Angio-associated migratory cell protein (AAMP) is a recently identified small protein with a molecular weight of 52 kDa. It is predominantly expressed in cells with a migratory phenotype, including endothelial cells, activated T-cells, and glia cells, as well as in malignant tissue such as human melanoma cells and adenocarcinoma cells of the colon. Thus, a role in angiogenesis and cancerogenesis was presumed (1- 2). Angio-associated migratory cell protein shares sequence homology with immunoglobulin superfamily members, including cellular adhesion molecules such as neural cell adhesion molecule (NCAM), platelet-endothelial cell adhesion molecule (PECAM), and leukocyte function antigen-2 (LFA-2); these and other immunoglobulin superfamily proteins mediate adhesion and migration of various malignant circulating cells (1,3).

Migration of smooth muscle cells (SMCs) into the developing neointima (NI) is a pivotal step during atherosclerosis development in native vessels as well as in restenosis formation after vascular intervention. After injury, a switch of the SMC phenotype from the quiescent to the secretory phenotype is followed by SMC migration and production of extracellular matrix in the NI (4- 5). During attachment and migration, SMCs polarize and extend protrusions in the direction of migration. These microextensions are driven by actin polymerization, representing a dynamic reorganization of the actin cytoskeleton. The regulatory mechanisms of actin restructuring remain unclear, but a complex interaction of actin-cytoskeletal associated proteins with GTPases might contribute (6). These processes possibly induce a change from a quiescent to a migratory SMC phenotype (7- 8). Various mediators such as interleukins or platelet-derived growth factor—which can be secreted from platelets, monocytes, or SMCs—can trigger the migration of SMCs (9). The expression of AAMP by SMCs during atherosclerosis and restenosis development, however, has not been studied previously. Thus, the rationale of this study was to elucidate the role of AAMP for the migration of SMCs and for the development of neointimal hyperplasia after vascular injury. To explore this, we quantified AAMP expression in SMCs derived from the NI and media as well as in atherosclerotic lesions of apolipoprotein E knockout (apoE^−/−) mice and restenotic lesions of porcine coronary arteries. Furthermore, the subcellular localization of AAMP and its effects on SMC migration and proliferation were analyzed. Eventually, we evaluated the in vivo expression of AAMP in the non-atherogenic porcine model of coronary balloon dilation and in the atherogenic mouse model of accelerated lesion formation. The effect of AAMP blockade on NI formation as well as SMC migration and proliferation was tested by treatment of wire-injured apoE^−/− mice with an inhibitory antibody against AAMP.

Medial rat smooth muscle cells (rSMCs) were isolated from the medial layer of the thoracic aorta of 6-week-old male Sprague-Dawley rats; neointimal rSMCs were derived from the neointimal thickening of the aorta 2 weeks after balloon angioplasty by microscopic dissection (10). Human smooth muscle cells (hSMCs) were isolated from the medial layer of mammary arteries after coronary artery bypass operation as described (11); informed written consent of all patients was obtained before the procedure. The SMCs were sparsely and densely grown and consecutively tested for their migratory potential with a transwell migration chamber system as previously described (11). Only SMCs with a distinct migratory phenotype were used for migration experiments; dense-grown SMCs, characterized by a significantly lower migratory potential, served as negative control.

An affinity-purified polyclonal antibody against recombinant AAMP was generated in rabbits (anti-rAAMP-ab; concentration 1 μg/ml) (1) and was used for detection of AAMP by Western blotting and immunohistochemistry. Due to its inhibitory capacity (12), the antibody was used for transwell migration chamber assays and for intraperitoneal administration in mice. Nonimmune rabbit immunoglobulin G serum (Sigma-Aldrich, St. Louis, Missouri) served as negative control. For detection, an alkaline phosphatase coupled secondary antibody (anti-rabbit, Chemicon, Temecula, California) was used. Antibody staining by monoclonal antibodies against alpha-smooth muscle actin (SMA) (Dako, Glostrup, Denmark), beta-actin (Sigma-Aldrich), von Willebrand factor (Dako), RhoA (Santa Cruz Biotechnology, Santa Cruz, California), and Ki67 (Dako) was detected by a secondary horseradish peroxidase-labeled antibody from Dako. For AAMP fluorescence staining, a fluorescein isothiocyanate (FITC)-conjugated antirabbit antibody was used (Chemicon).

