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From the Department of Biochemistry (C.S.H., C.P.S., H.-Y. J., P.J.S., P.T., B.M.S., G.E.S.), Boston University School of Medicine, Boston, Mass; and the Departments of Surgery (E.D.R.) and Medicine (I.C.), The Mount Sinai Medical Center, New York, NY.
ABSTRACT
Objectives— The function of B-Myb, a negative regulator of vascular smooth muscle cell (SMC) matrix gene transcription, was analyzed in the vasculature.
Methods and Results— Mice were generated in which the human B-myb gene was driven by the basal cytomegalovirus promoter, and 3 founders were identified. Mice appeared to develop normally, and human B-myb was expressed in the aortas. Total B-Myb levels were elevated in aortas of adult transgenic versus wild-type (WT) animals and varied inversely with 1(I) collagen mRNA expression. However, neonatal WT and transgenic aortas displayed comparable levels of 1(I) collagen mRNA, likely resulting from elevated levels of cyclin A, which ablated repression by B-Myb. Aortic SMCs from adult transgenic animals displayed decreased 1(I) collagen mRNA levels. To examine the role of B-Myb after vascular injury, animals were subjected to femoral artery denudation, which induces SMC-rich lesion formation. A dramatic reduction in neointima formation and lumenal narrowing was observed in arteries of B-myb transgenic versus WT mice 4 weeks after injury.
Conclusions— Data indicate that B-Myb, which inhibits matrix gene expression in the adult vessel wall, reduces neointima formation after vascular injury.
To analyze B-Myb function in the vasculature, mice overexpressing B-myb were generated. Neonates displayed normal 1(I) collagen mRNA levels, whereas adults expressed decreased collagen mRNA in aortas and isolated vascular SMCs. On femoral artery denudation, neointima formation was dramatically reduced in B-myb transgenic mice.
Key Words: Myb ? collagen ? cyclin A ? aorta ? femoral artery
Introduction
Smooth muscle cells (SMCs), the major cellular constituents of the medial layer of an artery, are responsible for synthesis and deposition of connective tissue proteins (including types I and V collagen, elastin, and proteoglycans) during artery development.1 After the artery is formed, SMCs differentiate into a contractile phenotype.1 During atherosclerosis development, a response to vascular injury is elicited. After monocyte invasion, SMCs migrate to the intima and dedifferentiate to a synthetic phenotype, displaying modest rounds of proliferation followed by matrix synthesis.2,3 Deposition of matrix proteins, lipids, and minerals results in atherosclerotic plaque formation. Rupture of the fibrous cap of plaque with resultant exposure of thrombogenic subendothelial plaque constituents is the critical event that leads to thromboembolic complications in atherosclerotic coronary and carotid artery disease.2–4
Atherosclerotic lesions are frequently treated by balloon angioplasty and stent placement. However, reoccurrence of arterial narrowing at the site of balloon angioplasty, termed restenosis, occurs in 30% to 50% of patients.1 Acute disruption of the protective endothelial lining at the site of angioplasty appears to trigger excessive SMC hyperplastic responses,5 extracellular matrix deposition,3 and local vessel remodeling.6,7
Cultured vascular SMCs from adult animals exhibit predominantly a synthetic phenotype, expressing genes encoding types I, III, and V/XI collagen at confluence or when deprived of serum growth factors,8–10 whereas during exponential growth, only low levels of matrix proteins are produced. Furthermore, basic fibroblast growth factor (bFGF), a potent inducer of SMC proliferation, decreased 1(I) and 2(V) collagen gene expression at the transcriptional level in bovine SMCs.11 Overall, an inverse relationship exists between the proliferative state of the adult SMC and matrix synthesis. Recently, we implicated B-myb, a member of the myb gene family, in repression of matrix gene expression in vascular SMCs.12 The B-myb gene was isolated on the basis of its homology with c-myb in its DNA-binding region and encodes a 3.3- to 3.5-kb mRNA and 704-aa protein.12 The consensus Myb-binding site (MBS) is . B-Myb also regulates promoters without MBS sequences.13,14 B-myb expression is linked tightly with proliferation, with mRNA and protein levels increasing in late G1 and S phase.12,15 Although B-Myb promotes G1/S phase transition in some cells, it does not induce proliferation of bovine SMCs and fails to cooperate with c-Myc to promote entry into S phase, unlike c-Myb and A-Myb.12 B-Myb functions as either a repressor or an activator of transcription in a cell-type and promoter-specific fashion.12,13,16–19 We demonstrated that B-Myb is a strong negative regulator of MBS-driven reporter activity and matrix gene promoters in cultured adult vascular SMCs. B-Myb repressed 1(I) and 2(I) collagen promoter activity12 and decreased bFGF-induced type I collagen gene transcription.11 Whereas phosphorylation of B-Myb by cyclin A enhanced its ability to transactivate, cyclin A greatly reduced the ability of B-Myb to repress matrix gene expression.20 B-Myb also inhibited c-Myb-mediated transactivation of the 2(I) collagen promoter in scleroderma fibroblasts, 21 and repressed the 1(I) collagen gene via interaction with Sp1 and CCAAT-binding factor (CBF) factors.22 Here, the hypothesis that B-Myb regulates SMC matrix gene expression in vivo was tested. A mouse model was generated in which the human B-myb gene was driven by the basal cytomegalovirus (CMV) promoter, which has been shown to express most highly in cells that are infected by the virus, such as the SMC.23 Aortas of adult transgenic animals displayed decreased type I collagen mRNA expression. Unchallenged mice appeared to develop normally, apparently because of elevated cyclin A expression in developing animals, leading to comparable levels of 1(I) collagen mRNA in neonatal transgenic and wild-type (WT) mice. Isolated SMCs from adult animals displayed reduced 1(I) collagen gene expression. Interestingly, adult transgenic animals subjected to femoral artery injury showed a dramatic reduction in neointima formation and matrix deposition compared with WT mice.
