Increased Neointima Formation in Cysteine-Rich Protein 2eCDeficient Mice in Response to Vascular Injury
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循环研究杂志 2005年第12期
the Pulmonary and Critical Care (J.W., X.L., B.I., A.T., M.D.L., S.-F.Y.) and Cardiovascular (Z.C., D.I.S.) Divisions, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass
the Division of Newborn Medicine (T.E.G.), Children’s Hospital, Boston, Mass.
Abstract
In response to arterial injury, medial vascular smooth muscle cells (VSMCs) proliferate and migrate into the intima, contributing to the development of occlusive vascular disease. The LIM protein cysteine-rich protein (CRP) 2 associates with the actin cytoskeleton and may maintain the cytoarchitecture. CRP2 also interacts with transcription factors in the nucleus to mediate SMC gene expression. To test the hypothesis that CRP2 may be an important regulator of vascular development or function we generated Csrp2 (gene symbol of the mouse CRP2 gene)-deficient (Csrp2eC/eC) mice by targeted mutation. Csrp2eC/eC mice did not have any gross vascular defects or altered expression levels of SM -actin, SM22, or calponin. Following femoral artery injury, CRP2 expression persisted in the vessel wall at 4 days and then decreased by 14 days. Intimal thickening was enhanced 3.4-fold in Csrp2eC/eC compared with wild-type (WT) mice 14 days following injury. Cellular proliferation was similar between WT and Csrp2eC/eC VSMC both in vivo and in vitro. Interestingly, Csrp2eC/eC VSMC migrated more rapidly in response to PDGF-BB and had increased Rac1 activation. Our data demonstrate that CRP2 is not required for vascular development. However, an absence of CRP2 enhanced VSMC migration and increased neointima formation following arterial injury.
Key Words: arterial wire injury vascular smooth muscle cells migration
Introduction
During blood vessel development, the vascular smooth muscle cell (VSMC) component arises from both neural crest and mesodermal origins. These cells proliferate at a high rate and synthesize extracellular matrix molecules that contribute to the structure and function of the blood vessel.1,2 As VSMC mature, they proliferate at a low rate and exhibit a differentiated contractile phenotype.3 Adult VSMCs are not terminally differentiated: in response to vessel injury, VSMCs within the vessel wall dedifferentiate and change from a quiescent and contractile phenotype to a proliferative and synthetic phenotype reminiscent of embryonic precursors.3eC5 The migration and proliferation of VSMCs from the media into the intima contribute to arterial intima thickening and subsequent arteriosclerosis.6,7 Arteriosclerosis and its complications, including heart attack and stroke, are major causes of death.4,8 Despite its importance, the molecular mechanisms that control VSMC development and differentiation and the phenotypic modulation of VSMC in vascular injury have not been elucidated completely.
The LIM protein family is characterized by a double zinc-finger structure that serves as a protein interaction module.9,10 Through binding of target proteins and assembly of multiprotein complexes, LIM proteins function in diverse biological processes.11eC13 The LIM-only cysteine-rich protein (CRP1eC3) family contains two tandem LIM domains, each followed by a short glycine-rich repeat.14eC19 CRP1 is expressed in most cell types.15,20 CRP2 is expressed primarily in arterial but not in venous smooth muscle cells (SMCs),18,21,22 and CRP3 is expressed only in striated muscle.14,23
CRPs associate with the actin cytoskeleton via interacting with the actineCcross-linking protein -actinin and the adhesion plaque protein zyxin.19,24 Gene deletion studies in mice revealed that CRP3 is essential in maintaining cardiomyocyte cytoarchitecture.14,23 Given the potential overlapping cellular functions of CRPs and their expression in different muscle types, CRP1 and CRP2 may maintain the cytoarchitecture of SMC and influence tissue development and cellular differentiation.19 In addition to their prominent association with cytoskeleton, CRPs also localize to the nucleus.9,18 CRP3 interacts with muscle-specific transcription factor MyoD and can promote myogenesis.25 Interestingly, Chang et al reported that CRP1 and CRP2 form complexes with serum response factor and GATA transcription factors, facilitating the expression of some smooth muscle (SM) marker genes.26
Through participation in multi-protein complexes, CRPs have important regulatory roles.19,25,26 However, nothing is known about the requirement for CRP2 in the vasculature. To gain insight into the biological functions of CRP2 in vivo, we generated Csrp2-deficient mice by targeted mutation.
Materials and Methods
Detailed methods are described in the expanded Materials and Methods in the data supplement, available online at http://circres.ahajournals.org.
Generation of Cysteine-Rich Protein 2eCDeficient Mice
We generated Csrp2 (gene symbol for the mouse cysteine-rich protein gene)-deficient (Csrp2eC/eC) mice by gene targeting. Mice were backcrossed 10 generations and fixed on a C57BL/6 background.
Antibody Production
We generated two CRP2-specific antibodies against amino acids 91 to 98 and 93 to 108, respectively.
Southern, Northern, and Western Blot Analysis
Southern analysis was performed using BglII-digested mouse genomic DNA. Total RNA isolated from mouse aorta was analyzed by Northern analysis. Protein extracts from adventitia-stripped aorta was analyzed by Western analysis with CRP2(91eC98) antiserum and a monoclonal SM -actin antibody (Sigma).
Blood Pressure Measurements
A tail-cuff method was used to measure systolic blood pressure of adult male conscious mice.27
Femoral Artery Injury
Endoluminal injury to the mouse left common femoral artery was performed as described.28
Histological Analysis and Immunohistochemistry
Femoral arteries were harvested for histological and morphometric analyses. Vessel sections were stained for elastin (Sigma) and the intimal and medial areas were measured using NIH Image software.
En Face VSMC Migration Assay
Four days after injury, en face VSMC migration was measured in femoral arteries.29 The total number of migrated VSMCs in the injured vessel was counted.
Proliferation and Migration Assays
We performed [methyl-3H]-thymidine incorporation assays to assess proliferation. To assess migration, serum-starved cells were placed in the upper chamber of transwell plates and the bottom chambers were filled with 0.5% FBS medium containing platelet-derived growth factor-BB.
