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Cardiovascular Research 2004 62(1):212-222; doi:10.1016/j.cardiores.2004.01.004
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation

Rüdiger Blindt*,a, Anja-Katrin Bosserhoffb, Julia Dammersa, Nicole Krottc, Lütfü Demircand, Rainer Hoffmanna, Peter Hanratha, Christian Webere and Felix Vogta,c

aMedical Clinic I, University Hospital Aachen, Pauwelsstr 30, 52074 Aachen, Germany
bInstitute of Pathology, University Regensburg, 93053 Regensburg, Germany
cInterdisciplinary Centre for Clinical Research in Biomaterials and Tissue–Material–Interaction in Implants "BIOMAT", University Hospital Aachen, Aachen, Germany
dDepartment for Cardiothoracic Surgery, University Hospital Aachen, Aachen, Germany
eDepartment of Molecular Cardiovascular Research, University Hospital Aachen, Aachen, Germany

* Corresponding author. Tel.: +49-241-8089300; fax: +49-2421-959262. Email address: ruediger.blindt{at}post.rwth-aachen.de

Received 7 October 2003; revised 12 December 2003; accepted 5 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The aim of the study was to analyze whether cadherin- and Rho-family GTPases-mediated dynamic rearrangement of cell–cell adhesion play an important role during human arterial smooth muscle cell (haSMC) migration. Methods: Expression patterns of N-cadherin and β-catenin were analyzed in a domestic pig restenosis model after 14, 28, and 90 days as well as in quiescent and migratory haSMCs in vitro. N-cadherin expression was upregulated by transient sense; downregulation was induced by antisense transfection. For functional inhibition, antibody GC-4 was used. Cell migration was quantified using Boyden chamber assays. Regulation of RhoA GTPase was tested by assessment of RhoA activity. Results: In vivo analysis of N-cadherin expression in a porcine restenosis model revealed downregulation in the neointima after 14 days. After 28 days, N-cadherin expression was slightly restored, while after 90 days, no difference between medial and neointimal expression was detectable. β-Catenin levels remained unchanged during the whole period. According to the in vivo situation, N-cadherin was significantly downregulated in migratory haSMCs compared to quiescent cells in vitro. After N-cadherin overexpression, haSMC migration was reduced by 87% (P<0.001). By contrast, inhibition of N-cadherin in quiescent haSMCs by GC-4 increased the migratory potential by 87% (P<0.01). In haSMCs overexpressing N-cadherin, a significant upregulation of RhoA activity was demonstrated, while RhoA activity was blocked by GC-4. Conclusions: These results indicate that the regulation of haSMC attachment by N-cadherins is essential for haSMC migration. Modification of N-cadherin expression and activity induces RhoA signaling with relevance for the reorganization of the actin cytoskeleton.

KEYWORDS Adhesion molecules; Restenosis; Smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cadherins comprise an important group of cell–cell adhesion molecules that mediate intercellular adhesion by Ca2+-dependent homophilic interactions [1–4]. Up to this time, more than 80 members of the cadherin superfamily have been identified that include classic cadherins, desmogleins, desmocollins, protocadherins, CNRs, fats, seven-pass transmembrane cadherins, and Ret tyrosine kinase. Cadherins cluster through a zipper-like mechanism to homodimers. The intracellular domains are conserved among the members of each subfamily, and in the case of classic cadherins, they interact with p120ctn and β-catenin at different portions of the cytoplasmic domain. The latter binds to {alpha}-catenin, and this molecular complex further associates with vinculin and other cytoskeletal proteins, resulting in the organization of adherens junction or zonula adherens in polarized epithelial cells. Dynamic rearrangement of cell–cell adhesion plays an important role in many physiological and pathological processes as embryonic and tissue development, cell scattering, wound healing, tumour metastasis, and cell migration [5,6].

