Published online before print March 17, 2005, doi: 10.1161/01.ATV.0000163183.27658.4b
© 2005 American Heart Association, Inc.
Vascular Biology |
N-Cadherin–Dependent Cell–Cell Contacts Promote Human Saphenous Vein Smooth Muscle Cell Survival
From Bristol Heart Institute (E.K., C.A.B., S.C.S., G.B.-N., S.J.G.), Bristol Royal Infirmary, Bristol, UK; Adherex Technologies Inc (O.W.B.), Ottawa, Ontario, Canada; and the Division of Urology (O.W.B.), Department of Surgery, McGill University, Montreal, Quebec, Canada.
Correspondence to Dr Sarah Jane George, Bristol Heart Institute, Level 7, Bristol Royal Infirmary, Bristol, BS2 8HW, UK. E-mail s.j.george{at}bris.ac.uk
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Abstract |
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Objective— Vascular smooth muscle cell (VSMC) apoptosis is thought to contribute to atherosclerotic plaque instability. Cadherin mediates calcium-dependent homophilic cell–cell contact. We studied the role of N-cadherin in VSMC apoptosis.
Methods and Results— Human saphenous vein VSMCs were grown in agarose-coated wells to allow cadherin-mediated aggregate formation. Cell death and apoptosis were determined after disruption of cadherins using several approaches (n
3 per approach). Calcium removal from culture medium or addition of nonspecific cadherin antagonist peptides significantly decreased aggregate formation and increased cell death by apoptosis (34±6% versus 75±1% and 19±1% versus 40±5%, respectively; P<0.05). Specific inhibition of N-cadherin using antagonists and neutralizing antibodies similarly increased apoptosis. Supporting this, overexpression of full-length N-cadherin significantly reduced VSMC apoptosis from 44±10% to 20±3% (P<0.05), whereas abolishing N-cadherin expression by overexpression of a dominant-negative N-cadherin significantly, even in the presence of cell–matrix contacts, increased apoptosis from 9±2% to 50±1% (P<0.05). Interestingly, cell–cell contacts provided a similar degree of protection from apoptosis to cell–matrix contacts. Finally, N-cadherin–mediated cell–cell contacts initiated anti-apoptotic signaling by increasing Akt and Bad phosphorylation.
Conclusions— Our results indicate that VSMC survival is dependent on N-cadherin–mediated cell–cell contacts, which could be important in the context of plaque instability.
We investigated the role of N-cadherin, a transmembrane protein that mediates cell–cell contacts, in VSMC survival. Inhibition of N-cadherin significantly decreased cell–cell contact and survival. Conversely, overexpression of N-cadherin significantly decreased VSMC apoptosis. We suggest that N-cadherin promotes VSMC survival by initiating survival signals via phosphorylation of Akt and Bad.
Key Words: apoptosis • atherosclerosis • N-cadherin • smooth muscle
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Introduction |
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Atherosclerotic plaques that have ruptured have thin fibrous caps that lack vascular smooth muscle cells (VSMCs). Stable plaques, by contrast, are rich in fibrous tissue and VSMCs. Thinning of the fibrous cap and loss of VSMCs in the late stages of atherosclerosis could therefore contribute to plaque instability.1 VSMCs are thought to contribute to plaque stability by producing the extracellular matrix that in conjunction with the VSMCs provides the fibrous cap tensile strength and encapsulates the lipid core.2 VSMC apoptosis is considered an important underlying mechanism that leads to reduced VSMC numbers and extracellular matrix in advanced atherosclerotic plaques, hence the thinning of the fibrous cap and plaque instability.3 In fact, a recent study in which induction of VSMC apoptosis in the fibrous cap of mouse lesions caused plaque rupture and thrombosis provides direct evidence for the involvement of VSMC apoptosis and plaque instability.4
Although abundant evidence indicates that VSMC apoptosis occurs in atherosclerosis, the mechanisms that initiate and regulate apoptosis are not fully understood. The presence of inflammatory cells, cytokines, modified low-density lipoprotein cholesterol, reactive oxygen species, and the systemic effects of altered blood pressure and flow are thought to promote apoptosis in atherosclerotic plaques.5 However, apoptosis of VSMCs appears to be a counterbalance of pro-apoptotic factors such as fas or tumor necrosis factor, and anti-apoptotic factors, known as survival factors. It has been proposed that all cells are programmed to die by default and that they require constant survival signaling to stay alive.6 The first survival signals described were soluble growth factors.7,8 More recently, it has been demonstrated that VSMCs also require normal cell–matrix interactions to maintain cell survival.9 The importance of cell–matrix attachment in inhibiting the default pathway and promoting cell survival has been extensively studied in adherent cells including VSMCs.10 Cell–cell contacts can also inhibit apoptosis in some cell types11,12; however, the extent to which cell–cell contact promotes survival of VSMCs is relatively unstudied.
