Prx1 Controls Vascular Smooth Muscle Cell Proliferation and Tenascin-C Expression and Is Upregulated With Prx2 in Pulmonary Vascular Disease
Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)–ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.
Homeobox transcription factors guide formation of the body plan during embryogenesis.1 Although homeobox genes also function during postnatal development and in adult disease,2–6 little is known about their roles in vascular remodeling.7 Prx1 and Prx2 represent paired-related homeobox genes,8–10 which characteristically bind class I homeodomain binding sites (HBSs) containing an ATTA core motif.11 Prx1 and Prx2 are expressed during embryogenesis, predominantly in mesenchyme-specific patterns.12–14 In the developing cardiovascular system, Prx1 and Prx2 are evident in the endocardial cushions and valves, the epicardium, and the wall of the great arteries and veins.14,15 In avian embryos, Prx1 and Prx2 are first expressed within the primary vessel wall of muscular coronary and pulmonary arteries (PAs), but as the vessels mature, their expression is restricted to nonmuscle cells in the adventitia and outer media.15
Among the targets that may be regulated by Prx proteins is the ECM protein tenascin-C (TN-C). In support of this, expression of Prx1, Prx2, and TN-C overlap in several settings including epithelial-mesenchymal transformation and vasculogenesis.16 TN-C is also expressed in remodeling adult tissues, including injured PAs, where it surrounds proliferating cells at the adventitial-medial boundary.17,18 Functionally, TN-C promotes growth and survival in cultured smooth muscle cells (SMCs) and in hypertensive PAs.19,20 On the basis of these findings, identifying the factors that control TN-C expression represents a potentially important step toward treating pulmonary vascular disease.
Multiple factors regulate TN-C, including ECM-degrading proteases.16 For example, inhibition of matrix metalloproteinase (MMP) activity suppresses TN-C expression and PA SMC growth, and reduces the severity of vascular lesions.19,20 These studies indicate that MMPs are upstream in an adhesion-dependent signaling pathway that controls TN-C. Consistent with this, we have shown that the TN-C gene promoter contains an ECM-responsive element that is silenced in SMCs cultivated on native type I collagen but is activated on the denatured form of this substrate.17,21 This ECM-responsive element harbors an HBS containing an ATTA core motif,22 which suggests that induction of TN-C expression by MMPs and denatured collagen might be controlled by homeobox proteins.
Here, we show that Prx1 and Prx2 are upregulated during the development of pulmonary vascular disease in adult rats, localizing to sites of TN-C expression. We also report that Prx gene expression is regulated by changes in SMC adhesion to type I collagen and that Prx1 controls SMC growth and TN-C gene transcription. These findings support the hypothesis that Prx proteins play key roles in the development of pulmonary vascular disease by controlling SMC proliferation and the composition of the vascular ECM.
Materials and Methods
A10 vascular SMCs were maintained in M199. All experiments were performed in triplicate, unless otherwise stated, in M199 containing 2% FBS. Collagen substrates were prepared as published.19 TN1-GFP cells were generated by transfecting A10 SMCs with the TN1-pEGFP construct. Cell lines were selected on the basis of resistance to G418 and via fluorescence-activated cell sorting for GFP. To assess proliferation, SMCs were maintained in medium supplemented with bromodeoxyuridine (BrdU) for the final 4 hours of the designated experiment.
Prx Expression Vectors
Prx cDNAs were isolated by reverse transcribing total RNA isolated from A10 SMCs. Truncated forms of rat Prx1 and Prx2 were also prepared using reverse transcriptase–polymerase chain reaction (RT-PCR) and Pfu polymerase. cDNAs were cloned into a modified pcDNA3 expression vector containing an N-terminal c-myc epitope tag.
