©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of gax, a Growth Arrest Homeobox Gene, Is Rapidly Down-regulated in the Rat Carotid Artery during the Proliferative Response to Balloon Injury (*)

(Received for publication, December 15, 1994)

Lawrence Weir (§) Dongfen Chen Christopher Pastore Jeffrey M. Isner Kenneth Walsh

From the Departments of Medicine (Cardiology) and Biomedical Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

gax is a recently described homeobox gene whose expression in the adult is largely confined to cardiovascular tissues. gax has been shown to be rapidly down-regulated in cultured vascular smooth muscle cells (VSMC) upon stimulation by serum or platelet-derived growth factor. The temporal profile of gax expression in vitro matches that of two families of growth arrest genes: the gas genes and the gadd genes. All of these genes are expressed at their highest levels in quiescent cells and are down-regulated following mitogen activation. Here we report that gax is also down-regulated in vivo in the vascular wall in response to endothelial denudation by balloon angioplasty. The reduction in steady state levels of gax mRNA is transient and occurs with a similar time course to that seen in vitro. The down-regulation of gax in response to balloon injury mirrors the up-regulation seen in a number of early response genes such as c-myc and c-fos. This report is the first to document the in vivo expression of a growth arrest gene which regulates proliferation of vascular smooth muscle cells. In addition, in contrast with previous reports which have demonstrated up-regulation of several genes following balloon injury and/or angioplasty, the present report demonstrates the down-regulation of a regulatory gene within hours of balloon injury. The characteristics of gax suggest it may be required to maintain the gene expression of proteins in VSMC that are associated with the nonproliferative or contractile phenotype in smooth muscle cells.


INTRODUCTION

Proliferation of smooth muscle cells in the vascular wall has a key role in the development of atherosclerosis and is generally held responsible for restenosis following angioplasty (1) and the failure of coronary bypass grafts(2) . Quiescent smooth muscle cells respond to a variety of growth factors by dedifferentiating and re-entering the cell cycle(3, 4) , and these changes in phenotype are mediated by changes in gene expression. Transcriptional modulation of a battery of genes, many coding for constituents of the contractile apparatus, is known to occur in this process. For example, proliferation of cultured VSMC (^1)has been shown to be associated with a major change in myosin isoform expression(5) , and similar changes have been reported in the development of atherosclerotic lesions in rabbits (6) and in humans(7, 8) . Similar switches in isoform expression have been observed for actin in VSMC after endothelial denudation (9, 10, 11) and in human arterial plaque(11) . Arterial injury also leads to transcriptional activation of genes encoding matrix proteins(12) . The expression of these genes is presumably regulated by transcription factors which are themselves regulated by mitogenic stimuli. In a number of cell types, proto-oncogene expression has been shown to be involved in the proliferative response (13, 14) and in the VSMC response to balloon injury(15) .

In skeletal muscle, several regulatory genes important in terminal differentiation have been isolated(16, 17) . Little is known, however, about the tissue-specific transcription factors in VSMC which must be required for their reversible differentiation. Attempts have been made to isolate specific markers of proliferating and differentiated smooth muscle cells by differential screening techniques, but to date no low abundance transcription factors have been found using these procedures (18) . Recently, however, several homeobox transcription factors have been shown to be expressed in cardiovascular tissues(19, 20) . Homeobox factors are known to be regulators of cell differentiation, proliferation, and migration. One of these factors, dubbed gax for growth-arrest-specific homeobox is expressed in adult cardiovascular tissues including VSMC of the blood vessel wall. gax is unusual among the homeobox class of transcription factors in that its expression is rapidly down-regulated in cultured smooth muscle cells upon stimulation to proliferate by serum or platelet-derived growth factor(20) . This down-regulation is dose-dependent and reversible. Removal of serum from growing VSMC induced expression of gax 5-fold within 24 h(20) .

In in vitro experiments, the timing of gax expression in response to mitogenic stimuli is remarkably similar to that of two families of genes which have been described previously: the growth arrest-specific (gas) genes (21, 22) and the growth arrest and DNA damage-inducible (gadd) genes(23) . Like gax, these families of genes are also expressed at their highest levels in quiescent cells and are down-regulated following mitogen activation. Some of these genes have been shown to be negative regulators of cell growth(24) , but little is currently known about the functions of this diverse group of genes. It is known, however, that gas1 and gas3 encode membrane proteins(25, 26, 27, 28) , and gas2 encodes a protein component of the microfilament network system(29) . gadd 153, the only gadd gene with a known function, is a member of the C/EBP family of transcription factors and is the human homolog of the murine CHOP-10(30) . The mechanisms by which these genes negatively regulate proliferation are currently unknown.

