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Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1397-1402
Published online before print June 3, 2004, doi: 10.1161/01.ATV.0000134529.65173.08
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1397.)
© 2004 American Heart Association, Inc.

Vascular Biology

Rat Aortic MCP-1 and Its Receptor CCR2 Increase With Age and Alter Vascular Smooth Muscle Cell Function

Gaia Spinetti; Mingyi Wang; Robert Monticone; Jing Zhang; Di Zhao; Edward G. Lakatta

From the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md.

Correspondence to Gaia Spinetti, PhD, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail spinettiga{at}grc.nia.nih.gov


*    Abstract
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Objective— With age, rat arterial walls thicken and vascular smooth muscle cells (VSMCs) exhibit enhanced migration and proliferation. Monocyte chemotactic protein-1 (MCP-1) affects these VSMC properties in vitro. Because arterial angiotensin II, which induces MCP-1 expression, increases with age, we hypothesized that aortic MCP-1 and its receptor CCR2 are also upregulated and affect VSMC properties.

Methods and Results— Both MCP-1 and CCR2 mRNAs and proteins increased in old (30-month) versus young (8-month) F344xBN rat aortas in vivo. Cellular MCP-1 and CCR2 staining colocalized with that of {alpha}-smooth muscle actin in the thickened aortas of old rats and were expressed by early-passage VSMCs isolated from old aortas, which, relative to young VSMCs, exhibited increased invasion, and the age difference was abolished by vCCI, an inhibitor of CCR2 signaling. MCP-1 treatment of young VSMCs induced migration and increased their ability to invade a synthetic basement membrane. The MCP-1–dependent VSMC invasiveness was blocked by vCCI. After MCP-1 treatment, migration and invasion capacities of VSMCs from young aortas no longer differed from those of VSMCs isolated from older rats.

Conclusions— Arterial wall and VSMC MCP-1/CCR2 increase with aging. MCP-1 enhances VSMC migration and invasion, and thus, MCP-1/CCR2 signaling may play a role in age-associated arterial remodeling.

This study demonstrates that MCP-1 and CCR2 are increased within the thickened aortas of older rats. In early-passage VSMCs from young rats, MCP-1 increased migration and invasion, imparting to these cells the characteristics of VSMCs from older rat aorta. Thus, MCP-1/CCR2 may be implicated in age-associated vascular remodeling.


Key Words: chemokines • aging • aorta • vascular smooth muscle cells • invasion


*    Introduction
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Aging is the major risk factor for the development of atherosclerosis and hypertension, which have an important impact on Western society because they lead to myocardial infarction, stroke, and heart failure.1–3 Arterial remodeling accompanies advancing age in all species, from rodents to nonhuman primates to humans, and the mechanisms involved in this remodeling likely confer on aging the status of the major risk factor for vascular diseases.1,2 Specific facets of age-associated remodeling include luminal dilation, thickening of the intimal and medial layers with cellular and extracellular matrix reorganization, increased stiffness, and endothelial dysfunction.4–6 Studies from our laboratory and others have shown that diffuse intimal thickening with aging is characterized by accumulation of fibronectin, collagen, and vascular smooth muscle cells (VSMCs), with an increase in matrix metalloproteinase (MMP)-2 and angiotensin II (Ang II) expression.7–11 In addition, the expression of aortic intracellular adhesion molecule and transforming growth factor-ß1 markedly increases with age, and these molecules localize to MMP-2–staining positive areas.12 Arterial aging is also characterized by an increase in NAD(P)H oxidase activity and reactive oxygen species production and a reduction in nitric oxide (NO) bioavailability.13–16 In addition, increased arterial levels of proinflammatory cytokines, such as tumor necrosis factor-{alpha} and interleukin-6, among others, accompany aging.17–20

