Published online before print December 19, 2002, doi: 10.1161/01.RES.0000052313.23087.3F
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© 2003 American Heart Association, Inc.
Molecular Medicine |
Local Monocyte Chemoattractant Protein-1 Therapy Increases Collateral Artery Formation in Apolipoprotein E–Deficient Mice but Induces Systemic Monocytic CD11b Expression, Neointimal Formation, and Plaque Progression
From the Department of Cardiology (N.v.R., M.V., J.J.P.), University of Amsterdam, Amsterdam, the Netherlands; the Department of Cardiology (N.v.R., I.H., M.B., J.H., S.G., C.B., I.B.), University of Freiburg, Freiburg, Germany; and the Department of Experimental Cardiology (W.S.), Max Planck Institute, Bad Nauheim, Germany.
Correspondence to Niels van Royen, Department of Cardiology, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands. E-mail n.vanroyen{at}amc.uva.nl
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Abstract |
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Monocyte chemoattractant protein-1 (MCP-1) stimulates the formation of a collateral circulation on arterial occlusion. The present study served to determine whether these proarteriogenic properties of MCP-1 are preserved in hyperlipidemic apolipoprotein E–deficient (apoE-/-) mice and whether it affects the systemic development of atherosclerosis. A total of 78 apoE-/- mice were treated with local infusion of low-dose MCP-1 (1 µg/kg per week), high-dose MCP-1 (10 µg/kg per week), or PBS as a control after unilateral ligation of the femoral artery. Collateral hindlimb flow, measured with fluorescent microspheres, significantly increased on a 1-week high-dose MCP-1 treatment (PBS 22.6±7.2%, MCP-1 31.3±10.3%; P<0.05). These effects were still present 2 months after the treatment (PBS 44.3±4.6%, MCP-1 56.5±10.4%; P<0.001). The increase in collateral flow was accompanied by an increase in the number of perivascular monocytes/macrophages on MCP-1 treatment. However, systemic CD11b expression by monocytes also increased, as did monocyte adhesion at the aortic endothelium and neointimal formation (intima/media ratio, 0.097±0.011 [PBS] versus 0.257±0.022 [MCP-1]; P<0.0001). Moreover, Sudan IV staining revealed an increase in aortic atherosclerotic plaque surface (24.3±5.2% [PBS] versus 38.2±9.5% [MCP-1]; P<0.01). Finally, a significant decrease in the percentage of smooth muscle cells was found in plaques (15.0±5.2% [PBS] versus 5.8±2.3% [MCP-1]; P<0.001). In conclusion, local infusion of MCP-1 significantly increases collateral flow on femoral artery ligation in apoE-/- mice up to 2 months after the treatment. However, the local treatment did not preclude systemic effects on atherogenesis, leading to increased atherosclerotic plaque formation and changes in cellular content of plaques.
Key Words: arteriogenesis • collateral circulation • atherosclerosis • monocytes • monocyte chemoattractant protein-1
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Introduction |
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Arteriogenesis refers to the outgrowth of preexisting collateral arteriolar connections into large-conductance arteries. Because of the high capacity of these vessels compared with the capillary networks that are formed during angiogenesis, arteriogenesis is believed to be the most efficient form of vessel growth for the restoration of tissue perfusion on arterial occlusion.1–3
Thus, proarteriogenic substances potentially can be used to increase the capacity of collateral arteries in patients suffering from arterial obstructive disease, thereby alleviating symptoms of intermittent claudication or angina pectoris.