In Situ Hybridization
Adult Sprague-Dawley rat lung tissues were used for in situ hybridization studies (a kind gift from Dr Marlene Rabinovitch, The Hospital for Sick Children, Toronto, Ontario, Canada). Riboprobes were generated using SP6 or T7 polymerase and [35S]dUTP. Sections were incubated with riboprobes before signal detection with photographic emulsion and were counterstained with Hoechst dye for visualization of nuclei by epifluorescence. Prx mRNA expression was visualized using dark-field microscopy.
mRNA Expression Studies
RT-PCR reactions were performed with cDNAs using primer pairs for Prx1, Prx2, and GAPDH. For Northern analysis, 1 μg of A10 SMC Poly(A+) RNA was separated and transferred to a nylon membrane. Hybridizations were performed with 32P–random-labeled Prx1 and Prx2 cDNA probes. Autoradiograms were analyzed using ImageQuant software. A loading control for RNA was also carried out by comparing Prx mRNA expression with that of rat GAPDH.
Protein was separated on 4% to 15% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The amount of protein loaded was determined on the basis of transfection efficiency. To detect GFP and c-myc, membranes were incubated with anti-GFP and anti-myc mouse monoclonal antibodies. Membranes were then incubated with a horseradish peroxidase–conjugated goat anti-mouse antibody and detected on Kodak X-Omat film by enhanced chemiluminescence.
For detection of Prx1, SMCs were incubated with an anti–c-myc antibody, or with control IgG, and thereafter with an FITC-conjugated species-specific antibody. Nuclei were detected with DAPI. For BrdU detection, a cell proliferation assay kit was used. BrdU-positive nuclei were scored in control- and Prx1-transfected SMCs, and the percentage increases in BrdU incorporation determined after accounting for differences in transfection efficiency.
A 4173-bp fragment of the murine TN-C gene promoter was ligated into pEGFP, a promoterless vector encoding GFP. This vector was designated TN1-pEGFP. To assess whether the ATTA HBS is involved in regulating TN-C promoter activity, site-directed mutagenesis was performed using mutant oligonucleotide primers.
Cotransfection and Luciferase Assays
The following plasmids were introduced using LipofectAMINE: Prx1-pcDNA3 myc, TN1-pEGFP (wild-type), TN1-pEGFP (mutant), pcDNAmyc empty vector, and pSV-β–galactosidase control reporter vector. Cells were harvested at 48 hours after transfection and evaluated for transfection efficiency on the basis of β–galactosidase activity. Western immunoblotting was then used for detection of GFP and myc proteins.
For luciferase reporter assays, A10 SMCs were transfected using Fugene reagent with either empty pcDNAmyc vector or the Prx1 expression vector, together with a TN-C promoter/luciferase reporter vector (TN7), containing the −247/+121 region of the murine TN-C gene containing the proximal promoter and a segment of the first exon.22 In these experiments, a lacZ reporter construct (CMVβ) was cotransfected to provide an internal reference standard for transfection efficiency. Luciferase activity was measured using the Millipore Cytofluor 2450 system. To demonstrate that comparable levels of different Prx proteins are expressed after transfection of A10 cells, Western blots using the myc-tag antibody were performed on lysates of SMCs transfected with each Prx expression construct and the CMVβ plasmid. In these experiments, the amount of lysate analyzed was first normalized to the β-galactosidase activity.
Wild-type and mutant oligonucleotides encompassing the TN-C promoter HBS were end-labeled with [γ-32P]ATP. 0, 0.5, 1.5, and 4.5 μg of nuclear protein were incubated with 20,000 cpm of 32-P labeled wild-type or mutant probe in binding buffer. For supershift assays, Prx1-transfected A10 SMCs were preincubated with anti-myc antibody before the addition of radiolabeled wild-type or mutant oligonucleotide probes. Samples were resolved on 7% nondenaturing acrylamide gels, and DNA:protein complexes were visualized by autoradiography.
Results were compared by 1-way ANOVA and Student-Newman-Keuls post hoc analysis. A P value of <0.05 was considered statistically significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Cloning of Rat Prx1 and Prx2
To isolate rat Prx genes, RT-PCR experiments were performed using A10 SMC total RNA. A full-length Prx1 cDNA of 810 bp was isolated containing an open reading frame of 651 bp, encoding a 217–amino acid protein. Comparison of this sequence with other Prx gene sequences indicated that it was 100% identical to rat Prx1 (Rhox) but also contained an additional exon of 72 bp that is found in several mammalian Prx1 genes. This additional sequence is inserted at position 927 in the Rhox sequence. The Prx1 cDNA sequence is 97% identical to that of mouse Prx1. The Prx2 cDNA from A10 cells was 570 bp and encodes a protein that is 94% identical to mouse Prx2. This cDNA represents a novel form of Prx2 that has a deletion of 57 amino acids within the N-terminal region.