Migration of human arterial smooth muscle cells (haSMCs) is an essential pathogenic process in the development of a broad spectrum of cardiovascular disorders like atherosclerosis, restenosis after percutaneous transluminal coronary angioplasty (PTCA), and stent implantation as well as transplant vessel disease [7,8]. These events are characterized by molecular mechanisms leading to proliferation of haSMCs with consecutive migration into the vessel intima. The process of haSMC migration is accompanied by a dedifferentiation of haSMCs into an extracellular matrix protein-producing phenotype causing vessel occlusion by atherosclerotic plaques or neointima formation in restenosis development [9,10]. Migration of haSMCs is characterized by dynamic reorganization of the actin cytoskeleton, changes in gene transcription with upregulation of new membrane components (integrins, junctional adhesion molecules), and controlled cell–matrix and cell–cell interaction [11–13]. The molecular control of actin filament assembly is regulated by the Rho family of small GTPases, particularly Rho, Rac, and Cdc42 [14]. It was described that RhoA mediates migration and DNA synthesis in haSMCs [15]. Recently, a potential role of cadherins in the process of haSMC proliferation and migration was suggested, although detailed mechanisms are still enigmatic [16,17]. Regulation of cadherin-mediated cell adhesion by Rho-family GTPases has been demonstrated [6]. Thus, homophilic N-cadherin adhesion causes activation of RhoA and deactivation of Rac and Cdc42 in skeletal muscle cells [18].

Therefore, the aim of this study was to investigate the regulation of N-cadherin and β-catenin expression during the process of haSMC migration. Firstly, expression was analysed in vivo and in vitro using a porcine restenosis model and haSMCs with a migratory or quiescent phenotype. Secondly, the effect of N-cadherin-mediated migration was analysed by transfection of haSMCs with sense and antisense expression vectors for N-cadherin. And finally, the role of RhoA during differential N-cadherin expression in haSMC migration was investigated.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animal model
A porcine model of coronary arterial injury was used as described previously [19]. The investigation conforms to the ‘Guide for the Care and Use of Laboratory Animals’ published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Domestic pigs (40 kg) were medicated with aspirin (500 mg) the evening before vascular intervention. For general anesthesia, pigs were sedated with intramuscular ketamine (20 mg/kg) and azaperone (5 mg/kg). After endotracheal intubation, they were ventilated with 1 l/min oxygen and 3 l/min nitrous oxide. By intravenous administration of pentobarbiturate, anesthesia was maintained throughout the experiment. Femoral arterial access was established by Doppler guided puncture of the vessel, a 7F sheath was placed, and an intra-arterial bolus of heparin (10,000 U) was administered. The left or right coronary artery was engaged with a guiding catheter and the artery was injured with an oversized balloon (1.2 overstretch) which was inflated for 30 s (12 atm), whereas the noninstrumented artery served as undilated control. Animals were maintained on a normal laboratory diet and 100 mg aspirin was administered until sacrification.

The pigs were sacrificed after 14, 28, and 90 days with a lethal dose of barbiturate. Immediately after euthanasia, the hearts were harvested and the epicardial coronary arteries were removed. The arteries were sectioned into 3- to 5-mm segments; tissue sections were performed as previously described [20].

2.2. Antibodies
For immunostaining an anti-N-cadherin antibody from Takara (1:250), and for Western blotting, an anti-N-cadherin antibody from BD Bioscience (1:2500) was used; both recognize the intracellular domain of N-cadherin. For functional inhibition, anti-N-cadherin antibody GC-4 (1:100, Sigma), which recognizes the extracellular domain of N-cadherin, was used. Anti-β-catenin and anti-E-cadherin antibodies were purchased from BD Bioscience (1:50 and 1:2500). Mouse monoclonal antibody against anti-smooth muscle actin (SMA, 1:400) and anti-von Willebrand factor VIII (vWF, 1:200) were purchased from Dako, anti-RhoA antibody (1:200) was used from Santa Cruz Biotechnology. TRITC-labelled phalloidin and nonimmune mouse IgG1 were purchased from Sigma.

2.3. Immunohistochemistry
Immunostaining was performed with 5-µm paraffin-embedded tissue sections as described previously [20]. Anti-N-cadherin and anti-β-catenin antibodies were used as primary antibodies; for detection, an HRP-labelled secondary antibody (1:50, Dako) was used. Negative controls were carried out with nonimmune IgG. Slides were analysed with a Leitz microscope.

2.4. Cell culture
Human mammary arteries were obtained from patients during coronary artery bypass operation with their informed written consent obtained prior to the procedure. haSMCs were isolated and cultured as previously described [21].

