Regional Differences in Integrin Expression
Role of α5β1 in Regulating Smooth Muscle Cell Functions
Abstract—There is increasing evidence to suggest that coronary smooth muscle cells (SMCs) differ from noncoronary SMCs. As integrin adhesion molecules regulate many SMC functions, we hypothesized that differences in integrin expression on coronary and noncoronary SMCs may account for cellular differences. Analysis of integrin expression on freshly isolated porcine coronary and noncoronary SMCs revealed that coronary SMCs express significantly less α5β1 than noncoronary SMCs, whereas the expression of total β1 and that of αvβ3 are similar. Consistent with these findings, coronary SMCs demonstrated significantly less adhesion to fibronectin, compared with carotid artery SMCs. As α5β1-mediated signaling has been associated with cellular proliferation, the effects of differential α5β1 expression on cell proliferation were examined by comparing primary coronary and carotid artery SMC proliferation. Coronary SMC growth was significantly lower than that of carotid artery SMCs when plated on fibronectin or type I collagen. Blocking α5β1 function on carotid artery SMCs produced a significant decrease in cellular proliferation, resulting in growth similar to that of coronary SMCs. Furthermore, blocking α5β1, but not αvβ3, inhibited loss of α-smooth muscle actin in proliferating SMCs. Proliferating coronary SMCs were found to upregulate α5β1 expression, further indicating a role for α5β1 in SMC growth. These results suggest that dissimilar α5β1 integrin expression may mediate regional differences in phenotype of vascular SMCs.
The integrin adhesion molecules, which are heterodimers consisting of noncovalently linked α and β subunits,1 mediate many of the smooth muscle cell (SMC) functions associated with atherosclerotic lesion formation, including SMC dedifferentiation, migration, and proliferation.2 At least 16 α and 8 β subunits have been identified, and members of both the β1 and β3 integrin families have been found to modulate vascular cell functions.1 2 3 Although SMCs express several heterodimers containing the β1 subunit, α5β1 has emerged as a potentially important mediator of SMC responses. α5β1, which is the primary vascular fibronectin receptor,4 plays a role in SMC modulation from a contractile to a synthetic phenotype,5 and α5β1-mediated fibronectin matrix assembly appears to be necessary for SMC proliferation.6 Increased α5β1 expression has been demonstrated on dedifferentiated intimal SMCs after balloon injury,7 and α5β1 has been implicated in constrictive vascular remodeling.8 9 The β3 integrin αvβ3 which is a vitronectin receptor, although it can also bind thrombospondin, fibronectin, von Willebrand factor, and osteopontin,3 10 11 has also been demonstrated to participate in fibronectin matrix assembly, cellular migration, and SMC proliferation.12 13 14 Similar to α5β1, αvβ3 expression has been shown to be upregulated in the media and adventitia during hyperplastic responses in injured arteries.15 16 In addition, the blockade of αvβ3 has been demonstrated to decrease intimal thickening after arterial injury.16
Despite the clinical relevance of coronary lesion formation, a limited number of studies have directly examined integrin expression and coronary SMC functions. Recent studies by our group and others have demonstrated that, unlike noncoronary vasculature, coronary arterial SMCs have a distinct phenotype and respond differently to in vivo and in vitro stimulation.17 18 19 20 For example, porcine coronary arteries lack a significant SMC response after endoluminal injury. Instead, activated adventitial fibroblasts proliferate and migrate, resulting in neointimal formation.17 18 21 Consistent with these in vivo findings, porcine and human coronary SMCs have been shown to behave differently in culture, demonstrating advanced differentiation and low proliferation when compared with SMCs from other vascular beds.19 20
In light of these findings, we hypothesized that differences in integrin expression, in particular expression of α5β1 and αvβ3, on coronary SMCs may account for the differential responses of these cells. Thus, the objectives of this study were (1) to determine whether integrin profile distinguishes porcine coronary SMCs from other vascular SMCs and (2) to determine whether integrin profile plays a role in the phenotype and proliferative functions observed in these cells.