Induction of Prx1 and Prx2 With Pulmonary Vascular Disease
To determine the expression patterns of Prx mRNAs, riboprobes were hybridized to pulmonary tissue isolated from adult rats injected with saline (control), or monocrotaline (MCT), an alkaloid toxin that induces pulmonary hypertension.17 Prx antisense riboprobes did not hybridize to normal tissue (Figure 1), whereas these genes were expressed at the adventitial–outer medial boundary in hypertensive animals by 21 days after injection (Figure 1). In addition, the airways and surrounding tissue expressed Prx1 mRNA. By 28 days, Prx1 and Prx2 mRNAs were expressed in the PA adventitia and within the media and subendothelium (Figure 1). Extensive Prx1 mRNA expression was also evident in the airways and lung interstitium (Figure 1). In contrast, Prx1 and Prx2 sense riboprobes did not hybridize with either control or hypertensive tissue (data not shown). Thus, Prx1 and Prx2 mRNAs are induced and upregulated with the progression of pulmonary vascular disease.
Changes in SMC Adhesion Regulate Prx1 and Prx2
Because remodeling of the PA ECM is critical to the progression of vascular disease,16,20 we next sought to determine whether alterations in vascular SMC-ECM interactions control Prx mRNA expression. As a model system, SMCs were cultured on native and denatured type I collagen. A10 SMCs were used for these studies because they behave in a manner that is identical to that of primary PA SMCs in terms of their phenotypic and gene expression responses to type I collagen.19,21 RT-PCR studies showed that Prx1 and Prx2 mRNA expression was suppressed by native collagen (Figure 2A). In contrast, on denatured collagen, high levels of both mRNAs were observed (Figure 2A). Northern analyses for Prx1 and Prx2 mRNAs revealed Prx1 and Prx2 mRNA transcripts of ≈4.5 and ≈1.3 kb, respectively (Figure 2B). Scanning densitometry of duplicate Northern blots, normalized to GAPDH, showed that Prx1 and Prx2 were both upregulated ≈3-fold in SMCs maintained on denatured collagen compared with those maintained on native collagen (Figure 2C). These results demonstrate that alterations in SMC adhesion to the ECM control Prx1 and Prx2 mRNA expression.
Prx1 Promotes SMC Growth
To begin to establish a function for Prx1, SMCs cultivated on denatured collagen were transiently transfected with a Prx1 expression plasmid containing an N-terminal c-myc tag. Protein expression was examined by Western blotting of A10 SMC nuclear extracts and by indirect immunofluorescence using an antibody against the c-myc tag. A Prx1 protein (≈26 kDa) was expressed in nuclei of transfected cells (Figure 3A). Immunofluorescence studies showed that Prx1 was expressed in SMCs that appeared to be undergoing division (Figure 3B). Taken together with our observation that Prx1 is upregulated in remodeling PAs, we hypothesized that Prx1 controls SMC growth. To test this, A10 SMCs cultivated on native collagen were cotransfected either with the parental c-myc vector or with the Prx1 vector and a β-galactosidase–encoding expression plasmid in the presence of BrdU to assess cell proliferation. When normalized for transfection efficiency, overexpression of Prx1 significantly increased BrdU incorporation by 78.1% (P=0.03) (Figure 3C).
Expression of TN-C Requires an HBS Within Its Gene Promoter
Previously, we showed that TN-C is first expressed at the adventitial-medial boundary of MCT-treated hypertensive rat PAs, as well as in the airways and the interstitium.17 This expression pattern is identical to that observed for Prx1 and Prx2 mRNAs (Figure 1). Also consistent with our present results obtained for Prx1 and Prx2 mRNA expression (Figure 2), TN-C mRNA expression is reduced by native collagen and is upregulated by denatured collagen.19,21 Collectively, these data indicate that TN-C is upregulated with Prx1 and Prx2 in pulmonary vascular lesions and that changes in vascular SMC adhesion alters the expression of all three genes.