All cells were maintained in Dulbecco's modified Eagle's medium (DMEM/Nut.Mix F-12 with Glutamax, Gibco-BRL), supplemented with penicillin (100 U/ml), streptomycine (10 µg/ml; both from Sigma-Aldrich) and 10% foetal calf serum (Gibco-BRL) and split 1:2 at confluency. Cells were detached by incubation with 0.05% trypsin, 0.04% EDTA (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 5 min at 37 °C. They were identified as vascular haSMCs by their spindle-cell-shaped morphology and their characteristic hill-and-valley growth pattern. Immunostaining confirmed the presence of smooth muscle actin (SMA) as positive marker for SMCs; vWF served as negative marker and confirmed the absence of endothelial cells. By this method, cultures of >95% haSMC purity were routinely obtained. The studies were performed with cells at passages 3–7. For each experiment, cells were explanted from at least three to four different donors to minimize donor dependency.

For the migration experiments, haSMCs with different migratory behaviours were used as previously described [22]. For quiescent haSMCs, a growth arrest was induced by plating 2 x 106 cells/ml in a 75-cm2 culture plastic flask. Cells were grown to confluency for at least 5 days and serum-deprivated 3 days prior to the assay. For the migratory phenotype, haSMCs (2 x 105 cells/ml) were plated in a 75-qcm flask, maintained in DMEM (10% FCS) and split at subconfluency 1:3. The subconfluent haSMCs were always split 2 days prior to the assay.

2.5. Immunofluorescence of haSMCs
Cells in culture were grown on glass slides, washed briefly in PBS, and fixated immediately with 3.7% formaldehyde/PBS for 5 min. Cells were dehydrated by immersion in acetone and permeabilized with 0.1% Triton X-100. Cells were stained with a 50 µg/ml fluorescent phalloidin conjugate solution in PBS for 40 min at room temperature. Unbound TRITC-phalloidin was removed by washing several times with PBS.

2.6. Chemotaxis assays
For chemotaxis experiments, Boyden-type blind well chambers were used (Costar). Polycarbonate filters (13 mm diameter, 8 µm pore size; Nucleopore) were coated with gelatine (5 mg/m) as previously described [22]. The lower compartment was filled with conditioned medium as a chemoattractant. For haSMCs, the conditioned medium was produced by proliferating HUVEC cells which were incubated for 24 h in serum-free medium. HUVEC-conditioned medium had the same proliferation stimulus as PDGF AB (10 ng/ml) [21,22]. The medium was cleared from cell debris by centrifugation, stored at –20 °C, and used as chemoattractant without adding further supplements. haSMCs were harvested by trypsinization, resuspended in medium without FCS and placed in the upper compartment of the chambers. After incubation for 4 h at 37 °C, the filters were removed. The cells adhering to the lower surface were fixed, stained and counted. Each sample was assayed sixfold.

2.7. Vector constructs and transient transfection
The coding region of the human N-cadherin gene was PCR amplified using the primers N-Cad for (5'-GAC GGA TCC CGG CCC CTC TCC GCC TCC ATG) and N-Cad rev (5'-GAC GGA TCC GTT CAC CCT GAA GTT CAG TC). Blunt ends of the PCR product were generated by the Klenow fragment of DNA Polymerase I and inserted into the pBluescript II KS vector (Gibco-BRL) via SmaI restriction site. For the sense expression vector, the Bam–XhoI fragment of pBluescript II KS was subcloned in pCMX-PL1. For the N-cadherin antisense vector, the EcoRV–XbaI fragment of pBluescript II KS was blunt-ended by the Klenow reaction and inserted into pCMX PL1, digested with EcoRV. The N-cadherin sense expression vector pCMV-cN-FLAG-pA was a kind gift of Prof. Tackeichi (University of Kyoto, Japan).

For transfection, either 3 x 105 cells were seeded into a T25 flask for functional assays or 5 x 104 cells were seeded into each well of a 6-well plate for protein assays. The Amaxa NucleofectorTM technology (Amaxa) was used for transfection of haSMCs as described earlier [19]. This method reveals a transfection efficacy of 50% [23]. Briefly, the cells were resuspended in 10 µl phosphate-buffered saline, pH 7.5 (PBS). 2 x 105 to 5 x 105 haSMCs were transfected with 5 µg cDNA according to the manufacturer's protocol and the mixture was transferred into the electroporation cuvette (Invitrogen). Immediately after electroporation, the cells were suspended in 4.9-ml cell culture medium and transferred to T25 flasks. The cells were harvested 24 or 48 h after transfection and used for further assays.