Materials and Methods
A detailed Materials and Methods section is available online at http://atvb.ahajournals.org.
Results
Characterization of the CMV-B-myb Mouse
To generate a mouse model in which B-myb is overexpressed in aortic SMCs, a construct was used containing full-length human B-myb cDNA (pCEP4-B-myb) driven by the basal CMV promoter, which expresses most highly in cells normally infected by the virus, such as vascular SMCs.23 Data demonstrate that the clone used to generate transgenic mice repressed collagen promoter activity in cultured SMCs and that 3 founder lines (lines 2, 4, and 16) overexpressing B-Myb were generated (Figure 1A and Figure I, available online at http://atvb.ahajournals.org). Western blot analysis revealed that expression of B-Myb was greatest in line 16, followed by line 4 and then line 2 (Figure 1A; and data not shown).
Figure 1. The aorta of adult but not neonatal transgenic mice display reduced 1(I) collagen mRNA levels. A, Immunoblot analysis. Whole-cell protein extracts (30-μg samples) from individual aortas of 6-week-old WT (n=3) and transgenic line 16 mice (n=5) prepared in radioimmunoprecipitation assay buffer were subjected to immunoblot analysis for levels of B-Myb (top) and -actin as a loading control (bottom). B, Northern blot analysis. Total RNA (12-μg samples), extracted from pooled (7–12) adult WT or transgenic line 2, 4, or 16 mouse aortas, was subjected to Northern blot analysis for 1(I) collagen and GAPDH mRNA levels. Densitometry was performed, and 1(I) collagen values were normalized to GAPDH. Values for the percentage relative to the WT signal are given below each lane. C, Quadruplicate experiments were analyzed as in B; data represent the mean±SD. Data were compared with WT by one group Student t test; asterisk indicates a statistically significant difference (P<0.05). D, Graphic representation of the expression of B-Myb protein versus 1(I) collagen mRNA. E, Total RNA was extracted from a pool of aortas (7–12) from neonatal WT or line 16 transgenic mice (6 days old) and samples (12 μg) subjected to Northern blot analysis for levels of 1(I) collagen and GAPDH mRNA.
Type I Collagen mRNA Expression, But Not Protein Deposition, Is Downregulated in Adult Transgenic Mouse Aortas
To compare 1(I) collagen steady-state mRNA levels in aortas of WT and B-myb mice, RNA was isolated from 7 to 12 pooled aortas of 6- to 10-week-old mice, and Northern blot analysis was performed (Figure 1B). Two bands of 1(I) collagen were detected resulting from alternative polyadenylation,24 as seen previously.9 To test for RNA integrity and equal loading, the blot was hybridized to a glyceraldehyde phosphate dehydrogenase (GAPDH) probe (Figure 1B), and some minor variability was seen. Densitometry was performed on 1(I) collagen and GAPDH mRNA levels, and normalized values are reported as a percentage of the WT value (Figure 1B). The 1(I) collagen mRNA expression levels in lines 4 and 16 were 77.0% and 50.4% relative to the WT, respectively, whereas in line 2, levels were essentially identical to those in the WT mice. The average of 4 independent experiments show that lines 2, 4, and 16 displayed 69.0% (±31.2%), 51.4% (±18.1%), and 46.5% (±17.5%) of WT levels, respectively (Figure 1C). These data indicate that lines 4 and 16 display a statistically significant difference in 1(I) collagen mRNA expression versus WT mice. When values for collagen mRNA were plotted against levels of B-Myb protein (Figure 1D), an inverse correlation was observed, consistent with the hypothesis that B-Myb represses expression of the COL1A1 gene within the aorta in vivo.
