Vascular remodeling is an important pathologic process in vascular injury for various vascular disorders such as atherosclerosis, postangioplasty restenosis and transplant arteriopathy, where the presence of hypercholesterolemia affects the development of the lesions.1,2 Hypercholesterolemia is a major risk factor that is associated with atherosclerotic disease and can modify the structure and function of the arterial wall in a manner that is consistent with an inflammatory response. The typical fatty streak, a characteristic vascular lesion of atherosclerosis, consists of macrophages, smooth muscle cells (SMCs), and varying numbers of T-lymphocytes that are recruited to the lesion site in response to chronic hypercholesterolemia.3
Recent evidence has shown that vascular function depends not only on cells within the vessels, but is also significantly modulated by circulating cells derived from the bone marrow. The roles of bone marrow (BM)-derived endothelial cells and SMCs have been extensively studied in the pathogenesis of atherosclerosis and restenosis.4-6 Our previous study has shown that BM-derived cells can differentiate into macrophage, SMCs, and endothelial cells (ECs) in the cuff-induced vascular remodeling lesion in C57BL/6 mice.7 However, the lesion formation was just occurred in the adventitia in the cuffed femoral artery in that normalcholesterolemic mouse model. Thus, we could not determine the role of BM-derived cells in the lesion formation in the intima after vascular injury. That study has left a question behind: whether hypercholesterolemia could affect the lesion formation and the mobilization of BM-derived cells after vascular injury.
To address this question, we established a new mouse model lacking low density lipoprotein receptor gene (LDLR-/-) and ubiquitously expressing green fluorescent protein (GFP+). We have utilized a vascular injury model induced by polyethylene cuff placement around the femoral artery after BM transplantation (BMT) from LDLR-/- and GFP+ mice to LDL-/- mice and tried to answer the question how hypercholesterolemia can modify the lesion formation in cuff-induced vascular remodeling.
All experimental protocols were performed in accordance with the guidelines of Kyoto University and Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medical College. Low density lipoprotein receptor knockout (LDLR-/-) mice with C57B1/6 background were a generous gift from Dr. S. Ishibashi (Jichi Medical School, Saitama, Japan). GFP transgenic mice with C57B1/6 background were a generous gift from Dr. M. Okabe (Osaka University, Osaka, Japan). The mice were housed and bred under specific pathogen-free conditions at the animal facility, kept in a temperature-controlled facility on a 14 hours light/10 hours dark cycle, with free access to food and water.
To obtain the GFP+ and LDLR-/- double donor mice for BMT, we bred GFP transgenic mice with LDLR-/- mice and screened using a portable UV checker (Model UVGL-58, USA) and PCR reaction. After being weaned at 4 weeks of age, mice were fed a normal chow diet (CMF, containing 8.7% (wt/wt) fat and 0.063% (wt/wt) cholesterol; Oriental Yeast, Chiba, Japan) until BMT.
Twenty two week-old female LDLR-/- mice were subjected to total body irradiation (9 Gy) using the Gammacell 40 Exactor Irradiator (Nordion International Inc., Ottawa, Canada). BM cell suspensions were isolated by pushing the femurs from GFP+ and LDL-/- double male and female mice. BM cell suspensions were prepared by passing the cells through a 70 μm nylon gauze. Each irradiated recipient received 5×105 bone marrow cells in 0.5 ml PBS by intravenous injection into the tail vein. While the LDLR-/- mice were subject to total body irradiation and perform BMT, they were switched to a high-fat diet containing 1.25% cholesterol, 7.5% (wt/wt) cocoa butter, 7.5% casein, and 0.5% (wt/wt) sodium cholate (Oriental Yeast) until the end of the experiment period. Drinking water was supplied with 0.1% hydrochloric acid. Four weeks after BMT, the recipient mice were phlebotomized and the circulating leukocytes were then checked for the expression of GFP by flow cytometry. Cuff placement was performed at least 4 weeks after BMT.
The surgical procedure of cuff placement was done according to the method as described below. Mice were anesthetized with barbiturate complex (propylene glycol 17.9% (v/v), ethanol 8.9% (v/v), sodium 5-ethyl-5-(1-methylbutyl) barbiturate 10.7% (v/v)). The right femoral artery was dissected from its surroundings. A non-constrictive polyethylene cuff (PE50, 0.58 mm inner diameter, 0.965 mm outer diameter, 2 mm length. Becton Dickinson Co., Sparks, NV, USA) was placed loosely around the right femoral artery. The control left femoral artery underwent isolation from the surrounding tissues without cuff placement.
