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【摘要】
Objective- Type VIII collagen is upregulated after vascular injury and in atherosclerosis. However, the role of type VIII collagen endogenously expressed by smooth muscle cells (SMCs) and in the context of the vascular matrix microenvironment, which is rich in type I collagen, is not known. To address this, we have compared aortic SMCs from wild-type (WT) mice to SMCs from type VIII collagen-deficient (KO) mice when plated on type I collagen.
Methods and Results- Type VIII collagen was upregulated after wounding of WT SMCs. KO SMCs exhibited greater adhesion to type I collagen than WT SMCs (optical density [OD 595 ]=0.458±0.044 versus 0.193±0.071). By contrast, the WT SMCs spread more (389±75% versus 108±14% increase in cell area), migrated further (total distance 80.6±6.2 µm versus 64.2±4.4 µm), and exhibited increased [ 3 H]-thymidine uptake (160 000±22 300 versus 63 100±12 100 counts per minute) when compared with KO SMCs. Gelatin zymograms showed that WT SMCs expressed latent matrix metalloproteinase 2, whereas KO SMCs did not. Addition of exogenous type VIII collagen returned levels of KO SMC adhesion (OD 595 =0.316±0.038), migration (79.5±5.8 µm), and latent matrix metalloproteinase 2 expression to levels comparable to WT SMCs.
Conclusions- This study suggests that SMCs can modify the matrix microenvironment by producing type VIII collagen, using it to overlay type I collagen, and generating a substrate favorable for migration.
Type VIII collagen is upregulated after vascular injury and in atherosclerosis. Using smooth muscle cells from wild-type and type VIII collagen knockout mice, we show that cells that are able to produce endogenous type VIII collagen, proliferate, spread, and migrate more when the cells are plated on a type I collagen matrix.
【关键词】 atherosclerosis restenosis collagen smooth muscle cell migration MMP
Introduction
Collagens compose a large portion of the extracellular matrix in the vessel wall and play important roles maintaining the strength and structural integrity of blood vessels. During atherosclerosis, collagen accumulation contributes to lesion growth and vessel contraction, and collagen degradation leads to plaque instability and rupture. 1 In addition to their roles providing structural support in the arterial wall, in vitro studies suggest that collagens can act as signaling molecules stimulating changes in the phenotype and behavior of smooth muscle cells (SMCs), endothelial cells, and macrophages. Recent research suggests that SMCs respond differently to different types or physical states of collagen. For example, intact type I collagen maintains SMC quiescence, whereas denatured or degraded collagen stimulates proliferation and migration. 2-4
Type VIII collagen is a member of the short chain collagen family, composed of 1 (VIII) and 2 (VIII) chains. It is produced by corneal and aortic endothelial cells, as well as mesangial cells, and is upregulated during the proliferation of these cells and during angiogenesis. 5-8 In normal arteries, it has been localized to the subendothelial intima and is present in very low amounts in the media and adventitia. 9 Type VIII collagen expression is dramatically increased after balloon injury of the rat carotid 10,11 or the porcine coronary artery. 12 In the rat balloon injury model, type VIII collagen was expressed by SMCs migrating and proliferating during intimal thickening. 10,11 It is also present in the atherosclerotic lesions of apolipoprotein E-deficient mice 13 and cholesterol-fed rabbits subject to balloon injury, 14 where expression is localized to intimal SMCs and to macrophage-rich areas of the plaque, suggesting that macrophages also produce type VIII collagen. Type VIII collagen was similarly localized in human atherosclerotic plaques. 15-17 Expression of mRNA for type VIII collagen is regulated by platelet-derived growth factor, fibroblast growth factor 2, and angiotensin II, all important factors in the pathogenesis of atherosclerosis. 11,18
Investigating the interaction of SMCs with exogenous type VIII collagen in vitro, we and others have shown that the protein acts as an attachment and chemotactic factor for SMCs. 11,18 SMCs attach to type VIII collagen, but it is a less adhesive substrate and promotes greater cell migration than type I collagen. In addition, type VIII collagen stimulates SMC matrix metalloproteinase synthesis, whereas type I collagen does not. 18 These studies were performed using exogenous type VIII collagen coated on tissue culture plates as a substrate for the SMCs. However, in the diseased vessel wall, type VIII collagen is expressed and deposited by SMCs in the presence of an existing matrix rich in type I collagen. The function of endogenously expressed type VIII collagen in this more complex matrix microenvironment has not been studied. We now hypothesize that after arterial injury, SMCs produce type VIII collagen and use it to overlay an existing extracellular matrix, providing a substrate more favorable for rapid migration. To address this hypothesis, we have compared aortic SMCs isolated from Col8a1 +/+ /Col8a2 +/+ mice [wild-type (WT)] to SMCs isolated from type VIII collagen-deficient mice, Col8a1 -/- /Col8a2 -/- (KO), to examine different components of the migratory process when the cells are plated on either uncoated or type I collagen-coated surfaces.
