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From Bristol Heart Institute, Department of Cardiac, Anesthetic, and Radiological Sciences, University of Bristol, Bristol Royal Infirmary, Bristol, UK.
ABSTRACT
Objective— Vascular smooth muscle cell (VSMC) proliferation is an important component of atherosclerosis, restenosis after angioplasty and stent placement, and vein graft failure. Outside-in signaling from the cadherin:?-catenin complex can increase transcription of the cell-cycle gene cyclin D1; however, its role in VSMC proliferation has only recently been considered.
Methods and Results— We examined the involvement of R-cadherin and ?-catenin in VSMC proliferation in balloon-injured carotid arteries in vivo and aortic rings in vitro. The number of medial VSMCs positive for R-cadherin was significantly reduced by 32%±5%, 52%±10%, and 23%±2% at 0.25, 24, and 48 hours after injury in vivo, respectively. These changes in cadherin expression coincided with the detection of nuclear ?-catenin and elevated cyclin D1 expression. Furthermore, loss of R-cadherin expression was associated with medial VSMC proliferation. Inhibition of classical cadherin function with a HAV peptide and R-cadherin neutralizing antibodies significantly increased proliferation by 4.3±1.0-fold and 4.1±0.98-fold, and increased the number of cells with ?-catenin in the nucleus and expressing cyclin D1 in aortic rings.
Conclusions— These results suggest that R-cadherin expression and ?-catenin signaling may be associated with increased cyclin D1 expression and VSMC proliferation and may therefore play an important role in vascular disease.
Human VSMCs that overexpress AIF-1 grow more rapidly and express G-CSF. G-CSF is capable of promoting VSMC proliferation, and AIF-1-transduced VSMCs are chemotactic for monocytes. This study indicates that AIF-1 enhances VSMC growth by autocrine production of G-CSF, and AIF-1 expression may influence VSMC-inflammatory cell communication.
Key Words: smooth muscle ? cadherin ? proliferation ? intimal thickening
Introduction
Vascular smooth muscle cell (VSMC) proliferation plays a key role in pathological processes characterized by neointimal thickening, such as atherosclerosis, vascular rejection, and restenosis after angioplasty and stent placement.1,2 Balloon injury of arteries causes quiescent, medial cells to lose their differentiated, contractile phenotype and proliferate for up to 3 days.3–5 After this first wave of medial VSMC proliferation, medial VSMCs migrate and appear in the intima 4 days after injury. A new proliferative wave is then observed in the intima, which peaks at 7 days. Increased synthesis of extracellular matrix components continues for at least 4 weeks after injury.6
The cadherins are a family of transmembrane glycoproteins that mediate calcium-dependent homophilic cell-cell interaction.7 One cell type can express multiple cadherins, and the expression pattern is cell type-specific.8 The extracellular domain of cadherins promotes the cell-cell adhesion through a binding site, which contains a HAV motif in the classical type I cadherins.9 The cytoplasmic region connects cadherins to the cytoskeletal components through the catenin proteins. In addition to serving a structural function by linking to the actin cytoskeleton, the classical cadherins act as signaling receptors that affect cell behavior, including cell proliferation, migration, and differentiation.10,11 In the absence of Wnt signals, ?-catenin is either coupled to cadherins and the cytoskeleton at the plasma membrane or targeted for proteosomal degradation by the adenomatous polyposis coli (APC), Axin, and glycogen synthase kinase 3? (GSK3?) complex. However, in the presence of Wnt signaling, the APC-Axin-GSK3? complex is inhibited because of inactivation of GSK-3? by Wnt. This results in increased free cytoplasmic ?-catenin, which translocates to the nucleus, binds lymphoid enhancer factor-1/T-cell factor (LEF-1/TCF), and activates Wnt target genes, including cell cycle activators, such as cyclin D1 and c-myc, or other genes such as MMP-712 and fibronectin.13
Although numerous studies have demonstrated that abnormal functioning of cadherins and ?-catenin promotes tumor invasiveness and metastasis,14 the role of cadherins in vascular pathologies is a relatively unstudied area. Wang et al recently demonstrated elevated levels of ?-catenin 7 days after balloon injury to rat carotid arteries and presented evidence for its role in intimal proliferation and protection from apoptosis in vitro.15 Furthermore, Jones et al showed upregulation of both N-cadherin and ?-catenin in the intima at the same time in the same model.16 They also demonstrated in vitro effects of N-cadherin on cell migration.16 We recently showed that dismantling of N-cadherin complexes in the first 24 hours after stimulation with fetal calf serum, and platelet-derived growth factor releases ?-catenin and stimulates proliferation of isolated human saphenous vein smooth muscle cells in culture.17
The possible roles of different cadherin:?-catenin complexes, particularly during the initial stages of the vascular responses to injury, are incompletely understood. In this study, the roles of R-cadherin expression and ?-catenin signaling in the first wave of VSMC proliferation, when cells are in contact with their native extracellular matrix, are examined. Firstly, the expression of R-cadherin, ?-catenin, and cyclin D1 was determined in the rat carotid balloon injury model. Secondly, the effect of cadherin inhibition on proliferation in cultured balloon-injured rat aortic rings was examined.