Measurement of plasma cholesterol levels
Blood samples were taken under general anesthesia from the tail vein at the time of operation and euthanization. Total plasma cholesterol concentrations were measured enzymatically using commercially available kits (TOYOBO Co., LTD., Japan)
Tissue preparation and immunocytochemistry
At euthanization, mice were anesthetized with barbiturate complex. Mouse thorax was opened and physiological pressure-perfusion-fixation (100 mmHg) with 4% paraformaldehyde in PBS for 10 minutes was performed by cardiac puncture. After the experiments, the mice were killed and bilateral femoral arteries were harvested. The tissue was snap-frozen in O.C.T. Compound (Sakura Finetek USA, Inc. Torrance, CA, USA). Serial cross sections (6-μm-thick) were obtained throughout the entire length of the cuffed femoral artery or equivalent portion of the contralateral artery for histological analysis.
Macrophages were detected in cuff-induced vascular injury lesion by immunohistochemistry with rat monoclonal antibody (mAb) MOMA-2 macrophage marker (BMA Biochemicals AG, Augst, Switzerland). Subsequently, sections were incubated with goat anti-rat IgG (BD science). Finally the sections were exposed to avidin and biotin complex (Vectastain ABC Kit, PK6100. Vector Laboratories) for 30 minutes for amplifying the signals. Lipid deposition was visualized with Oil red-O (Boehringer Mannheim) staining. For SMC staining, we used mouse monoclonal anti-human smooth muscle α-actin (SMA) antibody (clone 1A4) labeled with Cy3 (Sigma, St Louis, MO, USA).
Endothelial cells were identified by immunohistochemical staining with biotin-conjugated rat anti-mouse CD31 antibody (Southern Biotech). For CD31 staining, Tyramide Signal Amplification system (TSA Kit #24 with HRP-streptavidin and Alexa Flour 568 tyramide. Molecular Probes Europe BV, Netherlands) was employed to increase antigenicity of ECs.
TUNEL assay to detect cuff-induced apoptosis
The apoptotic cells were detected with TUNEL assay using VasoTACS in situ apoptosis detection kit (Trevigen, Inc.), according to the specifications and instructions recommended by the manufacturer. Briefly, after being washed in PBS, sections were incubated with proteinase K solution for 15 minutes and immerse slides in 3% H2O2 in methanol for 5 minutes. For detecting apoptotic DNA fragments in cuff-induced vascular remodeling cells, 50 μl of TUNEL reaction mixture (biotinylated TdT dNTP mix, 50×Mn2, TDT enzyme, 1×TDT labeling buffer) was added to each of them and incubate for 1 hour at 37°C. Then cover sample with 50 μl of Strep-HRP Solution (dilute Strep-HPP in Blue Streptavidin-diluent, 1:800) for 10 minutes at room temperature. Finally, visualized by Blue Label Solution for 10 minutes and counterstained with Red Counterstain C.
Quantification of vascular remodeling lesions
Atherosclerotic-like lesions in femoral vascular remodeling was evaluated for oil red O staining by Image-Pro Plus (Media Cybernetics), as previously described.8,9 Eight equally spaced cross sections were used in all mice to quantify lesions. The fraction areas of the lesion stained by oil red O were calculated by dividing the whole vessel areas including the lumen, intima, media, and adventitia, as previously described.10 The mean of the fraction area was calculated and expressed as a percentage.
Data are expressed as mean ± SD and were analyzed by analysis of variance (ANOVA) with SPSS. P < 0.05 was considered significantly different.
Accumulation of BM-derived progenitor cells in cuff-induced vascular remodeling
We used LDLR-/- mice as the recipient and performed cuff placement to examine the role of BM-derived cells on vascular injury. To eliminate the effect of the LDL receptor in recipient mice during vascular remodeling after BMT, we generated LDLR-/- mice which transgenically express GFP as a donor.