Methods
For a detailed account of the methodologies used in this article, please see http://atvb.ahajournals.org.
Mice with targeted deletion of both the Col8a1 and Col8a2 genes (KO) were generated in the laboratory of Bjorn Olsen (Harvard Medical School) as described 19 with WT littermate mice used as controls. Aortic vascular SMCs were isolated from the mice as described previously. 20 For all of the experiments, tissue culture plates/flasks were either left uncoated or coated with a solution containing 50 µg/mL of pepsin-solubilized bovine dermal type I collagen (Vitrogen 100; Collagen Biomaterials). Cell attachment assays were performed as described previously. 18 For spreading assays, 100 000 cells were seeded onto 25-cm 2 tissue culture flasks and imaged using a Nikon Eclipse TE200 inverted microscope equipped with a heated stage. A Hamamatsu digital camera (model #C4742-95) was used to capture images every 10 minutes for 4 hours after plating. Three to 6 cells were analyzed for each experiment. Migration assays were similar, with the following modifications: 100 000 cells/well were seeded onto 6-well plates, then grown until 50% confluence (0.5 to 2 days), and, subsequently, images of migrating cells were captured every 10 minutes for 8 hours. Six to 8 cells were analyzed in each experiment. SMC proliferation was estimated using [ 3 H]-thymidine incorporation. MMP activity in the SMC-conditioned medium was assayed using gelatin zymograms as described previously. 18 Rescue experiments using KO SMCs with the addition of exogenous type VIII collagen were also performed. Wells were first coated with a solution containing 37.5 µg/mL type I collagen/PBS then rinsed with PBS and coated with 6.6 µg/mL exogenous type VIII collagen/PBS (type VIII collagen was isolated from bovine Descemet?s membrane as described previously 21 ). This gives a coating composed of 75% type I collagen and 25% type VIII collagen, with the same total molar concentration as the 50 µg/mL type I collagen used in the first experiments. Adhesion and migration experiments were performed on this mixed collagen substrate as described above. Immunocytochemistry was done using an anticollagen 1 (VIII) monoclonal antibody (Clone 8C, Seikagaku America).
Results
SMC Morphology Differed Between KO and WT SMCs
Western blots probed with an antibody against type VIII collagen revealed a band of 240 kDa in the lysates from WT SMCs, whereas this band was absent in KO SMCs (Figure IA, available online at http://atvb.ahajournals.org). SMCs obtained from WT and type VIII collagen KO mice exhibited distinct morphologies in culture. When plated on uncoated wells, WT SMCs appeared small and elongated, usually displaying only 1 or 2 long cytoplasmic protrusions (Figure IB). By contrast, KO SMCs were larger and rounder, and they extended thin, short, stellate processes (Figure IC). Morphometric measurements demonstrated that KO SMCs were indeed significantly larger (2830±449 µm 2 versus 649±61 µm 2; P 0.001) and rounder (0.153±0.021 versus 0.112±0.008; P =0.039, with a value of 1 corresponding to a perfect circle) than WT SMCs. KO SMCs plated on polymerized type I collagen exhibited a similar morphology to those plated on plastic, whereas WT SMCs plated on type I collagen were more rounded with no protrusions visible (data not shown). There was no difference in viability between KO and WT SMCs, whether they were plated on uncoated or on type I collagen-coated wells (data not shown).
Production of Type VIII Collagen Was Upregulated After Injury
Confluent layers of SMCs were subject to a scrape wound, then immunostained with an antibody against type VIII collagen. Immediately after wounding, WT SMCs in the uninjured monolayer and in areas adjacent to the wound stained for type VIII collagen ( Figure 1 A), whereas KO SMCs did not stain ( Figure 1 B). Type VIII collagen was localized in the cytoplasm with a punctate staining pattern. Double staining with an antibody raised against a marker of the Golgi complex (58K Golgi protein marker) revealed that most of the intracellular type VIII collagen was localized in the Golgi (data not shown). A substantial increase in type VIII collagen was evident in the WT cells 24 hours after wounding ( Figure 1 C). By contrast, KO SMCs did not stain for type VIII collagen at 24 hours ( Figure 1 D).