Of all the known growth arrest genes, gax is unique in that it is the only homeobox transcription factor gene belonging to this class, and unlike most gas and gadd genes, its expression is highly cell type-specific, a feature that is commonly displayed by homeobox factors. Since the blood vessel wall is one of the adult tissues that expresses gax, we considered whether vascular smooth muscle cells would also demonstrate a down-regulation of gax expression when induced to proliferate via endothelial denudation. In this paper we utilized the rat carotid model of endothelial injury by a balloon catheter (31) to demonstrate the down-regulation of gax expression in vivo. These data indicate that gax, as a transcription factor presumably responsible for the activation or repression of a set of downstream genes, may hold a pivotal position in the control of smooth muscle cell phenotypic changes seen during normal vessel development and in proliferative blood vessel wall disorders.


MATERIALS AND METHODS

Rat Model of Balloon Injury

The model of balloon injury employed was based on that of Clowes et al.(31) . Sprague-Dawley rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg) (Abbot Laboratories, North Chicago, IL). The bifurcation of the left common carotid artery was exposed through a midline incision and the left common, internal, and external carotid arteries were temporarily ligated. A 2F Fogarty embolectomy catheter (Baxter Edwards Healthcare Corp., Irvine, CA) was introduced into the external carotid artery and advanced to the distal ligation of the common carotid. The balloon was inflated with saline and drawn toward the arteriotomy site three times to produce a distending, de-endothelializing injury of the left common carotid artery. The catheter was then withdrawn, and the proximal external carotid artery was ligated. The experimental protocol for this project was approved by the Institutional Animal Care and Use Committee of St. Elizabeth's Medical Center and complied with the ``Guide for the Care and Use of Laboratory Animals'' (National Institutes of Health Publication 86-23, revised 1985).

RNA Isolation

Total cellular RNA was isolated from segments of rat carotid artery using TRI Reagent (Molecular Research Center, Inc.). Specimens were removed from liquid nitrogen and immediately homogenized in 0.5 ml of denaturant solution using a Tekmar tissuemizer for 2 min. The concentration of purified RNA was determined by taking of the sample and measuring UV absorbance at 260 nm. All solutions for RNA isolation, cDNA synthesis, and PCR were made up with deionized DEPC water (i.e. treated with diethylpyrocarbonate) and aerosol-resistant micropipette tips were used throughout. RNA samples were checked for degradation by electrophoresis of 0.5 µg of sample on native agarose gels and visualizing ribosomal bands by ethidium bromide staining.

cDNA Synthesis

0.5 µg of each RNA sample was used to make cDNA in a reaction volume of 20 µl containing 0.5 mM of each deoxynucleotide triphosphate (Pharmacia Biotech Inc.), 10 mM dithiothreitol, 10 units of RNasin (Promega), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl(2), 1 µg random primers (Promega), and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). For greater accuracy and reproducibility, master mixes for a number of reactions were made up and aliquoted to tubes containing RNA. Reactions were incubated at 42 °C for 1 h, then at 95 °C for 5 min to terminate the reaction. 80 µl of DEPC water was added to increase the total volume to 100 µl before the PCR.

PCR Procedure

5 µl (i.e. ) of the diluted cDNA reaction (equivalent to the product of 25 ng of RNA) was used in each PCR. The optimized reaction in a total volume of 20 µl contained 20 nM of each deoxynucleotide triphosphate (Pharmacia), 1.5 mM MgCl(2), 2 µl of PCR II buffer, final concentrations, 50 mM KCl, 10 mM Tris-HCl (pH 8.3) (Perkin-Elmer), 5 ng of each primer, 10 µCi of [P]dCTP, and 0.1 units of AmpliTaq DNA polymerase (Perkin-Elmer). Master mixes of reagents were used when dealing with multiple samples. Amplification of gax and c-myc was for 30 cycles at 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 20 s, ending with one cycle at 72 °C for 5 min. The PCR was performed on a 9600 PCR system (Perkin-Elmer) using microamp 0.2-ml thin-walled tubes. Amplification of G3PDH was for 25 cycles using the same conditions. Coamplification of gax and G3PDH in the same reaction vessel involved stopping the PCR after five cycles before addition of the G3PDH primers, then continuing for a further 25 cycles. Concentration of all buffer constituents were appropriately adjusted to account for the slight increase in volume due to the addition of the second set of primers. Primer sequences (all written 5` to 3`) were as follows.

gax, c-myc, and G3PDH primer pairs yield PCR products at 313, 705, and 534 base pairs, respectively. In preliminary experiments (not shown) each primer pair was shown to yield a single band at the expected molecular weight. Coamplification yielded two bands which were easily identified and excised for counting. Primer pairs were designed using OLIGO(TM) version 4.0 primer analysis software (National BioSciences Inc.) to avoid unwanted duplex formation, false priming, and ensure similar annealing temperatures so that standard amplification conditions were possible.