2.8. Subcellular fractionation of RhoA
Adherent haSMC lysates were treated with ice-cold lysis buffer containing 20 mM Tris (pH 8.0), 250 mM sucrose, 1 mM PMSF, and 10 mM aprotinin and leupeptin. After 3 cycles of freeze-and-thaw, samples were centrifuged at 100,000 x g at 4 °C for 60 min. The cytosol-containing supernatant was removed as soluble fraction. Pellets were gently washed twice by the same lysis buffer and resuspended in 100 ml of lysis buffer supplemented with 1% Triton X-100 and 0.1% SDS. Cell debris was separated by centrifugation (14,000 rpm at 4 °C for 20 min), and the supernatant was saved as particulate fraction. Membrane and cytosolic fractions were then assayed for total protein. Equal amounts were analysed by Western blotting. Typically, 10 µg of protein was analysed representing 1–2% of the cytoplasmatic fraction and 10–20% of the membrane fraction.

2.9. Western blotting
Ten micrograms of protein per lane were separated on a SDS-PAGE gel and subsequently blotted onto a PVDF membrane. After blocking for 1 h with 3% BSA/PBS, the membrane was incubated for 2 h with the N-cadherin or RhoA antibodies. The membrane was washed three times in PBS, incubated for 1 h with an alkaline phosphatase-coupled secondary antibody and then washed again. For detection, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate staining (Sigma) was used.

2.10. Statistical analysis
For the chemotactic assays, all results are expressed as mean±S.D. Densitometric analysis of Western blots was performed with ‘Quantity One’ software from Bio-Rad Version 4.5.0. For pixel intensity analysis, RGB TIFF files of all images were converted to greyscale and further evaluated with ‘Analysis Software Version 3.1’ from Soft Imaging System. Statistical significance was evaluated using unpaired Student's t-test for comparison between 2 means, or ANOVA followed by Dunnett's post hoc test for more than 2 means. A P-value of <0.05 was considered to indicate statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Analysis of N-cadherin expression in vivo
Tissue sections from a porcine restenosis model were stained with N-cadherin or SMA-specific antibodies to analyse the expression of N-cadherin in medial and neointimal smooth muscle cells. An undilated artery always served as comparison for the analysis of N-cadherin expression patterns. Nonimmune mouse IgG was used as negative control. Tissue specimens were used from groups of four pigs after 14, 28, and 90 days, respectively. As reported earlier [19,24], in all dilated arteries, neointima formation was observable with a maximum after 90 days, whereas in nondilated arteries, no intimal proliferation was detectable.

In the undilated arteries, there was a strong N-cadherin staining in the medial wall, while no signal was detectable in the adventitia (Fig. 1A). N-cadherin expression was markedly downregulated in the neointima of dilated vessels after 14 days compared to the surrounding medial wall that further showed a strong expression of N-cadherin (48±1 vs. 71±4 intensity/mm2, P<0.001, Figs. 1B and 4AGo). In the adventitial layer, no relevant N-cadherin expression was observable. After 28 days, there was a slightly restituted N-cadherin expression in the neointima (54±3 vs. 72±2 intensity/mm2, P<0.01, Figs. 1C, D and 4AGo), while after 90 days, the neointimal N-cadherin expression level nearly reached the signal intensity of the uninjured media again (66±1 vs. 69±3 intensity/mm2, P>0.05, Figs. 1E, F and 4AGo). N-Cadherin and SMA were colocalised, indicating smooth muscle cell-specific N-cadherin staining (Fig. 2). Expression levels of SMA were similar in the neointima and media of all arteries (88±4 vs. 80±14, 68±4 vs. 66±4, and 80±5 vs. 86±3 intensity/mm2, P>0.05, Figs. 2 and 4BGo). Analysis of β-catenin expression in undilated and dilated arteries revealed equal expression in the neointima of dilated arteries and in the media of undilated vessels 14, 28, and 90 days after vascular injury (79±10 vs. 87±12, 81±14 vs. 90±6, and 84±4 vs. 88±6 intensity/mm2, P>0.05, Figs. 3 and 4CGo).