Figure 1. Immunostaining revealed type VIII collagen in WT SMCs as punctate cytoplasmic immunostaining on injury (A) that was upregulated 24 hours after injury (C). No staining was evident in KO SMCs on injury (B) or 24-hours later (D). W, wounded area; scale bar, 100 µm.
We had some difficulty staining type VIII collagen within the extracellular matrix, probably because the protein is tightly complexed with other matrix proteins. However, after lightly digesting the matrix with 10 µg/mL pepsin in 0.1 mol/L acetic acid, we were able to detect extracellular immunostaining for type VIII collagen ( Figure 2 A). Furthermore, after treating confluent cultures with a mixture of EDTA/EGTA to lift off cells, then lysing the underlying matrix, we were able to detect matrix-bound type VIII collagen produced by WT cells but not KO cells on a Western blot ( Figure 2 B).
Figure 2. Pepsin was used to digest the matrix in WT SMC cultures, and immunoflourescence staining revealed the presence of type VIII collagen in the matrix (A). The presence of type VIII collagen was confirmed on Western blots of matrix lysates of WT SMCs, but KO SMC lysates were negative (B). Scale bar, 100 µm.
Production of Type VIII Collagen Decreased Attachment of SMCs to Type I Collagen and Facilitated Spreading and Migration
SMCs must attach to substrate to gain traction for migration; however, too strong an attachment may fix the cells in place and prevent migration. To determine the effect of type VIII collagen production on SMC adhesion, we measured adhesion to uncoated wells or to wells coated with type I collagen. The WT SMCs adhered significantly less than KO SMCs to uncoated wells ( Figure 3 A) or to wells coated with type I collagen ( Figure 3 B). The difference in adhesion was especially large when the cells were plated on type I collagen (optical density [OD 595 ]=0.193±0.071 for WT cells versus OD 595 =0.458±0.044 for KO cells). The addition of exogenous type VIII collagen to the wells to rescue the phenotype resulted in the decreased attachment of KO cells (OD 595 =0.316±0.038) to a level that was not significantly different from WT cells ( Figure 3 B).
Figure 3. Adhesion of KO and WT SMCs to uncoated wells (A) or to wells coated with type I collagen (B). KO-R, rescue experiments with addition of type VIII collagen to wells. Values are mean±SEM. *Adhesion of KO SMCs was significantly greater than WT SMCs or KO-R SMCs.
Cell spreading and protrusion occur during migration and are also affected by adhesion strength, so we compared the spreading in WT and KO SMCs. Our preliminary experiments revealed that most spreading occurred during the first hour after plating. When plated on uncoated flasks, WT SMCs spread and increased cell area by &4-fold in 1 hour (370±61%), significantly more than KO SMCs, which increased in area by only 2-fold (206±37%; Figure 4 A). WT SMCs also spread &4-fold in 1 hour when plated on 50 µg/mL type I collagen (389±75%), significantly more than KO SMCs, which did not spread on type I collagen (108±14%; Figure 4 B).
Figure 4. Spreading of KO and WT SMCs after plating on either uncoated wells (A) or on wells coated with type I collagen (B). Values are mean±SEM. *Spreading was signficantly greater in the WT SMCs compared with KO SMCs.
We used time-lapse microscopy to measure cell migration. The total distance migrated by individual cells over an 8-hour period was calculated. Whether plated on uncoated or type I collagen-coated wells, the distance traveled by WT SMCs was significantly greater than that for KO SMCs ( Figure 5 ). When plated on uncoated wells, WT SMCs traveled a total distance of 115±9 µm compared with 72.7±3.8 µm for KO SMCs ( Figure 5 A). When plated on 50 µg/mL type I collagen, WT SMCs traveled a total distance of 80.6±6.2 µm compared with 64.2±4.4 µm for KO SMCs ( Figure 5 B). The addition of type VIII collagen to the plates rescued the KO SMC migration such that the KO rescue (KO-R) SMCs traveled a distance not different from the distance traveled by WT SMCs (79.5±5.8 µm; Figure 5 B).
Figure 5. Migration of KO and WT SMCs plated on uncoated wells (A) or on type I collagen (B). KO-R, rescue experiments with addition of type VIII collagen. Values are mean±SEM. *WT SMCs migrated a greater total distance than KO SMCs.
