Notch Signaling Regulates Endothelial Progenitor Cell Activity During Recovery From Arterial Injury in Hypercholesterolemic Mice
Background— Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (apolipoprotein E–null [ApoE−/−]) and wild-type (WT) mice to evaluate how cholesterol influences reendothelialization, atherosclerosis, and EPC function after arterial injury.
Methods and Results— Unexpectedly, reendothelialization (assessed by resistance to Evans blue staining) and circulating EPC counts (EPC culture assay) were greater in ApoE−/− mice than in WT mice, and transplantation of ApoE−/− bone marrow in WT mice accelerated endothelial recovery and increased recruitment of bone marrow–derived EPCs to the neoendothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE−/− and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I–, fibronectin-, and laminin-coated plates), migration (modified Boyden chamber assay), and antiapoptotic (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling stain) activity was biphasic. Cholesterol enhanced the messenger RNA expression (quantitative, real-time reverse-transcription polymerase chain reaction) of vascular endothelial growth factor and inhibited Notch1 messenger RNA expression in both ApoE−/− and WT EPCs, whereas endothelial nitric oxide synthase messenger RNA expression increased in ApoE−/− EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1+/− EPCs than in WT EPCs, and transplantation of Notch1+/− bone marrow accelerated endothelial recovery after arterial injury in WT mice.
Conclusion— The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity.
Received April 7, 2009; accepted December 15, 2009.
Hypercholesterolemia has repeatedly been identified as a risk factor for atherosclerosis; however, its influence on reendothelialization (ie, the recovery of endothelial integrity) after arterial injury is uncertain. Animal studies and clinical reports provide conflicting evidence relative to the importance of cholesterol after angioplasty or stenting and on the relationship between cholesterol levels and the risk of restenosis.1–4 The loss of endothelial integrity that occurs during angioplasty/stenting appears to induce the accumulation of inflammatory cells and the proliferation and migration of vascular smooth muscle cells, which can lead to neointimal thickening.5 Reendothelialization inhibits neointimal thickening, thereby suppressing the development of the substrate for lipid deposition and macrophage accumulation that is believed to induce the formation of atherosclerotic lesions and may contribute to restenosis. Drug-eluting stents have significantly reduced the rate of restenosis; however, drug-eluting stents also appear to delay reendothelialization.6 This delay results in excessive rates of thrombosis, which could increase the occurrence of acute coronary syndromes.
Clinical Perspective on p 1112
Endogenous reendothelialization occurs through the proliferation and migration of endothelial cells adjacent to the site of arterial injury and via the activity of endothelial progenitor cells (EPCs). Studies performed in our laboratory and others indicate that both exogenously infused EPCs and EPCs mobilized from the bone marrow (BM) are recruited to the sites of arterial injury, where they promote reendothelialization. Mobilization occurs in response to ischemia,7,8 physical training,9 and the administration of statins,10,11 estrogen,12,13 and a variety of cytokines.14–16 Then, the mobilized cells are recruited to ischemic tissue, where they form a structural component of the new vasculature and promote the proliferation and migration of local endothelial cells by secreting signaling molecules and growth factors.17–20
Kwon et al21 have recently reported that a knockout mutation of the Notch ligand Jagged-1 inhibits EPC-mediated angiogenesis by reducing EPC differentiation and bioactivity. Blood flow recovery and postnatal neovascularization after hind-limb ischemia are also impaired in haploinsufficient Notch1+/− mice22; however, similar results were not observed in mice lacking expression of another Notch ligand, Delta-like 1,21 and Notch1 activation in endothelial cells reduced the expression of vascular endothelial growth factor (VEGF) receptor 2 and VEGF-mediated endothelial cell proliferation.23,24 Thus, Notch signaling appears to modulate adult angiogenesis,23 but whether it acts as a positive or negative regulator is somewhat unclear. Mutations in Notch family members have also been linked to an adult-onset vascular dementia that is accompanied by an increased risk of coronary occlusion.25
The number and function of circulating EPCs are inversely correlated with many risk factors for atherosclerosis and cardiovascular disease26,27; however, the relationship between EPC function, gene expression, hypercholesterolemia, Notch1, and reendothelialization after arterial injury has not been completely characterized. Accordingly, we performed a series of investigations with hypercholesterolemic (apolipoprotein E–null [ApoE−/−]) and wild-type (WT) mice to evaluate the contribution of EPCs to atherosclerotic lesion formation, EPC-mediated reendothelialization in the setting of hypercholesterolemia, EPC function and gene expression under high cholesterol conditions, and the impact of reduced Notch1 expression on EPC function.