We have also shown that transcriptional induction of TN-C in SMCs cultivated on denatured collagen requires a 122-bp DNA element in the TN-C promoter containing an HBS.21 To determine whether the HBS is a key component in the ECM responsiveness of the TN-C promoter, GFP-reporter constructs containing either the wild-type or an HBS-mutated TN-C promoter were transiently transfected into SMCs cultivated on native or denatured collagen. Mutation of the TN-C HBS inhibited TN-C promoter activity in SMCs cultured on denatured collagen, as compared with wild type-transfected SMCs (Figure 4A). No GFP expression was observed in wild type- or mutant-transfected SMCs cultivated on native collagen (Figure 4A).
As a first step toward characterizing the protein(s) that may bind to the HBS, electrophoretic mobility-shift assays were performed. Radiolabeled oligonucleotides containing the wild-type and mutated HBS were incubated with nuclear extracts prepared from SMCs maintained on either native or denatured collagen. In SMCs cultured on denatured collagen, a high molecular weight complex was observed using the wild-type, but not the mutant, HBS (Figure 4B). With SMCs cultured on native collagen, a low molecular weight DNA:protein complex was observed when the wild-type, but not when the mutant HBS was used as probe (Figure 4B).
To demonstrate specificity of binding between the HBS and nuclear extracts, reactions were carried out using either different concentrations of nuclear extract or a 20- or 200-fold excess of unlabeled competitor oligonucleotide. In these experiments, the intensity of DNA:protein complexes observed with the wild-type HBS increased with greater concentrations of nuclear extract; formation of these complexes was abolished in reactions containing excess unlabeled HBS (Figure 4C). These experiments indicate that the TN-C promoter HBS interacts with different proteins in an ECM-dependent manner.
To determine whether Prx1 protein binds directly to the HBS, experiments were performed using radiolabeled HBS probes and nuclear extracts from cells transfected with the c-myc–tagged Prx1 expression vector. When compared with mock-transfected SMCs, no differences in DNA:protein complex formation were observed (data not shown). Preincubation of binding reactions with a c-myc antibody did not block or supershift DNA:protein complexes. Recombinant Prx1 protein also failed to bind to the wild-type HBS (data not shown). These results indicate that Prx1 either interacts weakly or does not bind directly to the core TN-C promoter HBS sequence.
Regions of the Prx1 Protein That Contribute Toward TN-C Promoter Activation
Although Prx1 protein did not directly bind to the probe containing the HBS, the coincident induction of Prx genes and TN-C in pulmonary vascular disease and in isolated SMCs prompted us to investigate whether Prx1 transactivates the TN-C gene promoter. For these experiments, an A10 SMC line (TN1-GFP) was generated in which a TN-C promoter-GFP reporter was stably integrated. As shown in Figures 5A and 5B, TN-C gene promoter activity was suppressed on native collagen and activated on denatured collagen. Transfection of TN1-GFP cells cultured on native collagen with the Prx1 expression plasmid led to increased Prx1 protein production and TN-C promoter activity (Figure 5C).
To quantify the level of TN-C promoter activation by Prx1 and to determine the regions of Prx1 required for this activation, cotransfection experiments were performed using a TN-C promoter/luciferase gene reporter plasmid (construct TN7)21,22 and three different myc-tagged Prx1 expression constructs (Figure 6A). These Prx constructs expressed either the full-length Prx1 protein, a truncated form (PNL) containing the N-terminal half of the protein with the Prx domain and nuclear localization sequence, or a truncated form (PHD) containing the N-terminal portion of Prx1 and the homeodomain.
To demonstrate that the constructs were expressed at equivalent levels, Western blots were performed on extracts from A10 cells transfected with Prx1, PNL, and PHD constructs. Cellular lysates were first adjusted to an internal reference standard of β-galactosidase activity. In a total of six experiments, no differences in Prx protein expression levels were noted. A representative Western blot is shown in Figure 6B.