Cadherins are a family of transmembrane proteins that mediate calcium-dependent homophilic cell–cell contacts and interact with the cytoskeleton through adaptor molecules called catenins.13 In addition to their crucial role in embryonic morphogenesis, cadherins modulate cell differentiation, migration, proliferation, and survival.14 In this study, we have considered whether cell–cell contacts provide a survival signal for VSMCs. First, we compared the contribution of the anti-apoptotic signals provided by cell–matrix and cell–cell contacts. Second, we determined the role of N-cadherin in VSMC survival using cells grown in the absence of cell–matrix contacts to exclude confounding effects of cell-matrix contacts.
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Methods |
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Additional information is provided in expanded Methods (see http://atvb.ahajournals.org).
Reagents
All tissue culture reagents were obtained from Cambrex (Wokingham, Berkshire, UK), and all other reagents were obtained from Sigma (Poole, Dorset, UK), except when noted.
Cell Culture
Surplus segments of human saphenous vein and radial artery were obtained from consenting patients undergoing coronary artery bypass surgery (Research Ethics Committee number 04/Q2007/6). VSMCs were grown from these segments by the explant method as previously described.15 In some experiments, VSMCs at 8x104 cells/mL were cultured in 24-well plates coated with 10% (weight/volume) agarose to allow cell–cell contacts in the absence of cell–matrix contacts. Each experiment was performed with VSMCs at passage 4 to 8 from at least 3 different segments of vein.
Overexpression of Full-Length and Dominant-Negative N-Cadherin
VSMCs grown in tissue culture wells were infected with a previously described adenovirus16 generously provided by Dr Gang Li and Professor Meenhard Herlyn (The Wistar Institute, Philadelphia, Pa) or the control adenovirus (RAd lacZ). Infected VSMCs were detached with trypsin and cultured in agarose-coated wells for 24 hours. VSMCs grown in agarose coated or tissue culture wells were infected with an adenovirus to overexpress a truncated form of N-cadherin that acts as a dominant-negative15 or control virus.
Inhibition of Cadherin Function
Cadherin function was inhibited in VSMCs grown on agarose-coated wells by supplementing culture media with the following reagents for 24 hours: (1) 2 mmol/L ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), in the presence or absence of 2 mmol/L CaCl2, 2 mmol/L MgCl2 or 50 µmol/L Ac-AAVALLPAVLLALLAP-DEVD-CHO (Calbiochem, Nottingham, UK); (2) 100 µg/mL linear peptide antagonist LRAHAVDVNG-NH2 (Peninsula Laboratories, St. Helens, Merseyside, UK); (3) 500 µg/mL cyclic peptide N-Ac-CHAVC-NH2 (pan cadherin), N-Ac-CHAVDC-NH2 (N-cadherin–specific), and the respective controls N-Ac-CHGVC-NH2 and N-Ac-CHGVDCAc-NH2 (Adherex Technologies Incorporated, Ontario, Canada); and (4) 80 µg/mL neutralizing anti-N-cadherin antibody or nonimmune mouse immunoglobulin G (MIgG). Hierarchy of cell–cell and cell–matrix contacts was examined by growing VSMCs on plastic or agarose-coated wells in the presence of 500 µg/mL of the pan cadherin or control peptide for up to 72 hours.
Calcium Switch Experiments
Adherent VSMCs were treated with 4 mmol/L EGTA for 40 minutes, then pre-incubated with 80 µg/mL anti-N-cadherin antibodies or MIgG for 30 minutes before addition of 4 mmol/L CaCl2 for 30 minutes. Lysates were analyzed by Western blotting and enzyme-linked immunosorbent assay for total and phosphorylated Akt and Bad.