Methods
A more detailed Methods section is provided online (please see http://atvb.ahajournals.org).
Materials
All reagents were purchased from Sigma (Poole, Dorset, UK) except when noted otherwise.
Balloon Injury of Rat Arteries
Male Sprague-Dawley rats (Charles River Laboratories, Manston, Kent, UK) were subjected to balloon injury of the carotid artery and aorta. Rats were euthanized at the designated time-points and then immediately perfused with phosphate-buffered saline. Control rats were subjected to a sham operation. Proliferating cells were labeled by treating the rats with bromodeoxyuridine (BrdU) for the last 24 hours before sacrifice.
Western Blotting for Cadherin Proteins
Rat aortas and carotid arteries were placed in RIPA protein extraction buffer. Reduced samples loaded at equal total protein concentrations were subjected to electrophoresis on 3% to 8% polyacrylamide gradient gels. Polyclonal antibodies for N- and R-cadherin (Santa Cruz, Calne, UK), and ?-catenin (Sigma) and a monoclonal antibody for cyclin D1 (Oncogene, Nottingham, UK) were used in Western blotting. Equal protein loading was checked with a monoclonal antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon, Harrow, UK).
Immunocytochemistry
Paraffin 3-μm sections were subjected to immunocytochemistry using polyclonal rabbit anticadherin antibodies for N- and R-cadherin, polyclonal rabbit anti-?-catenin antibody, and monoclonal cyclin D1 at 1 μg/mL for 1 hour. Cell proliferation was assessed by immunocytochemistry for incorporated BrdU using a monoclonal anti-BrdU antibody (ICN Pharmaceuticals Inc, Rickmansworth, UK) and for proliferating cell nuclear antigen (PCNA) as described previously.18,19 The percentage of positive cells per carotid section was quantified by 2 blinded observers. The reproducibility of assessments was demonstrated by unweighted test values of 0.75 and 0.73 for interobserver and intraobserver variation, respectively. Negative controls using nonimmune immunoglobulins were always included.
Dual Immunocytochemistry
Cadherin proteins and incorporated BrdU were colocated in balloon-injured carotids from animals exposed to BrdU for the last 24 hours before euthanization using dual immunocytochemistry.
Proliferation of VSMCs in Rat Aortic Rings
Rings (5 mm) of balloon-injured aorta (n=4 per condition) were cultured as described previously.20 Rings were incubated for 24 hours at 37°C and 5% CO2 in media supplemented with 10% fetal calf serum and 1 μCi/mL -thymidine (Amersham International, Little Chalfont, UK). Cadherin function was inhibited by the addition of 10 μg/mL rabbit antihuman R-cadherin antibody (Santa Cruz, Cambridge, UK), 80 μg/mL mouse antihuman N-cadherin (Clone A-CAM; Sigma) antibody, or 200 μg/mL HAV peptide (Leu-Arg-Ala-His-Ala-Val-Asp-Val-Asn-Gly-NH2; Bachem, Walden, UK) and compared with the controls (10 μg/mL nonimmune rabbit immunoglobulin G (IgG), 80 μg/mL nonimmune mouse IgG, or 200 μg/mL of the control HGV peptide, respectively). The HAV peptide inhibits cadherin function by binding to the HAV peptide within the extracellular binding site of cadherins that are not bound in cell-cell complexes. Consequently, the HAV peptide and the antibodies inhibit the formation of new cell-cell contacts.21 Neutralizing ability of this antibodies and the HAV peptide was examined by a VSMC adherence assay. Briefly, VSMCs were cultured in wells coated with 10% (wt/vol) agarose for 24 hours in the presence of the antibodies or peptides and the presence of cell aggregates noted. Proliferation of VSMCs in aortic rings was determined by autoradiography as described previously.22 Penetration of the antibody throughout the aortic ring segments was demonstrated by immunodetection (data not shown). Culture media was collected and cell viability measured using an assay kit for lactate dehydrogenase (Roche, Lewes, UK).