Structural and biochemical changes that occur within large arteries with aging are accompanied by a shift of the VSMC phenotype from the "contractile" to the "synthetic" state, characterized by an increased proliferative and migratory responsiveness to growth factors.21–23 The discrepancies in the literature about VSMC proliferation are possibly due to species specificity and the factors used experimentally to stimulate proliferation. Migration and proliferation of VSMCs, endothelial cells, or macrophages are also mediated by chemokines. These molecules are members of a family of low-molecular-weight cytokines, originally recognized for their role in the activation and trafficking of leukocytes but more recently implicated in the control of many aspects of vascular biology.24,25 Monocyte chemotactic protein-1 (MCP-1/CCL2), one of the initially identified chemokines, through the activation of CCR2, a 7-transmembrane G protein–coupled receptor, can induce migration of monocytes, lymphocytes, endothelial cells, and VSMCs, and in human fibroblasts, MCP-1 induces the production of matrix metalloproteinase-1.24–28 The aforementioned cells within the arterial wall express MCP-1 during the development of atherogenesis and after balloon injury,29–34 and both transgenic and knockout mice for MCP-1 and CCR2 have demonstrated a role for these molecules in the pathogenesis of atherosclerosis35,36 and intimal hyperplasia.37 MCP-1 levels are also elevated in the plasma of older persons in the apparent absence of cardiovascular disease.38

Because MCP-1 expression by vascular cells is induced by Ang II and the arterial Ang II level is upregulated with aging,10,39,40 we hypothesized that arterial MCP-1 and CCR2 also increase with aging and affect VSMC properties within the arterial wall. The specific aims of our study were to determine the expression levels of these molecules in the rat aorta in vivo and in early-passage VSMCs in vitro, and to analyze the functional role of MCP-1 in this cell type.


*    Methods
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Animals
Eight- and 30-month-old Fisher344 x Brown Norway (F344xBN) male rats (Harlan Sprague-Dawley, Indianapolis, Ind), were housed, acclimated, and studied with appropriately approved protocols as previously indicated.12 Animals were killed by an overdose of sodium pentobarbital, and thoracic aortas were immediately removed.

Real-Time PCR Analysis
Real-time polymerase chain reaction (PCR) was performed using the SYBRGreen PCR-based protocol in a 384-well plate format (Applied Biosystems) as previously described.41 See http://atvb.ahajournals.org for further details.

Western Blot Analysis
Western blotting of homogenates from thoracic aortas, VSMCs, or their supernatants was performed with antibodies used in accordance with the manufacturer’s instructions. See http://atvb.ahajournals.org for further details.

Immunofluorescence Microscopy
Thoracic aortic frozen sections were double stained with anti-CD31 (1:50) or anti–{alpha}-smooth muscle actin (1:80), and anti–MCP-1 (1:50) or anti-CCR2 (1:50). Slides were incubated overnight at 4°C with primary antibodies diluted in accordance with the manufacturer’s instructions. Tissue sections were further incubated with secondary fluorescence-conjugated antibodies; photomicrographs were obtained at x400 magnification (Zeiss).

VSMC Isolation and Culture
VSMCs were enzymatically isolated as previously described.42 See http://atvb.ahajournals.org for further details.

VSMC Migration and Invasion
We used modified Boyden chambers (Neuroprobe) to study migration in response to MCP-1 and invasion. See http://atvb.ahajournals.org for further details.

VSMC Proliferation
VSMC proliferation was measured either by cell counting or as the rate of DNA doubling with use of the CyQUANT cell proliferation assay kit and following the manufacturer’s instructions (Molecular Probes). See http://atvb.ahajournals.org for further details.

Statistical Analysis
Data are presented as mean±SEM. The statistical significance of age and treatment effect was tested by either Student’s t test or a 1-way ANOVA, as appropriate. Differences were considered statistically significant when P<0.05.


*    Results
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Aortic MCP-1 and CCR2 Transcriptome and Protein Abundance Increase In Vivo With Aging
The in vivo transcriptome abundance measured by real-time PCR analysis of aortic MCP-1 increased 48-fold, and its receptor CCR2 increased by 11-fold with aging (Figure 1A and 1B). Proteins encoded by MCP-1 and CCR2 genes also increased with age, by 8.5- and 1.9-fold, respectively (Figure 1C and 1D).



