Remodeling With Neointima Formation in the Mouse Carotid Artery After Cessation of Blood Flow
Abstract The ability of gene targeting in the mouse species presents a powerful tool to determine the role of specific molecules in vascular biology. Using a denuding-injury procedure, we recently reported that intimal lesions can be induced in the carotid artery of outbred mice. The technical challenge associated with achieving complete denudation and the relatively small size of the developing lesions prompted us to design the present model of neointima formation and vascular remodeling in the carotid artery of the inbred FVB mouse strain. Complete ligation of the vessel near the carotid bifurcation induced rapid proliferation of medial smooth muscle cells, leading to extensive neointima formation in the presence of an endothelial lining. Thrombus formation was not observed except in the most distal part of the vessel adjacent to the ligature. At 4 weeks after ligation, luminal area was reduced by ≈80% through a combination of decreased vessel diameter and neointima formation. Ultrastructural analysis provided evidence for cell death in the developing neointima as well as the remodeling media. The present model might be useful in identifying those genes important for neointima formation and vascular remodeling.
- Received January 22, 1997.
- Accepted April 8, 1997.
The ability to introduce transgenes or to disrupt endogenous gene expression has made the mouse an attractive species with which to study the function of specific genes in vascular biology. Unlike that in the rat, the study of SMC proliferation and intimal lesion formation in mice has been difficult owing to the lack of suitable experimental models. A few years ago, we developed a model in which the common carotid artery of mice was denuded with a flexible wire.1 Using an outbred strain, we found that within 2 weeks after injury, intimal lesions would form in areas that were still denuded. These intimal lesions, however, were not very extensive and usually did not exceed two or three cell layers in thickness. The relatively small proliferative response and the technical challenge to achieve complete denudation prompted us to explore other ways to induce extensive SMC proliferation in a reproducible manner.
A reduction in blood flow has been shown to increase intimal lesion formation in vascular grafts and balloon-injured vessels,2 3 4 thus indicating that alterations in blood flow will affect the proliferative response of SMCs. Furthermore, a number of studies have demonstrated that vessels adapt to chronic changes in blood flow by undergoing compensatory adjustments in their lumen size.5 6 Together these findings stimulated the development of the murine model presented here, in which blood flow in the common carotid artery was disrupted by ligating the vessel near the distal bifurcation. These ligated vessels did not contain clots, and pulsation was present at all times. Luminal narrowing occurred by formation of an extensive SMC-rich neointima and a reduction in vessel diameter. This model might be useful for studying events in SMC proliferation and vascular remodeling at the molecular level.
All animal studies were approved by the Institutional Animal Care and Use Committee. Forty female FVB mice (3 to 4 months old; The Jackson Laboratory, Bar Harbor, Me) weighing 22 to 30 g were used in all experiments. The animals were anesthetized by intraperitoneal injection of a solution of xylazine (5 mg/kg body weight, AnaSed; Lloyd Laboratories) and ketamine (80 mg/kg body weight, Ketaset; Aveco Co, Inc). The left common carotid artery was dissected and ligated near the carotid bifurcation. All animals recovered and showed no symptoms of a stroke. Groups of animals were killed at 0 and 3 hours and 2, 5, 8, 11, 14, 18, 21, and 28 days after ligation of the carotid artery. At least four mice were killed per time point. All animals received two subcutaneous injections of the thymidine analogue BrdU (2.5 mg per injection; Boehringer Mannheim). These injections were given 12 hours and 1 hour before the mice were killed. All animals were fixed for 3 minutes by perfusion with 4% p-formaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.3) as described.1 After excision of the left and right carotid arteries, the vessels were immersion fixed in 70% ethanol. Vessels used for electron microscopy were perfusion fixed with phosphate-buffered glutaraldehyde/p-formaldehyde (1%:4%, vol/vol). The common carotid arteries were ≈9 mm long, of which the proximal and distal 2 mm were discarded and the remaining portion (≈5 mm) was cut in half. The two segments were embedded in paraffin, and serial sections (5 μm thick) were cut for analysis by immunostaining and hematoxylin-eosin staining for morphometry. Five or more sections spanning most of the vessel segment were analyzed for morphometry.
Biotinylated secondary antibodies and the ABC-Elite kit (Vector Labs, Inc) with 3,3′-diaminobenzidine as the color substrate were used for all staining runs. Between incubations with antibodies and reagents of the ABC kit, four washes (5 minutes each) with TBS (0.8% NaCl, 25 mmol/L Tris; pH 7.6) were carried out. Controls with nonimmune immunoglobulins matching in species and concentration were run in parallel with every antibody staining experiment.