Statistical Analysis
After calculation of means±standard error of the mean (SEM), analyses were performed using 2-way analysis of variance (ANOVA) and Tukey post-tests. Significant differences were taken when P<0.05.
Results
Detection of R-Cadherin and N-Cadherin by Western Blotting
Western blotting detected only 1 band of 120 and 130 kDa for R-cadherin and N-cadherin, respectively, (Figure 1). Both cadherins were detected in aorta, carotid artery, and vena cava tissue lysates. However, significantly higher levels of R-cadherin were measured by densitometry in the aorta and carotid arteries (13.9±3.7 and 14.5±2.6 optical density xmm2, respectively) than vena cava (3.1±0.6 ODxmm2, n=3, P<0.05). In contrast, significantly a higher amount of N-cadherin was more abundant in vena cava (16.7±2.4 ODxmm2) than aorta and carotid arteries (8.1±0.9 and 7.4±2.0 ODxmm2, respectively, n=3, P<0.05).
Figure 1. Detection of cadherin proteins by Western blotting. Tissue extracts of rat aorta, carotid artery, and vena cava were loaded at 3 μg total protein/lane (n=3 animals). R- and N-cadherin were detected using polyclonal antibodies from Santa Cruz at 0.2 μg/mL. The molecular weights of the detected protein bands are indicated.
Effect of Balloon Injury of Carotid Arteries on R-Cadherin, N-Cadherin, ?-Catenin, and Cyclin D1 Protein Expression
Western blotting of tissue extracts of balloon injured carotid arteries revealed that R-cadherin protein levels were significantly lower in arteries at 0.25, 24, and 48 hours after injury compared with uninjured controls (Figure 2 and Figure I, available online at http://atvb.ahajournals.org). However, the amount of N-cadherin protein was unaffected by injury at the same time-points compared with uninjured controls (Figure II, available online at http://atvb.ahajournals.org). In contrast to R-cadherin, and in agreement with previous studies at later time-points in this model,15 the amount of ?-catenin was increased (Figures 2 and I). Levels of cyclin D1, a putative target of ?-catenin signaling, were also increased after injury (Figures 2 and I). Western blotting with an antibody for GAPDH revealed that equal protein was loaded (Figures 2 and I).
Figure 2. Detection of R-cadherin, ?-catenin, and cyclin D1 proteins in carotid arteries by Western blotting. A representative Western blot for R-cadherin, ?-catenin, cylcin D1, and GAPDH of carotids subjected to a sham operation (control) and at 0.25, 24, and 48 hours after balloon injury, n=7 (sham-operated) and n=4 (balloon-injured at all time-points).
Immunocytochemistry demonstrated that almost all (97%±2%) medial VSMCs were positive for R-cadherin (Figure 3A). Balloon injury significantly reduced the number of VSMCs positive for R-cadherin to 66%±11%, 47%±9% and 75%±6%, respectively (Figure 3B through 3D). At time-points beyond 48 hours, the percentage of R-cadherin-positive VSMCs was not significantly different from uninjured arteries (Table I, available online at http://atvb.ahajournals.org). In agreement with Western blotting results, the number of cells expressing N-cadherin was not significantly different from sham-operated controls at any time-point studied after injury (Table I). Furthermore, balloon injury did not affect the total number of medial cells at any time-point (Table I). The use of nonimmune immunoglobulins instead of primary antibody gave the expected negative result (Figure III, available online at http://atvbahajournals.org).