Quantitative Analysis of mRNA by RT-PCR

30 µl of DEPC water was added to each PCR to give a total volume of 50 µl. To remove unincorporated [P]dCTP, each sample was spun through a Biospin 6 column (Bio-Rad). One-third of each purified sample was electrophoresed in a 1% agarose gel in Tris borate/EDTA running buffer. Gels were dried onto Whatman No. 3MM paper and exposed to x-ray film (Kodak XAR). Bands visualized by autoradiography were excised from the dried gels and counted by liquid scintillation. Counts were adjusted by subtraction of background counts/min. Abundance of gax mRNA was expressed as the counts/min detected in the gax-specific band divided by the counts/min in the G3PDH-specific band. Preliminary experiments using serial dilutions of cDNA demonstrated that the conditions for PCR used for gax, G3PDH, and c-myc were within the linear range of the reaction. To obtain a more accurate estimate of the extent of gax down-regulation, we performed PCR on serial dilutions of cDNA derived from control arteries and from arteries harvested 4 h following balloon injury. The logarithm of the PCR yield (in counts/min) was plotted against the logarithm of the input RNA (in nanograms). The difference in gax mRNA abundance in the samples is reflected in the difference in PCR yield over a range of values for RNA input, and the differences are consistent during the exponential phase of the reaction. To take account of innaccuracies in measurement of RNA concentration by spectrophotometry, results were normalized to levels of G3PDH.

Northern Blots

RNA was subjected to electrophoresis through a 1% agarose gel containing formaldehyde as described(32) , transferred to a Hybond membrane (Amersham Corp.), and hybridized to the probes indicated in the figures. 40 µg of RNA (pooled from three rats) was loaded per lane. The three probes were for gax, c-myc, and G3PDH. Before hybridization to a different probe, the blot was stripped according to the manufacturer's protocol. Probes for gax and c-myc were generated by PCR using the same sets of primers described above. The probe for G3PDH was a cDNA fragment cloned into the EcoRI site of Bluescript SK- obtained from the American Type Culture Collection (ATCC 78463). The insert (1 kb) was excised by EcoRI. All three probes were purified from preparative agarose gels using Geneclean (BIO 101, Inc.) and labeled with P by random priming (U. S. Biochemical Corp.). Blots were hybridized in Rapid-hyb (Amersham) hybridization buffer. Following hybridization blots were washed for 20 min in 2 times SSC, 0.1% (w/v) SDS at room temperature followed by two 15-min washes in 0.1 times SSC,0.1% SDS at 65 °C. Blots were then exposed to X-Omat AR film (Eastman Kodak Co.) between intensifying screens at -80 °C for the times indicated in the text.

Statistical Analyses

Results are expressed as mean ± S.E. Statistical analysis of RT-PCR for pairs of data (one time point versus control) was performed using Student's t test where analysis of variance indicated significance for multiple comparisons. Statistical significance was defined as p < 0.05.


RESULTS

Time Course of gax Expression following Endothelial Denudation of the Rat Carotid Artery

Denudation of endothelial cells in the rat carotid artery by balloon injury reproducibly leads to an increase in intimal thickening when arteries are examined histologically within 2-4 weeks. Normal rat carotid contains no native intima, but at 2 weeks following balloon injury we found the intimal cross-sectional area was 0.2 mm^2 (±0.04), luminal narrowing was 56% (±12), and the intima/media ratio was 1.4 (±0.2) (n = 6 rats)(33) . The kinetics of smooth muscle proliferation in the rat carotid model of balloon injury have been previously determined by measurement of DNA synthesis as estimated by incorporation of tritiated thymidine(31) . These studies indicated that there was a burst of VSMC proliferation which reaches a maximum after 48 h in the media (46% of cells labeled) and 96 h in the intima (73% of cells labeled). The thymidine index did not return to base line until 4 weeks in the media and 8 weeks in the intima(31) . Segments of the intima which are not covered by regenerated endothelium still contain proliferating cells even 12 weeks after injury. Our studies have confirmed by staining with antibody against PCNA (proliferating cell nuclear antigen) that most of the cells at the luminal surface are still proliferating several weeks following injury (data not shown). Since we expected gax down-regulation to be one of the initial responses to injury, much earlier time points were examined in this study before evidence of proliferation would have been apparent.