The large majority of arterial obstructions are caused by atherosclerotic disease; therefore, substances that are envisioned to be of use for therapeutic purposes in this population should be tested for their "neglected, potential serious side-effects" on atherosclerosis.4 This is of particular interest regarding substances that stimulate angiogenesis because the process of angiogenesis is directly involved in atherosclerotic plaque progression.5 Although arteriogenesis as a process is not directly involved in atherogenesis, several pathophysiological entities, such as monocyte infiltration and increased expression of certain growth factors and cytokines, are displayed by arteriogenesis as well as atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a known proarteriogenic factor, accelerating significantly the formation of collateral arteries on arterial occlusion in rabbits.6,7 The proatherogenic properties of MCP-1 have been the subject of many studies, and it has been shown that overexpression of MCP-1 directly at the vessel wall leads to an increased macrophage infiltration and neointimal formation.8
Local and intravascular delivery of angiogenic/arteriogenic proteins most efficiently restores perfusion on arterial obstruction.9 Moreover, such local application limits systemic side effects and maximizes selectivity. However, even local application might lead to systemic side effects; therefore, the objective of the present study was to determine whether a local protein infusion of MCP-1 directly into the hindlimb collateral circulation, in a dosage that significantly stimulates arteriogenesis, promotes atherosclerosis systemically.
Apolipoprotein E (apoE)-deficient (apoE-/-) mice show high levels of serum lipids and formation of atherosclerotic plaques similar to human atherosclerotic plaques.10,11 In a newly developed mouse model, we delivered the MCP-1 protein directly into the hindlimb collateral circulation of apoE-/- mice. This enabled us to test simultaneously in one model the proarteriogenic effects of local MCP-1 protein therapy under hyperlipidemic conditions and the systemic negative side effects on atherosclerosis.
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Materials and Methods |
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Animals
A total of 50 apoE-/- mice aged 8 weeks (N10, backcrossed onto C57/Bl6, Jackson Laboratory, Bar Harbor, Maine) and 28 apoE-/- mice aged 6 months were used after securing the appropriate institutional approval conforming with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). The 8-week-old mice were treated with either low-dose MCP-1 (1 µg/kg per week, n=8), high-dose MCP-1 (10 µg/kg per week, n=21), or PBS as a control substance (n=21). Measurements were performed either at day 3 during treatment (high-dose MCP-1, n=5; PBS, n=5), directly after the 1-week treatment (low-dose MCP-1, n=8; high-dose MCP-1, n=8; and PBS, n=8), or 2 months after treatment (high-dose MCP-1, n=8; PBS, n=8). The 6-month-old mice were divided into two groups (high-dose MCP-1, n=14; PBS, n=14) and were analyzed directly after treatment (high-dose MCP-1, n=3; PBS, n=3) or after a 2-month period (high-dose MCP-1, n=11; PBS, n=11).
The treatment period with either PBS or MCP-1 was 1 week for all animals in all groups. The 1-week treatment period was ensured using osmotic minipumps (Alzet, 1007D, Alza Corp) that had been especially designed for delivery of content over 7 days.
Animal Microsurgery
Animals were anesthetized, and the femoral artery was dissected free under a stereoscopic microscope (Leica MZ6, Leica). A small incision was made in the femoral artery, distal to the arteria profunda femoris, and a catheter (inner diameter 0.28 mm, outer diameter 0.61 mm) was inserted into the proximal stump of the femoral artery with the tip of the catheter pointing upstream. The catheter was then secured with two ligations around the femoral artery, thereby also completely obstructing femoral artery flow. Before the catheter was introduced, it was connected to the osmotic micropump for local delivery of either MCP-1 or PBS over a 1-week period. Micropumps were then secured under the skin.
Collateral Flow Measurements
Collateral flow measurements were performed as previously described.12 In summary, a catheter was inserted in the abdominal aorta with the tip just proximal to the aortic bifurcation. Both hindlimbs were then perfused at four different pressure levels with differently labeled microspheres. Adequate mixing of the microspheres was ensured by vortexing for 30 seconds shortly before injection. After the performance of all four infusions, the animals were euthanized, and their distal hindlimb muscles (ie, gastrocnemius and peroneus muscles) were dissected. Tissue was digested and homogenized, and the number of accumulated microspheres in both hindlimbs was counted using flow cytometry (Epics XL-MCL, Beckman Coulter). Restoration of flow was then expressed as a percentage that was derived from the ratio between the flows in the occluded versus the nonoccluded hindlimb.