Figure 3. Detection of R-cadherin, ?-catenin, and cyclin D1 proteins in carotid arteries by immunocytochemistry. Representative immunocytochemistry for R-cadherin (A–D), ?-catenin (E–H), and cyclin D1 (I–L) of carotids subjected to a sham operation (control) and at 0.25, 24, and 48 hours after balloon-injury. Brown color indicates positive staining and nuclei are blue. Small arrows in panels (F–H) indicate some cells with ?-catenin protein in the nucleus. Scale bar in (A) represents 25 μm and applies to all panels.
In sham-operated carotids ?-catenin was detected as diffuse staining and was not detected in the nuclei (Figure 3E). In addition to detection of higher levels of ?-catenin after injury, ?-catenin protein was observed in nuclei at 0.25 hours (1.2%±0.1%), 24 hours (2.4%±0.4%), and 48 hours (1.8%±0.3%) after injury (Figure 3F through 3H; n=3 per time-point). The amount of cyclin D1 protein in sham-operated controls was low, but elevated levels were detected at 0.25, 24, and 48 hours after injury (Figure 3I through 3L).
Medial VSMC Proliferation After Balloon Injury
Proliferation assessed by incorporation of BrdU was observed in 4%±2% and 5%±0.5% of medial VSMCs between 0 and 24 and between 24 and 48 hours, respectively. This highlights that decreased R-cadherin expression and increased ?-catenin and cyclin D1 occurred rapidly after injury and, hence, concomitant with induction of the first wave of medial VSMC proliferation.
Dual Localization of Cadherin Protein and BrdU Incorporation in the Media
Dual localization of cadherin protein and BrdU was performed on vessels exposed to BrdU for the last 24 hours before removal. At 48 hours (Figure IV, available online at http://atvb.ahajournals.org), 8 days (data not shown), and 10 days (data not shown) after injury, all R-cadherin-negative medial VSMCs were positive for BrdU. Furthermore, linear regression demonstrated that the number of BrdU-positive medial cells at 48 hours, 8 days, and 10 days was significantly correlated (P=0.00102) with the number of R-cadherin-negative cells (correlation coefficient r=0.9165, r=0.84), illustrating an association between the loss of R-cadherin and proliferative rate. Interestingly, the percentage of proliferative cells (BrdU positive) that lack R-cadherin was significantly higher at 48 hours after injury than at 8 and 10 days (12%±6% and 16%±6%) when R-cadherin levels were higher. This illustrates that not all R-cadherin-negative cells were BrdU-positive and possibly reflects the re-expression of R-cadherin after completion of cell division within the 24-hour labeling period.
Effect of Cadherin Perturbation on VSMC Proliferation
Culture of VSMC in agarose coated wells for 24 hours led to the formation of large aggregates of cells (Figure V, available online at http://atvb.ahajournals.org). The addition of 200 μg/mL (Figure V) of the HAV peptide inhibited aggregation. Furthermore, the addition of 10 μg/mL anti-R-cadherin antibodies and 80 μg/mL anti-N-cadherin antibodies (Figure V), but not nonimmune rabbit and mouse immunoglobulins, inhibited the formation of these aggregates. This demonstrates, as expected, the ability of this HAV peptide to inhibit classical cadherin function and these antibodies to inhibit R-cadherin and N-cadherin function and therefore cell-cell contact.