The SMC replication indices in the media and intima were determined by staining the sections with a mouse monoclonal antibody against BrdU (1:200 dilution, Cappel). Before application of the antibody, the sections were treated with pepsin (0.1 mg/mL in 0.1N HCl) for 30 minutes at 37°C, washed with distilled water, and incubated in 1.5N HCl at 37°C for 15 minutes. The sections were rinsed in distilled water, washed twice for 5 minutes in 0.1 mol/L sodium tetraborate buffer (Borax, pH 8.5), and washed with TBS for 5 minutes before application of the anti-BrdU antibody (1 hour at 37°C). After three washes with TBS (5 minutes each), the sections were incubated for 30 minutes at room temperature with a biotinylated horse anti-mouse IgG (1:1000 dilution, Vector). Subsequent steps were carried out as described,7 except that the color reaction with diaminobenzidine was stopped after 30 to 45 seconds. The numbers of total and stained nuclei were counted separately for the media and intima, and the BrdU labeling indices [(stained nuclei/total nuclei)×100] were calculated for both the media and intima. At least three sections were analyzed per animal.
One question that we wished to answer was whether the endothelium would remain on the luminal surface of the carotid artery during intimal lesion formation. In addition to the ultrastructural analysis described below, a rabbit polyclonal antibody against factor VIII–related antigen (von Willebrand factor) was used to identify ECs. This antibody (Dako) was used at a 1:250 dilution, and subsequent steps of the staining protocol were carried out as described.8 At least 12 sections from each animal were stained.
The effects of carotid artery ligation on EC replication were determined in the ligated vessel as well as the contralateral carotid artery at 2, 5, and 8 days after ligation. The animals were injected with BrdU as described above, and the vessels were stained en face with the anti-BrdU antibody. Whole mounts were prepared, and the total number as well as the number of labeled endothelial nuclei was counted. Results were expressed as a replication index as described for SMCs.
The presence of inflammatory cells in this model was determined by using a rat monoclonal antibody against the mouse leukocyte common antigen CD45 (1:100 dilution, PharMingen). Mouse monocytes/macrophages were identified by immunostaining with a specific rat monoclonal antibody (1:200 dilution, BMA BM8; Accurate Chemical and Scientific Corp). A biotinylated mouse anti-rat antibody (adsorbed against mouse serum proteins, Jackson ImmunoResearch) was used at a 1:100 dilution. At least 12 sections from each animal were analyzed.
SMCs were identified by staining with a mouse monoclonal antibody specific for α-smooth muscle actin (clone 1A4, Sigma). This antibody was used at a 1:8000 dilution. A biotinylated horse anti-mouse immunoglobulin (Vector) was used as the secondary antibody at a 1:1000 dilution. At least 12 sections from each animal were stained.
Morphometric analysis was carried out on carotid arteries from unmanipulated mice and on carotid arteries harvested 4 weeks after ligation. In some animals morphometric analysis was also carried out on the contralateral vessel of the ligated carotid artery 4 weeks after ligation. All animals were perfusion fixed under physiological pressure. Digitized images of these vessels were analyzed with image analysis software for the Apple Macintosh computer (NIH Image 1.60). The circumferences (lengths) of the lumen, IEL, and EEL were determined by tracing along the luminal surface, IEL, and EEL. Under the assumption that the structures were circular, these measurements were used to calculate luminal area, intimal area, and medial area. The medial area was calculated by subtracting the area defined by the IEL from the area defined by the EEL, and intimal area was determined by subtracting the luminal area from the area defined by the IEL.
Student’s t test was used to compare the values between normal vessels and ligated vessels (intimal area, luminal area). ANOVA followed by Scheffés F test were used to compare the means of multiple groups (cell number). Means were considered significantly different if P<.05.
Transmission Electron Microscopy
For ultrastructural analysis, ligated carotid arteries from two or three animals per time point (2, 5, 8, 14, and 28 days after ligation) were examined. After perfusion fixation and immersion fixation with phosphate-buffered glutaraldehyde/p-formaldehyde (1%:4%, vol/vol), the specimens were placed in 1% OsO4 for 2 hours, dehydrated en bloc, stained with 3% uranyl acetate, and embedded in Epon. Thin sections were cut and poststained with uranyl acetate and Reynolds’ lead citrate.
