BACKGROUND
many vascular diseases have the tendency to manifest at specific sites in the vasculature.
aortic aneurysms are one such disease with approximately 90% developing between the renal arteries and iliac bifurcation.
aneurysms develop less frequently in the ascending and descending thoracic aorta, and are distinct from abdominal aortic aneurysms  in prevalence, risk factors, genetics and histology  <cit> .
several intrinsic structural differences between the abdominal and thoracic aorta may contribute to aneurysm susceptibility  <cit> .
the abdominal aorta has a narrower diameter and contains several major branch points, both of which result in turbulent blood flow and decreased shear stress.
areas of low shear stress are more susceptible to atherosclerotic lesions.
in addition, the abdominal aortic wall is thinner than that of the thoracic aorta, contains fewer elastic lamellae, as well as decreased amounts of the structural proteins, elastin and collagen.
these structural differences may confer greater susceptibility to injury and dilatation to the abdominal aorta.
there are also important embryological differences between the thoracic and abdominal aorta.
neural crest-derived smooth muscle cells  populate the ascending thoracic aorta, while mesoderm-derived cells are found in the abdominal aorta  <cit> .
these distinct cell populations respond differently to growth factors such as tgf-β  <cit> .
one group of genes that may contribute to regional heterogeneity in the vasculature is the homeobox  transcription factors, which are known to control embryonic spatial patterning.
in multiple adult cell types microarray analyses found a correlation between hox gene expression and anatomic site of origin  <cit> .
animal experiments have demonstrated distinct hox gene expression domains within the vasculature  <cit> .
finally, hox genes regulate proliferation and differentiation of vascular cells  <cit> .
characterization of regional heterogeneity in the aorta may help identify novel mechanisms of aaa formation.
in the current study we used microarray-based expression profiling to identify genes whose expression differed along the aorta in baboons.
we identified several members of the hox family of transcription factors with spatial expression changes, as well as reduced mrna levels in samples from human aaa.
further studies on hoxa <dig> with human aortic samples confirmed its spatial expression pattern and identified it as a novel gene of interest for human aaa pathogenesis.
methods
baboon samples
full thickness aortic wall tissue specimens were collected from baboons   euthanized for old age at the southwest national primate research center, san antonio, texas, usa.
the aortas were divided into 10- <dig> segments , placed in rnalater  solution and processed for rna isolation.
aortic segments were assigned similar numbers based on anatomical landmarks including the aortic arch , renal arteries  and iliac bifurcation .
human samples
full thickness aortic wall tissue specimens  were collected from patients undergoing aaa repair  operations at the harper university hospital, detroit, michigan or at geisinger medical center, danville, pennsylvania, usa, and from controls without aneurysms  at autopsies or through the national disease research interchange .
samples were stored in rnalater for rna isolations , fixed in phosphate-buffered formalin  for histological and immunohistochemical analyses, snap-frozen in liquid nitrogen for protein analyses, or embedded in o.c.t.-media  for immunohistochemical analyses.
the collection of the human tissues was approved by the institutional review boards of wayne state university, detroit, michigan, usa, and geisinger clinic, danville, pennsylvania, usa.
rna isolation, microarray studies, and rt-pcr
we isolated total rna from baboon and human blood vessels as described previously  <cit> .
quality of rna was assessed by bioanalyzer  and quantified using a nanodrop instrument .
our laboratory used two microarray platforms to generate global mrna expression profiles for both aneurysmal and non-aneurysmal human abdominal aorta.
the details on these studies have been described previously  <cit> , and the microarray data can be obtained at the gene expression omnibus  database .
details on the samples used for the microarray studies can be found in additional file  <dig>  table s <dig>  the aaa case  and control  groups were matched for age, sex and ethnicity .
for the baboon experiments  <dig> ng of total rna was reverse transcribed and  <dig>  μg of crna was hybridized to either sentrix human wg- <dig> version  <dig>  or humanref- <dig> version  <dig> microarrays  .
qc procedures included examination of raw and adjusted intensity histograms and principal component analysis for systematic bias.
we used the "detection score" to determine expression in the illumina platform.
the illumina data were adjusted  using a cubic spline function.
to identify genes with relative increases or decreases in expression along the length of the aorta, linear regression was performed using the r statistical package  <cit> .