The rSMCs and hSMCs grown on glass slides were washed in phosphate buffered saline (PBS), fixed immediately with 3.7% formaldehyde/PBS for 5 min, dehydrated by immersion in acetone, and permeabilized with 0.1% Triton X-100. Cells were stained with anti-rAAMP-ab and the respective FITC-conjugated antibody. After nuclear staining with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich), slides were analyzed by 2-photon laser-scanning microscopy using a Nikon Eclipse E600FN upright microscope (Tokyo, Japan), incorporated in the Bio-Rad Radiance 2100MP imaging system, and operated by Lasersharp2000 V6.0 (Bio-Rad, Hemel Hempstead, United Kingdom). Excitation was by the Tsunami Ti:sapphire laser (Spectra-Physics, Mountain View, California). Slides were observed through a water dipping 60× fluor objective with a 1.00 numerical aperture (Nikon, Tokyo, Japan) (13).

Flow cytometric analysis was performed as previously described (14). Cells were detached with accutase and subjected to flow cytometric analysis with or without Tween-induced membrane permeabilization and stained with anti-rAAMP-ab and the respective FITC-conjugated antibody. An FITC-conjugated immunoglobulin G isotype served as negative control.

For Western blotting of cultured SMCs, cells were suspended in radio-immunoprecipitation assay buffer (Roche, Mannheim, Germany); for analysis of SMC supernatants, concentration of supernatants was performed by freeze drying (Christ Alpha 2-4, Braun Biotech Int., Melsungen, Germany) before Western blotting. For in vivo expression analysis, porcine coronary arteries were prepared as previously described (15- 16). Always, 15 μg of protein was used. Equal loading of Western blots was assured by quantification of total protein content before each experiment (BCA Protein Assay Kit, Pierce, Rockford, Illinois) and by assessment of beta-actin expression. Blotted membranes were quantified with “ImageJ” image processing software (17).

To explore differential AAMP and RhoA localization by subcellular fractioning, a proteome extraction kit (Merck Chemicals, Nottingham, United Kingdom) was applied as described by the manufacturer. Membrane fractions that contain activated guanosine triphosphate-bound RhoA and cytosolic fractions that contain inactive guanosine diphosphate-bound RhoA were analyzed by Western blotting. For Rac analysis, a Rac activation assay kit (Upstate, Lake Placid, New York) was applied according to the manufacturer's instructions.

Searching for potential transmembrane domains was performed by computed AAMP sequence analysis (DNAMAN software, Lynnon BioSoft, Quebec City, Canada).

The AAMP complementary deoxyribonucleic acid was inserted into the vector pCMX-GL1 via EcoRI restriction sites. Mock and green fluorescent protein (GFP) vectors were used as controls. To induce AAMP knockdown in SMCs, siRNA was used. The AAMP siRNA, lamin controls, and fluo duplex transfection efficacy controls were purchased from Dharmacon (Lafayette, Colorado).

For transfection of AAMP vectors and siRNA, 3 × 10⁵ cells were seeded into T25 plates for functional assays or 5 × 10⁴ cells were seeded into each well of a 6-well plate for protein assays. The Amaxa NucleofectorTM technology (Amaxa, Cologne, Germany) was used for transfection of SMCs as previously described (11).

For chemotaxis experiments, transwell migration chambers were used (Neuro Probe Inc., Gaithersburg, Maryland). Polycarbonate filters (13 mm diameter, 8 μm pore size; Whatman International Ltd., Kent, United Kingdom) were coated with gelatine (5 mg/ml) as previously described (11). The upper compartment was filled with Dulbecco's modified Eagle's medium with or without anti-rAAMP-ab (100 μg/ml) (15). The rSMCs (2 × 10⁵ cells/ml) were placed in the upper compartment of the chamber and incubated for 4 h at 37°C. Transmigrated cells adhering to the lower chamber surface were fixed, stained, and counted (Leica DM RX microscope, Wetzlar, Germany).

For proliferation analysis, a 5-bromodeoxyuridine (BrdU) cell proliferation assay from Chemicon was applied according to the manufacturer's instructions.