Materials and Methods
Porcine hearts and carotid arteries were obtained from the local abattoir. SMCs were isolated as described previously.19 Briefly, coronary and carotid artery media was incubated with collagenase type II (0.75 mg/mL, Worthington) and elastase (0.75 mg/mL, Sigma), and cells were collected after terminating digestion by addition of DMEM (Gibco BRL) with 10% FBS. The cell viability was consistently between 85% and 92% as determined by erythrosine B dye (Sigma) exclusion. In some experiments, iliac artery SMCs were used and were obtained as described above.
Indirect Immunofluorescence and Flow Cytometry
Indirect immunofluorescence staining and flow cytometry were performed as previously described.22 Monoclonal antibodies (mAbs) cross-reactive with the following porcine cell surface markers were used: α5β1 (CD49e/CD29, mAb HA5 [10 μg/mL], mouse anti-human IgG2b, Chemicon International, Inc), β1 (CD29, mAb HB1.1 [10 μg/mL], mouse anti-human IgG1, Chemicon), and αvβ3 (CD41/CD61, JM2E5, 1/5 dilution of mouse anti-porcine IgG1, Serotec). Mouse IgG1 or IgG2b (Coulter-Immunotech) at 10 μg/mL was used as irrelevant isotype control. SMCs in PBS with 0.2% BSA were incubated with saturating concentrations of primary antibody or IgG control in the presence of excess porcine γ-globulin (4 mg/mL, Sigma) and then washed and incubated with excess goat anti-mouse FITC–conjugated secondary antibody (1/50 dilution, BioSource International). Cells were analyzed using a FACStarPlus flow cytometer (Becton-Dickinson), and the data analyzed using LYSIS II software. Mean fluorescence intensity (MFI) for IgG (ie, IgG1 or IgG2b) control was subtracted from the MFI measured with the integrin antibodies to derive net MFI.
Cell Adhesion Assay
Isolated SMCs were plated on 48-well plates (Falcon, Becton Dickinson, 10 000 cells per well) coated with either bovine plasma fibronectin (10 μg/mL, Sigma), human foreskin fibroblast–derived cellular fibronectin (10 μg/mL, Sigma), or bovine type I collagen (10 μg/mL, Becton Dickinson).19 Cells were allowed to adhere for 4 hours in DMEM with 0.1% BSA, and then the wells were washed free of nonadherent cells. Adherent cells were lifted with trypsin (Sigma) and counted in a Coulter counter (Coulter-Immunotech). Percentage adhesion was calculated by dividing the number of adherent cells by the total count of plated cells. Each experiment was carried out in triplicate and repeated at least 3 times on different occasions using cells isolated from multiple animals. In additional experiments, cells were plated as above and allowed to adhere for 48 hours in DMEM with 10% FBS. In these experiments, disintegrin antagonists were used to determine the role of α5β1 and αvβ3 in adhesion of carotid artery SMCs to plasma fibronectin and type I collagen. Antagonists included the α5β1 antagonist EMF-10, the αvβ3 antagonist kistrin, and the αIIβ3 antagonist eristostatin (all 1 μg/mL).23 24
Cell Proliferation Assays
SMCs were plated as described for 48 hours of adhesion. Proliferation was determined by counting cell numbers at 2, 4, 6, 8, and 10 days after plating, as described previously.19 Each experiment was carried out in triplicate and repeated 3 times on different occasions using cells isolated from multiple animals. To determine the role of α5β1 and αvβ3 in proliferation, the disintegrins EMF-10 (anti-α5β1, 15 to 150 μmol/L), echistatin (anti-α5β1, 150 μmol/L) kistrin (anti-αvβ3, 150 μmol/L), and eristostatin (anti-αIIβ3, 150 μmol/L) were added to the wells 48 hours after cell plating so as not to interfere with initial cell attachment.23 24 Antagonist was added every 48 hours when the growth medium was changed.
To verify SMC proliferation data obtained using cell-counting methods, additional experiments were performed in which a cell proliferation was analyzed via 5-bromo-2′-deoxyuridine (BrdU) incorporation (Cell Proliferation ELISA, BrdU Colorimetric, Boehringer Mannheim). In these experiments, coronary and carotid artery SMCs were plated in 96-well plates (3000 cells per well) and allowed to grow for 6 to 8 days (70% confluence). Cells then underwent 3 hours of incubation with BrdU, and then the ELISA assay was performed. In the case of treated cells (ie, EMF-10, eristostatin), cells were treated for 24 hours and then the ELISA assay was performed.