Assessment of ERK1/2, Akt, and Rac1 Activation
Mouse aortic smooth muscle cells were serum-starved and then stimulated with 10 ng/mL PDGF-BB. ERK1/2 and Akt activation was assessed by Western blot using antibodies (Cell Signaling Technology) for phospho-ERK1/2, total ERK1/2, phospho-Akt (Ser473), and total Akt. We assessed Rac1 activation by binding GTP-bound Rac1 to p21-binding domain of PAK-1 using a Rac1 activation assay kit.
Statistical Analysis
Data are presented as mean±SEM. Statistical significance was determined by Student t test. P0.05 was accepted as statistically significant.
Results
Generation of Csrp2eC/eC Mice
To examine the role of CRP2, we targeted the Csrp2 locus in mice by homologous recombination. The N-terminal LIM domain of CRP2 mediates interactions with its binding partners.26,30 Thus, we constructed a targeting vector to disrupt the N-terminal LIM domain (Figure 1A). Germ line transmission of the mutation and generation of Csrp2 heterozygous mice was demonstrated by Southern blot analysis with a 5'-external probe (Figure 1B, left panel, middle lane) or 3'-external probe (data not shown). Heterozygous mice, which were viable and fertile, were intercrossed to generate homozygous mutant mice (Figure 1B, left panel, right lane). Additionally, a neo probe hybridized to a single 11-kb mutated fragment in heterozygous and homozygous mutant but not in wild-type (WT) mouse genomic DNA (Figure 1B, middle panel), demonstrating that no additional integrations were present in the mutant mice. Southern analysis using deleted exon 3 as a probe revealed that a 4.8-kb WT fragment was present only in WT and heterozygous but not in Csrp2eC/eC mouse genomic DNA (Figure 1B, right panel), further demonstrating that we have deleted exon 3 and disrupted the Csrp2 allele.
Northern blot analysis with aortic RNA from WT, heterozygous, and homozygous mutant mice using exon 3 as a probe demonstrated that CRP2 message was absent in homozygous mutants (Figure 1C). To evaluate whether aberrant transcripts exist in the mutant mice, we performed RT-PCR using an upper and lower primer from exon 1 and exon 6, respectively. We were able to detect a shorter (0.83 kb) transcript. Sequence analysis revealed this transcript was produced by splicing of exon 2 to exon 4 and downstream exons. If the truncated transcript were translated, it would not encode any LIM domains due to a frame shift. By Western blot analysis, CRP2 was not detectable in protein isolated from the aorta of homozygous mutant mice (Figure 1D). In contrast, the SMC marker gene SM -actin expression was similar among the three genotypes (Figure 1D).
Arteries in Csrp2eC/eC Mice Are Structurally and Functionally Normal
Csrp2eC/eC mice were born alive at the expected Mendelian ratio, without any apparent abnormality. Adult Csrp2eC/eC mice were fertile and appeared grossly normal. To analyze the vasculature in greater detail, vessels were fixed at constant pressure and isolated from WT and Csrp2eC/eC mice. Immunostaining with the CRP2(93eC108) antiserum demonstrated strong CRP2 expression in the medial SM layers of WT aorta (Figure 2A) but not that from Csrp2eC/eC mice (Figure 2B). To evaluate the structure of the vessel, Verhoeff’s elastin stain was used to delineate the elastic lamina. At a similar anatomic level of the descending aorta, WT and Csrp2eC/eC mice exhibited a similar number of elastic and SM layers (Figure 2C and 2D, respectively). SM -actin expression was similar in the aortic SM layers from both WT (Figure 2E) and Csrp2eC/eC mice (Figure 2F).
Because one of the primary functions of VSMC is to regulate vascular tone, we examined whether an absence of CRP2 altered blood pressure. Systolic blood pressure was not statistically different between WT (111±3 mm Hg, n=7) and Csrp2eC/eC mice (115±2 mm Hg, n=8; P=0.30).
Several Characteristic SM Marker Genes Are Not Altered in the Absence of CRP2
A previous study has shown that overexpression of CRP2 results in translocation of the protein to the nucleus where it functions as a potent transcriptional coactivator with serum response factor and GATA to facilitate SMC-specific gene expression.26 Therefore, we wanted to test the hypothesis that SMC marker genes would be reduced in the absence of CRP2. CRP2 mRNA was not detectable in the aorta from Csrp2eC/eC mice (Figure 3). Surprisingly, the levels of calponin and SM22, whose expression is dependent on functional serum response factor complexes,31eC33 were not altered in the absence of CRP2 expression (Figure 3). Potentially this result could be explained by a compensatory upregulation of other CRP family members. Northern analysis revealed that CRP1 expression levels were not different between WT and Csrp2eC/eC mice (Figure 3). Additionally, CRP3 remained undetectable in the aorta from both WT and Csrp2eC/eC mice (Figure 3).
CRP2 Expression in the Arterial Wall After Vascular Injury
In response to vessel wall injury, SMCs undergo a phenotypic change and alter their gene expression patterns and their proliferative and migratory behavior.3eC5 To investigate the potential role of CRP2 in vascular injury, we first examined the temporal expression of CRP2 after femoral artery wire injury in WT mice. Verhoeff’s elastin stain of control femoral arteries revealed the endothelial layer (Figure 4A) abutting the internal elastic lamina (IEL) at the luminal surface (Figure 4A) and medial SM layers beneath the IEL (Figure 4A). Strong CRP2 expression was detected in the medial SM layers but not in adventitial fibroblasts or endothelial cells (Figure 4B), which was delineated with the endothelial cell marker PECAM-1 (Figure 4C). Four days after injury, CRP2 expression remained detectable in the medial layers (Figure 4D). Additionally, although very few cells were present in the intima, CRP2 expression was detectable (Figure 4D). Fourteen days after injury, CRP2 was present in some but not all medial (73.4±10.2%, n=4) and intimal (58.2±8.7%, n=4) areas (Figure 4E). No CRP2 staining was observed in adventitial fibroblasts (Figure 4E) or endothelial cells (Figure 4E and 4F). These data indicate that following wire injury CRP2 expression persisted in the first 4 days and decreased but was not abolished in the vessel wall by 14 days.