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  		Figure 1. CCR2 and MCP-1 transcript and protein expression increase during aging in vivo. A, Representative agarose gel showing the amplified cDNA fragments by real-time PCR analysis. B, Average transcriptome abundance based on amplification efficiency normalized to the expression of rRNA 18S. C, Representative Western blot analysis of protein extracts from rat aortas. CTR represents 50 ng of recombinant MCP-1 (upper panel) or protein extract from the THP-1 monocyte cell line (lower panel). D, Average densitometric analysis of the bands normalized to the level of expression of ß-actin. *P<0.05 old vs young samples. m indicates months of age.

Immunostaining showed that the increased MCP-1 and CCR2 protein expression within the old rat aortas was localized mainly in the intimal region (white arrows in Figure 2A through 2C). {alpha}-Smooth muscle actin staining of cells in the thickened intimas of the aged rats colocalized with that of MCP-1 and CCR2 (Figure 2A and 2C, right). Double staining with CD31, a marker for endothelial cells, and MCP-1 indicated the presence of MCP-1 within these cells (Figure 2B, right).



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  		Figure 2. Aortic cellular components associated with MCP-1 and CCR2 by immunofluorescence and double staining. A, MCP-1 (red, middle panels) increases in rat aortas with age and localizes mainly in the intima. Left-hand panels show (A) staining with {alpha}-smooth muscle actin (green), a VSMC marker, and (B) CD31 (green), an endothelial cell marker. Merged images show that MCP-1 staining colocalized with CD31- and {alpha}-smooth muscle actin–positive areas, as indicated by the white arrows (A and B, yellow areas, right-hand panels). C, Immunostaining with anti-CCR2 antibody (red, middle panels) of young (8-month) and old (30-month) rat aortas. Left-hand panels show {alpha}-smooth muscle actin (green) staining. Merged images show that CCR2 staining colocalized with {alpha}-smooth muscle actin–positive areas. L indicates lumen; M, media.

VSMC MCP-1 and CCR2 Transcriptome and Protein Abundance Increase In Vitro With Aging
To determine whether VSMCs produce MCP-1 and CCR2, we enzymatically isolated these cells from thoracic aortas and cultured them on plastic through early passages (3 to 5). Real-time PCR analysis showed that the in vitro transcriptome abundances of cellular MCP-1 and of its receptor CCR2 both increased by 1.8-fold with aging (Figure 3A and 3B). The conditioned media of VSMCs isolated from young (8-month-old) rats showed expression of MCP-1, and protein extracts from the same cells indicated the presence of CCR2 within VSMCs. The MCP-1 produced by VSMCs isolated from older rats (30-month-old) was increased by 8.4-fold (Figure 3C, upper and 3D, left) over that of VSMCs from younger rats, and CCR2 protein was increased by 1.7-fold (Figure 3C, lower and 3D, right).



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  		Figure 3. MCP-1 and CCR2 transcripts and proteins are expressed by rat aortic VSMCs. A, Representative agarose gel showing the amplified cDNA fragments by real-time PCR analysis. B, Average transcriptome abundance. C, Representative Western blot for MCP-1 and CCR2. For CCR2 analysis, the samples represent protein extracts from rat aortic VSMCs (passages 3 to 5) isolated from 3 different age-matched animals (lower panel). For MCP-1 measurement, supernatants from the same cells were concentrated 10 times before proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (upper panel). CTR represent 50 ng of recombinant MCP-1 (upper panel) or protein extract from the THP-1 monocyte cell line (lower panel). D, Average data of 3 experiments normalized to the level of expression of ß-actin. *P<0.05 old vs young samples. m indicates months of age.

MCP-1 Affects VSMC Migration and Invasion Through a Synthetic Basement Membrane In Vitro
Because MCP-1 and CCR2 were increased in the old rat aortas and early-passage arterial VSMCs from aortas of old rats produced an increased amount of MCP-1, we next determined the potential role of this chemokine/receptor pair in the modulation of various functions of these cells. In the absence of added MCP-1, VSMCs isolated from older rats showed an average 2.1-fold increase in proliferation potential compared with cells from young animals when measured by both cell counting and DNA content assays (Figure 4A and 4B). In addition, their migration ability increased by 1.7-fold (Figure 4C). MCP-1 had no effect on the proliferation rate of VSMCs from young rats (Figure 4A and 4B). In contrast, in response to MCP-1, the migration capacity of VSMCs from young rats increased by 1.8-fold, ie, to a level equivalent to that of untreated old cells (Figure 4C).