In some experiments, passage 3 coronary and carotid artery SMCs were plated in 48-well plates at a density of 5000 cells per well and proliferation was examined over 10 days. Cells were passaged by plating 0.5 to 1×106 freshly isolated cells in T75 flasks. At 15 to 25 days after plating, proliferating cells were lifted with trypsin and subcultured. Coronary SMCs routinely took longer to reach numbers sufficient for subculture as a result of their decreased rate of proliferation, and primary cells did not reach confluence before subculture.
Histochemical Detection of SMC Apoptosis
To determine whether disintegrin treatment of carotid artery SMCs results in cell apoptosis, carotid artery SMCs were plated on fibronectin-coated chamber slides and allowed to grow for 4 to 6 days. Cells were left either untreated or treated for either 2, 4, 6, 24, or 48 hours with EMF-10, kistrin, or eristostatin (150 μmol/L for each) and then fixed and apoptosis examined via DNA fragmentation methods (TdT-FragEl DNA Fragmentation Detection Kit, Oncogene Research Products).
Immunostaining for α-SM Actin
SMCs were plated on chamber slides (10 000 cells per well) coated with fibronectin (10 μg/mL) and allowed to grow for 8 days. Cells were either untreated or treated with EMF-10 or kistrin as described above. The Vectastain Elite ABC system was used (Vector Laboratories) to stain for α-SM actin (clone 1A4, IgG1, 1:50, Sigma) as previously described.19
All data are presented as mean±SEM. Data were compared by ANOVA using post hoc analysis with the Fisher corrected t test. Probabilities of 0.05 or less were considered statistically significant.
Regional Differences in Integrin Expression
Freshly Isolated Coronary SMCs Express Low Levels of Integrin α5β1 Compared With Other Vascular SMCs
Surface expression of α5β1, total β1, and αvβ3 was compared on freshly isolated coronary, carotid, and iliac artery SMCs by indirect immunofluorescence and flow cytometry. As seen in the Table⇓, coronary SMC α5β1 expression was significantly lower than that of carotid and iliac artery SMCs. The difference in α5β1 expression was not due to lower total β1 integrin, as total β1 expression was comparable among SMCs. In addition, expression of the other integrin of interest, αvβ3, was not significantly different among the cell types (Table⇓). Figure 1⇓ shows representative histograms for coronary and carotid artery SMC α5β1 and total β1 expression. Freshly isolated coronary artery SMCs showed a uniformly low level of α5β1 expression. Carotid artery SMC expression of α5β1 was less uniform; however, the vast majority of carotid artery cells show strong positive staining for α5β1. To assure that the lack of α5β1 surface expression was not a function of the isolation procedure, coronary SMCs were placed in culture for 24 or 48 hours and then examined. Expression of α5β1 remained low (data not shown), thus indicating that the lack of α5β1 was not likely the result of cell isolation procedures.
Coronary SMCs Demonstrate Decreased Adhesion to Fibronectin
To determine whether the low level of α5β1 on coronary SMCs affected their ability to adhere to fibronectin, the primary ligand for α5β1, adhesion (4 hours in DMEM+0.1% BSA) of coronary and carotid artery SMCs to plasma fibronectin, cellular fibronectin, and type I collagen was compared. Consistent with the low level of α5β1 expression, coronary SMCs demonstrated significantly less adhesion (34.6±3.5%, P<0.01, n=3) to plasma fibronectin than carotid artery cells (56.3±6.9%, n=3) (Figure 2⇓). There was no significant difference between adhesion of either SMC type to plasma or cellular fibronectin (data not shown); thus, the remainder of the experiments (ie, proliferation, differentiation, and apoptosis) were performed using only plasma fibronectin coating. The low level of adhesion to fibronectin was specific, as coronary and carotid artery SMCs adhered similarly to type I collagen (Figure 2⇓). When adhesion was examined over 48 hours, percentage adhesion to plasma fibronectin and type I collagen was very similar to values observed at 4 hours for both cell types (data not shown). At 48 hours, carotid artery SMC adhesion to fibronectin was significantly inhibited by the anti-α5β1 disintegrin EMF-10 (51.4±6.1% inhibition, n=4, P<0.01) but not by the anti-αvβ3 disintegrin kistrin (8.3±6.0% inhibition, n=4, NS) or the anti-αIIβ3 disintegrin eristostatin (−10.6±3.2% inhibition, n=4, NS). Carotid artery SMC adhesion to type I collagen was not significantly inhibited by any of the disintegrins used (n=3 for all, NS).