99% confidence intervals  were constructed for the calculated slope and those probes with a ci not crossing zero were considered to have increasing or decreasing expression.
real-time quantitative rt-pcr for hoxa <dig> on rna isolated from human aaa samples  and control  abdominal aorta samples  was performed using a commercially available assay  according to the manufacturer's instructions .
aaa case and aortic control groups did not differ significantly by age .
levels of 18s rna were also determined to standardize the results.
all experiments were run in triplicate.
after pcr, amplification plots were inspected and baselines and threshold values were set for signals using sequence detection system software according to the manufacturer's recommendations .
the threshold cycle numbers  were computed for each well.
for calculating the Δct values for each well and processing the results further, the raw data were transferred into a microsoft access database  <cit> .
briefly, 18s amplification ct values were subtracted from the hoxa <dig> ct values for each sample to calculate the Δct  <cit> .
the p-values were calculated using the wilcoxon rank-sum test.
the results of the statistical analysis were summarized using the program r  <cit>  and are presented as a box-and-whiskers plot.
western blot analysis of aortic proteins
between  <dig> and  <dig> mg of flash-frozen aortic tissue was homogenized in buffer consisting of  <dig> mm tris,  <dig> mm nacl,  <dig> mm edta,  <dig> mm egta, 1% triton x- <dig>   <dig> % sds, and 1x mammalian protease inhibitor cocktail , ph  <dig> .
additionally, either 1x halt phosphatase inhibitor  or phosphatase inhibitor cocktail ii  was included in the homogenization buffer.
homogenized samples were centrifuged and supernatants were used for analysis.
protein concentrations were determined using the bicinchoninic bicinchoninic acid  assay from pierce following the manufacturer's instructions .
a total of  <dig> μg of protein was used for hoxa <dig> and actb, and  <dig>  μg for acta <dig> analyses.
samples were loaded onto precast 4-15% gradient polyacrylamide gels  and electrophoresed for  <dig> minutes at  <dig> v. proteins were transferred to nitrocellulose membranes in a buffer containing 20% methanol,  <dig>  m glycine, and  <dig> mm tris in a bio-rad mini trans-blot cell at  <dig> v for  <dig> hour.
after protein transfer, blots were fixed at room temperature for  <dig> minutes in a solution containing 25% isopropanol and 10% acetic acid, rinsed briefly with  <dig>  m tris,  <dig>  m nacl, ph  <dig>  with  <dig> % tween  <dig>  and blocked for  <dig> minutes at room temperature in 5% skim milk in tbst.
blots were then transferred to heat-sealable bags and incubated overnight at 4°c with primary antibody  diluted in blocking solution.
following washes the blots were incubated with hrp-conjugated igg preabsorbed species-specific secondary antibody  diluted to 1: <dig>  in tbst containing 4% serum of the secondary antibody species.
after a final washing in tbst, blots were visualized using pierce supersignal west pico enhanced chemiluminescent substrate according to manufacturer's instructions and a bio-rad chemidoc imaging system.
autoradiography film exposures were also obtained for each blot .
the hoxa <dig> antibody  was produced against a synthetic peptide derived from residues  <dig> -  <dig> of human hoxa <dig>  the immunogen is specific for hoxa <dig> in ncbi blast search and shows no similarity to other hox proteins.
the same antibody has been used previously by ota et al.
<cit> .
blots were quantified using quantity one® software .
an intensity-per-area value was obtained for each sample band as well as for a control sample loaded on all blots.
band intensities were adjusted between blots using control sample intensities.
intensity values adjusted for expression of either acta or actb were input into the r statistical package  <cit> .
a wilcoxon signed rank test was used to compare the intensity values of protein bands from thoracic and abdominal aortic wall samples.
linear regression with stepwise backwards elimination was used to evaluate age, race and sex as predictors of atcb-adjusted hoxa <dig> protein levels.