Assessment of Cell Death
Trypsinized VSMCs were stained with 0.2% (v/v) trypan blue, stained by ISEL as described previously,17 5 mg/mL Hoechst 33342 dye, or propidium iodide as described previously.18
Western Blot Analysis
VSMC proteins were subjected to Western blotting as described previously.15
Immunoprecipitation
ß-Catenin protein was immunoprecipitated using 10 µg anti–ß-catenin agarose conjugate (C-18 AC; Autogen Bioclear) as described previously15 and subjected to Western blotting for ß-catenin and N-cadherin.
Immunocytochemistry for ß-Catenin and Cleaved Caspase 3
ß-Catenin protein was detected by immunocytochemistry as previously described.15 Cleaved caspase 3 was detected using anti-cleaved caspase 3 antibody (R&D Systems).
Akt Enzyme-Linked Immunosorbent Assay
Total and phosphorylated Akt was quantified using a commercial enzyme-linked immunosorbent assay (Active Motif, Rixensart, Belgium).
Caspase 3 Activity Assay
Cleaved caspase 3 activity was determined using a commercial kit (R&D Systems).
Statistical Analysis
Mean and standard error of the mean were calculated. Significant differences between means were determined using 2-tailed paired Student t tests for comparison of 2 groups and ANOVA and Student Newman Keuls post-tests for comparison of 3 groups. Significant difference was accepted when P<0.05.
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Results |
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Formation of VSMCs Aggregates
To deprive VSMCs of cell–matrix adhesion while allowing cell–cell interactions to occur, cells were suspended in agarose-coated wells. Small aggregates were visible within 4 hours (data not shown), and by 24 hours large aggregates were observed (Figure I, available online at http://atvb.ahajournals.org).
Disruption of Calcium-Dependent Intercellular Contacts
Addition of EGTA inhibited aggregation (Figure I), indicating disruption of cell–cell contacts. Furthermore, VSMC death was significantly increased (Table), demonstrating cell–cell contact disruption induced apoptosis. Addition of calcium to the EGTA-containing media reversed the effects of EGTA; cells aggregated and apoptosis was reduced to control levels (Table). By contrast, addition of magnesium had no effect (Table). Increased cleavage of ß-catenin was detected in EGTA-treated cells (Figure II, available online at http://atvb.ahajournals.org) compared with controls, suggesting cleavage of ß-catenin occurs in nonaggregated cells undergoing apoptosis. This cleavage was significantly reduced by 33±7% (P<0.05, n=3) by pre-incubation with the caspase inhibitor DEVD-CHO.
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Disruption of N-Cadherin–Mediated Cell–Cell Contacts
Culture of VSMCs in agarose-coated wells for 24 hours in the presence of a linear histidine alanine valine (HAV) peptide inhibited cell aggregation (data not shown) and significantly increased apoptosis compared with untreated control cells (Table). Similarly, elevated apoptosis was observed in VSMCs cultured for 24 hours on agarose-coated wells in the presence of the pan cadherin peptide N-Ac-CHAVC-NH2 compared with its control peptide N-Ac-CHGVC-NH2 (Table). Culture with the N-cadherin–specific peptide N-Ac-CHAVDC-NH2 also significantly elevated apoptosis compared with control peptide N-Ac-CHGVDC-NH2 (Table). The ability of N-cadherin to promote cell survival was confirmed by treatment with anti–N-cadherin antibodies that significantly increased death compared with mouse immunoglobulin G (MIgG) (Table).
Comparison of Cell–Cell and Cell–Matrix Contacts
The effects of cell–cell and cell–matrix contacts were compared by culturing on plastic or in agarose-coated wells in the presence of the pan cadherin HAV peptide or control peptide for up to 72 hours and cell death assessed by in situ end labeling (ISEL). At all time points, cell death was low in VSMCs grown in tissue culture plates in the presence of the control histidine glycine valine (HGV) peptide where cell–cell and cell–matrix contacts form (Figure 1). However, significantly increased cell death was observed at all time points when cell–cell and cell–matrix contacts were inhibited (agarose-coated wells plus HAV peptide). In addition, cell death was significantly higher at 8, 24, 48, and 72 hours when cell–cell contacts were maintained in the absence of cell–matrix contacts (agarose-coated wells plus HGV peptide) compared with cells with both contacts. Similarly, cell death was significantly increased when cell–matrix contacts were maintained in the absence of cell–cell contacts (plastic plus HAV peptide). Interestingly, the amount of cell death was similar in the absence of cell–cell contacts and the absence of cell–matrix contacts. Cell death was significantly lower in cells with only one contact compared with the absence of both contacts at 24, 48, and 72 hours.