Serum Measurements
A total of 1 mL blood was withdrawn from each animal shortly before euthanasia. Triglycerides, total cholesterol, VLDL, LDL, HDL, and C-reactive protein were measured enzymatically in serum using standard protocols.
FACS Analysis of CD11b Expression on Monocytes
CD11b expression on circulating monocytes was determined using fluorescence-activated cell sorter (FACS) analysis (Epics XL-MCL, Coulter). Blood (0.3 mL) was withdrawn from the left ventricle and stained with an R-phycoerythrin–labeled F4/80 marker (Caltag) as well as an FITC-labeled CD11b antibody (Serotec) using standard protocols for whole-blood staining. Monocytes were identified based on scatter properties and positive staining for F4/80. CD11b expression by monocytes was expressed as fluorescence intensity (arbitrary units).
Immunohistochemistry and Atherosclerotic Lesion Size Quantification
Aortas of 8-week-old mice were harvested and stored at -80°C, and 5-µm sections of the ascending aorta were placed on cationic coated slides (Superfrost Plus, MJ Research). For all histological examinations, a total of five slides per animal were analyzed, with 50-µm distance between the samples. All quantitative analyses were performed by two observers blinded to the experimental protocol. A mouse-specific marker for CD11b (Serotec) with FITC as a secondary antibody (Southern Biotechnologies) was used to detect monocytes. The number of adhering monocytes was quantified at a magnification of x400 at either day 3 or day 7 or after 2 months. Therefore, the total number of monocytes per aortic ring was counted visually, and endoluminal wall length was measured using Qfluoro software (Leica). Data were then expressed as monocytes per millimeter endoluminal wall to correct for different aortic diameters and cutting angles. To quantify neointimal formation, photographs were taken of the complete aortic ring at the level of the ascending aorta (7 to 9 photographs for each aortic ring, five rings per animal) at a magnification of x400 with a Leica DC 300F digital camera (Leica). Neointima was then quantified planimetrically using ImageJ software.13 Measurements of adhering monocytes and neointimal formation were performed on tissues derived from the 8-week-old apoE-/- mice.
Aortas from 6-month-old animals, euthanized 2 months after femoral artery ligation, were dissected and immersed in 4% formalin and stained with Sudan IV for detection of atherosclerotic plaques (PBS-treated animals, n=6; MCP-treated animals, n=6). Stained aortas were then photographed with a digital camera (Coolpix 900, Nikon), and the percentage of atherosclerotic surface compared with total aortic surface was calculated planimetrically.
To determine cellular content, plaques from the descending aortas of the remaining animals (PBS-treated animals, n=5; MCP-treated animals, n=5) were stained for monocytes/macrophages (CD11b; see above), lymphocytes (FITC-labeled mouse-specific CD3 marker, Serotec), and smooth muscle cells (FITC-labeled anti-human 
-smooth muscle marker with cross-reactivity for mouse tissue, Sigma Chemical Co). Nuclear staining was performed with Hoechst 33342 (Molecular Probes). Negative controls were performed for all immunologic stainings by omission of the primary antibody.
Six additional mice aged 6 months were treated with either high-dose MCP-1 or PBS, and tissue was harvested directly after the 1-week treatment period to detect the influence of MCP-1 on monocyte infiltration into aortic plaques and hindlimb tissue directly after treatment. Monocyte infiltration into plaques was performed as described above. Monocytes/macrophages around collateral vessels in hindlimb tissue (quadriceps and adductor muscles) were detected using a mouse monocyte/macrophage-specific monoclonal antibody against MOMA-2 (BMA Biomedicals) and Cy3 (DPC Biermann) as a secondary antibody. In addition, tissue was stained with the above-mentioned antibody against smooth muscle cells to ensure the arterial aspect of selected vessels. Photomicrographs were taken with a x400 magnification, and the number of monocytes/macrophages was counted in predefined squares of 273 µmx345 µm around muscular collateral arteries. Moreover, monocytes/macrophages were expressed as a percentage of total cell population in the predefined squares.