Morphological Changes After Cessation of Blood Flow
The common carotid artery in mice does not have side branches, and thus, vessels ligated near the bifurcation will have no net forward flow. Vessels were obtained from mice 4 weeks after ligation of the common carotid artery, and because of the ligature, blood components were still present in the lumen after perfusion fixation (Fig 1b⇓ and 1c⇓). Sections were cut from the entire length of the vessel. It was found that no intimal lesion had formed within the proximal 2 to 3 mm of the vessel adjacent to the aortic arch. Distal from the arch, the vessels consistently formed a neointima (Fig 1b⇓ and 1c⇓), and for all subsequent studies, sections from the middle portion of the vessel were analyzed. Compared with control vessels (Fig 1a⇓), the entire vessel diameter had decreased (Fig 1c⇓), as quantified by the outer circumference of the media.
The formation of an intimal lesion in response to ligation of the carotid artery suggested that SMC proliferation was a prominent feature of the model. To characterize the time course of SMC replication, mice were injected with BrdU and the replication index determined (Fig 2a⇓). Within 5 days after flow reduction, the SMC replication index in the media averaged 23.4%, and cells present in the intima also revealed high replication rates (27.6%). While cell replication in the media decreased dramatically after 8 days, intimal cells continued to replicate at a high rate until 2 weeks after flow reduction. Replicating SMCs were still found in the intima 4 weeks after carotid ligation.
Because cell death has been demonstrated in other models of intimal hyperplasia,9 10 we determined whether the increased SMC proliferation did indeed result in an increase in cell number by counting the number of cells present in the media and intima at various time points after vessel ligation (Fig 2b⇑). Because the vessels had not been denuded, cell counts in the intima included ECs. Within 2 days after ligation, a marked loss of SMCs from the media was evident. At 8 days and all later time points, the number of medial SMCs was not significantly different from that in control vessels. Cell numbers in the intima increased after ligation of the vessel, reaching the highest levels at 2 weeks. Intimal cell numbers were significantly increased over control values at 2 and 4 weeks after flow reduction.
The changes in vessel wall geometry in response to flow reduction were determined by measuring the luminal area of carotid arteries harvested 4 weeks after vessel ligation (Fig 3a⇓). The reduction in luminal area averaged nearly 80% in the ligated vessels compared with that in control vessels. To further determine whether this reduction in luminal area was the result of intimal lesion formation, vessel constriction, or both, we measured the intimal area as well as the outer circumference of the vessel as determined by the EEL. As shown in Fig 3b⇓, there was an ≈25% reduction in the circumference of the ligated vessel. This decrease in circumference represented a fixed change in vessel structure as opposed to active SMC contraction, since topical application of nitroglycerin prior to perfusion fixation did not relax the vessel (data not shown). In addition to the decrease in vessel diameter, there was also a thick intimal lesion (Fig 3c⇓) that led to narrowing of the lumen. Therefore, the adjustment of luminal area in response to the loss of net flow occurred due to both a decrease in vessel diameter and neointima formation. There was also a significant increase in cross-sectional area of the media in the ligated vessel (Fig 3d⇓). No significant change in vessel diameter was seen in the contralateral, unligated right carotid artery 4 weeks after ligation compared with vessels from unmanipulated animals (Fig 3e⇓).
Morphology and Immunocytochemistry
The mice were killed at various time points after ligation of the left common carotid artery to analyze the cellular composition and morphological changes in the vessel in response to blood flow reduction. SMCs were identified with an antibody against α-smooth muscle actin. Similar to the findings reported previously for proliferating SMC in the denuded mouse carotid artery,1 immunoreactivity for α-smooth muscle actin was reduced when SMCs were rapidly replicating (5 to 14 days; Fig 4a⇓ and 4b⇓). The mature 4-week-old intimal lesion, however, stained strongly for α-smooth muscle actin (Fig 4c⇓).
Inflammatory cells identified with an antibody directed against the common leukocyte antigen CD45 were frequently found in the adventitia and outer layer of the media (Fig 4d⇑). Some leukocytes were also present in the developing intima and near the luminal surface. At 4 weeks after ligation, inflammatory cells were usually not detectable. Immunostaining with an antibody that recognizes mouse macrophages and monocytes revealed similar results, indicating that the majority of inflammatory cells present were derived from this cell lineage.