As previously reported, organ culture of balloon-injured aortic rings led to cell proliferation during the next 24 to 96 hours.20 In addition, balloon injury of the aorta in the same manner as in the carotid artery significantly reduced R-cadherin protein levels by 45%±6% at 0.25 hours after injury (n=4, data not shown). Furthermore, the amount of R-cadherin remained significantly lower by 75%±16% than in uninjured controls in rings after culture for 24 hours (n=4, data not shown). HAV peptide and neutralizing antibodies to R- and N-cadherin were then used to inhibit cadherin function in the first 24 hours. Immunohistochemical analysis demonstrated that neutralizing antibodies permeated throughout the aortic rings (data not show). Proliferation was assessed using tritiated thymidine incorporation and autoradiography and confirmed by immunocytochemistry for PCNA antigen (Figure 4, n=4 per condition). The HAV peptide significantly increased the index of proliferation by 4-fold (0.69%±0.18%), compared with untreated controls (0.16%±0.05%, P=0.029) and control HGV peptide (0.21%±0.06%, P=0.025). VSMC proliferation was significantly increased by 4-fold in the presence of the polyclonal rabbit anti-R-cadherin antibodies (1.19%±0.24%) compared with nonimmune rabbit IgG (0.29%±0.02%, P=0.011). Similarly, increased proliferation was detected using immunocytochemistry for PCNA in HAV peptide and anti-R-cadherin antibody-treated aortic rings compared with controls (Figure 4). However, the number of proliferating cells, assessed by tritiated thymidine incorporation, in aortic rings treated with monoclonal anti-N-cadherin antibodies (0.21%±0.06%) was not significantly different from that observed in controls treated with nonimmune mouse immunoglobulin G (0.35%±0.07%). Using an lactate dehydrogenase assay, it was determined that cell viability was not affected by incubation of aortic rings under these conditions (data not shown). Very low levels of proliferation were detected in uninjured aortic rings after culture (<0.03%), and culture with neutralizing antibodies or HAV peptides did not affect proliferative rates (data not shown).
Figure 4. Effect of cadherin perturbation on proliferation. Autoradiography was used to detected proliferating SMC in control aortic rings (A) or those incubated in the presence of 200 μg/mL HAV (B), 10 μg/mL nonimmune rabbit immunoglobulin (RIgG) (C), and 10 μg/mL rabbit anti-R-cadherin antibody (D). Positive cells are those covered in black silver grains. Sections are counterstained with hematoxylin and eosin. Alternatively, immunocytochemistry for PCNA detected proliferating SMC in control aortic rings (E) or those incubated in the presence of 200 μg/mL HAV (F), 10 μg/mL RIgG (G), and 10 μg/mL rabbit anti-R-cadherin antibody (H). Brown color indicates positive staining and nuclei are counterstained blue with hematoxylin. Arrows indicate some of the positive cells. Scale bar in (A) represents 40 μm, n=4 per condition.
Effect of Cadherin Perturbation on ?-catenin Location and Cyclin D1 Expression
Diffuse ?-catenin immunolabeling was seen in balloon-injured cultured aortic rings under all conditions (Figure 5). However, an increased number of cells with nuclear-localized ?-catenin was observed after culture of aortic rings in the presence of HAV peptide compared with control segments (Figure 5A and 5B). This coincided with increased cyclin D1 immunolocalization in HAV-treated aortic rings (Figure 5C and 5D). Similarly, culture in the presence of anti-R-cadherin antibody increased the number of cells with ?-catenin in the nucleus and the presence of cyclin D1 protein was increased (data not shown). Immunocytochemistry with nonimmune control antibodies demonstrated the absence of nonspecific staining (Figure VI, available online at http://atvb.ahajournals.org).
Figure 5. Detection of ?-catenin and cyclin D1 proteins in aortic rings by immunocytochemistry. Representative immunocytochemistry for ?-catenin in control (A) and HAV-treated (B) aortic rings. Representative immunocytochemistry for cyclin D1 in control (C) and HAV-treated (D). Brown color indicates positive staining for cadherin protein and nuclei are stained blue. Small arrows in panels indicate some of the cells with ?-catenin protein in the nuclei. Scale bar in (A) represents 20 μm and applies to all panels.
Discussion
Based on previous studies with isolated VSMCs,17 we hypothesized that loss of cadherins and release of ?-catenin to the nucleus might modulate the first wave of medial VSMC proliferation after balloon injury. In support of our hypothesis, in vivo balloon injury led to a reduction in R-cadherin expression and increased appearance of ?-catenin in nuclei coinciding with increased VSMC proliferation. Furthermore, perturbation of cadherin function increased translocation of ?-catenin to the nucleus, cyclin D1 levels, and VSMC growth in balloon-injured aortic segments in organ culture. We also show that cadherin-mediated signaling modulates increased cyclin D1 protein expression and provides a possible mechanism for increased proliferation in injured arteries.