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  		Figure 4. MCP-1 induces VSMC migration but not proliferation. VSMCs isolated from old (30-month) rats exhibit increased proliferation (A and B) and migration (C) ability compared with cells from younger animals (8-month). MCP-1 increases migration in a dose-dependent manner (C) but not proliferation (A and B). All experiments were run in triplicate. Each experimental condition used cells derived from at least 3 rats of each age. *P<0.05 old vs young samples.

One mechanism to explain the increased number of VSMCs in the thickened intimas from aortas of older rats (as shown in Figure 2A, lower) is that they migrated from the media and accessed the intima by invading the internal elastic membrane.21 We simulated the extracellular matrix environment with a synthetic basement membrane (Matrigel) and measured the VSMC invasiveness.42 Untreated early-passage VSMCs from old rats were able to invade a Matrigel-coated filter barrier 1.6-fold more efficiently than those cells from young rats in the absence of a specific chemoattractant factor (Figure 5). The invasion potential of VSMCs from old rats was not maximal, because the invasion ability of these cells increased by 3-fold when platelet-derived growth factor-BB was used as a chemoattractant (not shown). We used the virally coded chemokine vCCI (150 ng/mL), a CCR2 antagonist, to determine whether the increased invasiveness of VSMCs from old rat aortas involved activation of the CCR2 receptor. Of note, vCCI reduced the increased invasion potential of cells from older rats (Figure 5).



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  		Figure 5. MCP-1 pretreatment affects VSMC invasion. VSMCs isolated from old rat aortas show increased basal invasiveness in the absence of a chemoattractant compared with cells from younger animals. Treatment with MCP-1 (50 ng/mL, 24 hours) increased the basal invasion potential of young (8-month) VSMCs to the level of untreated VSMCs from old rats (30-month). The CCR2 antagonist vCCI (150 ng/mL) blocked this effect.

We next determined whether MCP-1 confers an increased invasion potential to VSMCs. Treatment of VSMCs from young rats with MCP-1 (50 ng/mL for 24 hours) induced an increase in their invasion ability to a level comparable to that of the old untreated cells (1.8-fold, Figure 5). vCCI abolished the MCP-1–induced increase in the invasion potential of VSMCs of young rats. Interestingly, MCP-1 did not further enhance the invasion ability of VSMCs from old rat aortas above the increased level before MCP-1 treatment.


*    Discussion
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 up arrowResults
 *Discussion
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It has been hypothesized that the increased risk for vascular diseases with advancing age is linked to molecular alterations within the vessel wall that are intrinsic to the aging process.1 Some of the specific cellular and molecular mechanisms implicated in age-associated vascular remodeling (eg, Ang II, MMP-2, transforming growth factor-ß1, and intracellular adhesion molecule10,12; reduced NO bioavailability13,16; and increased reactive oxygen species15) have in fact been implicated in experimental models of vascular diseases in younger animals. There is increasing evidence of an age-associated shift in plasma and arterial proinflammatory cytokine expression.17,19,20,38

Recent studies have also implicated chemokines in vascular diseases.24,25 The chemokine family of low-molecular-weight cytokines is subdivided into groups based on the relative positions of 4 conserved residues of cysteine: the CXC, CC, C, and CX3C chemokines, which show differential activities on cellular subtypes.24 MCP-1, a CC chemokine, historically known to play an important role in leukocyte trafficking and activation, was among the first chemokines studied. Extensive research demonstrated its effect on monocyte and lymphocyte migration, proliferation, and activation and its role in every step of the development of atherosclerosis.24,43

The MCP-1 receptor CCR2 is expressed on monocytes, macrophages, activated lymphocytes, natural killer cells, and endothelial cells.27,44–46 In addition, CCR2 mRNA has been detected in human VSMCs.47 Prior studies have demonstrated that the overexpression of MCP-1 by VSMCs induces the shift to the synthetic phenotype.48 Recently, MCP-1, among other CC chemokines, has been demonstrated to play a role as an angiogenic factor and activator of endothelial cells.27,49