The presence of endothelium on the luminal surface was verified by immunostaining for von Willebrand factor (factor VIII–related antigen). Whole mounts of ligated and contralateral carotid arteries were prepared and stained en face with an anti-BrdU antibody to determine EC replication. No replicating cells were seen in normal vessels, but increased EC replication was apparent in vessels 5 and 8 days after ligation (Fig 5⇓). Examination of endothelial replication at later time points was hampered by increased narrowing of the lumen, which made it difficult to prepare whole mounts. Although the intimal lesions were covered by the endothelium (Fig 4e⇑), it was frequently found to have become detached from the underlying IEL, thereby forming spaces that were filled with red blood cells (Fig 4b⇑ and Fig 6a⇓). By transmission electron microscopy, evidence for SMC death was apparent, as indicated by the presence of condensed nuclei and vacuolized cytoplasm (Fig 6b⇓). Cells undergoing cell death were found in both the media and neointima. The electron micrograph in Fig 6c⇓ shows a thick, intimal lesion on top of a contracted IEL 4 weeks after ligation.
Proliferation of SMCs plays an important role in the failure of angioplasty procedures; more recently, the remodeling of vascular structures has also been implicated in restenosis after angioplasty.11 12 13 To study the molecular mechanisms that contribute to SMC proliferation and remodeling, the present study sought to establish an animal model in the mouse because this species is genetically well characterized and can be genetically manipulated. The chances of identifying key factors are more likely to succeed in a model that is aimed toward maximizing the adaptive changes and proliferative responses of SMCs. In the model described here, we chose to completely disrupt blood flow in the common carotid artery by ligation near the bifurcation, thus causing the lumen to decrease to ≈20% of its original size. Because the ligated vessel was still subject to arterial blood pressure, pulsation persisted in these vessels. With the exception of the most distal part of the vessel (within 1 to 2 mm of the ligature), clot formation did not occur within 1 to 2 mm of the ligature. However, when complete endothelial denudation was performed before ligation of the vessel, an occluding thrombus formed over the entire length of the carotid artery (data not shown). Thus, one role of the endothelium in this model is to maintain a nonthrombogenic surface. We observed two mechanisms that contributed to luminal narrowing in this model: one was by constriction or shrinkage of the vessel diameter and the other by neointima formation. Initial constriction has been described as a vasoactive response14 that is thought to depend on the presence of the endothelium.6 The fact that this constriction could not be reversed by topical application of nitroglycerin after 4 weeks demonstrates a fixed change in the vessel wall structure. The endothelium was present at all times, as evident by the presence of von Willebrand factor–immunoreactive cells on the luminal surface. Focal detachment of ECs from the underlying IEL, however, was observed during lesion formation. Detachment of the endothelium may in part result from persistent vessel constriction, as indicated by the presence of “wavy” elastic laminas. It is conceivable that endothelial detachment may lead to discontinuities in the endothelial sheet, which may allow penetration of blood cells and platelets into the subendothelial space (Fig 5a⇑). Large-scale endothelial degeneration and denudation have been reported in rat models of vessel segments isolated between two ligatures.15 16 17 These models, however, differ from the one described here in that they were no longer exposed to hemodynamic forces.
When SMC replication rates and numbers in the intima are compared at 14 and 28 days, it is apparent that there is no further increase in cell number in the intima despite the fact that the intimal cell replication index is still ≈11% at 4 weeks. Between days 5 and 14, cell replication rate in the intima exceeded 22% per day. This would amount to an approximate sixfold increase in cell number (1.229) during this 9-day period, an amount that was considerably more than actually determined by counting cell numbers in the intima. Thus, these data strongly argue for ongoing cell death and turnover. Apoptosis has also been reported in the balloon-injured rat carotid artery,9 10 in which we and others have observed numerous inflammatory cells, particularly in the adventitia (Lindner, 1995, unpublished data).18 Therefore, the presence of inflammatory cells in the present model does not necessarily contradict ongoing apoptosis, despite the fact that apoptosis is not usually associated with inflammation.
In our mouse model the endothelium was not removed, and it is possible that the altered flow conditions might have played a role in expression of endothelial-leukocyte adhesion molecules and chemokines, with a subsequent influx of inflammatory cells. This explanation is supported by a recent study by Walpola and coworkers,19 who demonstrated that a reduction in flow caused upregulation of vascular cell adhesion molecule-1 expression in a rabbit model. It was interesting to note that a neointima did not develop in the proximal part of the ligated vessel adjacent to the aortic arch. Because the common carotid artery is an elastic vessel and does not possess side branches, it is likely that oscillations of blood flow and therefore shear stress (bidirectional) are higher at this location than at regions closer to the ligature. The near-stasis conditions in more distal segments of the ligated vessel may have additional implications for platelet and leukocyte activation, which in turn may affect SMC proliferation at this site. Complete blood stasis is more likely to prevail only in the immediate vicinity of the ligature where thrombus formation is seen.