Role of α5β1 in Regulating SMC Functions
Coronary SMCs Demonstrate Decreased Proliferation Compared With Carotid Artery SMCs, a Function Associated With α5β1 Expression
As α5β1 has been associated with cell proliferation, experiments were performed to determine whether the low level of α5β1 expression on coronary SMCs affected their ability to proliferate. As seen in Figure 3⇓, coronary SMC numbers at 10 days after plating on fibronectin (22 578±6531 cells per well) and type I collagen (22 455±5852 cells per well) were significantly lower than carotid artery SMC numbers plated on the same matrix coatings (72 291±3766 and 55 013±2343 cells per well, respectively, P<0.05). When cell proliferation was examined using BrdU incorporation, very similar results were observed; BrdU incorporation in coronary SMCs was ≈45% of that measured in carotid artery SMCs (data not shown). Differences in cell proliferation could not be attributed solely to cell adhesion, as there was no difference in coronary and carotid artery adhesion to type I collagen (Figure 2⇑).
As seen in Figure 4⇓, blocking α5β1 function on carotid artery SMCs with the disintegrin EMF-10 (15 to 150 μmol/L) or echistatin (150 μmol/L) resulted in a concentration-dependent decrease in cellular proliferation, thus confirming a role for α5β1in SMC proliferation. As expected, the effects of α5β1 antagonism were greater when cells were plated on fibronectin (≈70% inhibition) versus type I collagen (≈40% inhibition). Although the αvβ3 antagonist kistrin (150 μmol/L) also significantly inhibited cell proliferation (Figure 4⇓), for cells plated on fibronectin, α5β1 blockade was more effective than αvβ3 blockade in inhibiting cell proliferation. The effects of EMF-10 on SMC proliferation were also verified using a BrdU incorporation assay (data not shown). Similar inhibition of proliferation was observed with both assays. The effects of EMF-10 and kistrin on proliferation cannot be attributed to adhesion blockade, as disintegrin treatment was initiated 48 hours after plating and none of the disintegrins inhibited SMC adhesion to type I collagen (see adhesion results, above). As expected, the anti-αIIβ3 antagonist eristostatin had no effect on SMC proliferation regardless of matrix coating. These data indicate that in the presence of fibronectin matrix, α5β1, and not αvβ3, may provide the primary integrin signal for cell proliferation.
As shown in Figure 5⇓, α5β1 blockade on carotid artery SMCs resulted in cell numbers similar to those of slowly growing coronary SMCs at day 10 (25 505±2688 and 29 563±2428, respectively, n=3). Thus, the low proliferative rate of coronary SMCs was mimicked by blocking α5β1 function on carotid artery SMCs. Interestingly, blocking α5β1 function on coronary SMCs resulted in a significant reduction in cell numbers, when cells were plated on fibronectin, suggesting that either the low level of α5β1 present at the time of isolation is sufficient to signal for cell proliferation or that α5β1 may be upregulated on coronary SMCs in response to serum stimulation.
Antagonism of α5β1 Does Not Result in SMC Apoptosis
To determine whether the decreased cell numbers observed with treatment of carotid artery SMCs with either of the peptide antagonists, EMF-10 or kistrin, was in part the result of cell apoptosis, cell apoptosis was examined via DNA fragmentation methods. Carotid artery SMCs were plated on fibronectin-coated chamber slides and allowed to grow for 4 to 6 days. Cells were then either left untreated or were treated for either 2, 4, 6, 24, or 48 hours with EMF-10, kistrin, or eristostatin (150 μmol/L for each) and then fixed, and apoptosis was examined via DNA fragmentation methods (TdT-FragEl DNA Fragmentation Detection Kit, Oncogene Research Products). Although even short-term treatment of carotid artery SMCs with EMF-10 resulted in significant shape change (ie, rounding) of cells, the cells remained firmly attached and did not undergo apoptosis. For untreated cells and all treatments examined, <1% of cells were found to be apoptotic at the times observed. These results in conjunction with BrdU incorporation studies appear to indicate that inhibition of proliferation is the primary means by which α5β1 antagonism results in decreased cell numbers.