BM-cells from donor mice were transplanted into lethally irradiated LDLR-/- mice before cuff placement. After 4 weeks of BMT, we confirmed the reconstitution of the hematopoietic system by checking the fluorescence of blood leukocytes by flow cytometry. We found that more than 85% of the cells were positive for GFP (data not shown), indicating that most of the leukocytes were derived from the donor BM. After 2 weeks of cuff placement, cuffed or sham-operated femoral arteries were examined under a fluorescence microscope. In the cuffed artery, most of the cells accumulating in the lesion were GFP-positive (Figures 1A), suggesting that those cells were derived from the donor BM. In contrast, in the sham-operated artery, GFP-positive cells were hardly detected (Figure 1B). Although numerous GFP-positive cells were involved in the lesion formation, we found no significant change of these cells at the time points of 1 and 2 weeks after cuff placement (data not shown).
Plasma lipid levels and remodeling lesions
As shown in Figure 2A, total plasma cholesterol levels in the mice fed normal chow were (285.3 ± 19.9) mg/dl (before), (287.8 ± 22.1) mg/dl (after 2 weeks), and those fed a high fat diet were (288.0 ± 14.0) mg/dl (before) and (2652.0 ± 102.7) mg/dl (after 2 weeks). These data indicated that the total plasma cholesterol levels were significantly increased after high-fat diet (n=4, P<0.01 vs normal chow diet). Corresponding to the increased cholesterol levels, the fractions of the cross-sectional area covered by remodeling lesions were significantly larger in mice fed a high-fat diet than those fed a normal chow diet ((23.0 ± 8.68)% vs (2.11 ± 1.17)%, P<0.01, n=4 in each group, Figure 2B). These results indicated that hypercholesterolemia could increase intimal lesion formation in cuff-induced vascular injury in a short time period.
Cuff placement induced foam cell formation in the intima and media in LDL-/- mice
In this study, we found that atherosclerotic-like lesions, predominantly consisting of a massive accumulation of macrophage foam cells in the intima and media at 2 weeks after cuff placement (Figure 3B). Although we did not find a visible change of intimal thickening after cuff placement in C57B1/6 mice in our previous study, this finding clearly showed that hypercholesterolemia was a key factor for the intimal lesion formation after cuff placement. However, in the earlier time point at 1 week after cuff placement, we could hardly detect any neointima formation, where we just observed some foam cells in the adventitia (Figure 3A). We also found that the media was hyperplastic and clearly stained with Oil Red O (Figure 3B), indicating a plenty of cholesterol accumulation in the media while the intima thickening was formed 2 weeks after cuff placement. The cell number in the media was markedly reduced and the cell shape looked different from normal SMCs.
Cuff-induced remodeling lesion was similar to human atherosclerotic plaque
Up to now, there is no suitable animal model of plaque rupture that develops in atherosclerosis. In the present study, we observed that the accumulation of MOMA-2 and Oil Red O positive cells at neointima and media (Figure 4A and 3B). However, 1A4 positive SMCs were localized on top of macrophages (Figure 4B), as a sign of cap formation. The deposition of foam cells and SMCs occurred between the endothelial layer and the inner elastic lamina.
BM cells could differentiate into vascular SMCs
To examine whether BM-derived cells can differentiate into SMCs in the vascular remodeling lesion, we stained the tissue with Cy3-labeled anti-SMA (clone 1A4) antibody. We found a number of 1A4-positive cells in the intimal lesion area (Figures 5B and 5E) and that many 1A4-positive cells were also positive for GFP (Figures 5C and 5F), indicating that BM-derived cells could also differentiate into SMCs in the cuff-induced vascular remodeling lesion.
Endothelial progenitor cells (EPCs) were recruited to the cuffed vascular remodeling lesion
Because it is unknown whether hypercholesterolemia can increase EPC recruitment to the cuff-induced vascular remodeling lesion, we performed endothelial staining by anti-CD31 antibody. We found that the endothelial lining of the intima was clearly stained with anti-CD31 antibody, and that plenty of small vessels in the adventitia were also stained (Figures 6B and 6E). Many clustered cells in small vessels in the adventitia were positive for CD31 and GFP (Figures 6C and 6F), indicating the involvement of angiogenesis from BM-derived cells.