In a previous study, we have demonstrated that dismantling of N-cadherin-mediated cell-cell contacts and ?-catenin signaling modulates proliferation of isolated human saphenous vein smooth muscle cells.17 In this study we have examined the involvement of cadherin:catenin complexes in regulation of VSMC proliferation in situ to determine whether the complex can modulate VSMC proliferation even in the presence of native extracellular matrix. We have focused on R-cadherin because N-cadherin levels were not affected at early time-points after balloon injury and higher levels of this cadherin were detected in uninjured aorta and carotid arteries than N-cadherin. In support of our previous findings with human saphenous vein VSMCs, the expression of N-cadherin was significantly higher in vena cava than arteries.17 Because the classical type I cadherins, N-cadherin and R-cadherin, have distinct roles despite their 74% amino acid sequence identity,23 the difference in amount of these cadherins may reflect the inherent differences in function of arteries and veins.
To examine whether R-cadherin modulates VSMC proliferation in vivo, we investigated the expression of R-cadherin, N-cadherin, ?-catenin, and cyclin D1 after balloon injury of carotid arteries. R-cadherin expression was reduced at time-points early enough such that its loss could be involved in the initial wave of medial VSMC proliferation. However, the level of N-cadherin protein was unaffected by balloon injury. The use of immunohistochemistry in addition to Western blotting confirmed these findings for R-cadherin and N-cadherin. It also demonstrated that this change in R-cadherin expression occurred in a subset of medial VSMCs rather than a generalized reduction of R-cadherin protein levels on all cells. The discrepancy in the degree of loss observed with Western blotting and immunohistochemical analysis most probably reflects the fact that cells do not contain a fixed amount of cadherin. Total protein is analyzed by Western blotting; while using immunohistochemistry, we counted positive and negative cells, which is relatively insensitive to the level of expression in individual cells. Because the total number of medial cells was unaffected by the injury conditions, we determined it is unlikely that VSMC death was responsible for the changes in R-cadherin expression. Furthermore, macrophages are largely absent from the media of injured rat carotids (data not show), indicating that these cells do not contribute to the change in R-cadherin protein levels in the media. Removal of the endothelium alone was not responsible for the reduction in R-cadherin, because endothelial denudation with no detectable damage to the medial layer of smooth muscle cells using a guide wire, as described previously by Jackson et al,24 did not affect R-cadherin protein levels (data not shown). The loss of R-cadherin was rapid, indicating that changes in protein synthesis are unlikely to be responsible. We propose that the observed loss of R-cadherin protein after balloon injury may be mediated by internalization and proteosomal degradation25 or by shedding of R-cadherin by proteases, such as metalloproteinases, in the same manner as we and others have observed in isolated cells.17,26,27 Metalloproteinase activity is increased after balloon injury of the rat carotid.28 In addition, loss of cadherins by proteolysis has been observed rapidly after treatment.29
Previously, Wang et al illustrated that ?-catenin is elevated in rat carotids at 7 days after injury.15 In this study, we detected significantly increased levels of ?-catenin as early as 15 minutes after injury. The rapid effect on the levels of ?-catenin protein at 0.25 hours appears too rapid to be explained by changes in protein synthesis. This elevation of total ?-catenin may be a result of decreased activity of the APC-Axin-GSK3? complex caused by Wnt signaling, as proposed by Wang et al from their observations at later time-points in the same model.15 The GSK-3?-Axin-APC complex targets free ?-catenin for proteosomal degradation and therefore maintains free ?-catenin protein at very low levels. However, Wnt signaling inhibits this complex and can lead to rapid changes in ?-catenin levels30 and translocation to the nucleus after very short time periods, such as 15 minutes.31 The ability of overexpression of active GSK3? to reduce proliferation and intimal thickening in the same model 14 days after injury further supports the involvement of Wnt signaling in VSMC proliferation in this model.32 In addition, we observed increased numbers of medial VSMCs containing nuclear ?-catenin after balloon injury in vivo, suggesting that ?-catenin signaling may participate in upregulation of Wnt target genes and proliferation. The detection of ?-catenin in the nuclei was detected at 0.25 hours but this time-point is too short to detect proliferation by BrdU incorporation. However, the number of cells with nuclear ?-catenin at 24 and 48 hours after injury (2.4%±0.4% and 1.8%±0.3%, respectively) was slightly lower than the number of proliferative cells determined by BrdU incorporation during 24 hours at 24 and 48 hours after injury (4%±2% and 5%±0.5%, respectively). The discrepancy may reflect that the ?-catenin staining is a snapshot whereas proliferation determined by BrdU incorporation is monitored over the last 24 hours.