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Significant difference from cell–cell. 
Significant difference from cell–matrix, n=3, ANOVA, and Student Newman Keuls post-test, P<0.05.
Effect of Full-Length N-Cadherin
To investigate the ability of full-length N-cadherin to promote VSMC survival, cells were infected with an adenovirus to express full-length N-cadherin (RAd FL-N-cad). Adenoviral infection of VSMCs using 100, 300, and 1000 pfu/cell resulted in a dose-dependent overexpression of the full-length form of N-cadherin (Figure III, available online at http://atvb.ahajournals.org). Immunoprecipitation of ß-catenin showed elevated levels of ß-catenin in VSMCs overexpressing full-length N-cadherin compared with controls (Figure 2A). This was confirmed by immunocytochemistry when elevated ß-catenin protein was detected compared with controls (Figure 2B and 2C). Furthermore, increased coimmunoprecipitation of N-cadherin and ß-catenin (Figure 2A) and elevated ß-catenin levels at the cell membrane (arrowheads) were detected in cells overexpressing N-cadherin, suggesting increased numbers of N-cadherin:ß-catenin–mediated cell–cell junctions (Figure 2B and 2C). VSMC apoptosis assessed by ISEL was significantly decreased by 
50% in cells infected with 1000 pfu/cell RAd FL-N-cadherin (20±3%) compared with cells infected with control virus (44±10%) or control uninfected cells (36±11%, P<0.05, n=3).
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Effect of Dominant-Negative N-Cadherin
To further clarify the role of N-cadherin in VSMC survival, cells were transfected with an adenovirus to express a truncated form of N-cadherin (RAd dn-N-cadherin) that lacks the extracellular domain. This acts as a dominant-negative11,15,19–21 by preventing formation of functional cadherin dimers and cell–cell contact. In addition, it reduces the presence full-length N-cadherin.15 Western blotting revealed that adenoviral infection of adherent VSMCs (Figure 3A) or agarose coated wells (data not shown) with higher doses (100, 300, and 1000 pfu/cell) than used previously15 resulted in a dose-dependent overexpression of the truncated N-cadherin and abolished expression of full-length N-cadherin. In addition, expression of the dominant-negative N-cadherin reduced ß-catenin protein (Figure 3A) and aggregation in cells cultured on agarose (Figure IV, available online at http://atvb.ahajournals.org) compared with cells infected with the control adenovirus. Moreover, increased cell death (ie, cell rounding and membrane blebbing) was observed in adherent VSMCs infected with RAd dn-N-cadherin compared with controls (Figure IV). Apoptosis was significantly increased in adherent VSMCs infected with RAd dn-N-cadherin compared with VSMCs infected with RAd lacZ (Figure 4). Furthermore, a significant increase (2.1±0.3-fold, n=3, P<0.05) in cytochrome c release into the cytoplasm was detected in VSMCs infected with RAd dn-N-cadherin compared with controls (Figure V, available online at http://atvb.ahajournals.org). Western blotting for the pro-form (inactive) of caspase-3 revealed that this inactive form was significantly reduced by 59±1% in VSMCs infected with RAd dn-N-cadherin compared with controls (n=3, P<0.05; Figure V). This occurred concurrently with detection of significantly increased (2.5±0.4-fold, n=3) caspase-3 activity using an activity assay and increased detection of cleaved caspase-3 protein by immunocytochemistry (Figure V).