Animals and Surgical Procedure
All procedures were approved by St Elizabeth’s Institutional Animal Care and Use Committee. Male ApoE−/− (B6.129P2-Apoetm1Unc/J), WT (C57BL/6J), and FVB-TgN(Tie2-LacZ)182Sato mice were obtained from The Jackson Laboratories (Bar Harbor, Me). Tie2-LacZ mice were generated in our laboratory by backcrossing FVB-TgN(Tie2-LacZ)182Sato mice with C57BL/6J mice for >10 generations. Tie2-Cre Notch1+/− mice22 were provided by Dr Liao. Wire-induced carotid denudation was performed as described previously28 in animals 10 to 12 weeks of age. Mice were euthanized for histological analyses via cervical dislocation.
The number of EPCs in the BM and circulation of ApoE−/− and WT mice was determined via the EPC culture assay as described previously.14,29
Donor mice were strain and age matched to recipient mice 6 to 8 weeks of age. The BM transplantation procedure was performed as described previously14,30 and as summarized in the Methods section of the online-only Data Supplement.
Incorporation of BM-Derived EPCs
The incorporation of BM-derived EPCs was evaluated in ApoE−/− and WT mice transplanted with BM from Tie2-LacZ donor mice, which express LacZ from the endothelium-specific Tie2 promoter. Carotid arteries were harvested from mice euthanized 14 days after carotid denudation for en face histological assessments. BM-derived endothelial cells were identified and quantified by X-gal staining as described previously.10,12,30
Incorporation of Infused EPCs
The incorporation of systemically infused ApoE−/− and WT EPCs was evaluated in both ApoE−/− mice and WT mice. The infused EPCs were cultured from mouse BM,14,30 and EPC transfusion was performed as described previously28 and as summarized in the Methods section of the online-only Data Supplement. Carotid arteries were harvested 14 days after carotid denudation for en face histological assessments; transfused EPCs were visualized with fluorescent microscopy as described previously.28
Reendothelialization was evaluated as described previously31,32 and as summarized in the Methods section of the online-only Data Supplement; assessments were performed in ApoE−/− and WT mice, ApoE−/− mice transplanted with BM from ApoE−/− or WT donor mice, WT mice transplanted with BM from ApoE−/− or WT donor mice, and WT mice transplanted with BM from Tie2-Cre Notch1+/− or Tie2-Cre donor mice.
Cellular Contributions to Atherosclerotic Lesion Formation
The formation of atherosclerotic lesions was evaluated in ApoE−/− and WT mice transplanted with BM from Tie2-LacZ donor mice. Assessments of lesion size, apoptosis, β-galactosidase expression (signaling the presence of BM-derived endothelial cells), and CD68 expression (signaling the presence of monocytes/macrophages) were performed as summarized in the Methods section of the online-only Data Supplement.
EPC Function and Apoptosis
Proliferation, adhesion, migration, and apoptosis were assessed in EPCs isolated from the BM of ApoE−/− mice, WT mice, Tie2-Cre mice, and Tie2-Cre Notch1+/− mice. The effect of cholesterol on EPC function was determined by repeating the assays in the presence of 0, 50, 150, 300, and/or 500 mg/dL cholesterol (Sigma-Aldrich Co, St Louis, Mo). Assays were performed as summarized in the Methods section of the online-only Data Supplement.
Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Messenger RNA (mRNA) expression was evaluated in EPCs isolated from the BM of ApoE−/− and WT mice as summarized in the Methods section of the online-only Data Supplement (primer and probe sequences are also provided). Relative mRNA expression was calculated with the comparative CT method (relative expression=2ΔCT) and normalized to the expression of the endogenous 18S gene.
All values were expressed as mean±SEM. Statistical analyses were performed with commercially available software (Statview, Abacus Concepts, Berkeley, Calif). Comparisons between 2 groups were tested for significance with the Student t test; comparisons between multiple groups were tested by ANOVA followed by posthoc testing with the Tukey procedure; measurements obtained at multiple time points were evaluated via repeated-measure analysis. A value of P<0.05 was considered significant.