The initial loss of SMCs from the media 2 days after ligation raises the question of whether hypoxia may have been a contributing factor. Although we cannot rule out this possibility, there are several reasons that argue against it. One is that a thick neointima can be supported in the absence of flow, and second, blood within the ligated vessels consistently had the color of arterial blood. A significant amount of cell death can also be expected to occur in the endothelial population of the ligated vessel, since replication of these cells was increased despite the fact that the reduction in luminal area would require a net loss of ECs.
The histology of neointima formation in a single-ligation model of the rat carotid artery has previously been described by Wexler.20 The morphological findings reported for normotensive rats are similar to those that we observed in the mouse model with regard to intimal lesion formation. These models differ from others in that they do not require mechanical trauma and widespread endothelial denudation to induce SMC proliferation. For example, in the balloon-injury model of the rat carotid artery, proliferation of SMCs stops in the denuded vessel as soon as reendothelialization occurs. Similarly, in the denudation model of the mouse carotid artery, no SMC replication was seen underneath regenerated endothelium.1 On the other hand, development of intimal lesions in atherosclerosis models and in human vascular disease occurs in the absence of noticeable endothelial denudation.21 The model presented here may not mimic a physiological situation. However, it should be pointed out that vascular lesions in humans often develop at sites of altered hemodynamics associated with low shear stress.22 23 Therefore, it is conceivable that the factors responsible for intimal lesion formation at these sites might differ from those involved in intimal hyperplasia after arterial injury associated with endothelial denudation. Further studies are required to determine how the present model relates to clinical situations of vascular disease and restenosis in humans.
In summary, the present study describes a model of vascular remodeling in the FVB mouse strain that is characterized by rapid proliferation of SMCs in an endothelialized artery in response to cessation of mean flow. It should be a useful model to study events in SMC proliferation and vascular remodeling at the molecular level.
Selected Abbreviations and Acronyms
|EEL||=||external elastic lamina|
|IEL||=||internal elastic lamina|
|SMC||=||smooth muscle cell|
This work was supported by a Grant-in-Aid from the American Heart Association and funds contributed in part by the American Heart Association, Alaska Affiliate, Inc, awarded to Dr Lindner. The excellent technical assistance provided by Veronica Poppa and Rei Port is greatly appreciated.
Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ Res. 1993;73:792-796.
Geary RL, Kohler TR, Vergel S, Kirkman TR, Clowes AW. Time course of flow-induced smooth muscle cell proliferation and intimal thickening in endothelialized baboon vascular grafts. Circ Res. 1994;74:14-23.
Kohler TR, Kirkman TR, Kraiss LW, Zierler BK, Clowes AW. Increased blood flow inhibits neointimal hyperplasia in endothelialized vascular grafts. Circ Res. 1991;69:1557-1565.
Guyton JR, Hartley CJ. Flow restriction of one carotid artery in juvenile rats inhibits growth of arterial diameter. Am J Physiol. 1985;248:H540-H546.
Langille BL, O’Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science. 1986;231:405-407.
Lindner V, Giachelli CM, Schwartz SM, Reidy MA. A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-B chain mRNA. Circ Res. 1995;76:951-957.
Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest. 1990;85:2004-2008.
Mintz G, Popma J, Pichard A, Kent K, Satler L. Mechanisms of late arterial responses to transcatheter therapy: a serial quantitative angiographic and intravascular ultrasound study. Circulation. 1994;90(suppl I):I-24. Abstract.
Losordo DW, Rosenfield K, Kaufman J, Pieczek A, Isner JM. Focal compensatory enlargement of human arteries in response to progressive atherosclerosis: in vivo documentation using intravascular ultrasound. Circulation. 1994;89:2570-2577.
Buck RC. Intimal thickening after ligature of arteries: an electron-microscopic study. Circ Res. 1961;9:418-426.
Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler Thromb. 1995;15:2-10.
Wexler BC. Histopathological reactivity of carotid arteries of normotensive Sprague-Dawley vs spontaneously hypertensive rats to ligation injury. Stroke. 1979;10:674-679.
Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis. 1985;5:293-302.
Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990;66:1045-1066.