Changes in gax mRNA levels in the rat carotid artery following endothelial denudation by injury with a balloon catheter were estimated using RT-PCR of RNA isolated from control (untreated) arteries and arteries harvested at various time points following injury. Electrophoresis of RNA samples on native agarose gels and visualization of ribosomal bands by ethidium bromide staining indicated that the RNA was undegraded during isolation (data not shown). However, it was apparent that the measurement of RNA concentration by UV absorbance at 260 nm is not sufficiently reliable to ensure equal loading of RNA samples for RT-PCR, since small inaccuracies may tend to be amplified in this procedure. As a better indicator of input RNA, therefore, we used G3PDH as an internal control gene and coamplified gax alongside G3PDH in the same reaction vessel (see ``Materials and Methods''). Since G3PDH mRNA is much more abundant than gax mRNA, amplification was for 25 and 30 cycles, respectively. Abundance of gax mRNA was expressed in arbitrary units derived from dividing gax expression (in counts/min contained in the band excised from the agarose gel) by that obtained for G3PDH. Fig. 1indicates the levels of gax mRNA in the rat carotid artery compared with the control untreated artery (0 h time point). Expression of gax mRNA significantly decreased after only 2 h following balloon injury (p < 0.001 versus controls) and reached its lowest point around 4 h. The mean value for gax mRNA at 4 h was 0.23 (±0.027) of the control untreated artery. Levels of gax started to recover by 24 h and were indistinguishable from controls after 7 days. This temporal pattern of gax down-regulation was consistent in five independent experiments. The small increase in gax at 6 h and decrease at 12 h were not statistically significant.


Figure 1: Time course of expression of gax following endothelial denudation by balloon angioplasty. Levels of gax mRNA were calculated in arbitrary units obtained by dividing the PCR yield of gax (in counts/min contained in band excised from agarose gel) by that of G3PDH. Each time point represents the mean value (±S.E.) of 6-10 PCR determinations expressed as a proportion of the control untreated arteries. Results are derived from five independently carried out experiments with three to five rats used for each time point.



Comparison of gax and c-myc Expression following Balloon Injury

To confirm that the results obtained did not merely reflect changes in the expression of the G3PDH gene, we included analysis, using the same RT-PCR method, of a gene which has been previously been documented to be up-regulated in response to balloon injury, namely c-myc(34) . Fig. 2shows the time course of c-myc expression relative to that of gax in the same set of rats. Up-regulation of c-myc appears to follow a similar time course as the down-regulation of gax, with the peak of c-myc expression matching the nadir of gax expression at 4 h following balloon injury.


Figure 2: Comparison of time course of gax and c-myc expression following endothelial denudation of the rat carotid artery by balloon angioplasty. Levels of gax were calculated in arbitrary units obtained by dividing the PCR yield of gax (in counts/min contained in band excised from agarose gel) by that of G3PDH. Determinations of gax and c-myc expression were made on cDNA from the same set of RNA samples.



Determination of -Fold Reduction in gax mRNA

Coamplification of gax and G3PDH provides a useful method for determining changes in mRNA abundance in many samples at different time intervals, but it may not be sufficiently precise to measure the exact extent of the change. To obtain an alternative estimate of the extent of gax down-regulation, therefore, we performed PCR on serial dilutions of cDNA derived from control arteries and from arteries harvested 4 h following balloon injury (Fig. 3). The difference in gax mRNA abundance is reflected in the PCR yield, and this difference is consistent during the exponential phase of the amplification and for the range of RNA input shown. When the logarithm of the PCR yield (in counts/min) is plotted against the logarithm of the input RNA (in nanograms) a straight line is obtained in the exponential phase of the reaction. Higher amounts of RNA input cause the reaction to approach plateau but only the exponential phase of the reaction is shown. The fact that the two lines are essentially parallel demonstrates that the efficiency of the amplification is similar for each RNA. The difference in gax mRNA abundance in the two samples is reflected in the ratio between PCR yields over a range of values for RNA input. This analysis indicated an approximately 5-fold decrease in gax mRNA abundance in response to balloon injury.