Deposition of fibrillar collagen in aortas of B-myb versus WT mice was examined. The adventitia was removed from 4 aortas per line, and samples were lyophilized, hydrolyzed, and subjected to amino acid analysis to determine the levels of insoluble collagen. Lines 2, 4, and 16 displayed 117.8% (±4.1%), 96.9% (±14.0%), and 109.3% (±13.9%) of WT levels, respectively. Thus, no significant differences in collagen deposition in aortas of WT and transgenic lines are observed.
Neonatal Transgenic and WT Mice Display Elevated Levels of Cyclin A and Comparable Collagen Gene Expression
Because vessel wall synthesis occurs early in development,25–27 collagen expression in neonatal animals was assessed. RNA was isolated from pooled aortas of neonatal (6 days old) WT and line 16 mice, and Northern blot analysis was performed (Figure 1E). Levels of collagen mRNA normalized to GAPDH were no less in transgenic compared with WT animals (0.68 versus 0.5 for line 16 and WT, respectively), in contrast to the decreased expression in aortas of adult animals (Figure 1B). RT-PCR analysis confirmed overexpression of human B-myb in neonatal transgenic animals (data not shown). Thus, overexpression of B-myb does not reduce 1(I) collagen gene expression in neonatal mice.
We showed recently that the ability of B-Myb to function as a repressor of type V collagen promoter activity is abolished by cyclin A-regulated phosphorylation,20 so it was of interest to evaluate cyclin A expression in neonatal versus adult mouse aortas. The murine cyclin A subtype corresponding to human cyclin A is known as cyclin A2. To compare cyclin A2 levels in neonates and adults, RNA was isolated and Northern analysis performed using human cyclin A as probe (Figure 2A). Two bands that arise from alternative polyadenylation were detected.28 Although cyclin A2 expression was substantial in both WT and transgenic neonatal mice, levels were very low in aortas of adult animals. The ability of cyclin A to alleviate B-Myb-mediated repression of the 1(I) collagen promoter was assessed using transient transfection analysis (Figure 2B). Bovine aortic SMCs were cotransfected with ColCAT3.6, a chloramphenicol acetyltransferase (CAT) reporter construct driven by a 3.5-kb upstream sequence of the human 1(I) promoter and 115 bp of the first exon, in the absence or presence of vectors expressing B-myb and cyclin A. B-Myb repressed 1(I) collagen-promoter activity to 8.3% of the control, and addition of 0.5 and 1 μg cyclin A re-established 79.6% and 105.1% of control activity, respectively. Thus, cyclin A alleviates B-myb-mediated repression of the 1(I) collagen promoter, similar to its effects on the 2(V) promoter.20 Because the bulk of matrix synthesis occurs early during development,25–27 the presence of cyclin A likely explains, at least in part, why no significant differences were observed in collagen deposition in the vessel wall of transgenic animals.
Figure 2. Effects of cyclin A and animal age on collagen gene expression. A, Cyclin A mRNA is expressed at higher levels in neonatal vs adult mice. Total RNA was extracted from pooled aortas (7–12) from neonatal (7 days old) or adult (7 to 8 weeks old) WT and line 16 transgenic mice and samples (20 μg) subjected to Northern blot analysis for cyclin A and GAPDH mRNA levels. B, Cyclin A ablates repression of the 1(I) collagen promoter by B-Myb. Bovine aortic SMC cultures, plated in triplicate at a density of 6x104 cells per well in a 6-well dish, were cotransfected with 0.6 μg ColCAT 3.6 in the absence or presence of 1 μg pB14 and 0.5 or 1 μg of human cyclin A expression vector and pBluescript (to make up a total of 2.6 μg DNA) using Lipofectamine reagent. After 72 hours, CAT activity, normalized to total protein, was determined. C, Cultured aortic SMCs from adult transgenic mice display reduced 1(I) collagen mRNA levels. Left, Radiolabeled RT-PCR analysis. Total RNA (5-μg sample) extracted from aortic SMCs isolated from adult WT or transgenic mice was DNase treated and subjected to radiolabeled RT-PCR in the absence (–) or presence (+) of RT for human B-myb (hB-myb) and ?-actin. Right, Northern blot analysis. Total RNA was extracted from aortic SMCs isolated from adult WT or transgenic mice and samples (6 μg) subjected to Northern blot analysis for mRNA levels of 1(I) collagen, B-myb, and GAPDH.