Circulating EPCs Are More Prevalent in ApoE−/− Mice
Before carotid artery injury, the number of EPCs in the BM of ApoE−/− and WT mice was similar (ApoE−/−, 113.3±9.3 cells per high-powered field [HPF]; WT, 106.3±4.7 cells per HPF; n=5 in each group; Figure 1A), but the number of circulating EPCs was significantly greater in ApoE−/− mice than in WT mice before injury (ApoE−/− mice, 73.9±8.4 cells per HPF; WT, 48.9±3.4 cells per HPF; P<0.05; n=10 in each group) and at all subsequent time points (Figure 1B). Circulating EPC counts increased after injury in both groups; however, ApoE−/− mice maintained this increase for 4 weeks after carotid injury, whereas counts in WT mice declined to preinjury levels by day 14. Serum cholesterol levels did not change significantly in either mouse strain during the course of the experiment (see the Table in the online-only Data Supplement).
EPC Recruitment to the Site of Carotid Artery Injury Is Enhanced in ApoE−/− Mice
Circulating EPC counts were higher after carotid artery injury in ApoE−/− mice transplanted with BM from Tie2-LacZ WT mice (LacZBMApoE−/− mice; 102.8±10.6 cells per HPF; n=5) than in WT mice transplanted with WT Tie2-LacZ BM (LacZBMWT mice; 74.0±6.6 cells per HPF; n=5; Figure 2A), but the difference did not reach statistical significance, and circulating EPC levels in LacZBMApoE−/− mice declined to preinjury levels by day 14. Nevertheless, significantly more (P<0.001; n=5 in each group) X-gal–positive cells were observed on the luminal surface of carotid arteries from LacZBMApoE−/− mice (60.4±5.4 cells per HPF) than in arteries from LacZBMWT mice (20.2±4.1 cells per HPF; Figure 2B and 2C). When DiI-labeled ApoE−/− or WT EPCs were administered systemically, significantly more (P<0.001; n=5 in each group) ApoE−/− EPCs than WT EPCs were observed in the neoendothelium of either mouse strain (ApoE−/− mice, 35.0±3.5 ApoE−/− EPCs per HPF versus 20.4±3.3 WT EPCs per HPF; WT mice, 41.4±4.9 ApoE−/− EPCs per HPF versus 22.0±3.9 WT EPCs per HPF; Figure 2D and 2E); however, ApoE−/− EPC recruitment in ApoE−/− and WT mice was similar, as was WT EPC recruitment. Collectively, these observations suggest that the enhanced EPC mobilization observed in ApoE−/− mice (Figure 1B) could be largely dependent on the genotype of the BM cells, whereas both the genotype of the circulating ApoE−/− EPCs and the physiological environment associated with the ApoE−/− mutation (eg, hypercholesterolemia) may increase EPC biopotency.
Reendothelialization Is Accelerated in ApoE−/− Mice and Is Dependent on EPC Genotype
Evans blue staining in whole-mount carotid arteries indicated that reendothelialization was slightly but significantly (P<0.05; n=5 in each group) elevated in ApoE−/− mice on day 7 (ApoE−/− mice, 77.7±4.1%; WT mice, 63.4±4.7%) and day 14 (ApoE−/− mice, 99.3±1.6%; WT mice, 94.8±5.3%) after carotid artery injury (Figure 3A and 3B). The difference between groups was more apparent in mice that had undergone BM transplantation surgery; reendothelialization was significantly greater (P<0.001; n=5 in each group) in ApoE−/− mice transplanted with ApoE−/− or WT BM (69.5±3.6% and 67.1±2.2%, respectively) and in WT mice transplanted with ApoE−/− BM (64.3±1.9%) than in WT mice transplanted with WT BM (48.6±0.6%; Figure 3C and 3D). Thus, both the physiological environment in ApoE−/− mice and the ApoE−/− BM genotype appear to improve reendothelialization.
BM-Derived EPCs Are Present in Atherosclerotic Lesions
Advanced atherosclerotic lesions were observed in Masson trichrome–stained carotid artery sections from ApoE−/− mice transplanted with WT Tie2-LacZ BM (Figure 4A), and the lesions were significantly larger than those observed in WT mice transplanted with WT Tie2-LacZ BM (week 4: LacZBMApoE−/− mice, 143.5±9.0%; LacZBMWT mice, 18.1±6.7%; week 6: LacZBMApoE−/− mice, 136.3±13.7%; LacZBMWT mice, 31.5±5.0%; P<0.001 at both time points; n=4 in each group; Figure 4B). Two weeks after carotid artery injury, double-immunostained sections identified apoptotic (ie, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling [TUNEL]–stained cells) and BM-derived (ie, cells stained positively for Tie2-β-galactosidase expression) cells on both the internal elastic lamina and the luminal surface (Figure 4C), suggesting that the plaque was surrounded by BM-derived EPCs shortly after the onset of reendothelialization. Evidence of apoptosis and EPC accumulation persisted through week 4, but the plaque core contained only amorphous β-galactosidase protein, and no viable EPCs, at week 6. Similar staining patterns were observed in sections double immunostained for evidence of apoptosis and monocytes/macrophages (ie, cells stained positively for CD68 expression); macrophages accumulated through week 4 but were not present in the plaque core by week 6 (Figure 4D). Thus, both EPCs and inflammatory cells contributed to the formation of the atherosclerotic lesion.