Apoptosis occurred in cuff-induced vascular remodeling lesion
To detect cells undergoing apoptosis after cuff placement, we conducted an in situ TUNEL assay. After 1 or 2 weeks of cuff placement, the intima and media showed a large number of TUNEL-positive signals. Especially, after 2 weeks after cuff placement, apoptotic cells were found in most of the lesion area (Figures 7D, 7E and 7F). In contrast, we could just observe some apoptotic cells in the adventitia 1 week after cuff placement (Figures 7A, 7B and 7C).
Hypercholesterolemia is associated with increased cardiovascular mortality and is known to promote the advancement of atherosclerotic lesions in experimental animal models. In this study, we have shown that hypercholesterolemic conditions in LDL-/- mice can induce intimal lesions after placement of a nonconstrictive polyethylene cuff around the femoral artery. Although other investigators have demonstrated intimal thickening in the cuff-induced vascular injury model,11-13 but we failed to reproduce their results in our earlier experiments in normal C57BL/6 mice.7 Likewise, Ilana et al14 also reported that placing a loose cuff around the carotid artery did not induce detectable changes in medial tissue mass nor constrained the circumference of the native vessels. Hereby, in the present study, we report a successful intimal hyperplasia and vascular remodeling in an atherogenic mouse model in a period of 2 weeks time.
After 1 week of cuff placement in hypercholesterolemic animals, numerous foamy macrophages were observed in the adventitia, indicating that hypercholesterolemia can accelerate foam cells formation and its accumulation in the initial stages of vascular injury. However, during this time period despite the high plasma cholesterol levels the intimal leisions were not evident. But after two weeks of cuff placement, numerous foam cells accumulated in both intima and media. Additionally, atherosclerotic lesions, develop spontaneously in transgenic mice, and are predominantly located in the central part of the arterial tree, therefore it is difficult to apply local antiatherosclerotic strategies. Thus, it is important to induce atherosclerotic-like lesions within 2 weeks, at a predefined, easily accessible site in the arterial tree, with known onset time period. This will allow to analyze the factors responsible for early onset of atherosclerotic lesions and also for assessing the effect of systemic and local antiatherosclerotic therapies. In the present study we have demonstrated that the cuff injury is a suitable model for accelerating the atherosclerotic lesions and also to study the initial steps involved in the atherosclerotic plaque formation.
In the previous study, we observed numerous BM-derived macrophages and recruitment of SMCs to the adventitia following vascular injury in C57BL/6 mice.7 Now we clearly demonstrate that BM-derived macrophages, SMCs, and ECs contribute to the lesion formation after cuff placement. It has been noted that almost all of the SMCs in the intima and most of the ECs in adventitia tissues of the perivascular remodeling lesion are derived from BM. These results indicate that the hypercholesterolemia not only accelerates the intimal lesion formation, but also affects the recruitment of BM-derived cells following vascular injury. Sata et al15 reported that bone marrow serves as the source for arterial SMC upon mechanical vascular injury. Although the role of BM-derived cells in lesion formation in femoral injury in C57BL/6J mice with normal cholesterolemia has been reported by Sata et al, our findings indicated more detail pathologic changes after vascular injury in hypercholesterolemic LDL-/- mice lacking low density lipoprotein receptor gene. Furthermore, the lesion formation occurred in a short time period, and the lesion area is significantly different from C57BL/6 mice undergone mechanical vascular injury. This result indicated our model is favorable to analyze the vascular injury during lesion formation.
In response to injury, BM-derived endothelial progenitors have also been implicated in neointimal formation. Werner et al16 described that bone marrow-derived endothelial progenitors participated in reendothelialization of injured carotid artery in mice. It has been noted by Schmeisser et al17 that BM-derived macrophages may also contribute to neovascularization by in situ transdifferentiation to EC-like cells. In our experiments it was noted that the staining of femoral tissue with anti-CD31 antibody numerous positive cells were detectable in the lesions following 1 week and 2 weeks of cuff placement. Furthermore, most of the cells forming small vessels were also positive for CD31 as well as GFP, indicating that the BM is the source of these ECs in the adventitia present in small vessels.