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Survival Signals Induced by N-Cadherin
Calcium switch experiments were used to begin to examine the mechanisms by which N-cadherin reduced VSMC death. Calcium chelation led to disruption of cell–cell contact observed as rounding up of cells grown on plastic (Figure VI, available online at http://atvb.ahajournals.org). Re-addition of calcium restored cell–cell contact. However, pre-incubation with neutralizing anti–N-cadherin antibodies inhibited re-formation of cell–cell contacts, whereas MIgG had no effect. Reformation of cell–cell contacts after restoration of calcium increased phosphorylated Akt and Bad, whereas total levels of Akt and Bad were unaffected (Figure 5A). Pre-incubation with neutralizing anti–N-cadherin antibodies reduced phosphorylation of Akt and Bad, whereas MIgG had no effect. The anti–N-cadherin and MIgG did not affect the level of total Akt and Bad proteins (Figure 5A). In support of these findings, re-addition of calcium significantly increased the level of phosphorylated Akt detected by enzyme-linked immunosorbent assay (Figure 5B). However, this was significantly inhibited by pre-incubation with anti–N-cadherin antibody, whereas pre-incubation with MIgG had no effect (Figure 5B).
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Discussion |
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The results obtained from this study provide new insights into the regulation of apoptosis by cell–cell and cell–matrix contacts in VSMCs. We observed that in a similar manner to cell–matrix, cell–cell contacts regulate VSMC apoptosis. Cell–cell contacts mediated by N-cadherin modulates cell survival when VSMCs are grown in suspension. Interestingly, we observed that abolishing N-cadherin from VSMCs induces apoptosis even in the presence of cell–matrix contact. In fact, the resistance to apoptosis provided by cell-cell contacts is of a similar magnitude to that provided by cell–matrix contacts. We found that N-cadherin homophilic cell–cell contact initiates Akt signaling and thereby reduces apoptosis. We therefore propose that N-cadherin–mediated cell–cell contact is important for VSMC survival.
To focus on cell–cell contacts in regulating survival, VSMCs were cultured in agarose-coated wells that permit cell–cell contacts and exclude cell–matrix attachment. Inhibition of N-cadherin, the predominant cadherin in human saphenous vein VSMCs,15 was then determined. Calcium chelation, which inhibits cadherin-mediated cell–cell contacts,22 prevented VSMC aggregation and significantly induced cell death. This was reversed by the addition of CaCl2, whereas MgCl2 had no effect, indicating the importance of calcium-dependent cell–cell contacts for survival. Interestingly, caspase-dependent cleavage of ß-catenin was detected in VSMCs with elevated apoptosis caused by calcium chelation, indicating that caspase activity modulates the disorganization of N-cadherin:ß-catenin junctions in VSMCs in a similar manner to that previously observed in endothelial cells.23–26
Classical cadherins contain the binding sequence HAV in the extracellular domain. The amino acids that flank this sequence differ between the cadherins and confer cadherin specificity.27,28 Inhibitory peptides containing the HAV sequence inhibit cadherin function, affecting a variety of processes including neurite outgrowth, myoblast fusion, calcium-dependent cell aggregation, and cell death, whereas the negative control peptides containing HGV have no effect22,29–33 and specificity can be manipulated by the inclusion of flanking amino acids.28 In our study, addition of nonspecific HAV peptides also resulted in loss of cell aggregation and survival. Interestingly, the addition of the N-cadherin–specific cyclic peptide antagonist N-Ac-CHAVDC-NH2 and neutralizing anti-N-cadherin antibodies demonstrated the importance of N-cadherin in promoting VSMC cell–cell contact and survival. The use of the N-cadherin–specific cyclic peptide antagonist N-Ac-CHAVDC-NH2 produced similar results to that observed with calcium removal and the nonspecific peptide N-Ac-CHAVC-NH2, which inhibits both N-cadherin and E-cadherin function. This observation indicates that N-cadherin is the predominant cadherin to provide survival signals in human saphenous vein VSMCs. This is confirmed by the lack of detection of E-cadherin in cultured human VSMCs using reverse-transcription polymerase chain reaction and Western blotting (George and Uglow, unpublished observations, 2004).
Quantification of cell death by all 4 methods generated similar results. This could be considered surprising because trypan blue exclusion detects both necrosis and apoptosis as it assesses membrane permeability, whereas the other techniques detect DNA fragmentation, which is characteristic of apoptosis. We suggest the detection of similar levels of death with these methods indicates that the majority of death occurred by apoptosis. Because these methods, particularly ISEL, have been criticized as nonspecific,34 we have also examined caspase 3 activity and cytochrome c release into the cytoplasm. The detection of apoptosis with these techniques confirmed the initiation of apoptosis. Furthermore, comparison of the results obtained from ISEL and cleaved caspase 3 immunocytochemistry revealed no significant difference in detection of death (45.3±3.9% and 46.5±3.6%, respectively; n=15, P=0.98). Therefore, we are confident of the involvement of apoptosis in our studies.