Dual immunohistochemistry results suggested that loss of R-cadherin expression occurs in a small subset of VSMCs and that this is correlated with the proliferative rate. In fact, all R-cadherin-negative cells were positive for BrdU, indicating that they are proliferative. We suggest that the injury-induced loss of R-cadherin contributes to the increase in free ?-catenin observed in the nuclei early after injury. It is of note, however, that the number of cells negative for R-cadherin is dramatically higher than the number of cells with nuclear ?-catenin and identified as proliferative by BrdU incorporation. It is well-documented that GSK-3?-Axin-APC complex that tightly regulates the levels of free ?-catenin by targeting it for proteosomal degradation. ?-Catenin can escape degradation if this complex is inhibited by Wnt signaling. We therefore suggest that when R-cadherin is lost that the ?-catenin is released into the cytoplasm, but in the majority of cells the GSK-3?-Axin-APC complex degrades the released ?-catenin. It may be only in a small subset of cells in which R-cadherin is lost that Wnt signaling is also activated, permitting the escape of ?-catenin from degradation and allowing translocation to the nucleus.
In addition, we observed elevation of the ?-catenin target gene, cyclin D1, after injury. This highlights that translocation of ?-catenin to the nucleus induced by regulation of cadherin levels may stimulate transcription of this ?-catenin target gene and stimulate VSMC proliferation after injury and supports the recent demonstration of the involvement of ?-catenin/TCF signaling cascade in VSMC proliferation after injury and in cyclin D1 promoter activity in a rat aortic-derived cell line.15 The increase in cyclin D1 protein levels at 0.25 hours may be too rapid to be caused by increased protein synthesis. It is possible that cyclin D1 protein levels are also regulated rapidly by proteolysis by GSK-3?,33 whereas they continue to increase over the next 2 days because of increased protein synthesis. Inhibition of ?-catenin signaling using strategies such as small cadherin sequences34 may therefore be a useful approach for inhibition of VSMC proliferation.
Although we have not examined the role of R-cadherin and ?-catenin signaling in VSMC migration in this study, it is possible that they also modulate migration. Our future studies will determine whether R-cadherin is a positive modulator of migration as seen with N-cadherin in porcine aortic VSMCs16 or a negative modulator as seen in other cell types.35,36
To determine whether cadherin regulation was necessary for or merely a consequence of proliferation, we used an organ culture of balloon-injured rat aorta as previously described.20,37 Inhibition of classical cadherin function by incubation in the presence of a HAV peptide or R-cadherin function with a neutralizing antibody increased VSMC proliferation. This increased proliferation was associated with increased presence of ?-catenin in nuclei and expression of cyclin D1, supporting our in vivo findings. We suggest that inhibition of cadherin function in these aortic ring cultures permits the translocation of ?-catenin to the nucleus, where it may participate in increased transcription of target genes, including cyclin D1.
In summary, this study has illustrated that inhibition of R-cadherin function augments the early proliferative response of VSMCs in rat aortic tissue. Furthermore, dynamic modulation R-cadherin levels and nuclear localization of ?-catenin are temporally associated with increased expression of a Wnt target gene, cyclin D1, and VSMC proliferation in rat carotid arteries. These findings imply that maintenance of R-cadherin expression and inhibition of ?-catenin signaling may all be useful strategies to reduce VSMC proliferation in pathologies such as in-stent restenosis and vein bypass graft failure.
Acknowledgments
We thank Jason Johnson for his excellent technical assistance. This work was supported by the British Heart Foundation.
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