Cholesterol Modulates EPC Function
In the absence of cholesterol, proliferation (ApoE−/− EPCs, 1.15±0.02 OD; WT EPCs, 1.00±0.00 OD; P<0.01) and adhesion (ApoE−/− EPCs, 57.4±2.4 cells per HPF; WT EPCs, 41.3±3.2 cells per HPF; P<0.05) were significantly greater in ApoE−/− EPCs than in WT EPCs (Figure 5A and 5B), but the migration of ApoE−/− and WT EPCs (51.3±1.5 and 47.0±3.1 cells per HPF, respectively) was similar (Figure 5C). Cholesterol enhanced the proliferation of both ApoE−/− EPCs and WT EPCs in a concentration-dependent manner (Figure 5A), whereas the effect of progressively greater cholesterol concentrations on EPC adhesion and migration was biphasic (Figure 5B and 5C). Peak adhesion and migration were observed at 150 mg/dL, and measurements were significantly lower (P<0.05) at 500 mg/dL than in the absence of cholesterol.
Cholesterol Influences the Expression of VEGF, Endothelial Nitric Oxide Synthase, and Notch1 mRNA in EPCs and EPC Apoptosis
The influence of cholesterol on the mRNA expression of VEGF and endothelial nitric oxide synthase (eNOS), which promote angiogenesis and EPC activity, and Notch1, which determines cell fate (including apoptosis), was assessed via real-time reverse-transcription polymerase chain reaction. In the absence of cholesterol, there was no significant difference between ApoE−/− and WT EPCs in VEGF, eNOS, or Notch1 expression (Figure 5D through 5F). Cholesterol concentration-dependently increased VEGF expression (Figure 5D) and decreased Notch1 expression (Figure 5E) in both ApoE−/− EPCs and WT EPCs, whereas eNOS expression was significantly higher (P<0.05) in ApoE−/− EPCs but declined in WT EPCs after exposure to cholesterol (Figure 5F).
The effect of progressively greater cholesterol concentrations on EPC apoptosis was biphasic (Figure 5G). Apoptotic cells were significantly less frequent (P<0.05) in 50 mg/dL cholesterol (29.0±2.9% of ApoE−/− EPCs and 22.3±3.4% of WT EPCs) and 150 mg/dL cholesterol (33.1±2.8% of ApoE−/− EPCs and 25.9±1.9% of WT EPCs) than in the absence of cholesterol (44.5±8.1% of ApoE−/− EPCs and 35.8±2.7% of WT EPCs); however, apoptosis was significantly higher (P<0.0001) in 300 mg/dL (67.2±6.4% of ApoE−/− EPCs and 60.4±3.7% of WT EPCs) or 500 mg/dL (90.3±3.1% of ApoE−/− EPCs and 84.2±8.1% of WT EPCs) cholesterol than in 0 mg/dL cholesterol.