Recently, apoptosis has been demonstrated in various atherosclerotic lesions.18-20 In this study, after 1 week of cuff placement, almost all of the TUNEL-positive cells in the vascular remodeling lesion were observed in the adventitia. Since, most of the cells in the adventitia were MOMA-2 positive, a marker for macrophages, thus implying that TUNEL-positive cells were derived from macrophages. After 2 weeks of cuff placement, these TUNEL-positive cells appeared from adventitia to media, suggesting that apoptosis has occurred in wide areas in cuff-induced vascular remodeling lesions. Macrophagederived apoptotic cells were primarily present in the adventitia whereas, SMC-derived TUNEL-positive cells were only present in the medial side. These results clearly indicate that apoptosis is actively involved in the development of all phases of vascular remodeling lesion formation, particularly during the early stages.
In summary, atherosclerotic-like lesions can be developed after cuff-induced vascular injury under hypercholesterolemic conditions. Indeed, hypercholesterolemia appears to play an important role in the recruitment of BM-derived cells to vascular remodeling lesions.
1. Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell
proliferation. J Am Coll Cardiol 1990; 15: 1667-1687.
2. Davies MG, Hagen PO. Pathophysiology of vein graft failure: a review. Eur J Vasc Endovasc Surg 1995; 9: 7-18.
3. Ross R. Atherosclerosis: an inflammatory disease. N Eng J Med 1999; 340: 115-126.
4. Han CI, Campbell GR, Campbell JH. Circulating bone marrow
cells can contribute to neointimal formation. J Vasc Res 2001; 38:113-119.
5. Saiura A, Sata M, Hirata Y, Nagai R, Makuuchi M. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 2001; 7: 382-383.
6. Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, et al. Host bone-marrow cells are a source of donor intimal smoothmuscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001; 7: 738-741.
7. Xu Y, Arai H, Zhuge X, Sano H, Murayama T, Yoshimoto M, et al. Role of bone marrow
-derived progenitor cells in cuff-induced vascular injury in mice. Arterioscler Thromb Vasc Biol 2004; 3:477-482.
8. Murayama T, Yokode M, Kataoka H, Imabayashi T, Yoshida H, Sano H, et al. Intraperitoneal administration of anti-c-fms monoclonal antibody prevents initial events of atherogenesis but does not reduce the size of advanced lesions in apolipoprotein E-deficient mice. Circulation 1999; 99: 1740-1746.
9. Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson GK. Immunoglobulin treatment reduces atherosclerosis in apoE knockout mice. J Clin Invest 1998; 102: 910-918.
10. Sano H, Sudo T, Yokode M, Murayama T, Kataoka H, Takakura N, et al. Functional blockade of platelet-derived growth factor receptor-B but not of receptor-a prevents vascular smooth muscle cell
accumulation in fibrous cap lesions in apolipoprotein E-deficient mice. Circulation 2001; 103: 2955-2960.
11. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, et al. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest 1998; 101:1225-1232.
12. Liu HW, Iwai M, Takeda-Matsubara Y, Wu L, Li JM, Okumura M, et al. Effect of estrogen and AT1 receptor blocker on neointima formation. Hypertension 2002; 40: 451-457; discussion 448-450.
13. Suzuki J, Iwai M, Nakagami H, Wu L, Chen R, Sugaya T, et al. Role of angiotensin II-regulated apoptosis through distinct AT1 and AT2 receptors in neointimal formation. Circulation 2002; 106: 847-853.
14. Ilana M, Bayer Isabella Caniggia S, Lee Adamson B, Lowell Langille. Experimental angiogenesis of arterial vasa vasorum. Cell Tissue Res 2002; 307:303-313.
15. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002; 8: 403-409.
16. Werner N, Priller J, Laufs U, Endres M, Bohm M, Dirnagl U, et al. Bone marrow
-derived progenitor cells modulate vascular reendothelialization and neointimal formation: effect of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition. Arterioscler Thromb Vasc Biol 2002; 22: 1567-1572.
17. Schmeisser A, Garlichs CD, Zhang H, Eskafi S, Graffy C, Ludwig J, et al. Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovasc Res 2001; 49: 671-680.
18. Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation 1995; 91: 2703-2711.
19. Durand E, Mamat Z, Addad F, Addad F, Vilde F, Desnos F, et al. Time courses of apoptosis and cell proliferation and their relationship to arterial remodeling and restenosis after angioplasty in an atherosclerotic rabbit model. J Am Coll Cardiol 2002; 39: 1680-1685.
20. Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med Indones 2007; 39: 86-93.