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Results |
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Collateral Flow Measurements
In the 8-week-old apoE mice, collateral flow did not increase in the low-dose MCP-1–treated animals compared with control animals 7 days after femoral artery ligation (PBS 22.6±7.2%, low-dose MCP-1 20.3±4.3%; P=NS).
When the high-dose MCP-1 group was compared with the control group, no statistically significant difference was observed at day 3 (PBS 10.2±2.7%, high-dose MCP-1 11.8±3.3%). However, at day 7 after femoral artery ligation, a significant difference could be observed between treated and control animals (PBS 22.6±7.2%, high-dose MCP-1 31.3±10.3%; P<0.05). Two months after the ligation and the 1-week treatment, the difference in collateral conductance between the MCP-1–treated and the control animals was maintained (PBS 44.3±4.6%, high-dose MCP-1 56.5±10.4%; P<0.001) (Figure 1). This increase in collateral flow was accompanied by an increased number of monocytes/macrophages around muscular arteries in the quadriceps and adductor muscles of the ligated leg (PBS 26.9±19.3, high-dose MCP-1 64.2±37.1; P<0.01) (Figure 2). Also when expressed as a percentage of total cells around collateral vessels, an increase of monocytes/macrophages was detected in MCP-1–treated animals compared with control animals (PBS 19.0±6.5%, high-dose MCP-1 34.9±9.8%; P<0.001).
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Serum Measurements
No increase in serum levels of C-reactive protein was found in any of the groups. High values for triglycerides, total cholesterol, VLDL, and LDL as well as low levels of HDL were found in all groups. However, the treatment with MCP-1 had no influence on any of these values at either 7 days or 2 months after initiation of the 1-week treatment (Table).
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FACS Analysis of CD11b Expression on Monocytes
The local infusion of high-dose MCP-1 directly in the peripheral collateral circulation led to an increased expression of CD11b by circulating monocytes that were withdrawn from the left ventricle. Fluorescence intensity (given in arbitrary units) was 214.5±8.7 in the control mice and 256.7±11.4 (P<0.001) in the high-dose MCP-1 mice. No increase in CD11b expression by circulating monocytes was detected in the low-dose MCP-1 mice (data not shown).
Immunohistochemistry and Atherosclerotic Lesion Size Quantification
On MCP-1 treatment of the 8-week-old animals, an increase in endoluminal monocytes/macrophages in the ascending aorta was observed as early as day 3 (PBS 12.4±4.0 monocytes/mm endoluminal vessel wall, MCP-1 20.1±9.9 monocytes/mm; P<0.001) as well as at day 7 (PBS 13.7±3.1 monocytes/mm, MCP-1 21.2±9.6 monocytes/mm; P<0.001). Two months after femoral artery ligation, the difference between the treated and the control group had further increased (PBS 16.5±5.6 monocytes/mm, MCP-1 41.7±9.8 monocytes/mm; P<0.0001) (Figure 3). This was accompanied by an increased neointimal formation in the MCP-1–treated animals (intima/media ratio: PBS 0.097±0.011, MCP-1 0.257±0.022; P<0.0001) (Figure 4).