All animal study protocols were approved by the local Institutional Animal Care and Use Committee and conformed to the tenets of the American Heart Association on research animal use.

An atherogenic mouse model of accelerated lesion formation was applied as previously described (18). In brief, a 0.014-inch flexible angioplasty guide wire was advanced into the carotid artery of apoE^−/− mice (C57BL/6 background, M&B, Ry, Denmark) via an incision in the external carotid artery, and endothelial denudation was achieved by 3 rotary passes along the common carotid artery.

After intraperitoneal administration of anti-rAAMP-ab at different concentrations, serum concentration of anti-rAAMP-ab was determined by dot blot reactions at 24, 48, and 72 h. At 72 h, an anti-rAAMP-ab serum concentration of 2 μg/ml was achieved after application of 50 μg anti-rAAMP-ab/animal. Subsequently, apoE^−/− mice were intraperitoneally treated with 50 μg anti-AAMP-ab or control nonimmune serum (n = 9 each) every 72 h starting 1 week before and up to 4 weeks after injury. One week (n = 3) or 4 weeks (n = 6) after injury, the animals were killed and the arteries were perfusion-fixed with 4% paraformaldehyde and embedded in paraffin.

A porcine restenosis model after coronary arterial injury was performed as previously described (15- 16). Briefly, after establishing arterial access by Doppler guided puncture of the femoral artery, 7,500 IU heparin were administered and the left anterior descending coronary artery was engaged with a 6-F guiding catheter. After intracoronary administration of nitrate (0.1 mg), the vessel diameter was calculated online by quantitative coronary angiography. The balloon/artery ratio was 1.1 to 1.2. The noninstrumented left circumflex artery served as uninjured control. Angioplasty was performed in a total of 6 animals by balloon inflation for 2 × 30 s (10 to 12 atm). Animals were maintained on a normal laboratory diet until being killed at 1 week (n = 6) or 4 weeks (n = 6). Hearts were harvested, and the epicardial coronary arteries were removed.

For histomorphometrical evaluation of murine arteries, modified Movat's pentachrome stainings were performed on paraffin-embedded sections as previously described (17). For histopathological evaluation, cell nuclei were counterstained with Mayer's hemalaun (Merck, Darmstadt, Germany). The AAMP expression intensity in SMCs was quantified by pixel analysis of AAMP staining intensity (Vectastain ABC-AP, Vector Laboratories, Burlingame, California). Results were expressed as cumulative intensity divided by total SMC area. Total AAMP⁺ or α-SMA⁺ vessel wall area of the media and NI was quantified and expressed as positive area percentage.

For proliferation analyses, Ki67-positive cell nuclei were counted, divided by total cell numbers, and displayed as percentage index of cell proliferation. Tissue sections from the proximal, mid, and distal part of the injured vessel segment were analyzed as previously described (15- 17). Tissue analysis was performed on digitized images (Leica DM RX microscope; “Diskus” analysis software, version 4.30, Hilgers, Koenigswinter, Germany) (15); for pixel analysis, “ImageJ” image processing software was applied.

All results are expressed as mean ± SEM. For in vitro experiments, statistical significance was evaluated with the unpaired Student t test or analysis of variance, followed by Dunnett's post hoc test for more than 2 means. For histomorphometric measurements, the Kruskal-Wallis test was used to determine overall statistical significance, followed by the Mann-Whitney U test with Bonferroni correction for subsequent pairwise comparison if the overall significance was <0.05; p values <0.05 were considered significant.

In sparsely grown hSMCs, AAMP expression was enhanced and preferentially localized along the cell membrane, whereas only weak staining for AAMP was detected in dense hSMCs and medial rSMCs (Figure 1A), implying a phenotype-related change in localization and strength of expression. Subcellular fractioning of hSMCs confirmed that AAMP is located primarily at the cellular membrane (55.5 ± 4.4 intensity/area) but also in the cytosolic fraction (23.1 ± 3.9; p < 0.01), whereas the nuclear fraction did not contain AAMP (0.2 ± 0.1; p < 0.001) (Figure 1B). Flow cytometric analysis of hSMCs also demonstrated that AAMP is presented preferentially at the cell membrane (without Tween treatment: 1,906 ± 185 FITC-A intensity) but can also be found in the cytosol (permeabilized cells with Tween treatment: 3,967 ± 203; nonimmune control: 345 ± 25; p < 0.01) (Figure 1C). Accordingly, computed analysis of AAMP sequence revealed a potential transmembrane domain between amino acids 322 and 345 (Figure 1D).