The present study investigated a potential role of MCP-1 and CCR2 in age-associated vascular remodeling in the absence of atherosclerosis in the F344xBN rat model, which exhibits a moderate age-associated increase in systolic arterial pressure9 but does not develop atherosclerosis with advancing age.50 The first novel finding of the present study is that the transcriptome and protein levels of the chemokine MCP-1 and its receptor CCR2 in the rat aorta markedly increase with advancing age. Although monocytes and macrophages are important cellular components in the inflammatory response to vascular injury and in atherosclerosis,43 these cell types are not present in the arterial wall remodeled by aging in rodents and nonhuman primates.9,10

A second finding of this study is that MCP-1 protein is increased in endothelial cells and VSMCs within the arterial wall of aged rats in vivo. Moreover, we have demonstrated that CCR2 is present in vivo in the intimal VSMCs of old rat aortas. The effects of MCP-1 and the engagement of CCR2 as a mechanism for MCP-1 function in VSMCs are controversial.51–54 A third novel finding of the present study is that early-passage aortic VSMCs produce MCP-1 and express CCR2 and that both are increased in VSMCs isolated from old versus young rats. Ang II is one factor that is likely to control MCP-1 expression in VSMCs, because prior studies have shown that arterial Ang II is upregulated with aging and other downstream targets of Ang II type 1 receptor signaling also increase with aging.10,39,40,55 Additionally, these early-passage VSMCs isolated from old rat aortas exhibited enhanced proliferative, chemotactic, and invasive capacities relative to those from younger rats.21,22,56 A fourth new finding of the present study is that the CCR2 antagonist vCCI inhibited the increased invasiveness of early-passage VSMCs isolated from old rat aortas, suggesting a role for the MCP-1/CCR2 signaling pathway in the age-associated changes in VSMC invasion potential. vCCI is a viral chemokine-binding protein expressed by the vaccinia virus strain Lister that blocks MCP-1 activity by masking the receptor binding site.57 This molecule has been shown to ameliorate some aspects of inflammatory disease.58 A fifth novel finding of the present study is that MCP-1 enhances migration and the ability of VSMCs from young aortas to invade a Matrigel-coated filter that mimics the extracellular matrix and that after MCP-1 treatment, these features of young VSMCs are indistinguishable from those of early-passage VSMCs from older rats.

The increased invasiveness of young VSMCs in culture after treatment with MCP-1 was not observed in cells isolated from old rat aortas. Because receptor desensitization is a known regulatory mechanism of chemokine receptor activity,24 one explanation for the lack of response may be the high level of expression of MCP-1 by the old cells in situ, with the consequent desensitization of the receptor. In addition, the signaling response downstream from CCR2 could be reduced with aging. This has been reported for other vascular G protein–coupled receptors, such as the ß-adrenergic receptor.59 Another possibility is that the invasion potential of old VSMCs is already at a maximum. The use of the most potent agonist for VSMC invasion, platelet derived growth factor-BB, as a chemoattractant ruled out this explanation, because old VSMCs responded with a marked increase in invasion.

In summary, the present results demonstrate an age-associated increase in aortic MCP-1 and CCR2 gene transcript and protein levels that colocalized with aortic VSMCs and endothelial cells, combined with an increased proliferation, migration, and invasion capacity of early-passage VSMCs from the aortic walls of older versus younger rats. Treatment with MCP-1 in a CCR2-dependent manner increased the invasion capacity of VSMCs from young rats, rendering these cells indistinguishable from untreated cells isolated from older animals. These results support the hypothesis that MCP-1 and CCR2 are involved in the age-associated shift to the VSMC synthetic phenotype, including an increased invasiveness and number of VSMCs within the thickened aortic intimas of older rats. Thus, the MCP-1/CCR2 ligand/receptor pair represents a potential therapeutic target for age-associated vascular remodeling that underlies diseases such as hypertension and atherosclerosis.


*    Acknowledgments
 
Acknowledgments

This work was sponsored by the National Institutes of Health, National Institute on Aging, Intramural Program. The authors wish to thank Bruce Ziman for his assistance in the surgical procedures. We thank Maria Volkova and Kirill Tarasov for thoughtful discussions on the real-time PCR method.

Received May 4, 2004; accepted May 24, 2004.


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