Isolated Vascular SMCs From Adult Animals Display Decreased Collagen Gene Expression
To determine whether collagen expression was downregulated in cultured aortic SMCs from adult transgenic animals, total RNA was isolated from aortic SMCs from 12-week-old WT and line 16 mice in exponential growth. Human B-myb expression was confirmed by RT-PCR analysis using the transgene specific primer pair (Figure 2C). In the presence of RT, a band corresponding to human B-myb was amplified selectively with RNA isolated from line 16, but not from WT mice, as expected. Analysis of ?-actin confirmed integrity of the RNA samples. To determine the effects on collagen mRNA levels, RNA isolated from subconfluent cultures was subjected to Northern blot analysis for B-myb, 1(I) collagen, and GAPDH (Figure 2C). B-myb mRNA levels were higher (4.3-fold after normalization to GAPDH) in the vascular SMCs from the transgenic mice, whereas 1(I) collagen gene expression showed a decrease to 65.6% of WT levels. A comparable decrease was seen in synthesis of type I collagen during an 8-hour labeling period with 14C-proline (data not shown). Thus, aortic SMCs from transgenic animals display reduced type I collagen gene expression.
B-Myb Protects Against Neointima Formation After Femoral Artery Injury
Although B-myb overexpression did not appear to affect normal vascular development, it was of interest to evaluate whether it might alter vascular remodeling or neointima formation after injury to the developed vasculature. A model of guidewire-induced denudation of the femoral artery was used. Northern blot analysis confirmed the decrease in 1(I) collagen mRNA levels in femoral artery RNA isolated from pools of 10 to 12 line 16 versus WT adult animals (46.9% normalized to GAPDH; data not shown). The response to injury was compared in femoral arteries of WT versus B-myb transgenic (line 16) mice (n=9). Four weeks after bilateral injury, injured and sham-operated arteries were harvested, fixed, and 5-μm sections were made throughout the entire guidewire-injured region of each artery. Every twentieth section was stained with Masson’s trichrome, and the images were quantified using Image-Pro Plus software. As shown previously,29 the typical lesion of a WT mouse displayed a large neointima rich in matrix (blue-green color) and SMCs (red color); however, these large lesions were not apparent in B-myb mice (Figure 3). The lesions with the largest neointima/media in each group are depicted in Figure 3. As expected, no lesion formation was evident in sham-injured arteries.30
Figure 3. Neointima formation is inhibited in B-myb transgenic mice. Four weeks after endothelial denudation or sham surgery of the mouse femoral artery, 5 μm sections of the fixed arteries were stained with Masson’s trichrome. Top, Injured WT arteries. Middle, Injured B-myb line 16 arteries. Bottom, Sham-injured WT (left) and B-myb line 16 (right) arteries. Blue-green color, indicates extracellular matrix; red color, SMC; black color, elastin. Arrowheads mark the internal elastic lamina.
Image analysis of the lesions demonstrated that compared with WT mice, B-myb mice displayed a significant decrease in the area of the neointima (Table). Similarly, the ratio of the areas of the neointima and the medial layers and the percentage of lumenal narrowing were significantly lowered in the B-myb mice (Table; Figure 4). Some of the lesions were extremely small, and if one took a value of >0.5 to indicate a substantial neointima/media ratio, then 8 of the 9 injured arteries from the WT mice developed significant lesions, whereas only 2 of the 9 B-myb mice did so. The data also show that there was no significant difference between the areas of the entire vessel, lumen, or medial layers of the femoral arteries in WT versus B-myb transgenic mice (Table). Likewise, the outer perimeters of the vessels (ie, the length of the external elastic laminae) were not significantly different in injured arteries of the 2 mouse lines. Finally, the lesions were rich in SMCs and negative for monocyte/macrophage invasion as judged by immunohistochemistry for -actin, and MOMA-2, respectively (Figure II, available online at http://atvb.ahajournals.org). Thus, circulating monocytes are not likely to be responsible for the observed inhibition of lesion formation in B-myb transgenic mice.
Quantitative Analysis of Injured Femoral Arteries
Figure 4. The formation of a neointima and luminal narrowing are reduced in B-myb transgenic mice. Averages of the ratio of the areas of neointima and media (, left axis) and percentage of lumenal narrowing (, right axis; n=9) are plotted.