In medial sparse-grown hSMCs, AAMP expression was significantly increased compared with dense-grown medial hSMCs (33.3 ± 4.0 and 11.2 ± 3.3 intensity/area, p < 0.01). Similarly, sparse medial rSMCs displayed significantly higher AAMP levels compared with dense medial rSMCs (64.9 ± 6.0 and 21.4 ± 4.1, p < 0.01). Both, sparse and dense rSMCs derived from the NI displayed a pronounced AAMP expression (72.5 ± 6.1 and 66.2 ± 7.1, p = 0.54), indicating that AAMP expression in neointimal cells can not be downregulated (Figure 2A).

In cellular supernatants of rSMCs as well as hSMCs, AAMP expression was very low or not detectable (data not shown).

Treatment with the inhibitory anti-rAAMP-ab reduced SMC migration. Migration of both medial and, even more profoundly, neointimal rSMCs was inhibited by AAMP blockade in comparison with control (21.5 ± 4.0 and 74.4 ± 11.6 cells/area vs. 49.7 ± 3.8 and 164.5 ± 20.6 cells/area, respectively; p < 0.001) (Figure 2B). After transfection with AAMP expression vectors, SMCs displayed an enhanced migratory potential compared with mock or GFP transfected cells (hSMCs: 48.5 ± 3.5, 26.3 ± 2.7, and 27.3 ± 3.0; p < 0.01; rSMCs: 55.9 ± 4.2, 31.2 ± 2.4, and 34.4 ± 2.9; p < 0.01) (Figure 2C). The AAMP vector transfection resulted in AAMP overexpression as compared with mock and GFP transfected cells (hSMCs: 34.1 ± 2.0, 13.2 ± 2.1, and 16.0 ± 2.8 intensity/area; rSMCs: 41.1 ± 2.0, 18.0 ± 3.0, and 17.5 ± 2.8 intensity/area; p < 0.01) (Figure 2C).

Accordingly, AAMP knockdown by siRNA reduced the migratory potential of hSMCs compared with untreated and lamin controls (24.9 ± 2.0, 57.9 ± 3.5, and 55.6 ± 3.5 cells/area; p < 0.01) (Figure 2D), and the cellular AAMP content was reduced compared with controls (13.1 ± 2.2, 33.3 ± 2.8, and 31.3 ± 3.5 intensity/area; p < 0.01) (Figure 2D). Fluo duplex transfection efficacy checks confirmed good transfection efficacy (data not shown).

Treatment with anti-rAAMP-ab (0.094 ± 0.003 absorbance), AAMP sense-vectors (0.090 ± 0.007), and AAMP siRNA (0.096 ± 0.007) did not alter hSMC proliferation compared with controls (untreated: 0.098 ± 0.005; nonimmune serum: 0.099 ± 0.005; mock transfection: 0.095 ± 0.008; GFP transfection: 0.087 ± 0.002; lamin: 0.094 ± 0.005; p = 0.738) (Figure 2E).

These data imply that AAMP is crucially involved in the regulation of SMC migratory activity.

By proteome extraction, the effect of AAMP knockdown by siRNA was assessed for membrane and cytosolic hSMC fractions. The AAMP knockdown effectively reduced the membranous AAMP content (11.3 ± 1.5 intensity/area) compared with untreated (24.3 ± 2.0) and lamin controls (23.3 ± 2.7; p < 0.01) (Figure 3A). In the cytosolic fraction, AAMP content was not significantly altered by siRNA (14.2 ± 2.5) compared with untreated (18.5 ± 2.0) and lamin controls (19.2 ± 1.8; p > 0.05) (Figure 3A). The AAMP knockdown (0.5 ± 0.1) and anti-rAAMP-ab treatment (0.4 ± 0.0) both reduced RhoA content, whereas AAMP sense-vector transfection (9.1 ± 1.0) enhanced RhoA content in the membrane compared with controls (untreated: 4.6 ± 1.2, mock: 4.2 ± 1.0, GFP: 5.4 ± 0.9, lamin: 5.1 ± 0.8; p < 0.01) (Figure 3B). Cytosolic RhoA content was not altered by the different treatment modalities (AAMP siRNA: 11.0 ± 1.0; anti-rAAMP-ab: 11.3 ± 1.0; AAMP sense-vector: 10.2 ± 1.0; untreated: 11.3 ± 0.9; mock: 11.3 ± 0.8 intensity/area; GFP: 10.2 ± 0.9; lamin: 11.0 ± 1.0; p < 0.01) (Figure 3B). Analysis of Rac activation did not reveal significant differences (data not shown).