Figure 3: Quantification of gax mRNA in injured and control arteries by RT-PCR of dilution series of cDNA. PCR was performed on serial 1:2 dilutions of the cDNA obtained by reverse transcription of arterial RNA. Following agarose gel electrophoresis, radioactive bands were excised and counted. For each sample the logarithm of the PCR yield (in counts/min) was plotted against the logarithm of the RNA (in nanograms) put into the RT-PCR after normalization for G3PDH levels. The difference in PCR yield reflects the difference in RNA input within the exponential phase of the PCR. For the four values of input RNA compared here, the mean PCR yield from the control RNA was 5.06 ± 0.27-fold higher than that from the injured artery. box, control; bullet, injured.



Northern Blot Analysis

To confirm the results of RT-PCR we performed Northern blots on RNA taken from arteries which PCR analysis had indicated contained the highest and lowest levels of gax mRNA, namely, untreated control arteries and arteries harvested 4 h following injury, respectively. In order to detect gax by Northern blot, it was necessary to overload the sample lanes with 40 µg of total RNA, and three rats were required for each sample. The same blot was hybridized sequentially with probes for gax, c-myc, and G3PDH. A low abundance gax transcript of approximately 2.3 kb was detected in RNA isolated from untreated rat carotid arteries (Fig. 4). The size of the c-myc transcript is very close to that of gax at 2.4 kb, and the G3PDH transcript appears around 1.4 kb. It is important to note that the times of exposure of the blot required to visualize the mRNA bands following each hybridization were very different, which reflects the relative abundance of each mRNA species. Thus, G3PDH mRNA was visible after only 1-h exposure, c-myc after 12 h, but gax required 5 days before a clear band was detectable on the autoradiogram. It is apparent in Fig. 4that gax mRNA is down-regulated following balloon injury. The gax transcript is clearly detectable in the untreated arteries but falls to undetectable levels 4 h following injury. In contrast, c-myc mRNA is almost undetectable in the RNA from the uninjured artery in which the gax transcript was clearly visible, whereas 4 h following injury the c-myc band appears. This result confirms that the gene encoding gax is down-regulated by the same stimulus which causes an up-regulation of the gene for c-myc. We used G3PDH to control for possible differences in loading or degradation of RNA. Relatively constant levels of G3PDH mRNA were seen in all RNA samples. Since gax mRNA was not detectable in the treated arteries, we could make no estimate of the extent of its down-regulation by densitometric analysis.


Figure 4: Northern blot to demonstrate expression of gax, c-myc, and G3PDH following endothelial denudation by balloon angioplasty. Each lane contains total RNA pooled from three rat carotid arteries (40 µg). The three lanes on the left contain control RNA from uninjured arteries, whereas the three lanes on the right contain RNA from injured arteries harvested 4 h following balloon angioplasty. The blot was hybridized to each probe sequentially and stripped after each hybridization.




DISCUSSION

Arterial smooth muscle cells proliferate in response to endothelial denudation(35) . This process is key to the development of atherosclerotic lesions and restenosis following treatment by balloon angioplasty. Associated with proliferation is a switch in phenotype toward the neonatal or embryonic genetic program. A number of phenotypic markers have been identified which are re-expressed in proliferating VSMC. Most of these markers are presumably regulated by control genes which may activate cells to enter (or exit) the cell cycle. In addition to oncogenes, homeobox genes have been implicated in such processes during development and differentiation. In quiescent VSMC, nuclear proto-oncogenes such as c-fos, c-myc, and c-myb are transcriptionally induced by growth factors. Previous work has shown that gax is expressed at low levels in quiescent vascular smooth muscle cells, and its expression falls to levels undetectable by Northern blot 4 h following stimulation of the cells to proliferate using serum or platelet-derived growth factor. To find out if the same phenomenon could be demonstrated in vivo, we used the rat carotid model of balloon injury which has been used to demonstrate changes in the expression of a number of genes in response to balloon injury(3, 12, 15, 36, 37) .