Moderate Reduction in Notch1 Expression Enhances EPC Function, Reduces EPC Apoptosis, and Accelerates Reendothelialization
The cholesterol-independent effects of Notch1 were investigated by comparing EPCs obtained from Tie2-Cre Notch1+/− mice, in which Notch1 expression is reduced by ≈50%,22 with EPCs from Tie2-Cre (ie, Notch1+/+) mice. All EPC functional measures were significantly greater (P<0.05) in Tie2-Cre Notch1+/− EPCs than in Tie2-Cre EPCs (proliferation: Tie2-Cre Notch1+/−, 1.57±0.12 OD; Tie2-Cre, 1.0±0.0 OD; adhesion: Tie2-Cre Notch1+/−, 102.6±4.0 cells per HPF; Tie2-Cre, 38.8±3.9 cells per HPF; migration: Tie2-Cre Notch1+/−, 115.6±13.2 cells per HPF; Tie2-Cre, 27.8±3.9 cells per HPF); Tie2-Cre Notch1+/− EPCs also exhibited less apoptosis (Tie2-Cre Notch1+/−, 13.1±0.7%,; Tie2-Cre, 47.0±5.9%; P<0.05; Figure 6A through 5⇑D). Furthermore, exposure to 150 mg/dL cholesterol, which decreased Notch1 expression by ≈50% (Figure 5E), increased Tie2-Cre EPC function (proliferation, 1.65±0.43 OD; adhesion, 89.6±8.8 cells per HPF; migration, 139.3±22.5 cells per HPF) and decreased Tie2-Cre EPC apoptosis (18.4±2.0%) to levels similar to those measured in Tie2-Cre Notch1+/− EPCs. Treatment of Tie2-Cre Notch1+/− EPCs with 150 mg/dL cholesterol enhanced proliferation, impaired adhesion and migration, and increased apoptosis, which is consistent with the results obtained when Notch1 expression declined to <50% of baseline levels in WT and ApoE−/− EPCs treated with 300 to 500 mg/dL cholesterol (Figure 5A through 5C and 5G).
The potential contribution of Notch1 expression to endothelial recovery was evaluated in WT mice transplanted with BM from Tie2-Cre Notch1+/− or Tie2-Cre mice. Evans blue staining of whole-mount carotid arteries revealed significantly greater (P<0.01; n=4 in each group) evidence of reendothelialization on day 7 after carotid artery injury in mice transplanted with Tie2-Cre Notch1+/− BM (75.2±3.2%) than in mice transplanted with Tie2-Cre BM (48.8±5.5%) (Figure 6E and 6F), suggesting that a moderate reduction in Notch1 expression in BM-derived cells promotes endothelial recovery.
Results from the series of experiments presented here indicate that the number of circulating EPCs and their recruitment to the site of arterial injury are greater in ApoE−/− mice than in WT mice and that the enhanced recruitment likely evolves from the properties of the EPCs themselves as well as the physiological environment. ApoE−/− mice also displayed greater evidence of reendothelialization than WT mice, which was somewhat surprising because ApoE−/− mice are prone to atherosclerosis, and accelerated reendothelialization is commonly believed to inhibit the formation of atherosclerotic lesions.33–35 In general, ApoE−/− and WT EPCs responded similarly to the presence of cholesterol in vitro: Progressively greater cholesterol concentrations enhanced VEGF expression and EPC proliferation, whereas Notch1 expression declined and the relationship between cholesterol concentration and EPC adhesion, migration, and apoptosis was biphasic. However, eNOS expression increased with cholesterol exposure in ApoE−/− EPCs and declined in WT EPCs. In vivo, the recruitment of WT EPCs derived from transplanted BM was higher in ApoE−/− mice than in WT mice, whereas the recruitment of systemically administered WT EPCs was similar in both strains, perhaps because the duration of exposure to high serum cholesterol levels (ie, the length of time between BM transplantation or EPC injection and the evaluation of recruitment) was 6 to 8 weeks for the transplanted cells compared with just 2 weeks for the injected cells.
The concentration-dependent enhancement of VEGF expression by cholesterol could explain the greater EPC proliferation observed at high cholesterol levels. The secretion of VEGF and other growth factors by EPCs is believed to enhance angiogenesis in ischemic tissues by promoting the proliferation and migration of local endothelial cells,17–20 and this paracrine mechanism could contribute to reendothelialization in response to arterial injury. However, VEGF has also been linked to lesion formation and the progression of atherosclerosis.36–38
Although mild impairment of Notch1 expression was accompanied by declines in apoptosis both in Tie2-Cre Notch1+/− EPCs and in EPCs exposed to low cholesterol levels, excessive impairment of Notch1 expression by high cholesterol levels increased apoptosis. This potential biphasic relationship between Notch1 expression and apoptosis has been implied by results from previous investigations indicating that both Notch1 deletion39 and overexpression40 increase apoptosis in endothelial cells. Notch1 may also regulate EPC activity in the presence of cholesterol because moderate cholesterol-induced reductions in Notch1 expression were associated with greater EPC adhesion and migration; however, confirmation of this potential regulatory mechanism requires additional investigation.