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In the 6-month-old apoE mice, an increase in monocyte content of atherosclerotic plaques could be appreciated directly after MCP-1 treatment. Several plaques of MCP-1–treated animals consisted almost exclusively of monocytes/macrophages accompanied by aortic wall invasion of monocytes/macrophages. This was encountered solely in MCP-1–treated animals, whereas control animals showed normal cellular content of plaques directly after the 1-week PBS infusion (Figure 5). In the 6-month-old apoE mice, treatment with high-dose MCP-1 led to an increased percentage of atherosclerotic plaque surface in total aortas 2 months after initiation of the treatment (PBS 24.3±5.2%, MCP-1 38.2±9.5%; P<0.01) (Figure 6). This increased total plaque surface could be attributed almost completely to increased plaque formation in the thoracic and abdominal aorta (PBS 14.8±5.1%, MCP-1 30.9±7.8%; P<0.05), whereas plaque percentage in the aortic arch remained almost unchanged (PBS 54.4±3.9%, MCP-1 60.1±7.8%; P=NS).
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However, cellular content of plaques from the aortic arch was changed on MCP-1 treatment. Two months after MCP-1 treatment, a significant decrease in the percentage of smooth muscle cells was observed in MCP-1–treated animals (PBS 15.0±5.2%, MCP-1 5.8±2.3%; P<0.001). No significant change was found in either monocyte/macrophage or lymphocyte content of atherosclerotic plaques in the 6-month-old animals 2 months after treatment (Figure 7).
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Discussion |
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As a first step, we have determined the dosage of MCP-1 required to induce arteriogenesis in apoE mice. Our data show that the arteriogenic potency of MCP-1 was preserved under hyperlipidemic conditions in the apoE-/- mice up to 2 months after ligation at a dosage of 10 µg/kg per week. At the same time, when this dosage was used, a systemic increase in monocytic CD11b expression was observed on the local MCP-1 treatment. This was accompanied by an increased monocyte adhesion to the aortic endothelium and an increased neointimal formation. Finally, in 6-month-old mice, the treatment with MCP-1 increased total plaque surface in the aorta and also modulated the cellular composition of plaques toward a morphology that is potentially more prone to rupture.
The potential to form a collateral circulation on arterial obstruction is distributed very heterogeneously among the population. Several factors influencing this potential, such as age14 or the presence of diabetes,15 have been identified in recent years. Hyperlipidemia also negatively influences the formation of a collateral circulation16; therefore, we first determined the natural time course of arteriogenesis in apoE-/- mice and the additive proarteriogenic effects of MCP-1.
The natural arteriogenic response in the PBS-treated control group restored flow to 
45% of normal. This seems to be in contradiction to the observed 60% restoration of flow that had previously been observed 5 weeks after femoral artery ligation (Couffinhal et al17). This difference most probably relates to the different methodological approaches. In contrast to the previous study, we performed measurements of tissue perfusion using fluorescent microspheres instead of laser Doppler flowmetry. In a previous study, we showed that compared with microsphere-based measurements, laser Doppler flowmetry in the mouse hindlimb model leads to overestimation of tissue perfusion.12 This is in agreement with the observed differences between the present study and the study of Couffinhal et al.
Compared with the control group, the group given MCP-1 at 1 µg/kg per week showed no increase in collateral flow. A dosage of 10 µg/kg per week significantly increased the flow ratio by 30% at 7 days after femoral artery ligation. Interestingly, this positive effect of the 1-week treatment on collateral flow was still observed 2 months after initiation of the therapy, demonstrating an ongoing benefit of the treatment.
The issue remains whether the beneficial effects of MCP-1 in the present model can be attributed to the enlargement of preexisting collateral vessels or the de novo formation of new arteries. The mouse model of femoral artery ligation as performed in the present study is designed for the study of arteriogenesis specifically. Therefore, the arteria profunda is left intact, and the ligation is at a relative distal site of the femoral artery. By use of this model, 6 preexisting collateral arterioles are readily recruitable and macroscopically visible.18 Immediately on ligation, these preexisting vessels partially restore flow to jeopardized tissues as a natural escape mechanism from massive ischemic tissue damage on acute femoral artery ligation. Over time, the lumen of these preexisting vessels widens via active proliferation of vascular wall cells, thereby restoring flow to values of up to 45% of normal in the present model. Finally, when analyzed histologically, these arteries are always in close anatomic relation to veins and nerves. It can be postulated that arteries and nerves develop in a coordinated fashion, as shown recently in a different experimental setting in embryonic mouse limb skin19; however, the simultaneous development of veins in the present model seems redundant because the femoral vein is left intact. Moreover, proliferation markers such as KI-67 were found to be positive only in the arterial wall and not in accompanying veins or nerves (authors’ unpublished data, 2002). Taken together, in the present model, the proliferation of preexisting collateral vessels seems to be the dominating form of vessel growth rather than the de novo formation of collateral arteries.