Antagonism of α5β1 Results in Decreased SMC Dedifferentiation
As fibronectin binding through α5β1 has been hypothesized to be the primary signal for SMC dedifferentiation, the effect of α5β1 function blocking on α-SM actin immunostaining was examined. Carotid artery SMCs were plated on fibronectin-coated slides in the absence of disintegrin or in the presence of EMF-10 or kistrin. As shown in Figure 6⇓, under normal growth conditions, only 48.7±3.5% of carotid artery SMCs continue to express α-SM actin by day 8. In contrast, carotid artery SMCs grown in the presence of the α5β1 disintegrin EMF-10 continue to express high levels (>80% positive) of α-SM actin, similar to coronary SMCs, which have low baseline levels of α5β1. This is in contrast to carotid artery SMCs grown in the presence of kistrin, which did not prevent SMC dedifferentiation (Figures 6⇓ and 7⇓). As seen in Figure 7⇓, blockade of α5β1 also altered cell morphology. Untreated carotid artery SMCs (Figure 7A⇓) plated on fibronectin show a spindle-shaped morphology, with cells generally possessing long, thin projections. However, when cells were grown in the presence of EMF-10, the cells were larger (Figure 7B⇓), with dense cell bodies lacking spindle projections. These cells look very similar to coronary SMCs (Figure 7D⇓) both in their morphology and in their expression of α-SM actin.
Proliferating Coronary SMCs Upregulate α5β1 and Demonstrate an Increased Rate of Proliferation
Despite the slower growth of coronary SMCs, a subset of these cells proliferate in culture, and proliferation was inhibited by blocking α5β1 (Figure 5⇑). To determine whether one of the mechanisms by which coronary SMC begin to grow is through upregulation of α5β1, coronary SMC integrin phenotype was examined on primary (nonpassaged, 14 days after culture) and passaged (passages 1 to 3) coronary SMCs. Primary coronary SMCs significantly upregulate α5β1. Freshly isolated coronary SMCs had net MFI of 5.5±3.4 for α5β1 expression, whereas net MFI for α5β1 expression at day 14 after plating was 121.5±32.3 (n=3, P<0.01). This value was not significantly different from α5β1 expression on primary carotid artery SMCs at day 14 (net MFI 110.0±12.5, n=3). In addition, passaged coronary SMCs continue to express α5β1 at levels similar to carotid artery SMCs (data not shown). To determine whether the upregulation of α5β1 on coronary SMCs conferred an increased rate of proliferation and thus a rate similar to carotid artery SMCs, passage 3 coronary and carotid artery SMCs were plated in 48-well plates at a density of 5000 cells per well and proliferation was examined over 10 days. Passage 3 coronary and carotid artery SMCs demonstrated similar levels of proliferation (9.4±1.7- versus 10.7±0.4-fold increase in cell number, respectively), further indicating a role for α5β1 in SMC proliferation.
There is a growing body of evidence to suggest that coronary SMCs differ from noncoronary SMCs.17 18 19 20 The data presented herein suggest an explanation for these differences by demonstrating low basal expression of α5β1 on coronary SMCs, compared with SMCs from other vascular beds, which may account for the highly differentiated phenotype of coronary SMCs (retention of α-SM actin and decreased proliferation). Our finding that α5β1 expression is not uniform among vascular beds may be of importance to the understanding of vascular lesion formation, for two main reasons. First, α5β1 has been linked to phenotypic changes in SMC function associated with lesion development, including SMC dedifferentiation5 7 and proliferation.6 Second, our data imply that unlike SMCs from other vascular beds, upregulation of α5β1 by coronary SMCs may be a prerequisite to coronary SMC proliferation and thus intimal lesion formation.