Type VIII Collagen Production Increases MMP Activity
Because MMPs facilitate migration of SMCs by allowing the clearance of matrix barriers, gelatin zymograms were used to measure MMP-2 and MMP-9 activity in conditioned media from WT and KO SMCs ( Figure 6 A). Conditioned media from mouse embryonic fibroblasts (MEFs) was used as a positive control and to identify the lytic bands on zymogram gels. MEF-conditioned media contained distinct lytic bands at 95 kDa (active MMP-9), 70 kDa (latent MMP-2), and 61 kDa (active MMP-2). Media from WT and KO mouse SMCs contained lytic bands of 106 kDa (latent MMP-9), 95 kDa (active MMP-9), 84 kDa (unknown), 77 kDa (unknown), and 70 kDa (latent MMP-2). There was increased lysis in the latent MMP-2 band in the conditioned media of WT SMCs compared with the conditioned media of KO SMCs. The addition of exogenous type VIII collagen to the plate led to an increase in latent MMP-2 production by KO cells, showing a complete rescue of the KO phenotype. By contrast, there were no apparent differences in the activity of MMP-9 or the unidentified bands, comparing WT and KO SMCs. Within each cell type, there were no differences in MMP activity between cells plated on plastic or on type I collagen (data not shown).
Figure 6. Gelatin zymogram containing conditioned media samples obtained from KO and WT SMCs (A). MEF lane contains conditioned media from MEFs. KO-R, conditioned media from KO SMCs rescued with the addition of type VIII collagen. (B) Proliferation of KO and WT SMCs plated on uncoated wells or wells coated with type I collagen. Values are mean ± SEM. *WT SMCs incorporated significantly more [ 3 H]-thymidine than KO SMCs when plated on type I collagen.
Type VIII Collagen Facilitates SMC Proliferation
To assess cell proliferation, [ 3 H]-thymidine incorporation was measured. Thymidine incorporation was similar in WT and KO SMCs plated on plastic ( Figure 6 B). By contrast, thymidine uptake was increased in WT SMCs plated on type I collagen (160 000±22 300 counts per minute) compared with KO SMCs (63 100±12 100 counts per minute).
Discussion
Knowledge about the function of type VIII collagen is scarce. However, it is expressed at high levels in injured arteries and in the atherosclerotic lesions of humans. Previous studies using differential display PCR described the upregulation of type VIII collagen in injured compared with uninjured rat carotid arteries. 10,11 Type VIII collagen was deposited in copious amounts by SMCs immediately subjacent to the vessel lumen and in SMCs forming thickened neointimal lesions after injury, a pattern that correlated with SMC migration. 11 Previously, we performed in vitro experiments to study the interactions of SMC with exogenous type VIII collagen and demonstrated that the protein was an adhesive and chemotactic substrate and that it also stimulated MMP synthesis by neointimal SMCs. 18 Taken together, these data suggested an important role for type VIII collagen in promoting SMC migration.
However, the vascular extracellular matrix is a complex mixture composed of several different types of molecules, and it is particularly rich in type I collagen. In fact, type I collagen and type VIII collagen are both upregulated and colocalized during plaque development. 14 In vitro studies have shown that SMCs can attach to type I collagen; nonetheless, a substantial body of evidence shows that intact polymerized type I collagen inhibits cell migration and proliferation and downregulates the expression of many genes. 2-4 By contrast, type VIII collagen appears to stimulate opposite responses. Although the effects of exogenous type VIII collagen on SMCs have been studied, the importance of endogenously produced type VIII collagen is not known. Furthermore, the effect of type VIII collagen in the presence of a polymerized type I collagen matrix has not been examined. In the current study, we hypothesized that SMCs produce type VIII collagen, lay it down on top of type I collagen, and use this modified, less adhesive matrix to facilitate migration. To investigate this, we studied SMCs harvested from the aortas of WT and type VIII collagen KO mice. We compared the ability of these cells to migrate on dishes coated with polymerized type I collagen, which was used to mimic the natural environment encountered in the vascular media.
KO SMCs displayed significantly stronger attachment than WT SMCs to both tissue culture plastic and to wells coated with type I collagen substrate. This suggests that cells that are able to produce type VIII collagen adhere less, and these results are in accordance with our previous studies where we found that SMC attachment to type VIII collagen was less than attachment to type I collagen. 18 In fact, when KO SMCs were plated on a mixture of type VIII and type I collagen, their levels of attachment were reduced to a level comparable to WT SMCs, showing a partial rescue of the KO phenotype.