Figure 1
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  		Fig. 1 Photomicrographs of tissue sections from the porcine restenosis model (L=lumen, arrows indicate the border between the neointima and the media). (A) N-cadherin expression in undilated arteries (50-fold magnification). (B) N-cadherin expression in dilated vessels after 14 days (50-fold magnification). (C) and (D) N-cadherin expression in dilated arteries after 28 days (50- and 200-fold magnification) and after 90 days [(E) and (F), 50- and 200-fold magnification].

 

Figure 4
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  		Fig. 4 Expression of N-cadherin (A), SMA (B), and β-catenin (C) was quantified by pixel intensity analysis. The neointimal and medial areas were quantified separately 14, 28, and 90 days after vascular injury.

 

Figure 2
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  		Fig. 2 Photomicrographs of tissue sections from the porcine restenosis model (L=lumen, arrows indicate the border between the neointima and the media). (A) SMA expression in undilated arteries (50-fold magnification). (B) SMA expression in dilated vessels after 14 days (50-fold magnification). SMA expression in dilated arteries after 28 days [(C) and (D) 50- and 200-fold magnification] and after 90 days [(E) and (F) 50- and 200-fold magnification].

 

Figure 3
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  		Fig. 3 Photomicrographs of tissue sections from the porcine restenosis model (L=lumen, arrows indicate the border between the neointima and the media). β-catenin expression was analysed in the media of undilated vessels [(A) 50-fold magnification] as well as in the neointima and media of dilated vessels 14 days [(B) and (C) 50- and 200-fold magnification], 28 days [(D) and (E) 50- and 200-fold magnification], and 90 days after vascular injury [(F) 200-fold magnification].

 
3.2. Analysis of cadherin and catenin expression in quiescent and migratory haSMC phenotypes in vitro
According to the significant downregulation of N-cadherin expression in the early stage of restenosis development, when migration of SMCs essentially contributes to neointima formation, the regulation of cadherins and its role in haSMC migration was analysed in vitro. For this reason, a model was used that is based on different migratory potentials of primary haSMCs. As it had been demonstrated earlier [21,22], the migratory potential of haSMCs depends on their phenotype that is schematically characterized either as contractile and quiescent, or as synthetic. The quiescent phenotype can be induced in vitro by growth arrest through long-time confluent cell culture and serum depletion, while synthetic haSMCs are cultured in the presence of serum or growth factors. If both phenotypes are attracted by promigratory chemokines, the quiescent one only shows a low migratory potential with nearly random mobility while synthetic haSMCs migrate approximately 10-fold more towards the stimulus (Fig. 5A). N-cadherin expression was analysed in both phenotypes. According to the in vivo expression pattern, N-cadherin was strongly expressed in the quiescent phenotype, while a marked downregulation was observed in the migratory phenotype (91±17 vs. 11±6 intensity/mm2, P<0.001, Fig. 5B). By contrast, expression of E-cadherin, which was formerly described to be upregulated in atherosclerotic lesions [25], and the intracellular cadherin-binding partner, β-catenin, was unchanged in both phenotypes (118±14 vs. 112±17, 111±13 vs. 120±19 intensity/mm2, P>0.05, Fig. 5B). All assays were performed in triplicate with pooled haSMCs explanted from three to four different donors.


Figure 5
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  		Fig. 5 (A) Comparison of the migratory potential of quiescent and synthetic haSMCs. Both phenotypes were stimulated by conditioned medium of proliferating HUVEC cells (ECM) or by PDGF AB (10 ng/ml) in the Boyden chamber. The quiescent haSMC phenotype only showed a low migratory potential with nearly random mobility while synthetic, migratory haSMCs migrated approximately 10-fold more towards the stimulus. HUVEC conditioned medium had the same proliferation stimulus as PDGF AB. (B) Representative Western blots of E-cadherin, β-catenin, and N-cadherin expression in quiescent (Q) and migratory (M) haSMCs. Quantification was performed by densitometric analysis.