To compare the effect of cell–cell and cell–matrix in the regulation of apoptosis, we cultured cells in the presence or absence of these contacts. As expected, cell death was low in cells with both contacts, whereas in the absence of both contacts death significantly increased in a time-dependent manner. However, cell death was reduced by the presence of cell–cell or cell–matrix contacts. Interestingly, the presence of one contact reduced cell death 20% to 30% over the time course, suggesting that the presence of either contact is sufficient to limit cell death. It is of note that after 24 to 72 hours, the sum of cell death in cells with only one contact is approximately equal to that observed in cells in the absence of both contacts. To our knowledge, this is the first estimation of the hierarchy of cell contacts and clearly highlights that in addition to cell–matrix contacts, cell–cell contacts play a similar role in cell survival. It is thought that during VSMC proliferation and migration, cell–matrix and cell–cell contacts are modulated without induction of apoptosis. We suggest from our findings that maintenance of at least one type of contact is sufficient to reduce the incidence of death. In support of this, we have observed that N-cadherin expression is reduced during proliferation but not abolished.15 Furthermore, basement membrane degradation is not complete during VSMC proliferation.35
Further confirmation of the ability of N-cadherin to promote VSMC survival was obtained by increasing the level of the full-length form of N-cadherin in VSMCs, which increased cell survival. Interestingly, overexpression of the full-length form of N-cadherin increased ß-catenin at the cell membrane, as well as its association with N-cadherin, suggesting that increased numbers of N-cadherin:ß-catenin contacts form and may increase survival. In contrast, abolishing the expression of full-length N-cadherin with an adenovirus carrying a mutated form of N-cadherin reduced cell survival. The mutated form of N-cadherin lacking the extracellular domain of N-cadherin acts as a dominant-negative ablating N-cadherin function.11,15,19–21 As reported previously,15 the dominant-negative N-cadherin reduced the presence of the full-length N-cadherin protein. In this study, we used higher amounts of adenovirus (up to 1000 pfu/cell compared with 5 and 10 pfu/cell) to increase the expression of the dominant-negative protein and abolish the full-length N-cadherin. Interestingly, the dominant-negative N-cadherin also affected cells grown on plastic. This suggests that abolishing expression of the full-length N-cadherin increases cell death even in the presence of 2 known survival signals for VSMCs, cell–matrix contacts and growth factors, confirming the involvement of cell–cell contacts in promoting survival.
To investigate the mechanism by which N-cadherin provides a survival signal for VSMCs, we performed calcium switch experiments as described previously.36 Reformation of cell–cell contacts by re-addition of calcium increased Akt and Bad phosphorylation, suggesting that formation of cell–cell contacts induces these anti-apoptotic signals. These anti-apoptotic signals were reduced by pre-incubation with anti-N-cadherin neutralizing antibodies, indicating that N-cadherin–mediated cell–cell contact initiates anti-apoptotic signals by Akt activation and Bad phosphorylation.
We have used human saphenous vein VSMCs because of availability of material; however, we are interested in the potential role of N-cadherin in cell death in arterial VSMCs in atherosclerotic plaques. Therefore, to exclude the possibility that the effect of N-cadherin differs in venous and arterial VSMCs, we compared the effect of overexpression of full-length N-cadherin and the dominant-negative N-cadherin on cell death in VSMCs from saphenous vein and radial artery. We observed almost identical effects on cell death in both venous and arterial VSMCs (Figure VII, available online at http://atvb.ahajournals.org), indicating extrapolation of our findings to the atherosclerotic plaque is valid.
In summary, we demonstrate for the first time to our knowledge that aggregation of VSMCs act as a potent survival factor. Our data indicate that cell aggregation mediated by N-cadherin and subsequent activation of Akt promote cell survival. We suggest that in addition to cell–matrix contacts, cell–cell contacts are involved in maintaining VSMC survival. Taken together, our results suggest a potential mechanism in which N-cadherin-mediated cell–cell contact can contribute to the regulation of VSMC survival.
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Acknowledgments |
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We thank Jill Tarlton for her excellent technical assistance and the British Heart Foundation for funding this research.
Received July 29, 2004;
revision received March 3, 2005;
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