The first evidence that α5β1 may play a significant role in vascular lesion formation was demonstrated by Hedin and Thyberg,5 who showed that fibronectin was the primary serum factor mediating modulation of SMCs from a contractile to a synthetic phenotype. The fibronectin receptor α5β1 or very late–appearing antigen (VLA)–5 was soon identified as a member of the integrin family of adhesion molecules.3 25 Herein we show data that support the role of α5β1 in mediating SMC dedifferentiation by demonstrating that blocking α5β1, but not αvβ3, inhibits loss of α-SM actin in proliferating SMCs (Figures 6⇑ and 7⇑). This is consistent with the finding of Hedin and Thyberg,5 as they demonstrated that rat aortic SMCs plated on fibronectin reduced their myofilament bundles, even in the absence of serum. Most recently, Pickering et al7 showed that α5β1 expression was strongly associated with dedifferentiated cells (ie, reduced α-SM actin) and fibronectin in the injury-induced intima. In contrast, coronary SMCs, which express low levels of α5β1, maintained expression of SMC markers in culture.19 20
In addition to its role in SMC phenotype switching, α5β1 has been demonstrated to play a role in SMC proliferation6 as well as proliferation of other cell types.26 The role of α5β1 as the primary mediator of fibronectin matrix assembly has been demonstrated by several studies,13 27 28 29 which has led to the hypothesis that signaling through α5β1 binding to fibronectin stimulates cell growth.6 13 27 28 29 Mercurius and Morla6 recently demonstrated that peptide antagonists that block α5β1-mediated fibronectin matrix assembly, but not α5β1 function, effectively prevent SMC proliferation. The data presented herein show for the first time that directly blocking SMC α5β1 function is also very effective in inhibiting SMC proliferation. Importantly, αvβ3 has also been shown to mediate fibronectin matrix assembly in the absence of α5β1.13 These findings may explain the inhibition of cell growth observed with the αvβ3-antagonist kistrin and the ability of anti-αvβ3 antagonists to partially inhibit intimal formation in injured porcine coronary arteries.16 Together, these data suggest that multiple integrins are likely involved in the cell proliferation associated with intimal formation. However, a significant difference between α5β1 and αvβ3 may be their impact on constrictive remodeling. Yee et al8 have recently demonstrated that α5β1, but not αvβ3, plays a crucial role in SMC migration into and contraction of the fibrin clot, possibly contributing to geometric remodeling of the vessel.
Given the importance of α5β1 to SMC functions, our observation that α5β1 is expressed at very low levels on quiescent coronary SMCs may provide new insight into why these cells respond differently to both in vivo and in vitro growth stimulation.17 18 19 20 Data presented herein suggest that the failure of coronary SMCs to respond to in vivo stimulation may be due in large part to their low-level expression of α5β1. It remains to be determined whether the differences in integrin expression arise from the heterogenous lineages of vascular cells, with coronary vessels originating from proepicardial cells in situ rather than from ectodermal or mesodermal parts of the aorta.30 The differential integrin phenotype of coronary SMCs may confer a protective effect, preventing rapid formation of occlusive coronary lesions in response to noxious stimuli, and further supports a role of “invasive” fibroblast in coronary vascular remodeling.17 21 The roles of α5β1 and αvβ3 in coronary fibroblast functions remain to be determined.
As cultured coronary SMCs did upregulate α5β1, phenotypic switching of coronary SMCs from low to high α5β1 expression may play a role in the loss of protective homeostasis in these cells. It is likely that only a subset of coronary SMCs are capable of upregulating α5β1 and thus proliferating, given that the majority of coronary SMCs that adhere to fibronectin and type I collagen fail to spread or take on a dedifferentiated phenotype. Clonal analysis of coronary cells will be necessary to more fully elucidate this phenomenon. Clonal heterogeneity of medial SMCs has been observed in noncoronary vascular beds31 ; however, this has not been examined in the coronary media.
In conclusion, we have demonstrated that freshly isolated porcine coronary SMCs express low levels of α5β1 compared with SMCs from other vascular beds, and this difference in α5β1 may contribute to the observed differences in coronary SMC responses to in vivo and in vitro stimulation. Furthermore, unlike other SMCs, coronary SMCs appear to have to upregulate α5β1to proliferate. Increasing our understanding of the importance of α5β1 to coronary SMCs may provide important new insight into our understanding of coronary vascular disease.
This study was supported by NIH Grants HL-44150 and HL-60672 and the John S. Sharpe Foundation. We acknowledge Dr Rosario Scalia, Department of Physiology, Thomas Jefferson University, for providing generous use of flow cytometry facilities.
Original received July 28, 2000; revision received November 8, 2000; accepted December 18, 2000.
- © 2001 American Heart Association, Inc.
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