Absence of CRP2 Increases Neointima Formation in Response to Vascular Injury
Under basal conditions, SMC gene expression and blood vessel morphology were similar in WT and Csrp2eC/eC mice. Given that CRP2 was expressed initially after injury and decreased but not diminished by 14 days, we hypothesized that an absence of CRP2 might influence neointima formation after injury. Fourteen days after wire injury, vessel size (area inside external elastic lamina [EEL]) of the injured femoral arteries was not different between WT (40500±2406 e2, n=12) and Csrp2eC/eC mice (39993±2654 e2, n=11; P=0.44). The medial areas were also similar between WT (11441±511, n=12) and Csrp2eC/eC mice (10243±1032, n=11; P=0.16). In WT mice, intimal thickening was evident, although small (3897±912 e2 or 97±15 cells, n=12), 14 days after injury (Figure 5A and 5C). In contrast, there was a robust 3.4-fold increase in intimal thickening in Csrp2eC/eC mice (13438±2905 e2 or 311±43 cells, n=11; P<0.05) (Figure 5B and 5C). An absence of CRP2 increased the intima/media ratio &4-fold to 1.58±0.40, compared with 0.36±0.10 of WT mice (Figure 5D, P<0.05). Because endothelial regeneration after arterial injury affects neointima formation, we measured the extent of endothelial regeneration by PECAM-1 staining of arteries 14 days after wire injury. At this time point injured arteries have &50% endothelial coverage as reported previously.28 The endothelial regeneration was similar between WT (61.7±21%, n=5) and Csrp2eC/eC mice (61.8±12.2%, n=5; P=0.50 versus WT), suggesting that the increased neointima formation in Csrp2eC/eC injured arteries was not likely due to alterations in endothelial regeneration.
Characterization of the neointima revealed that WT (n=8) and Csrp2eC/eC (n=8) neointima had similar cell densities (19.6±0.5 and 20.3±1.7 nuclei/1000 e2, P=0.34). Interestingly, in contrast to the barely detectable SM -actin staining in the WT neointima (14.2±6.2%, n=6) (Figure 6A), Csrp2eC/eC neointima were composed mainly of SM -actin positive cells (63.3±10.6%, n=3; P<0.05 versus WT) (Figure 6B). Very few CD45 positive inflammatory cells were present in either WT (0.8±0.4%, n=3) (Figure 6C) or Csrp2eC/eC (0.5±0.3%, n=3; P=0.56 versus WT) (Figure 6D) intima. Proliferation and migration contribute to neointima formation in the injured vessel wall. To assess cellular proliferation, we quantified incorporation of BrdUrd in the arteries 14 days after injury. Proliferation was evident in WT vessels (n=5) (Figure 6E) with 10.2±4.6% BrdUrd incorporation in the neointima and 5.4±1.6% in the media. In Csrp2eC/eC mice (n=9) (Figure 6F), we observed 7.1±2.7% of BrdUrd incorporation in the neointima (P=0.63 versus WT) and 6.0±1.9% in the media (P=0.82 versus WT), suggesting similar cellular proliferation in WT and Csrp2eC/eC mice. TUNEL staining revealed that very few apoptotic cells were observed in the injured vessels (Figure 6G and 6H). WT had even lower apoptosis (0.6±0.6%, n=4) than Csrp2eC/eC (2.8±0.6%, n=7; P<0.05) vessels, indicating that the increased neointima in Csrp2eC/eC mice was not due to decreased apoptosis in Csrp2eC/eC mice.
CRP2 Deficiency Promotes VSMC Migration But Not Proliferation
To investigate potential mechanisms by which an absence of CRP2 leads to increased neointima formation in response to vascular injury, we isolated VSMC from 18.5 dpc embryos of WT and Csrp2eC/eC mice and assessed their proliferation in vitro. PDGF-BB dose-dependently increased 3H-thymidine incorporation in both WT and Csrp2eC/eC VSMC (Figure 7A). The increase in 3H-thymidine incorporation by 10 ng/mL PDGF-BB was comparable to 20% fetal bovine serum (Figure 7A). Consistent with in vivo findings (Figure 6E and 6F), WT and Csrp2eC/eC VSMC exhibited similar increases in 3H-thymidine incorporation (Figure 7A). PDGF-BB induced similar degree of ERK1/2 and Akt phosphorylation (Figure 7B), suggesting the signaling pathways linked to cellular proliferation stimulated by PDGF-BB were normal in Csrp2eC/eC VSMC.
In addition to proliferation, SMC migration contributes to the development of the neointima after injury.3,6,7 Given that the cellular proliferation was similar between WT and Csrp2eC/eC mice both in vivo and in vitro, we hypothesized Csrp2eC/eC VSMC would have altered migratory behavior. PDGF-BB is a potent chemoattractant for VSMC and is released at sites of vessel injury.6,7,34 We measured the migratory responses of WT and Csrp2eC/eC VSMC toward the chemoattractant PDGF-BB. Two hours after stimulation, PDGF-BB at 0.1 or 1 ng/mL minimally stimulated cell migration (Figure 7C), whereas 10 ng/mL significantly stimulated cell migration (Figure 7C). Interestingly, 1 hour after stimulation with PDGF-BB (10 ng/mL), Csrp2eC/eC VSMC showed increased migration (1.1±0.1%) compared with WT cells (0.6±0.1%, P<0.05) (Figure 7D). Two hours after stimulation, 11.0±1.7% of Csrp2eC/eC cells had migrated through the filters. In contrast, only 5.5±0.1% of WT cells migrated toward PDGF (Figure 7D, P<0.05). At 3 hours after stimulation, migrated Csrp2eC/eC cells increased to 18.0±2.1%, whereas only 12.0±0.6% of WT cells migrated (Figure 7D, P<0.05). These results indicate that in the absence of CRP2, VSMC migrate more rapidly in response to PDGF-BB. The Rho GTPases play key roles in regulating cell migration.35 In particular, Rac1 regulates VSMC migration in response to factors released at sites of vessel injury.36 Interestingly, in response to PDGF-BB stimulation Rac1 activation was enhanced in the Csrp2eC/eC VSMC compared with WT cells despite similar total Rac1 levels (Figure 7E), correlating with increased migration rate.