In luciferase reporter assays, Prx1 induced a 20-fold induction of TN-C promoter activity relative to that observed in cells transfected with empty pcDNA3-myc vector (Figure 6C). The PHD construct led to a significant 10-fold activation of the TN-C promoter, whereas the PNL construct produced a <4-fold induction (Figure 6B). These data indicate that the C-terminal portion of Prx1 contributes ≈50% of the level of TN-C promoter activation. However, the remaining segment of Prx1 containing the N-terminal portion and homeodomain of Prx1 is required for high levels of TN-C promoter activation. These data were collected from four separate experiments performed in triplicate (n=12).
Although homeobox transcription factors control a range of cellular activities during embryonic development,1–8 little is known about their regulation and functions in adult tissues. We have shown that expression of two paired-related homeobox genes, Prx1 and Prx2, is induced during the development of pulmonary vascular disease in adult rats. This induction of Prx1 and Prx2 coincides with that of the ECM protein, TN-C. In addition, alterations in SMC adhesion were shown to regulate Prx gene expression. Because structural remodeling of the ECM also regulates SMC proliferation, TN-C biosynthesis, and the severity of pulmonary vascular disease,16,19,21 we assessed whether Prx1 could modulate these functions. Expression of Prx1 promoted SMC growth and induced TN-C expression. Finally, we showed that the ability of Prx1 to transactivate the TN-C promoter relies on distinct regions of this homeobox protein. These experiments support the idea that Prx proteins and concomitant alterations in the expression of particular ECM proteins (ie, TN-C) are likely to be important factors in the genesis of pulmonary vascular disease.
A tenable hypothesis based on this study is that expression of Prx and TN-C genes is controlled by the same factors. In keeping with this, bone morphogenetic proteins and angiotensin II23–25 have each been shown to regulate Prx and TN-C expression. In this study, we focused on the role of changes in SMC adhesion as a factor that controls Prx genes and TN-C. This direction was based on a growing body of evidence implicating cell adhesion as an important factor regulating vascular disease.16 Moreover, our previous work demonstrated that remodeling of native collagen activates a β3 integrin–dependent extracellular signal–regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling cascade that results in TN-C gene transcription.21 The present study indicates that Prx genes are also regulated by changes in the structure of type I collagen. It will therefore be important to determine whether β3 integrin and ERK MAPKs also control the expression and/or posttranslational processing of Prx genes and proteins.
The expression of other homeobox proteins has also been shown to depend on the surrounding ECM. For example, endothelial cell HoxD3 expression is suppressed by basement membrane proteins during acquisition of an angiogenic phenotype.6 Similarly, HoxB7 expression in mammary epithelial cells is incompatible with basement membrane–directed lactational differentiation.2 Because HoxB7 is also expressed in fetal but not adult SMCs,4 it would be interesting to determine whether alterations in SMC HoxB7 expression are also influenced by changes in the vascular ECM.
Our studies show that Prx1 activates TN-C transcription. This suggests that Prx proteins not only respond to changes in cell adhesion, but they can also act in a reciprocal manner to regulate the composition of the ECM. This type of “inside-out” control has already been described for the Gax homeobox gene, which promotes a quiescent SMC phenotype by suppressing expression of αvβ3 and αvβ5 integrins.5 Given that SMC αvβ3 integrins also interact with denatured collagen19 (which we have now shown promotes Prx1 and Prx2 gene expression), it is possible that an inverse relationship exists between Gax and Prx genes in developing and remodeling arteries.
The appearance of Prx1 and Prx2 in the adventitia of hypertensive PAs suggests that these genes might regulate the behavior of nonmuscular cells. Consistent with this, developmental studies show that Prx gene expression and recruitment of SMCs first takes place in the surrounding loose mesenchyme or primordial adventitia.15,26 Whether further growth of the vessel wall involves proliferation of medial SMCs or recruitment and growth of undifferentiated adventitial cells is presently unknown. However, cell labeling studies clearly show that adventitial fibroblasts represent a component of the neointimal layer within injured adult systemic arteries.27 In addition to Prx1 and Prx2 expression, induction of α–smooth muscle (SM) actin represents another hallmark of activated fibroblasts in different remodeling tissues,28 and it has been shown that Prx1 can transactivate the α–SM actin gene promoter.9 In light of the present results, it will be important to determine whether Prx genes also control adventitial fibroblast behavior in hypertensive PAs via their ability to modulate α–SM actin.