immunohistochemical staining of aortic tissues
immunohistochemical staining of o.c.t-embedded frozen tissues was carried out using the vectastain elite avidin:biotinylated enzyme complex  kit .
air-dried slides with 8-μm sections were fixed in methanol with 3% hydrogen peroxide to quench endogenous peroxidases and then blocked with serum for  <dig> minutes.
incubation with hoxa <dig> primary antibody , secondary antibody , abc reagent, and diaminobenzidine substrate were performed according to the manufacturer's instructions.
each treatment was preceded by a 10-minute pbs wash. immunostained slides were counterstained with hematoxylin and mounted with a coverslip.
non-specific igg antibody in lieu of primary antibody served as a negative control.
when using formalin-fixed paraffin embedded aortic tissue, 5-μm sections were used in a protocol described previously  <cit> .
the slides were incubated with hoxa <dig> primary antibody  <cit>   on an automatic immunostainer .
a secondary antibody with avidin-biotin peroxidase amplification from dako was used and the signal detected using diaminobenzidine as a chromogen.
images were obtained using either a leica dm4000b  or nikon  microscope.
western blot analysis of protein lysates from cultured cells
human aortic smcs , human aortic endothelial cells , and monocyte/macrophage cells  listed in additional file  <dig>  table s <dig> were cultured in appropriate medium according to suppliers' recommendations.
aortic ecs and smcs were stimulated for  <dig> hours using  <dig> ng/ml ifn-γ   <cit> .
monocyte/macrophage cell lines were stimulated  <dig> hours using  <dig> ng/ml lps , and  <dig> ng/ml ifn-γ  <cit> .
during stimulation, serum concentrations were half the concentrations used for maintenance culture.
this stimulation was considered relevant to aaa, since mice lacking ifn-γ are resistant to aaa formation in the cacl <dig> model  <cit>  and ifn-γ-producing t cells are present in the blood and aortic wall of most aaa patients  <cit> .
cells were washed twice with cold pbs and lysed in ripa buffer with included protease inhibitors .
cytoplasmic and nuclear extracts were also prepared using ne-per nuclear and cytoplasmic extraction reagents  according to the manufacturer's recommendations.
cell lysates were subjected to sds-page, transferred to pvdf membrane and immunoblotted with anti-hoxa <dig> rabbit polyclonal antibody  or anti-actb mouse monoclonal antibody .
blots were quantified to obtain an intensity-per-area value for each band.
intensity values were adjusted for actb protein levels.
actb-adjusted band intensity values were input into the r statistical package  <cit> .
repeated measures mixed effects linear model , was used to evaluate the effect of inflammatory stimulation on hoxa <dig> levels.
fluorescence microscopy on cultured cells
human aortic smcs  and human aortic ecs  were grown on 2-well permanox chamber slides , fixed with 3% paraformaldehyde and permeabilized with  <dig> % triton x- <dig> in pbs, and then incubated with rhodamine-phalloidin  at 1: <dig> dilution, and anti-hoxa <dig> with 1: <dig> dilution , followed by incubation with fluorescein-conjugated affinity-purified donkey anti-rabbit igg .
the nuclei were stained with dapi.
the stained cells were examined using a zeiss observer z <dig> inverted light microscope equipped for epifluorescence.
RESULTS
regional differences in the expression of hox genes in the baboon aorta
to identify novel candidate genes with spatial expression differences in the aorta, genome-wide expression profiling was undertaken using segments along the entire aorta  from baboons , which have been used as a model organism for vascular disease  <cit> .
aortic tissue from the baboons was preserved immediately following euthanasia and high-quality rna was subsequently extracted.
as no baboon-specific microarrays were available, rna was hybridized to human arrays.
without a completed baboon genome the full extent of sequence conservation is not known, but over 95% conservation would be expected based on sequence similarity between humans and other primate species.
for highly conserved genes such as the hox family of genes, this degree of conservation is expected; hoxa <dig> coding region sequence identity is  <dig> %  and protein sequence identity is  <dig> %  between humans and baboons.
genes with increasing or decreasing trends in expression along the baboon aorta included several members of the hox gene family.
figure  <dig> shows the regional expression of hoxa <dig> in two baboon aortas separately .