Having determined the proarteriogenic properties of both the low-dose and the high-dose MCP-1, we then used MCP-1 at 10 µg/kg per week for all further experiments, focusing on the proatherogenic properties of MCP-1. In recent years, numerous preclinical and clinical studies have been conducted to identify proangiogenic or proarteriogenic strategies, but only few have addressed the possible negative side effects of such therapies, as have recently been stressed by Epstein et al.4 Barger et al20 first proposed that angiogenesis is an integral part of atherosclerotic plaque formation. Subsequently, Moulton et al5 showed that the inhibition of angiogenesis via TNP-470 or endostatin diminishes plaque formation. Furthermore, basic fibroblast growth factor, either as gene or as protein therapy, induces neointimal hyperplasia as well as an increased neovascularization of the intima in porcine arteries.21,22 More recently, it has been shown that the exogenous application of a low dose of vascular endothelial growth factor, an angiogenic factor, strongly stimulates plaque formation in both cholesterol-fed rabbits and knockout mice, doubly deficient in apoE/apolipoprotein B-100.23
Arteriogenesis plays no direct role during atherogenesis. However, arteriogenesis and atherogenesis share numerous common features. Shear stress upregulates the expression of endothelial cell adhesion receptors such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1,24 and this occurs during both atherogenesis and arteriogenesis.25,26 Also, the subsequent monocyte/macrophage invasion plays a pivotal role in both arteriogenesis and atherogenesis. Other features shared by arteriogenesis and atherogenesis include smooth muscle cell mitosis and elastolysis. A direct role for MCP-1 in atherogenesis was suggested by two studies showing that a deficiency in either MCP-1 or its receptor, CCR2, leads to diminished plaque formation in mice.27,28 In addition, in irradiated apoE-/- mice that were repopulated with bone marrow cells from MCP-1 transgenic mice, the localized overexpression of MCP-1 by macrophages resulted in an amplification of atherosclerosis.29 Finally, local overexpression of MCP-1 in the vessel wall of rabbits on a high cholesterol diet has been shown to lead to a local increase in monocyte/macrophage infiltration as well as lesion formation.8
In the present study, we have shown for the first time that a local treatment with MCP-1, administered as a protein, clearly affects the several steps in systemic atherogenesis. First of all, we could show that the local treatment induced activation of circulating monocytes as measured by CD11b expression, even in the absence of detectable systemic MCP-1 levels as measured by ELISA (data not shown). Most probably, monocytes are activated by high local MCP-1 levels. Some of these activated monocytes will adhere to the endothelium of collateral arteries, increasing the arteriogenic response and explaining in part the beneficial effects of MCP-1 on the development of the collateral circulation. However, another fraction of the activated monocytes will recirculate and adhere to the endothelium at distant sites, such as the atherosclerosis-prone regions in the aortic arch. Indeed, the increased expression of CD11b on circulating monocytes was accompanied by a strongly increased amount of adhering monocytes in the aortic arch, both directly as well as 2 months after treatment. Moreover, monocyte infiltration in existing advanced lesions in 6-month-old apoE mice was observed directly after the 1-week treatment period. Two months after MCP-1 treatment, an increase in the intima/media ratio was observed in the treated animals compared with the control animals, showing that local MCP-1 treatment did not result in a transient effect on monocyte adhesion but rather induced atherogenesis.