Because gax is a low abundance mRNA, as are other homeobox gene transcripts, it is difficult to detect by Northern blot analysis unless large amounts of RNA are used. For time course studies, therefore, gax mRNA levels in the rat carotid artery were determined by RT-PCR. A number of different protocols have been described for using PCR to obtain quantitative information regarding mRNA levels(38, 39, 40, 41, 42, 43, 44) , and each of these protocols has its own particular drawbacks. We elected, therefore, to use two different methods of quantitative PCR: in the first method, G3PDH was coamplified as an internal control gene. This method of quantitative RT-PCR is useful when the amount of RNA available for analysis is very small. Only 1 µg was needed for the analysis of each artery. A similar method was recently used to demonstrate decreased levels of mRNA encoding myotonin-protein kinase in adults with myotonic dystrophy (45) , although in that case a different internal control gene (the transferrin receptor) was used. The time course obtained here was similar to that obtained in vitro with cultured smooth muscle cells(19) . The second method, which utilizes PCR analysis of serial dilutions of RNA(38) , indicated that there was an approximately 5-fold decrease in gax mRNA abundance 4 h following balloon injury. The -fold decrease was considerably less than that seen in cultured cells (5-fold as compared with 20-fold), and this difference may be due to the heterogeneity of the cells in the artery wall as compared with a cell culture with respect to their response to local injury.

The down-regulation of gax mRNA in response to balloon injury was confirmed by Northern blot analysis using only the time points deemed to contain the highest and lowest amounts of gax mRNA, namely the uninjured control arteries and those harvested 4 h following balloon angioplasty. Fig. 4clearly demonstrates the difference in gax mRNA abundance between injured and uninjured arteries. We showed that this difference is not a result of differences in RNA loading or RNA degradation by hybridizing the same blot with G3PDH. Finally, by hybridizing the blot with a probe for c-myc, we confirmed that c-myc is up-regulated in the injured artery at the same time that gax is down-regulated. It should be noted that the results shown here do not indicate how the decrease in gax mRNA abundance is achieved. This could conceivably come about as a result of transcriptional regulation or decreased mRNA stability.

The time course of gax down-regulation and recovery in vivo in response to balloon injury is remarkably similar to that seen in vitro when quiescent VSMCs are stimulated by mitogens. Our knowledge of the two systems is currently limited to the mRNA level, because immunochemical detection of the Gax protein has been largely unsuccessful, presumably because levels of the transcription factor are normally so low.

Down-regulation of gax is one of the earliest events in the proliferative response. In the rat carotid model the maximal intima/media ratio and luminal narrowing is observed histochemically 2 weeks following injury. Measurement of DNA synthesis as estimated by incorporation of tritiated thymidine indicated that VSMC proliferation reached a maximum after 48 h in the media and 96 h in the intima(31) . Our own studies using staining with antibody against PCNA (proliferating cell nuclear antigen) confirmed that most of the cells at the luminal surface are still proliferating 1 and 2 weeks following injury. (^2)gax expression, therefore, reaches its nadir 4 days before maximal proliferation in the intima and returns to normal levels, while proliferation is still extant. If, as has been proposed(19) , down-regulation of the gax gene is in fact required for VSMCs to enter the cell cycle, then this down-regulation should be detectable in these arteries given these large values for proliferation. Nonproliferating cells, however, which would still be expressing gax, would be expected to dilute the apparent effect. The extent of the down-regulation detected, therefore, is a function of the proportion of nonproliferating cells in the whole artery segment which includes intima, media, and adventitia. This explains the fact that we only see a modest 5-fold down-regulation in vivo as compared with a >20-fold down-regulation in synchronized cells in vitro. Levels of gax mRNA in the artery segment apparently return to normal after 7 days, even though there are still proliferating cells near the luminal surface. It is possible that these cells have down-regulated the gax gene but that this effect is undetectable in the midst of the large number of quiescent VSMCs which are expressing or reexpressing gax. Using in situ hybridization we were unable to detect the presence of local concentrations of cells not expressing gax (data not shown), as might be expected for instance on the luminal surface where PCNA-positive cells are known to accumulate. This may be related to the low resolution of this technique when used to detect a low abundance mRNA. In summary, the present report is the first documentation of a growth arrest-specific gene in vivo. While previous reports have demonstrated up-regulation of several genes following balloon injury and/or angioplasty, the present report establishes for the first time down-regulation of a regulatory gene within h of balloon injury. The data presented support the concept that gax is an important gene regulating proliferation of vascular smooth muscle cells.


FOOTNOTES

*
This work was supported by Grants AR 40580 (to L. W.), HL 40518 and HL 02428 (to J. M. I.), and AR40197 and HL50692 (to K. W.) from the NHLBI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cardiology, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 01748. Tel.: 617-789-3156; Fax: 617-789-5029, LWEIR{at}Tufts.edu.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cell(s); G3PDH, glyceraldehyde 3-phosphate dehydrogenase; RT, reverse transcription; kb, kilobase pair(s); PCNA, proliferating cell nuclear antigen.

(^2)
C. Pastore, unpublished data.


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