of the left EC completely eliminates the perfusion field, thus markedly decreasing blood flow in the ipsilateral LC but not in the contralateral RC. The unperturbed hemodynamics and lumen diameters in the RC relative to control mice permit the RC to serve as an internal control for any morphological changes in the LC. Consistent with observations in rabbit,¹⁷ the decrease in lumen diameter after 3 days of EC ligation is reversed by perfusion of a VD cocktail (consisting of adenosine and papaverine). The reversible changes in vessel diameter are observed only in LCs, but not in RCs, consistent with an increase in vasomotor tone before structural changes during low-flow remodeling. At day 7 after ligation and onward, structural remodeling occurs, because the reduction in lumen diameter triggered by the remodeling stimulus is not influenced by VDs in the perfusate. Collectively, these data support the idea that the summation of many short-term vasomotor events (ie, persistent vasoconstriction) will initially lead to a reversible diameter reduction that is followed by a more permanent reduction in lumen diameter and, thus, structural remodeling.

Although lumen diameters were reduced in remodeled LCs (at 7 and 14 days after ligation), increases in isometric tension in response to PGF₂ were virtually identical compared with nonremodeled LCs or RCs. This suggests that the cellular rearrangements and apoptosis leading to a reduced vessel size in response to a chronic hemodynamic change do not significantly influence the contractile machinery in remodeled carotid arteries. This is in contrast to a recent study in low-flow, remodeled rat mesenteric arteries in which diameter changes to exogenous norepinephrine were markedly diminished, whereas the responses to arginine vasopressin were identical in sham and remodeled arteries.¹⁹ However, in our experiments, by day 7 after ligation, ß-actin expression increased in remodeled LCs compared with contralateral RCs and controls, indicative of structural reorganization of the blood vessel, consistent with observations in cultured vascular smooth muscle cells exposed to angiotensin II and arginine vasopression,²⁰ the neointima of aortic vascular smooth muscle cells after balloon injury,²¹ and human atheromatous plaques.²²

Functional responses to Ach were not different between RCs and remodeled LCs, indicating that agonist-triggered endothelium-derived NO release and bioactivity is intact in remodeled arteries. However, vasodilation induced by the NO donor drug SNP is increased in LCs (day 7), reminiscent of the ability of endothelial denudation, chronic NOS inhibition,²³ or eNOS deficiency due to disruption of the gene,^{18 24} to increase vasorelaxant responses to NO donors. The mechanisms for enhanced responsiveness to NO donors are not clear, but they may be mediated through regulation of soluble guanylyl cyclase expression/activation or via NO-stimulated, non–cGMP-dependent mechanisms of vasorelaxation. Interestingly, in the face of normal Ach-stimulated relaxations and enhanced SNP responses in LCs, basal NO-dependent tone is reduced in remodeled LCs at day 7 and begins to normalize by day 14. Our results demonstrating a deficit in basal, but not stimulated, release of endothelium-derived NO in remodeled carotid arteries are similar to findings in aortic rings from estrogen receptor knockout (ERKO) mice.²⁵ In remodeled LCs, the deficit in basal NO production occurred without changes in eNOS mRNA or protein levels, which is also consistent with the above studies in ERKO mice. Furthermore, the dissociation between basal and stimulated NO production has been observed in human studies. Long-term cigarette smokers showed a reduction in basal NO production with no differences in Ach-stimulated NO production,²⁶ suggesting that endothelial dysfunction may precede the appearance of symptomatic cardiovascular disease.

With these findings in mind, what are the potential mechanisms that can account for differential suppression of basal, but not Ach-stimulated, relaxations in remodeled vessels? Although Ach can trigger the release of several endothelium-derived vasodilatory substances (NO, prostaglandins, and endothelium-derived hyperpolarizing factors) to compensate for the loss of basal NOS activity in remodeled vessels, this is unlikely in our studies for the following reasons: (1) eNOS mRNA and protein levels are not different in control carotid arteries versus RCs or remodeled LCs; (2) ibuprofen, a cyclooxygenase inhibitor (used at a concentration that completely suppresses Ach-driven prostaglandin I₂ production in mouse aortic rings [M. Bucci, J.P. Gratton, and W.C. Sessa, unpublished observations, 1999]), was present in the Krebs solution; and (3) L-NAME inhibited Ach-mediated vasorelaxations to the same extent in both control RCs and remodeled LCs.

An alternative possibility is that post-translational control of eNOS is influenced by a remodeling stimulus, thereby blunting basal, but not stimulated, eNOS activation. Indeed, eNOS can be negatively regulated by interactions with caveolin-1²⁷ and intracellular domains of several G protein–coupled receptors²⁸ and positively regulated via interaction with Hsp90²⁹ and phosphorylation by the serine kinase Akt (protein kinase B)^{30 31} and AMP kinase.³² The levels of caveolin-1 (not shown), Hsp90, and Akt are not different in extracts from RCs and LCs, arguing against an overall change in the expression of these regulatory proteins. Whether remodeling influences the complex interaction between these proteins is not known. Fleming et al³³ have shown that L-NAME–induced increases in vascular tone occur independently of changes in cytoplasmic calcium, whereas Ach-induced NO requires a calcium flux. This finding, and recent observations that Akt or AMP kinase phosphorylation of eNOS stimulates NO synthesis at resting levels of calcium, suggests potential dysregulation of these pathways in remodeled vessels. However, to date, all of the above post-translational control mechanisms do not appear to unequivocally discriminate basal from agonist-dependent NO production.

In conclusion, we show that in normal mice flow-initiated vascular remodeling is accompanied by medial cell death associated with endothelial dysfunction. These findings raise the possibility that other remodeling events that occur secondary to an atherosclerotic lesion, after angioplasty or vein grafting, can trigger local endothelial dysfunction in the vessel undergoing active remodeling. In this scenario, the loss of NO may synergize with other etiologic factors present during the disease process, thus leading to noncompensated remodeling and progression of vascular disease.

	Acknowledgments

 
This work was supported by NIH Grants HL51948 and HL50974 (to W.C.S.) and HL10183 (to D.F.) and a Grant-in-Aid from the American Heart Association. W.C.S. and S.S.S. are Established Investigators of the American Heart Association. We thank Dr Phil Marsden for the murine eNOS cDNA, Dr Vijay Shah for help with the Western blot analysis, Dr Joseph A. Madri for PECAM antibody, and Dr Ray Fontana (Multi-Arc, Inc) for the zirconium nitride coating of mortar and pestle sets.

Received March 14, 2000; accepted April 14, 2000.

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