The positive correlation between increased CD11b expression by circulating monocytes, the increased monocyte infiltration, and the progression of atherosclerotic disease after MCP-1 treatment confirm recently published data showing the reversed phenomenon after leukotriene B4 receptor antagonism, leading to decreased CD11b expression of circulating monocytes, decreased monocyte infiltration, and the reduction of lesion progression.30 It should be noted that CD11b expression was used in the present study as a marker of monocyte activation. A direct role of CD11b in MCP-1–induced atherogenesis remains to be elucidated but seems less probable because it has been shown recently that atherosclerosis develops normally in LDL receptor–deficient mice also when CD11b expression on leukocytes is absent, as was achieved in a chimera model using CD11b-deficient bone marrow.31 However, it could still be postulated that the induction of CD11b overexpression, as was the case in the present study, does influence monocyte trafficking to atherosclerotic lesions directly, and we hope to further unravel the exact mechanistic background of MCP-1–induced atherogenesis in future studies.
Interestingly, 2 months after MCP-1 treatment, we also observed an increased expression of ICAM-1 on the aortic endothelium (data not shown). ICAM-1 is essential for monocyte adhesion to atherosclerosis-prone regions,32 and ICAM-1 upregulation is one of the earliest events occurring in atherogenesis. A correlation exists between ICAM-1 expression on the aortic endothelium and atherosclerotic disease progression in apoE-/- mice.33 Because in our model increased ICAM expression on the aortic endothelium was detected only 2 months after MCP-1 treatment, we postulate that this was also merely an indicator of more progressed atherosclerotic disease in treated animals rather than a direct effect of the MCP-1 treatment. This can also be concluded from the earlier mentioned ELISA data, which showed no increase in circulating MCP-1 after treatment and excluded the direct effects of MCP-1 on ICAM expression on the aortic endothelium.
To test whether MCP-1 treatment leads to increased lesion progression via induction of plaque neovascularization, we performed a CD31 staining of plaques. Although neovascularization was present, especially in large-sized advanced lesions, no obvious difference between treated and nontreated animals could be detected when size-matched plaques (data not shown) were compared.
The proatherogenic effects of MCP-1 treatment were further confirmed by the data from the 6-month-old apoE mice showing an increase in total plaque surface on treatment. This increase in plaque surface of the whole aorta could be attributed mainly to increased plaque formation in the abdominal and the thoracic aorta, whereas the plaque surface in the aortic arch remained unchanged. Cellular content of plaques in the aortic arch did change though, leading to a 3-fold decrease in relative smooth muscle cell content of plaques on MCP-1 treatment. It can be postulated that this decrease in smooth muscle cell content drives plaques toward a more rupture-prone form of atherosclerotic lesions.
Taken together, MCP-1 exerts a strong arteriogenic effect, even under hyperlipidemic conditions. The beneficial effects of MCP-1 were ongoing; ie, they were still present 2 months after the treatment. However, the local treatment with MCP-1 did not preclude negative systemic effects on atherogenesis. These proatherogenic properties of MCP-1 confirm earlier observations of the proatherogenic properties of the proangiogenic substances basic fibroblast growth factor and vascular endothelial growth factor. Strategies need to be identified, focusing at either different dosage regimens to minimize the proatherogenic effects or a combination with other substances that might act in an antiatherogenic fashion. Of interest in this regard is the recent identification of two other proarteriogenic substances, granulocyte-macrophage colony–stimulating factor34,35 and transforming growth factor-ß1,36 which have also been reported to exert antiatherogenic properties.37–39
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Acknowledgments |
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The present study was supported by the Volkswagen Foundation. The authors thank Caroline Kempf and Susanne Hartmann for their expert technical assistance.
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Footnotes |
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*Both authors contributed equally to this study.
Received July 26, 2002; revision received November 27, 2002; accepted December 4, 2002.
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References |
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