To provide additional evidence that the increased neointima formation in Csrp2eC/eC mice may be due to increased migration of medial VSMC into intima, we examined neointima formation 4 days after wire injury by two independent methods: (1) en face migration assays29 and (2) histological analysis of paraffin cross-sections. The cells observed in the neointima at the 4-day time point might reflect migrated rather than proliferating cells.6,7 En face preparations revealed that compared with WT mice (Figure 8A), more cells migrated through IEL onto the luminal surface in Csrp2eC/eC mice (Figure 8B). In WT mice, 144±9 cells (n=5) migrated onto the injured luminal surface, whereas in Csrp2eC/eC mice 326±52 cells (n=4; P<0.05 versus WT) migrated (Figure 8E). We also assessed the neointima formation by counting the number of nuclei between the lumen and IEL from histological sections. Consistent with the en face results, there were fewer neointimal cells in WT mice (6.1±1.2 nuclei/section, n=11) (Figure 8C and 8F). A 2.3-fold increase of neointimal cells was observed in Csrp2eC/eC mice (13.7±1.5 nuclei/section, n=6; P<0.05 versus WT) (Figure 8D and 8F).
Discussion
To examine the function of CRP2 in vivo, we generated Csrp2eC/eC mice by targeted mutation. The LIM domains of CRP2 mediate interactions with its binding partners including zyxin, -actinin, and CRP2BP.12,19,30 Thus, we targeted the first LIM domain by disrupting exon 3, the largest coding exon. No message was detected in Csrp2eC/eC mouse RNA when exon 3 was used as a probe, although a smaller transcript was detected by RT-PCR. Nevertheless, no CRP2 was detected in protein isolated from Csrp2eC/eC mouse aorta by Western blot analysis using CRP2(91eC98) antiserum. Furthermore, immunostaining of aortic sections with CRP2(93eC108) antiserum did not detect CRP2 expression in Csrp2eC/eC mice. We cannot exclude the possibility that a truncated protein could be generated because the two antisera used in this study were against epitopes C-terminal to those encoded by exon 2. However, even if the truncated transcript were translated, it would not encode either LIM domain due to a frame shift. Therefore, we believe that the observed phenotype in the Csrp2eC/eC mice is a result of the absence of a functional CRP2 protein.
Our finding that Csrp2eC/eC mice do not have apparent developmental vascular defects was unexpected. Chang et al26 proposed that in progenitor proepicardial cells CRP2 might function as a transcriptional coactivator to facilitate smooth muscle differentiation and the specification of the smooth muscle lineage. It is possible that CRP1, which is also expressed in VSMCs (Figure 3), may functionally compensate for the absence of CRP2 expression. Furthermore, the expression levels of SMC marker genes SM -actin (Figure 2), calponin, and SM22 (Figure 3) were not changed, indicating that a CRP2 expression is not required for the transcriptional regulation of these genes in the mouse aorta. Mice that are deficient in both CRP1 and CRP2 may be needed to address these questions.
Vascular injury downregulated, but did not abolish, CRP2 expression in blood vessels (Figure 4), suggesting CRP2 may be required to maintain VSMC in the quiescent and differentiated state. Alternatively, an absence of CRP2 may render cells more primed to the migratory phenotype in response to arterial injury. Consistent with this hypothesis, one major finding of our current study was that Csrp2eC/eC mice developed larger neointima than WT mice in a femoral artery wire injury model (Figure 5), whereas endothelial regeneration was similar between WT and Csrp2eC/eC vessels. Furthermore, serum cholesterol levels and peripheral leukocyte counts, factors that may potentially affect neointimal thickening, were not different between WT and Csrp2eC/eC mice before and 4 days after vascular injury (data not shown). Additionally, cellular proliferation was similar between WT and Csrp2eC/eC VSMC both in vivo (Figure 6) and in vitro (Figure 7A). Migration of medial SMC into intima also contributes to neointima formation.3,6,7 Therefore, one possible mechanism for increased neointima formation is that medial SMC of Csrp2eC/eC mice migrate into intima at a faster rate than WT mice. Indeed, we found that Csrp2eC/eC VSMC migrate toward PDGF-BB, a key chemoattractant in vascular injury,6,7,34 at a faster rate than WT cells in vitro (Figure 7D). Further supporting this concept, we found 2.3-fold more neointimal cells in Csrp2eC/eC than WT vessels 4 days after injury assessed either by en face migration assays or histological sections (Figure 8). The cells observed in the neointima at the 4-day time point reflect migrated rather than proliferating cells.6,7
We demonstrated that the increased cell motility in the absence of CRP2 was not a result of defect in PDGF signaling. PDGF-BB stimulated ERK1/2 and Akt phosphorylation to a similar degree in both WT and Csrp2eC/eC cells (Figure 7B). The increased cell motility might be due in part to an increased Rac1 activation (Figure 7E), a pathway linked to cell migration. Lamellipodia extension and focal adhesion formation are the initial steps for cell migration.37 The signaling complex p130Cas-Crk-DOCK180 activates Rac,38,39 which in turn promotes lamellipodia extension and cell migration.35 Interestingly, the zyxin family of LIM proteins associate with p130Cas, and may regulate Cas-Crk interactions and downstream signaling.40,41 Displacement of zyxin from its normal subcellular localization inhibits cell migration,42 suggesting an important role of zyxin in cell motility. Given the interaction between CRP2 and zyxin,19,24 it is possible that CRP2 may function as a negative regulator of zyxineCassociated multiprotein signaling complexes and cell motility.
In summary, we demonstrated that an absence of CRP2 did not alter vascular development, morphology, or the expression of several characteristic SMC-specific genes. However, in response to mechanical arterial injury, an absence of CRP2 increases neointima formation, correlating with increased VSMC migration.