Discussion
Using a transgenic mouse model in which the human B-myb cDNA was driven by the basal CMV promoter, this study demonstrates for the first time that B-Myb leads to markedly reduced neointima formation after mechanical injury to the vasculature and to decreased 1(I) collagen mRNA expression in the aorta and femoral artery of adult animals. Three independent transgenic mouse lines were generated, all of which apparently developed and bred normally. An inverse relationship between levels of B-Myb protein and expression of 1(I) collagen mRNA in the adult aorta was demonstrated. A decrease was also seen in 2(V) collagen mRNA levels in the aorta and in cultured aortic SMCs isolated from adult transgenic B-myb mice (data not shown). Importantly, when a femoral artery model of endothelial denudation was used, a dramatic reduction in neointima formation was observed in arteries of transgenic versus WT mice 4 weeks after injury. After injury, the neointimal area and the ratio of the areas of the neointima and media were significantly reduced in transgenic animals to 12.7% and 15.4%, respectively, of the levels observed in WT injured mice. As expected, the lesions were rich in SMCs, whereas invading monocytes/macrophages were not detected. Furthermore, Masson’s trichrome staining was consistent with the lack of lesion formation and deposition of collagen and other matrix proteins. Thus, B-myb overexpression inhibits neointima formation after vascular injury.
Dysfunction of adult vessel endothelium causes a phenotypic switch in the normally quiescent, contractile SMCs. Mitogenic signals can promote this switch to the synthetic phenotype and migration to the intima, where the cells reinitiate production of matrix proteins. Microarray analysis of neointimal SMCs in primate bypass graft neointima showed that 6 of the 13 genes expressed more highly in the neointima than in the aorta encoded collagens, suggesting the importance of transcriptional regulation of collagen genes in formation of a collagenous lesion within the neointima.31 Interestingly, there is evidence that type I collagen promotes SMC migration in vitro.32,33 De novo synthesis of collagen has been shown to occur as an early response to injury,3 and may be required for normal migration of SMCs during arterial remodeling. After injury in transgenic mice, inhibition of type I collagen gene expression could affect 2 critical steps in the response to injury: migration and collagen deposition. Our studies do not exclude the possibility that B-Myb-mediated effects on other genes may also play a role in the observed inhibition of neointima formation (including matrix synthesis or degrading enzymes that might alter the matrix metalloproteinase/tissue inhibitor of metalloproteinase balance). A phenotype similar to the B-myb mouse was seen after overexpression of MMP 1, which has the capacity to degrade newly synthesized collagen.34 Thus, injury of the vessel wall in a situation in which there is reduced collagen synthesis or increased collagen degradation protects against lesion formation.
No differences in the vessel structure were observed in the unchallenged animal, and no change in collagen protein levels in the adult vessel wall was noted. Cyclin A levels were higher in aortas of the neonatal versus adult mice, and cyclin A reversed the B-Myb-induced downregulation of the 1(I) collagen promoter (as shown previously for 2(V) collagen).20 This suggests that elevated cyclin A expression during development prevents B-Myb-mediated repression of collagen gene expression. In addition, other post-translational modifications, coactivators, and corepressors that affect B-Myb may also be subject to developmental regulation.14,35–37 Recent work by Baskar et al using the lacZ reporter gene showed that CMV-driven expression is not as ubiquitous as originally thought. In developing embryos38 and in adult animals,23 high levels of CMV-driven lacZ expression were noted specifically in tissues that are naturally targeted and infected by CMV in humans (eg, vascular SMCs). In contrast, lacZ expression was not detectable in adult endothelial cells or in blood-borne cells (including peritoneal macrophages). Our data, which show that expression of B-myb is evident in adult aortas and isolated vascular SMCs of the transgenic mice, are in agreement with these observations and suggest that B-Myb overexpression in SMCs contributed to inhibition of lesion formation in transgenic mice. The guidewire-induced femoral artery injury in the FVB mouse produces an SMC-rich lesion without extensive monocyte/macrophage infiltration.29 Because the injury is performed on unaffected vessels, it does not recapitulate exactly human restenosis, which occurs after balloon angioplasty or stenting of an atherosclerotic lesion; however, it may provide information on the SMC response to vascular injury. Overall, our findings suggest that vascular SMC expression of B-Myb protects against the formation of a neointima in response to vascular injury.
Acknowledgments
This work was supported by grants from the National Institutes of Health (PO1 HL13262; B.M.S., G.E.S.) and the American Heart Association (0256215T; B.M.S.).We thank K. Ravid for generously providing cloned DNA, and V. Verbitski for excellent technical assistance. We thank R. Romieu-Mourez for comments on this study and B. Smith for help with Image Pro analysis.
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