Pixel analysis of immunofluorescence staining in injured apoE^−/− mice by calculating AAMP expression intensity/cellular area revealed a slightly stronger expression of AAMP in 1- and 4-week groups in the media compared with the uninjured group (25.3 ± 2.8, 23.1 ± 2.2, and 18.0 ± 1.4 intensity/cellular area, p = 0.18) ((Figure 4)A and Figure 4B), whereas neointimal AAMP expression was significantly increased at 1 and 4 weeks as compared with the media of the same group (40.2 ± 2.8 and 36.8 ± 2.6, p < 0.01) ((Figure 4)A and Figure 4B).

Similar AAMP expression patterns were observed in the porcine model of balloon-induced coronary restenosis (Figure 5A). Again, pixel analysis revealed a substantial increase in AAMP expression in the NI at 1 and 4 weeks after injury (39.1 ± 3.1 and 35.0 ± 2.5, p < 0.01), whereas medial expression of AAMP was slightly enhanced at 1 and 4 weeks compared with control (24.5 ± 2.6, 21.9 ± 1.9, and 17.5 ± 1.1, p = 0.22) (Figure 5B). Sections stained with nonimmune serum showed only faint nonspecific background staining. Additionally, vessel wall protein lysates of injured and non-injured arteries were analyzed by Western blotting and quantified by subsequent densitometry. A significantly higher AAMP protein content was demonstrated at 1 and 4 weeks (41.5 ± 2.2 and 22.2 ± 1.2 intensity/area, respectively) after balloon dilation compared with control vessels (2.8 ± 0.6, p < 0.01) (Figure 5C).

An AAMP/α-SMA-colocalization analysis was performed to examine how SMCs contribute to AAMP expression.

In the murine model, medial AAMP/α-SMA double-positive areas as percentage of the total medial area were similar for AAMP and α-SMA in uninjured (44.0 ± 3.6% vs. 44.4 ± 2.9%, p = 0.87) and in injured specimen (at 1 week: 42.0 ± 3.9% vs. 39.6 ± 2.7%, p = 0.87; at 4 weeks: 37.0 ± 3.0% vs. 36.0 ± 3.2%, p = 0.87). Also, the neointimal double-positive percentage area was similar within the groups (at 1 week: 32.0 ± 3.4% vs. 33.0 ± 3.2%, p = 0.82; at 4 weeks: 29.7 ± 3.8% vs. 28.3 ± 3.2%, p = 0.82) ((Figure 6)A and Figure 6B); due to reduced neotintimal cell density, absolute values for AAMP/α-SMA double-positive percentage area were lower compared with the media.

The apoE^−/− mice were treated with inhibitory anti-rAAMP-ab or nonimmune serum intraperitoneally. At 4 weeks after injury, neointimal hyperplasia of the carotid artery was significantly reduced in anti-rAAMP-ab–treated mice compared with nonimmune serum-treated control mice (42,100 ± 2,800 and 78,700 ± 3,100 μm², p < 0.01), whereas medial areas were not different (19,237 ± 3,572 and 18,423 ± 815 μm², p = 0.86) ((Figure 7)A and Figure 7B). The reduction in neointimal area was associated with a significant reduction in the relative density of α-SMA⁺ cells in the NI compared with nonimmune serum-treated mice at 1 week (23.5 ± 3.2% and 39.6 ± 2.7%, p < 0.01) but not at 4 weeks (37.8 ± 3.9% and 36.0 ± 3.2%, p = 0.73) after injury ((Figure 7)C and Figure 7D). Neointimal cell proliferation, as quantified by Ki67 staining, was similar in both groups at 1 (anti-rAAMP-ab: 12.2 ± 0.9%; uninjured: 12.9 ± 1.0%, p = 0.92) and at 4 weeks (anti-rAAMP-ab: 4.2 ± 0.9%; uninjured: 5.1 ± 1.0%; p = 0.57) (Figure 7E). In the media of uninjured and injured arteries, there was only minimal Ki67 staining of about 1 percentage point (data not shown). These data imply that neutralization of AAMP protects from NI remodeling by early reduction of SMC migration into the developing lesion.