 
3.3 Overexpression of N-cadherin in haSMCs induces a dose-depending reduction of the migratory potential
Migratory haSMCs were transfected with full-length N-cadherin or E-cadherin expression vectors in parallel to mock control. Depending on the amount of transfected plasmids, a dose-relating upregulation of N-cadherin was observed resulting in either an N-cadherin signal intensity of 45±11/mm2 (N-cadherin sense) and 54±16/mm2 (pCMV-cN, Fig. 6A), or an N-cadherin signal intensity of 92±7/mm2 (Fig. 6B). No changes in N-cadherin expression were observed in mock control or after transfection of E-cadherin sense expression vector. N-cadherin intensity levels of 45±11 and 54±16/mm2 decreased migration by 25% compared to mock control (mock control 100%, N-cadherin sense 75±24%, and pCMV-cN 73±29%, P<0.05 for both, Fig. 6A), while an approximately twofold stronger signal of 92±7/mm2 decreased the migratory potential by 87% compared to mock control (mock control 100%, N-cadherin 13±10%, P<0.001, Fig. 6B). The E-cadherin expression plasmid did not alter the migratory capacities of haSMCs (E-cadherin 102±28% and 91±16%, Fig. 6A and B). All assays were performed in triplicate with pooled haSMCs explanted from three to four different donors.


Figure 6
Figure 6
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  		Fig. 6 Analysis of the migratory potential of N-cadherin sense-transfected haSMCs in the Boyden chamber. Migratory haSMCs were transfected with different amounts of N-cadherin sense expression vector and the migratory potential of these cells was evaluated [(A) and (B)]. Mock control was set as 100% migration. Results are always given compared to mock control. (C) Analysis of the migratory potential of quiescent (Q) and migratory (M) haSMCs in the Boyden chamber. Quiescent haSMCs were treated with GC-4 during the migration assay. Nonimmune IgG1 isotype served as negative control.

 
3.4 Reversal of the quiescent haSMC phenotype by N-cadherin blockade
Anti-N-cadherin antibody GC-4, which binds to the extracellular N-cadherin domain and which was formerly shown to effectively inhibit N-cadherin adhesion [26], was used to verify the hypothesis that downregulation of N-cadherin activity facilitates cell migration. Therefore, quiescent haSMCs were treated with GC-4 during the migration assay. N-cadherin blockade strongly increased the migratory potential of these cells by 87%. A reversal of the quiescent phenotype to a migratory potential equally to the untransfected migratory control was revealed (migratory haSMCs 100%, quiescent haSMCs 42±26%, P<0.05, GC-4-treated quiescent haSMCs 113±58%, P>0.05). Treatment of quiescent haSMCs with nonimmune IgG1 did not change their migratory potential (32±10%, Fig. 6C). All assays were performed in triplicate with pooled haSMCs explanted from three to four different donors.

3.5 N-cadherin affects haSMC migration by modulation of RhoA activity
Activated GTP-bound RhoA is mainly located at the cellular membrane, while inactive, GDP-bound RhoA is found in the cytosolic fraction. Thus, Rho partially translocates from the soluble to the particulate fraction during its activation [27]. In order to analyse the activity level of RhoA in quiescent and migratory haSMCs as well as in cells treated with inhibitory antibody GC-4 and in N-cadherin-transfected cells, the subcellular localization of RhoA was examined after 2, 6, and 12 h.

First, RhoA activity was analysed in quiescent haSMCs with strong intrinsic N-cadherin expression (see Fig. 5B) and in migratory haSMCs that express significantly less N-cadherin. Consistent with previous reports [28], the amounts of RhoA in the cytosolic fraction did not change significantly (58±17 vs. 52±4 intensity/mm2, P>0.05, Fig. 7A), while RhoA activity in the particulate fraction of migratory haSMCs significantly decreased (61±22 vs. 9±8 intensity/mm2, P<0.05, Fig. 7A). Furthermore, RhoA activity in quiescent haSMCs was also reduced significantly after treatment with inhibitory antibody GC-4 comparable to RhoA activity in migratory haSMCs (61±22 vs. 4±8 intensity/mm2, P<0.01, Fig. 7A). Again, RhoA amounts in the cytosolic fraction remained unchanged (58±17 vs. 56±11 intensity/mm2, P>0.05, Fig. 7A).


Figure 7
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  		Fig. 7 (A) Analysis of RhoA activation after 12 h in quiescent and migratory haSMCs as well as in quiescent haSMCs treated with inhibitory antibody GC-4. Quiescent haSMCs showed a significantly increased activation of RhoA (particulate fraction) compared to migratory haSMCs, which was nearly completely reversible by GC-4. (B) Overexpression of N-cadherin in migratory haSMCs after 12 h led to marked activation of RhoA compared to mock control while N-cadherin antisense transfection did not alter RhoA activity. RhoA expression levels were evaluated by densitometric analysis.