We also performed cellular transfection studies in SMCs cultivated on native collagen, a culture condition that suppresses endogenous Prx gene expression (present study) and proliferation.29 Overexpression of Prx1 produced significant increases in BrdU uptake. Although other studies indicate that specific Hox genes regulate cell growth during tumorigenesis,30,31 the present work is the first to show that paired-related homeobox genes control growth in a nontransformed adult tissue. In support of a growth-modulatory role for Prx1 protein, a yeast 2-hybrid screen with the N-terminal portion of p130, a member of the retinoblastoma (RB) gene family, identified Prx1 as an interacting transcription factor.32 Whether Prx1 inhibits or promotes RB function has not been determined. In vascular cells and tissues, however, overexpression of RB attenuates growth and neointimal formation.33 Perhaps interaction of Prx1 with RB favors cell cycle progression during vascular remodeling? Also in keeping with a growth-related role for Prx genes, selective expression of Prx2 in proliferating fibroblasts and the developing dermis correlates with TN-C expression and wound healing in fetal tissue.34,35
Our study also demonstrates that different Prx1 domains contribute to TN-C gene transcription. One way that the N- and C-terminal portions of Prx1 might participate in this transactivation event is via interaction with a protein domain on another transcription factor, or they might modulate the interaction of the Prx1 homeodomain with other factors. Also, Prx1 activation of TN-C promoter activity with lack of direct binding of Prx1 protein to the HBS suggests several possibilities for transactivation. First, Prx1-HBS interactions might require an accessory DNA element in the −247-bp TN-C promoter, which binds a protein cofactor that is important to stabilize interaction of Prx proteins with the HBS. Alternatively, Prx1 might not bind to the HBS but instead interact with other proteins that are assembled at other elements within the TN-C promoter. Such elements in the −247/+121 TN-C promoter include binding sites for nuclear factor-1, POU homeodomain proteins, nuclear factor-κB, and a TRE/AP-1 element that binds to fos, jun, and other bZip proteins.22 A tenable hypothesis is that Prx1 activates TN-C transcription via protein-protein interactions with these factors. For instance, Prx1 is known to form complexes with the Maf oncoprotein, a bZip transcription factor family member.36 A Maf-recognition element, which is also a consensus TRE/AP-1 element, is found at −114 bp in the TN-C promoter. Maf forms heterodimeric combinations with Prx1, as well as with fos and jun; such interactions may modulate the activity of TN-C and other genes that are targets of these factors. Additionally, Prx1 may interact directly with components of the basic transcription machinery assembled at the TATA box. For instance, Prx1 is known to interact with the serum response factor and RB,32,37 proteins that are capable of interacting with the basic transcription machinery. Alternatively, it is possible that Prx1 transactivates TN-C via its ability to modulate the expression of transcription factors that bind directly to the TN-C gene promoter.
Mice bearing null mutations in both Prx1 and Prx2 have been informative in elucidating the role of these genes during development.38–42 Mutant animals die 24 hours after birth and display skeletal and limb defects, as well as vascular anomalies, including abnormal positioning and awkward curvature of the aortic arch, and a misdirected and elongated ductus arteriosus. These defects are conceivably due to deregulated ECM synthesis.42 Whether this relates to altered TN-C expression, however, has not been determined.
In summary, our findings support the hypothesis that changes in SMC adhesion to the ECM are related to the expression and functions of Prx genes. In addition, the present work provides a foundation for studies aimed at deciphering the gene networks and signal-transduction events that are responsible for Prx gene expression through changes in the vascular ECM, as well as the mechanisms by which TN-C gene transcription and cellular proliferation are controlled under these conditions.
This work was supported by a grant from the National Science Foundation (to F.S.J.) and an American Heart Association Grant-in-Aid (to P.L.J.). We are grateful for the excellent technical assistance of Tom Moller.
Original received January 24, 2001; revision received May 22, 2001; accepted May 22, 2001.
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