differences in the expression of hox genes in human abdominal aortic aneurysms compared to matched control aortas
the regional expression of hox genes in the baboon aorta led us to hypothesize that hox gene expression might be altered in human aaa.
indeed, using microarray data generated previously  <cit> , we found  <dig> homeodomain-containing genes which were expressed in the human aorta ;  <dig> of these had significantly different mrna levels  in aaa compared to control abdominal aortic tissues from an age-, sex-, and ethnicity-matched group of individuals .
ten of the  <dig> differentially expressed homeodomain-containing genes were classic hox-genes; the mrna levels of all of them were significantly decreased in aaa .
hoxa <dig> was the most significantly decreased hox gene in human aaa tissue based on microarray results.
in a different set of  <dig> aaa cases and  <dig> similarly-aged controls  real-time quantitative rt-pcr was used to confirm the decrease in hoxa <dig> mrna expression .
we also detected hoxa <dig> mrna in cultured human aortic smcs and aortic ecs .
regional differences in the abundance of hoxa <dig> protein in the human aorta
western blot analysis using paired human thoracic and abdominal aortic samples  was performed to determine whether the observed spatial differences in baboon mrna expression were reflected in differences in protein levels in human aorta.
in non-aneurysmal aortic tissue hoxa <dig> was detected using a specific antibody  <cit>  as a single band at ~ <dig> kda .
after adjusting for β-actin , the level of hoxa <dig> protein was significantly higher in thoracic than the abdominal aorta , consistent with the baboon microarray analysis .
smooth muscle α-actin  protein levels were also investigated to assess whether the difference in the abundance of hoxa <dig> was a result of higher smc density in the thoracic aorta.
after adjusting for α-actin levels, hoxa <dig> remained significantly higher in the thoracic aorta than in the abdominal aorta .
the potential effects of age, race and sex  on hoxa <dig> protein levels were also investigated.
linear regression with stepwise backwards elimination showed age to be the only significant predictor of hoxa <dig> protein abundance, with evidence that protein levels decrease with age .
immunohistochemical localization of hoxa <dig> in aortic tissue samples
formalin-fixed paraffin-embedded samples of human aortic tissues were used for immunohistochemical staining.
hoxa <dig> staining was observed in the nuclei and perinuclear regions of several cell types in all three layers of the aorta .
staining patterns appeared to change with age.
in samples from both young and old individuals staining was observed in and around the nuclei, but staining tended to concentrate more in the nuclei of samples from young individuals whereas samples from older individuals showed more diffuse perinuclear staining .
additionally, an intense punctate staining pattern was observed in samples from older individuals that was not present in younger samples .
similar staining patterns, including the punctate staining, were also observed with frozen tissue sections , demonstrating that it was not an artifact related to tissue preparation.
characterization of hoxa <dig> expression in cultured vascular cells
since inflammation is a hallmark of aaa pathogenesis  <cit> , we examined whether inflammatory stimuli altered levels of hoxa <dig> in cultured human vascular cells.
human macrophage/monocytes  were stimulated using both ifn-γ and lps, whereas human aortic ecs , and human aortic smcs  were stimulated with ifn-γ.
hoxa <dig> protein levels were analyzed by western blots and compared with unstimulated control cells.
this stimulation was considered relevant to aaa, since mice lacking ifn-γ are resistant to aaa formation in the cacl <dig> model  <cit>  and ifn-γ-producing t cells are present in the blood and aortic wall of most aaa patients  <cit> .
all but one of the cell lines  exhibited downregulation of hoxa <dig> levels within  <dig> hours of exposure to inflammatory stimuli .
the decrease in hoxa <dig> levels was statistically significant .
when investigating the cytoplasmic and nuclear fractions of proteins separately, the relative amount of hoxa <dig> protein  in the nucleus decreased with inflammatory stimulation  in all cell types.
immunofluorescent staining for hoxa <dig> was also performed using cultured human aortic ec and human aortic smcs.
in both cell types the strongest signal was observed in the perinuclear region with a small amount of nuclear staining .