Acknowledgments
This work was supported by National Institutes of Health grants HL-057977 (S.-F.Y.) and AR-047861 (M.D.L). We thank the late Arthur Mu-En Lee and Mark A. Perrella for enthusiasm for our work.
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the Division of Newborn Medicine (T.E.G.), Children’s Hospital, Boston, Mass.
Abstract
In response to arterial injury, medial vascular smooth muscle cells (VSMCs) proliferate and migrate into the intima, contributing to the development of occlusive vascular disease. The LIM protein cysteine-rich protein (CRP) 2 associates with the actin cytoskeleton and may maintain the cytoarchitecture. CRP2 also interacts with transcription factors in the nucleus to mediate SMC gene expression. To test the hypothesis that CRP2 may be an important regulator of vascular development or function we generated Csrp2 (gene symbol of the mouse CRP2 gene)-deficient (Csrp2eC/eC) mice by targeted mutation. Csrp2eC/eC mice did not have any gross vascular defects or altered expression levels of SM -actin, SM22, or calponin. Following femoral artery injury, CRP2 expression persisted in the vessel wall at 4 days and then decreased by 14 days. Intimal thickening was enhanced 3.4-fold in Csrp2eC/eC compared with wild-type (WT) mice 14 days following injury. Cellular proliferation was similar between WT and Csrp2eC/eC VSMC both in vivo and in vitro. Interestingly, Csrp2eC/eC VSMC migrated more rapidly in response to PDGF-BB and had increased Rac1 activation. Our data demonstrate that CRP2 is not required for vascular development. However, an absence of CRP2 enhanced VSMC migration and increased neointima formation following arterial injury.
Key Words: arterial wire injury vascular smooth muscle cells migration
Introduction
During blood vessel development, the vascular smooth muscle cell (VSMC) component arises from both neural crest and mesodermal origins. These cells proliferate at a high rate and synthesize extracellular matrix molecules that contribute to the structure and function of the blood vessel.1,2 As VSMC mature, they proliferate at a low rate and exhibit a differentiated contractile phenotype.3 Adult VSMCs are not terminally differentiated: in response to vessel injury, VSMCs within the vessel wall dedifferentiate and change from a quiescent and contractile phenotype to a proliferative and synthetic phenotype reminiscent of embryonic precursors.3eC5 The migration and proliferation of VSMCs from the media into the intima contribute to arterial intima thickening and subsequent arteriosclerosis.6,7 Arteriosclerosis and its complications, including heart attack and stroke, are major causes of death.4,8 Despite its importance, the molecular mechanisms that control VSMC development and differentiation and the phenotypic modulation of VSMC in vascular injury have not been elucidated completely.
The LIM protein family is characterized by a double zinc-finger structure that serves as a protein interaction module.9,10 Through binding of target proteins and assembly of multiprotein complexes, LIM proteins function in diverse biological processes.11eC13 The LIM-only cysteine-rich protein (CRP1eC3) family contains two tandem LIM domains, each followed by a short glycine-rich repeat.14eC19 CRP1 is expressed in most cell types.15,20 CRP2 is expressed primarily in arterial but not in venous smooth muscle cells (SMCs),18,21,22 and CRP3 is expressed only in striated muscle.14,23
CRPs associate with the actin cytoskeleton via interacting with the actineCcross-linking protein -actinin and the adhesion plaque protein zyxin.19,24 Gene deletion studies in mice revealed that CRP3 is essential in maintaining cardiomyocyte cytoarchitecture.14,23 Given the potential overlapping cellular functions of CRPs and their expression in different muscle types, CRP1 and CRP2 may maintain the cytoarchitecture of SMC and influence tissue development and cellular differentiation.19 In addition to their prominent association with cytoskeleton, CRPs also localize to the nucleus.9,18 CRP3 interacts with muscle-specific transcription factor MyoD and can promote myogenesis.25 Interestingly, Chang et al reported that CRP1 and CRP2 form complexes with serum response factor and GATA transcription factors, facilitating the expression of some smooth muscle (SM) marker genes.26
Through participation in multi-protein complexes, CRPs have important regulatory roles.19,25,26 However, nothing is known about the requirement for CRP2 in the vasculature. To gain insight into the biological functions of CRP2 in vivo, we generated Csrp2-deficient mice by targeted mutation.
Materials and Methods
Detailed methods are described in the expanded Materials and Methods in the data supplement, available online at http://circres.ahajournals.org.
Generation of Cysteine-Rich Protein 2eCDeficient Mice
We generated Csrp2 (gene symbol for the mouse cysteine-rich protein gene)-deficient (Csrp2eC/eC) mice by gene targeting. Mice were backcrossed 10 generations and fixed on a C57BL/6 background.
Antibody Production
We generated two CRP2-specific antibodies against amino acids 91 to 98 and 93 to 108, respectively.
Southern, Northern, and Western Blot Analysis
Southern analysis was performed using BglII-digested mouse genomic DNA. Total RNA isolated from mouse aorta was analyzed by Northern analysis. Protein extracts from adventitia-stripped aorta was analyzed by Western analysis with CRP2(91eC98) antiserum and a monoclonal SM -actin antibody (Sigma).
Blood Pressure Measurements
A tail-cuff method was used to measure systolic blood pressure of adult male conscious mice.27
Femoral Artery Injury
Endoluminal injury to the mouse left common femoral artery was performed as described.28
Histological Analysis and Immunohistochemistry
Femoral arteries were harvested for histological and morphometric analyses. Vessel sections were stained for elastin (Sigma) and the intimal and medial areas were measured using NIH Image software.
En Face VSMC Migration Assay
Four days after injury, en face VSMC migration was measured in femoral arteries.29 The total number of migrated VSMCs in the injured vessel was counted.
Proliferation and Migration Assays
We performed [methyl-3H]-thymidine incorporation assays to assess proliferation. To assess migration, serum-starved cells were placed in the upper chamber of transwell plates and the bottom chambers were filled with 0.5% FBS medium containing platelet-derived growth factor-BB.