Cholesterol increased the expression of eNOS in ApoE−/− EPCs and decreased eNOS expression in WT EPCs. eNOS is essential for the mobilization of stem and progenitor cells from the BM,41 and reendothelialization after carotid artery injury is retarded in both eNOS knockout mice12 and diabetic mice,28 in which eNOS expression is attenuated. Thus, the higher circulating EPC counts and accelerated reendothelialization observed in ApoE−/− mice might be attributable to enhanced eNOS expression under hypercholesterolemic conditions. However, the recruitment of transplanted WT (Tie2-LacZ) EPCs was greater in the hypercholesterolemic ApoE−/− mice than in WT mice despite the cholesterol-induced inhibition of eNOS expression, so the potential relationship between cholesterol, eNOS expression, and EPC recruitment remains uncertain.
Transplanted, BM-derived WT (Tie2-LacZ) EPCs were found at the border of atherosclerotic lesions in ApoE−/− mice and were accompanied by ample evidence of apoptosis. Although these results could imply that EPCs participate in the formation of atherosclerotic lesions, the mechanism and consequences associated with the cholesterol-mediated modulation of EPC activity have yet to be determined. Our in vitro results indicate that hypercholesterolemia likely enhances EPC adhesion, migration, and VEGF expression, but these effects might be offset (at least partially) by cholesterol-induced EPC apoptosis, perhaps caused by very high local cholesterol concentrations at the luminal surface. Furthermore, EPCs comprise a heterogeneous cell population, and individual subpopulations of EPCs may function differently during recovery from vascular injury.
This investigation provided novel insights into the role of EPCs during recovery from arterial injury and identified the potential importance of cholesterol and Notch1 for the regulation of EPC activity. EPCs appear to participate in reendothelialization, which is enhanced in hypercholesterolemic mice, and BM-derived EPCs and monocyte/macrophages may also contribute to the formation of atherosclerotic lesions. Both cholesterol and Notch1 influence EPC function and viability; however, the influence of cholesterol on many EPC functions is biphasic, so the consequences of EPC activity under hypercholesterolemic conditions warrant further investigation.
We thank W. Kevin Meisner, PhD, ELS, for editorial support and M. Neely and Ashley Peterson for administrative assistance.
Sources of Funding
This study was supported in part by National Institutes of Health grants HL-53354, HL-57516, HL-77428, HL-95874, HL-80137, PO1HL-66957 (to Dr Losordo), and HL-052233 (to Dr Liao).
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Abundant evidence has linked hypercholesterolemia with the advent and progression of atherosclerosis. To facilitate the study of the mechanisms by which hypercholesterolemia leads to atherosclerosis, animal models have been developed. Most notably, the apolipoprotein E (ApoE)–null and low-density lipoprotein receptor–null mice have been used extensively in attempts to gain a better understanding of the effects of high lipid levels on atheroma formation. In recent years, traditional cardiovascular risk factors have also been associated with decreased numbers of circulating endothelial progenitor cells (EPCs), which have been suggested to participate in endothelial repair and maintenance. Ample experimental evidence suggests that EPCs are mobilized from the bone marrow to the peripheral circulation after arterial injury (eg, percutaneous transluminal coronary angioplasty or stent implantation) and promote reendothelialization at the injury site, thereby speeding endothelial recovery and reducing the risk of restenosis and atherosclerotic plaque formation. Here, we compared EPC recruitment, reendothelialization, and plaque formation after carotid artery injury in wild-type mice and ApoE−/− mice. Our findings indicate that EPC recruitment was higher in ApoE−/− mice than in wild-type mice and that the enhanced recruitment likely evolves through a moderate decline in Notch expression. Reendothelialization was also greater in ApoE−/− mice, which was somewhat surprising because ApoE−/− mice are prone to atherosclerosis. Furthermore, transplanted, bone-marrow–derived EPCs were found at the border of the atherosclerotic lesions, which could suggest that EPCs contribute to the early stage of plaque formation. However, the clinical implications of these observations must be interpreted with caution, particularly because circulating EPC levels were higher in ApoE−/− mice than in wild-type mice, whereas hypercholesterolemia, hypertension, and many other risk factors for cardiovascular disease are associated with declines in human EPC levels. Collectively, our findings emphasize the need for additional experiments in other hypercholesterolemic animal models and underscore the potential for genetic mouse models of human disease to yield data that do not necessarily reflect clinical reality. To clarify the paradoxical observation on the direct effect of cholesterol on EPC contribution to reendothelialization versus plaque formation in Apo E knockout mice after arterial injury, further investigations using different types of hypercholesterolemic animals and, importantly, additional clinical correlation are required.
Guest Editor for this article was James T. Willerson, MD.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.553917/DC1.