 
In further experiments, migratory haSMCs were transfected with N-cadherin sense expression vector to confirm that upregulation of N-cadherin induces RhoA activation in this haSMC phenotype. The RhoA intensity in the membrane fraction of sense-transfected cells was significantly higher compared to mock control (47±4 vs. 26±4 intensity/mm2, P<0.001, Fig. 7B). RhoA intensity after control transfection with antisense constructs did not induce RhoA activation and was similar to mock control (21±4 vs. 26±4 intensity/mm2, P>0.05, Fig. 7B). The intensity of RhoA in the cytosolic fractions was similar (48±4 vs. 51±3 vs. 53±1 intensity/mm2, P>0.05). Two and 6 hours after N-cadherin sense transfection only a slight activation of RhoA was detectable (data not shown).

3.6. Structural changes of the actin cytoskeleton related to N-cadherin expression and activity
To analyse the effect of RhoA activity modification on structural changes of the actin cytoskeleton, actin stress fibre formation was analysed by immunofluorescence-staining with fluorescence-labelled phalloidin. In quiescent haSMCs, intense formation of stress fibres was observed that were orientated along the spindle-shaped cell axis (Fig. 8A and B). In the migratory haSMC phenotype, haSMCs showed a different morphology with broader cell bodies and increased cell extensions like lamellipodia and filopodia. In these cells, a marked loss of stress fibres could be observed. In quiescent haSMCs treated with GC-4, there was a morphology that was very similar to the migratory phenotype with a marked reduction of stress fibres (Fig. 8C and D).


Figure 8
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  		Fig. 8 Stress fibre formation was analysed by TRITC-conjugated phalloidin staining. Quiescent haSMCs [(A) 100-fold magnification, (B) 1000-fold magnification]. Quiescent haSMCs treated with GC-4 antibody for 12 h [(C) 100-fold magnification, (D) 1000-fold magnification]. Migratory haSMCs transfected with mock control [(E) 100-fold magnification, (F) 1000-fold magnification]. Migratory haSMCs transfected with N-cadherin sense expression vector after 12 h [(G) 100-fold magnification, (H) 1000-fold magnification].

 
Migratory haSMCs that were transfected with mock control had a morphology comparable to untransfected migratory haSMCs and showed reduced stress fibre formation (Fig. 8E and F), while actin fibres were markedly increased in N-cadherin sense-transfected haSMCs (Fig. 8G and H). The amount of stress fibres in antisense-transfected cells was comparable to mock-transfected cells (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
This study has demonstrated that (1) N-cadherin is downregulated in haSMCs during cell migration in vivo and in vitro, while expression levels of its intracellular binding partner β-catenin remain equal; (2) downregulation or blockade of N-cadherins stimulates haSMC migration in vitro; and (3) downregulation or functional blockade of N-cadherin in migratory haSMCs is associated with reduced RhoA activity.

4.1 Differential N-cadherin expression of haSMCs and its role for cell migration and proliferation
As we demonstrated, downregulation of N-cadherin during restenosis development led to a significant increase of the haSMC migratory potential while expression levels of the intracellular cadherin-binding partner, β-catenin, remained unchanged. A shift of the cadherin expression profile is well known from the process of epithelial to mesenchymal transition in cancerogenesis, which is mainly characterized by downregulation of cadherins leading to increased invasiveness of transformed tumour cells that finally results in tumour progression [29]. Due to the fact that, after vascular injury, haSMCs undergo a phenotypic modulation that is in many respects similar to the process of epithelial to mesenchymal transition, the mechanisms between cadherin downregulation and increased migratory capacities are potentially similar. Because E-cadherin was not differentially regulated in this study, N-cadherin seems to be the relevant cadherin for the migratory behaviour of haSMCs. Relevance of N-cadherin for cell migration has already been described in developmental biology. It was found that cadherin-type switching from N-cadherin to cad7 with decreased expression of N-cadherin caused increased emigration from the neural tube [30]. Overexpression of N-cadherin by adenoviral expression vectors in this system prohibited neural crest emigration, as it was shown for N-cadherin overexpression in haSMCs in this study. The role of N-cadherins in smooth muscle cell proliferation was recently analyzed by Uglow et al. [16]. Interestingly, this group demonstrated the downregulation of N-cadherins in proliferating smooth muscle cells in VSMCs of human saphenous veins that were stimulated with FCS and PDGF in vitro while β-catenin in this model was not differentially regulated. In another study, Jones et al. [17] have examined expression of N-cadherins in a rat injury model of carotid arteries. This group has observed an upregulation of N-cadherin in the neointima at 1 and 3 weeks after injury. It was discussed very early during the development of animal restenotic models [31] that the rat neointima mainly consists of proliferated smooth muscle cells with a significantly lower percentage of extracellular matrix compared to the porcine neointima. Thus, the rat model might be more adequate to address problems of SMC proliferation. Nevertheless, as described by Uglow et al., N-cadherin seems to be downregulated in proliferating human SMCs compared to quiescent ones. Therefore, in this point, these observations seem to conflict with the results by Jones et al. As a potential explanation, N-cadherin regulation might differ between various species.