Assessment of ERK1/2, Akt, and Rac1 Activation
Mouse aortic smooth muscle cells were serum-starved and then stimulated with 10 ng/mL PDGF-BB. ERK1/2 and Akt activation was assessed by Western blot using antibodies (Cell Signaling Technology) for phospho-ERK1/2, total ERK1/2, phospho-Akt (Ser473), and total Akt. We assessed Rac1 activation by binding GTP-bound Rac1 to p21-binding domain of PAK-1 using a Rac1 activation assay kit.
Statistical Analysis
Data are presented as mean±SEM. Statistical significance was determined by Student t test. P0.05 was accepted as statistically significant.
Results
Generation of Csrp2eC/eC Mice
To examine the role of CRP2, we targeted the Csrp2 locus in mice by homologous recombination. The N-terminal LIM domain of CRP2 mediates interactions with its binding partners.26,30 Thus, we constructed a targeting vector to disrupt the N-terminal LIM domain (Figure 1A). Germ line transmission of the mutation and generation of Csrp2 heterozygous mice was demonstrated by Southern blot analysis with a 5'-external probe (Figure 1B, left panel, middle lane) or 3'-external probe (data not shown). Heterozygous mice, which were viable and fertile, were intercrossed to generate homozygous mutant mice (Figure 1B, left panel, right lane). Additionally, a neo probe hybridized to a single 11-kb mutated fragment in heterozygous and homozygous mutant but not in wild-type (WT) mouse genomic DNA (Figure 1B, middle panel), demonstrating that no additional integrations were present in the mutant mice. Southern analysis using deleted exon 3 as a probe revealed that a 4.8-kb WT fragment was present only in WT and heterozygous but not in Csrp2eC/eC mouse genomic DNA (Figure 1B, right panel), further demonstrating that we have deleted exon 3 and disrupted the Csrp2 allele.
Northern blot analysis with aortic RNA from WT, heterozygous, and homozygous mutant mice using exon 3 as a probe demonstrated that CRP2 message was absent in homozygous mutants (Figure 1C). To evaluate whether aberrant transcripts exist in the mutant mice, we performed RT-PCR using an upper and lower primer from exon 1 and exon 6, respectively. We were able to detect a shorter (0.83 kb) transcript. Sequence analysis revealed this transcript was produced by splicing of exon 2 to exon 4 and downstream exons. If the truncated transcript were translated, it would not encode any LIM domains due to a frame shift. By Western blot analysis, CRP2 was not detectable in protein isolated from the aorta of homozygous mutant mice (Figure 1D). In contrast, the SMC marker gene SM -actin expression was similar among the three genotypes (Figure 1D).
Arteries in Csrp2eC/eC Mice Are Structurally and Functionally Normal
Csrp2eC/eC mice were born alive at the expected Mendelian ratio, without any apparent abnormality. Adult Csrp2eC/eC mice were fertile and appeared grossly normal. To analyze the vasculature in greater detail, vessels were fixed at constant pressure and isolated from WT and Csrp2eC/eC mice. Immunostaining with the CRP2(93eC108) antiserum demonstrated strong CRP2 expression in the medial SM layers of WT aorta (Figure 2A) but not that from Csrp2eC/eC mice (Figure 2B). To evaluate the structure of the vessel, Verhoeff’s elastin stain was used to delineate the elastic lamina. At a similar anatomic level of the descending aorta, WT and Csrp2eC/eC mice exhibited a similar number of elastic and SM layers (Figure 2C and 2D, respectively). SM -actin expression was similar in the aortic SM layers from both WT (Figure 2E) and Csrp2eC/eC mice (Figure 2F).
Because one of the primary functions of VSMC is to regulate vascular tone, we examined whether an absence of CRP2 altered blood pressure. Systolic blood pressure was not statistically different between WT (111±3 mm Hg, n=7) and Csrp2eC/eC mice (115±2 mm Hg, n=8; P=0.30).
Several Characteristic SM Marker Genes Are Not Altered in the Absence of CRP2
A previous study has shown that overexpression of CRP2 results in translocation of the protein to the nucleus where it functions as a potent transcriptional coactivator with serum response factor and GATA to facilitate SMC-specific gene expression.26 Therefore, we wanted to test the hypothesis that SMC marker genes would be reduced in the absence of CRP2. CRP2 mRNA was not detectable in the aorta from Csrp2eC/eC mice (Figure 3). Surprisingly, the levels of calponin and SM22, whose expression is dependent on functional serum response factor complexes,31eC33 were not altered in the absence of CRP2 expression (Figure 3). Potentially this result could be explained by a compensatory upregulation of other CRP family members. Northern analysis revealed that CRP1 expression levels were not different between WT and Csrp2eC/eC mice (Figure 3). Additionally, CRP3 remained undetectable in the aorta from both WT and Csrp2eC/eC mice (Figure 3).
CRP2 Expression in the Arterial Wall After Vascular Injury
In response to vessel wall injury, SMCs undergo a phenotypic change and alter their gene expression patterns and their proliferative and migratory behavior.3eC5 To investigate the potential role of CRP2 in vascular injury, we first examined the temporal expression of CRP2 after femoral artery wire injury in WT mice. Verhoeff’s elastin stain of control femoral arteries revealed the endothelial layer (Figure 4A) abutting the internal elastic lamina (IEL) at the luminal surface (Figure 4A) and medial SM layers beneath the IEL (Figure 4A). Strong CRP2 expression was detected in the medial SM layers but not in adventitial fibroblasts or endothelial cells (Figure 4B), which was delineated with the endothelial cell marker PECAM-1 (Figure 4C). Four days after injury, CRP2 expression remained detectable in the medial layers (Figure 4D). Additionally, although very few cells were present in the intima, CRP2 expression was detectable (Figure 4D). Fourteen days after injury, CRP2 was present in some but not all medial (73.4±10.2%, n=4) and intimal (58.2±8.7%, n=4) areas (Figure 4E). No CRP2 staining was observed in adventitial fibroblasts (Figure 4E) or endothelial cells (Figure 4E and 4F). These data indicate that following wire injury CRP2 expression persisted in the first 4 days and decreased but was not abolished in the vessel wall by 14 days.