Initially, the expression of AAMP was described for a variety of cell types, including endothelial cells, activated T-cells, and malignant cells, which are all characterized by a migratory phenotype (12). In further studies, upregulation of AAMP was observed during cancerogenesis in gastrointestinal stromal tumors (19) and ductal carcinoma in situ of the breast (20). On matrigel, endothelial tube formation and cell migration could be blocked by an inhibitory AAMP antibody (12). Thus, it was speculated that AAMP was involved in tumor formation by supporting tumor angiogenesis (21). Although cell migration is also integral during the development of accelerated atherosclerosis and restenosis (4), data investigating the role of AAMP are not available.

In the present study, a significant upregulation of AAMP expression in vascular SMCs derived from the NI and in SMCs from different atherosclerotic and restenotic animal models was demonstrated. Moreover, AAMP overexpression promoted SMC migration, and blockade of AAMP by an inhibitory antibody or by siRNA reduced the migratory activity of vascular SMCs without affecting proliferation in vitro and in vivo. The study elucidated that, in line with these observations, blockade of AAMP results in a highly significant reduction of NI development in an apoE^−/− mouse injury model. Concomitantly, the blockade of AAMP generated an early substantial decrease of SMC density in the NI.

In summary, these data strongly suggest that AAMP plays an important role for SMC migration during the development and progression of accelerated atherosclerosis and restenosis. Notably, this was conclusively demonstrated for the non-atherogenic porcine restenotic model and the atherogenic mouse model of accelerated lesion formation. This is of importance, because both models are characterized by different strengths and weaknesses. The pathophysiologic mechanisms of neointimal development in the porcine coronary model are most similar to restenotic lesions in humans, but the model has a nonatherogenic background. The atherogenic mouse model overcomes this limitation, but the character of the restenotic lesions in the smaller mouse carotid arteries as well as the mechanisms of applied mechanical injury vary significantly between rodents and the situation in humans.

Although prior studies supported a relevance of AAMP for cellular migration in angiogenesis and cancerogenesis, only limited data regarding the mechanistic role of AAMP are available. Here, it could be demonstrated that AAMP is localized on the cellular membrane and in the cytosol of SMCs and that the membrane-associated pool of AAMP seems to be decisive for the regulation of SMC migration. Thus, a blockade of membranous AAMP by an inhibitory antibody or by downregulation of AAMP decreases SMC migration and, concomitantly, results in a decreased activity of the small GTPase RhoA, which is known to play a key role for control of cellular migration (6,22). Computed protein structure analysis revealed a potential transmembrane domain between amino acids 322 and 345 and thereby supports the significance of AAMP as a membrane-associated protein. Because homology analysis shows that AAMP shares sequence homology with immunoglobulin superfamily members like cellular adhesion molecules NCAM, PECAM, and LFA-2, which are known to mediate adhesion and migration of various malignant circulating cells (1), a potential binding of AAMP to heparin or to beta-actinin was suggested. Applying advanced imaging and cell fractioning techniques, this study could clearly demonstrate that AAMP is localized in the cellular membrane and regulates SMC migration via the RhoA pathway. (Figure 8) summarizes these findings schematically. Due to our data, AAMP can also be found in the cytosol but a functional role for the regulation of cell migration could not be attributed to cytosolic AAMP. Furthermore, secreted AAMP to the extracellular area as suggested previously could not be detected.

In summary, we have identified AAMP, abundantly produced by neointimal SMCs after vascular injury, as a potential regulator of SMC migration in atherosclerotic and restenotic disease.

For supplementary material regarding the culture of smooth muscle cells, please see the online version of this article.

Blockade of Angio-Associated Migratory Cell Protein Inhibits Smooth Muscle Cell Migration and Neointima Formation in Accelerated Atherosclerosis