To accomplish migration in a defined direction, cells first extend leading lamellopodia, which attach to the substrate. After this is cell contraction, then attachments to the substrate at the rear of the cell are broken, allowing tail retraction and forward translocation of the cell body. 22,23 The ability to spread and subsequently migrate depends on a critical value of adhesive strength between cell and substrate: high or low levels of substrate attachment inhibit spreading and migration, whereas maximum migration occurs at intermediate adhesion strengths. 24 We found that WT SMCs migrated a greater distance than KO SMCs on both uncoated and type I collagen-coated wells. This suggests that the ability to produce type VIII collagen allowed the cells to overcome strong adhesion to type I collagen and, thus, enabled migration. The WT SMCs extruded well-defined protrusions and translocated efficiently on the substrate. By contrast, the KO cells displayed membrane ruffling with repeated extensions and retractions of stellate processes in all directions. Importantly, we were able to rescue KO SMC migration by adding exogenous type VIII collagen to the wells. Interestingly, Rocnik et al 25 reported that new collagen synthesis is required for SMC migration on polymerized type I collagen. They did not identify the type of collagen produced; however, we can speculate that it is type VIII collagen.
Another important difference was that WT cells exhibited higher proliferation rates than KO cells when plated on type I collagen. This suggests that endogenously produced type VIII collagen allows cells to overcome the inhibitory effects of type I collagen on proliferation. Likewise, type VIII collagen has recently been implicated in stimulation of the proliferation of corneal endothelial cells. 19,26 The production of matrix-degrading enzymes, such as the MMPs, is required for SMCs to detach from matrix to migrate or proliferate and to facilitate the clearance of matrix barriers. Gelatin zymograms revealed MMP-2 activity in the media from WT SMCs, whereas there was less MMP-2 activity in the media from the KO SMCs. However, the addition of exogenous type VIII collagen to the KO SMCs increased the MMP-2 production by these cells. These results confirm previous studies where we showed that type VIII collagen stimulated the production of both MMP-2 and MMP-9 by rat SMCs. 18 However, we did not see a difference in MMP-9 activity in the mouse cells, suggesting that there may be species-specific differences.
Type VIII collagen interacts with SMCs via the 1 ß 1 and 2 ß 1 integrins 18 and the recently discovered discoidin domain receptor tyrosine kinases (DDR1 and 2). 20 Both integrins and DDR1 control cell proliferation on collagenous substrates and can activate intracellular signaling pathways leading to the upregulation of MMPs. 18,27-32 Furthermore, both DDR1, 20 and the 1 ß 1 integrin 33 are upregulated after arterial injury in coincidence with type VIII collagen.
Our studies have concentrated on the SMC as the source of type VIII collagen and focused on SMC interactions with this protein. However, other cell types in the vessel wall produce type VIII collagen in the atherosclerotic plaque, including endothelial cells 5 and macrophages. 15 At this time, very little is known about the interactions of these cells with the protein, but these interactions are also likely to be important in mediating the injury response in vascular disease.
The results presented here show that vascular SMCs derived from mice with targeted deletion of type VIII collagen exhibit critical defects in migration and proliferation. Furthermore, the ability of these cells to express MMP-2 is reduced compared with WT SMCs, which produce type VIII collagen. This reduction in MMP-2 activity and migration over a type I collagen matrix was reversed with the addition of exogenous type VIII collagen. These studies suggest that SMCs are able to produce type VIII collagen and use it to overlay type I collagen, providing a provisional substrate favorable for migration. Thus, type VIII collagen may be an important mediator of SMC responses in vascular diseases that involve cell migration, including atherosclerosis, restenosis, vein graft, and transplant atherosclerosis.
Acknowledgments
This work was funded by grant MT-37847 from the Canadian Institutes of Health Research, grant B4009 from the Heart and Stroke Foundation of Ontario (to M.B.), and grant AR36820 from the National Institutes of Health (to B.O.). E.A. was supported by a Heart and Stroke/Richard Lewar Centre of Excellence studentship, a Heart and Stroke Foundation of Ontario Government Scholarship in Science and Technology, and a Premier?s Research Excellence Award. M.B. was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario. We thank Dr. Rama Khokha (University of Toronto) for providing conditioned media from MEFs.
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作者单位：Departments of Laboratory Medicine and Pathobiology and Medicine (E.A., G.H., D.M., M.B.), University of Toronto, Ontario, Canada; the Department of Medicine (U.H.), University of Hamburg, Germany; and the Department of Oral and Developmental Biology (N.F., B.O.), Harvard Medical School, Boston, Mas