4.2 Regulation of N-cadherin induced cell migration
Cell migration is a complex process that comprises attachment and detachment of cells from each other or the extracellular matrix as well as reorganization of the actin cytoskeleton [1–4]. It is well known that cadherins exhibit homophilic and heterophilic cell–cell adhesion capacities and that cadherin–cadherin binding differs considerably in its adhesion strength and specificity. In this study, N-cadherin upregulation by N-cadherin sense transfection caused a lower migratory potential of haSMCs. Thus, the influence of N-cadherin on cell migration can partially be explained by increased N-cadherin expression and by increased binding affinity as it was described for other cell systems [32]. Nevertheless, the migratory potential of haSMCs with differentially regulated N-cadherin expression was significantly changed even in the setting of the Boyden chamber model, where cells have little or no cell–cell contact. A potential explanation for this phenomenon is that different cell-signaling pathways are activated by cadherin–cadherin binding. In an elegant flow model based on cadherin–IgG–Fc–fragment fusion proteins, Niessen and Gumbiner [33] elucidated that cells expressing different cadherins can sort from each other. The ability of cell sorting did not simply depend on the extent of sequence homology, but was determined by other mechanisms than adhesive-binding specificity and strength. Thus, cadherin-dependent cell–cell affinity seems to be regulated by extracellular or intracellular signaling as it has been described for integrins before [34]. It was shown by Zeitvogel et al. [35] that altered N-cadherin expression in endometric tumour cells caused increased invasiveness that was determined by the authors with a Boyden chamber model. In this model, the authors could find unaltered expression of β-catenin but differential subcellular location and speculated on formation of β-catenin complexes with Tcf/Lef in the nucleus.

One important family of signal transduction molecules with decisive impact for cellular migration processes are Rho GTPases [6]. As we could show, quiescent haSMCs exhibited significantly increased RhoA activity compared to migratory cells. Furthermore, after downregulation of N-cadherins or after blockade of N-cadherin attachment by an inhibitory antibody, RhoA activity of quiescent haSMCs decreased. By contrast, RhoA activity was markedly inducible by overexpression of N-cadherins. These observations indicate that in haSMCs, N-cadherin activation induces the RhoA pathway leading to reorganization of the actin cytoskeleton which could be confirmed by structural changes of the actin cytosceleton. Activation of RhoA was also demonstrated by Charasse et al. [18] who detected that N-cadherin-mediated adhesion increased RhoA activity in skeletal smooth muscle cells with activation of p21 and p27 promotors during their differentiation process. Furthermore, Wickstrom et al. [36] showed that decreased RhoA activity in endothelial cells reduced migration by disassembly of actin stress fibres and focal adhesions. Nevertheless, detailed regulation of the N-cadherin-triggered RhoA signal transduction pathway has to be determined in further studies.


   Acknowledgements
 
Dr. Vogt was supported by a grant of the IZKF ‘BIOMAT’—‘Interdisciplinary Centre for Clinical Research in Biomaterials and Tissue–Material Interaction in Implants (BMBF project No. 01 KS 9503/9)’.


   Notes
 
Time for primary 20 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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F. Vogt, A. Zernecke, M. Beckner, N. Krott, A.-K. Bosserhoff, R. Hoffmann, M. A.M.J. Zandvoort, T. Jahnke, M. Kelm, C. Weber, et al.
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J. Am. Coll. Cardiol., July 22, 2008; 52(4): 302 - 311.
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