Absence of CRP2 Increases Neointima Formation in Response to Vascular Injury
Under basal conditions, SMC gene expression and blood vessel morphology were similar in WT and Csrp2eC/eC mice. Given that CRP2 was expressed initially after injury and decreased but not diminished by 14 days, we hypothesized that an absence of CRP2 might influence neointima formation after injury. Fourteen days after wire injury, vessel size (area inside external elastic lamina [EEL]) of the injured femoral arteries was not different between WT (40500±2406 e2, n=12) and Csrp2eC/eC mice (39993±2654 e2, n=11; P=0.44). The medial areas were also similar between WT (11441±511, n=12) and Csrp2eC/eC mice (10243±1032, n=11; P=0.16). In WT mice, intimal thickening was evident, although small (3897±912 e2 or 97±15 cells, n=12), 14 days after injury (Figure 5A and 5C). In contrast, there was a robust 3.4-fold increase in intimal thickening in Csrp2eC/eC mice (13438±2905 e2 or 311±43 cells, n=11; P<0.05) (Figure 5B and 5C). An absence of CRP2 increased the intima/media ratio &4-fold to 1.58±0.40, compared with 0.36±0.10 of WT mice (Figure 5D, P<0.05). Because endothelial regeneration after arterial injury affects neointima formation, we measured the extent of endothelial regeneration by PECAM-1 staining of arteries 14 days after wire injury. At this time point injured arteries have &50% endothelial coverage as reported previously.28 The endothelial regeneration was similar between WT (61.7±21%, n=5) and Csrp2eC/eC mice (61.8±12.2%, n=5; P=0.50 versus WT), suggesting that the increased neointima formation in Csrp2eC/eC injured arteries was not likely due to alterations in endothelial regeneration.
Characterization of the neointima revealed that WT (n=8) and Csrp2eC/eC (n=8) neointima had similar cell densities (19.6±0.5 and 20.3±1.7 nuclei/1000 e2, P=0.34). Interestingly, in contrast to the barely detectable SM -actin staining in the WT neointima (14.2±6.2%, n=6) (Figure 6A), Csrp2eC/eC neointima were composed mainly of SM -actin positive cells (63.3±10.6%, n=3; P<0.05 versus WT) (Figure 6B). Very few CD45 positive inflammatory cells were present in either WT (0.8±0.4%, n=3) (Figure 6C) or Csrp2eC/eC (0.5±0.3%, n=3; P=0.56 versus WT) (Figure 6D) intima. Proliferation and migration contribute to neointima formation in the injured vessel wall. To assess cellular proliferation, we quantified incorporation of BrdUrd in the arteries 14 days after injury. Proliferation was evident in WT vessels (n=5) (Figure 6E) with 10.2±4.6% BrdUrd incorporation in the neointima and 5.4±1.6% in the media. In Csrp2eC/eC mice (n=9) (Figure 6F), we observed 7.1±2.7% of BrdUrd incorporation in the neointima (P=0.63 versus WT) and 6.0±1.9% in the media (P=0.82 versus WT), suggesting similar cellular proliferation in WT and Csrp2eC/eC mice. TUNEL staining revealed that very few apoptotic cells were observed in the injured vessels (Figure 6G and 6H). WT had even lower apoptosis (0.6±0.6%, n=4) than Csrp2eC/eC (2.8±0.6%, n=7; P<0.05) vessels, indicating that the increased neointima in Csrp2eC/eC mice was not due to decreased apoptosis in Csrp2eC/eC mice.
CRP2 Deficiency Promotes VSMC Migration But Not Proliferation
To investigate potential mechanisms by which an absence of CRP2 leads to increased neointima formation in response to vascular injury, we isolated VSMC from 18.5 dpc embryos of WT and Csrp2eC/eC mice and assessed their proliferation in vitro. PDGF-BB dose-dependently increased 3H-thymidine incorporation in both WT and Csrp2eC/eC VSMC (Figure 7A). The increase in 3H-thymidine incorporation by 10 ng/mL PDGF-BB was comparable to 20% fetal bovine serum (Figure 7A). Consistent with in vivo findings (Figure 6E and 6F), WT and Csrp2eC/eC VSMC exhibited similar increases in 3H-thymidine incorporation (Figure 7A). PDGF-BB induced similar degree of ERK1/2 and Akt phosphorylation (Figure 7B), suggesting the signaling pathways linked to cellular proliferation stimulated by PDGF-BB were normal in Csrp2eC/eC VSMC.
In addition to proliferation, SMC migration contributes to the development of the neointima after injury.3,6,7 Given that the cellular proliferation was similar between WT and Csrp2eC/eC mice both in vivo and in vitro, we hypothesized Csrp2eC/eC VSMC would have altered migratory behavior. PDGF-BB is a potent chemoattractant for VSMC and is released at sites of vessel injury.6,7,34 We measured the migratory responses of WT and Csrp2eC/eC VSMC toward the chemoattractant PDGF-BB. Two hours after stimulation, PDGF-BB at 0.1 or 1 ng/mL minimally stimulated cell migration (Figure 7C), whereas 10 ng/mL significantly stimulated cell migration (Figure 7C). Interestingly, 1 hour after stimulation with PDGF-BB (10 ng/mL), Csrp2eC/eC VSMC showed increased migration (1.1±0.1%) compared with WT cells (0.6±0.1%, P<0.05) (Figure 7D). Two hours after stimulation, 11.0±1.7% of Csrp2eC/eC cells had migrated through the filters. In contrast, only 5.5±0.1% of WT cells migrated toward PDGF (Figure 7D, P<0.05). At 3 hours after stimulation, migrated Csrp2eC/eC cells increased to 18.0±2.1%, whereas only 12.0±0.6% of WT cells migrated (Figure 7D, P<0.05). These results indicate that in the absence of CRP2, VSMC migrate more rapidly in response to PDGF-BB. The Rho GTPases play key roles in regulating cell migration.35 In particular, Rac1 regulates VSMC migration in response to factors released at sites of vessel injury.36 Interestingly, in response to PDGF-BB stimulation Rac1 activation was enhanced in the Csrp2eC/eC VSMC compared with WT cells despite similar total Rac1 levels (Figure 7E), correlating with increased migration rate.
