(Received for publication, December 15, 1994)
From the
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.
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 ()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.
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.
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.
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.
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. , control;
,
injured.
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.
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. ()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.