To provide additional evidence that the increased neointima formation in Csrp2eC/eC mice may be due to increased migration of medial VSMC into intima, we examined neointima formation 4 days after wire injury by two independent methods: (1) en face migration assays29 and (2) histological analysis of paraffin cross-sections. The cells observed in the neointima at the 4-day time point might reflect migrated rather than proliferating cells.6,7 En face preparations revealed that compared with WT mice (Figure 8A), more cells migrated through IEL onto the luminal surface in Csrp2eC/eC mice (Figure 8B). In WT mice, 144±9 cells (n=5) migrated onto the injured luminal surface, whereas in Csrp2eC/eC mice 326±52 cells (n=4; P<0.05 versus WT) migrated (Figure 8E). We also assessed the neointima formation by counting the number of nuclei between the lumen and IEL from histological sections. Consistent with the en face results, there were fewer neointimal cells in WT mice (6.1±1.2 nuclei/section, n=11) (Figure 8C and 8F). A 2.3-fold increase of neointimal cells was observed in Csrp2eC/eC mice (13.7±1.5 nuclei/section, n=6; P<0.05 versus WT) (Figure 8D and 8F).
Discussion
To examine the function of CRP2 in vivo, we generated Csrp2eC/eC mice by targeted mutation. The LIM domains of CRP2 mediate interactions with its binding partners including zyxin, -actinin, and CRP2BP.12,19,30 Thus, we targeted the first LIM domain by disrupting exon 3, the largest coding exon. No message was detected in Csrp2eC/eC mouse RNA when exon 3 was used as a probe, although a smaller transcript was detected by RT-PCR. Nevertheless, no CRP2 was detected in protein isolated from Csrp2eC/eC mouse aorta by Western blot analysis using CRP2(91eC98) antiserum. Furthermore, immunostaining of aortic sections with CRP2(93eC108) antiserum did not detect CRP2 expression in Csrp2eC/eC mice. We cannot exclude the possibility that a truncated protein could be generated because the two antisera used in this study were against epitopes C-terminal to those encoded by exon 2. However, even if the truncated transcript were translated, it would not encode either LIM domain due to a frame shift. Therefore, we believe that the observed phenotype in the Csrp2eC/eC mice is a result of the absence of a functional CRP2 protein.
Our finding that Csrp2eC/eC mice do not have apparent developmental vascular defects was unexpected. Chang et al26 proposed that in progenitor proepicardial cells CRP2 might function as a transcriptional coactivator to facilitate smooth muscle differentiation and the specification of the smooth muscle lineage. It is possible that CRP1, which is also expressed in VSMCs (Figure 3), may functionally compensate for the absence of CRP2 expression. Furthermore, the expression levels of SMC marker genes SM -actin (Figure 2), calponin, and SM22 (Figure 3) were not changed, indicating that a CRP2 expression is not required for the transcriptional regulation of these genes in the mouse aorta. Mice that are deficient in both CRP1 and CRP2 may be needed to address these questions.
Vascular injury downregulated, but did not abolish, CRP2 expression in blood vessels (Figure 4), suggesting CRP2 may be required to maintain VSMC in the quiescent and differentiated state. Alternatively, an absence of CRP2 may render cells more primed to the migratory phenotype in response to arterial injury. Consistent with this hypothesis, one major finding of our current study was that Csrp2eC/eC mice developed larger neointima than WT mice in a femoral artery wire injury model (Figure 5), whereas endothelial regeneration was similar between WT and Csrp2eC/eC vessels. Furthermore, serum cholesterol levels and peripheral leukocyte counts, factors that may potentially affect neointimal thickening, were not different between WT and Csrp2eC/eC mice before and 4 days after vascular injury (data not shown). Additionally, cellular proliferation was similar between WT and Csrp2eC/eC VSMC both in vivo (Figure 6) and in vitro (Figure 7A). Migration of medial SMC into intima also contributes to neointima formation.3,6,7 Therefore, one possible mechanism for increased neointima formation is that medial SMC of Csrp2eC/eC mice migrate into intima at a faster rate than WT mice. Indeed, we found that Csrp2eC/eC VSMC migrate toward PDGF-BB, a key chemoattractant in vascular injury,6,7,34 at a faster rate than WT cells in vitro (Figure 7D). Further supporting this concept, we found 2.3-fold more neointimal cells in Csrp2eC/eC than WT vessels 4 days after injury assessed either by en face migration assays or histological sections (Figure 8). The cells observed in the neointima at the 4-day time point reflect migrated rather than proliferating cells.6,7
We demonstrated that the increased cell motility in the absence of CRP2 was not a result of defect in PDGF signaling. PDGF-BB stimulated ERK1/2 and Akt phosphorylation to a similar degree in both WT and Csrp2eC/eC cells (Figure 7B). The increased cell motility might be due in part to an increased Rac1 activation (Figure 7E), a pathway linked to cell migration. Lamellipodia extension and focal adhesion formation are the initial steps for cell migration.37 The signaling complex p130Cas-Crk-DOCK180 activates Rac,38,39 which in turn promotes lamellipodia extension and cell migration.35 Interestingly, the zyxin family of LIM proteins associate with p130Cas, and may regulate Cas-Crk interactions and downstream signaling.40,41 Displacement of zyxin from its normal subcellular localization inhibits cell migration,42 suggesting an important role of zyxin in cell motility. Given the interaction between CRP2 and zyxin,19,24 it is possible that CRP2 may function as a negative regulator of zyxineCassociated multiprotein signaling complexes and cell motility.
In summary, we demonstrated that an absence of CRP2 did not alter vascular development, morphology, or the expression of several characteristic SMC-specific genes. However, in response to mechanical arterial injury, an absence of CRP2 increases neointima formation, correlating with increased VSMC migration.
Acknowledgments
This work was supported by National Institutes of Health grants HL-057977 (S.-F.Y.) and AR-047861 (M.D.L). We thank the late Arthur Mu-En Lee and Mark A. Perrella for enthusiasm for our work.
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