consistent with previously published results, stimulated smcs had fewer large actin stress fibers visualized by co-staining with rhodamine-phalloidin, whereas stress fibers in ecs did not decrease with stimulation  <cit> .
discussion
the results of the current study provide evidence of differences in gene expression between the descending thoracic and abdominal aorta, particularly among the hox family of transcription factors.
additionally, several hox transcription factors demonstrated altered expression in human aaa compared to a group of age-, race- and sex-matched control aorta, with hoxa <dig> being the most downregulated.
quantitative rt-pcr using a separate set of samples confirmed that hoxa <dig> mrna levels were decreased in aaa and western blots demonstrated that protein levels were lower in the human abdominal than thoracic aorta.
immunohistochemical staining suggested expression of hoxa <dig> in smcs and ecs in aortic tissue sections.
finally, hoxa <dig> was observed in the nuclear and perinuclear regions of cultured aortic ecs and smcs with decreased protein levels after stimulation with inflammatory cytokines.
hox genes were originally identified for their functions in embryonic patterning.
increasing evidence suggests, however, that these genes also have a role in maintaining spatial identity of tissues, including vasculature, as demonstrated by microarray analyses  <cit>  and animal models  <cit> .
the results of our microarray analyses using the baboon aortas were consistent with these studies, demonstrating that the expression of hox genes varies spatially, in particular between the more aneurysm-susceptible abdominal aorta and the less aneurysm-susceptible thoracic aorta.
several of these hox genes have been implicated in vascular function and disease, supporting the hypothesis that regional gene expression could alter disease susceptibility  <cit> .
a recent study supports this hypothesis, with hoxa <dig> exhibiting higher expression at atherosusceptible sites in the porcine aorta  <cit> .
to assess the potential role of hox genes in aaa pathogenesis, their expression profiles from human aaa and control aorta were compared, demonstrating significant downregulation of  <dig> classic hox genes in aaa tissue.
these findings are consistent with previous array-based studies, which demonstrated downregulation of hox genes in atherosclerosis and ulcerative colitis compared with non-diseased aorta and colon, respectively  <cit> .
protein analyses described here using cultured human vascular cells demonstrated a decrease in hoxa <dig> protein levels after in vitro stimulation with ifn-γ or lps and show that inflammatory stimuli alter hox gene expression.
previously, treatment with interleukin 1ß , an inflammatory cytokine implicated in aaa pathogenesis  <cit> , was shown to downregulate several hox genes  <cit> .
downregulation of hox genes could have several detrimental effects.
hox genes have well-established roles in regulating cell proliferation and differentiation  <cit> ; downregulation of hox genes could alter cell phenotype in aaa.
decreased hox gene expression could alter transcriptional regulation; hox gene targets include spp <dig>  <cit>  and the matrix metallopeptidases  <cit> , which have been implicated in aaa pathogenesis  <cit> .
in the current study we concentrated on hoxa <dig>  since it exhibited the most significant downregulation in human aaa tissue, had a distinct regional expression pattern along the aorta, and a specific antibody against it was available.
western blots confirmed regional variation in hoxa <dig> protein abundance, with significantly higher hoxa <dig> levels in human thoracic aorta compared with abdominal aorta.
immunohistochemical staining suggested that multiple cell types in the aorta express hoxa <dig>  primarily ecs, and smcs, as well as some adventitial fibroblasts.
immunofluorescent staining of cultured ecs and smcs showed nuclear and perinuclear protein localization of hoxa <dig>  hoxa <dig> expression in both ecs and smcs is interesting, as human hoxa <dig> and murine hoxa <dig> are also expressed in both endothelium and smooth muscle, whereas murine hoxc <dig> is expressed in the smc only, suggesting that expression in both ec and smc is characteristic of the hoxa gene cluster  <cit> .
similar to other hox genes, hoxa <dig> is involved in embryonic patterning.
its function in embryogenesis does not appear to be critical, since targeted disruption of hoxa <dig> results in a relatively mild phenotype; hoxa <dig> knockout mice are viable and exhibit only some deformations of the cervical spine  <cit> .
conversely, transgenic mice overexpressing the hoxa <dig> gene develop congenital megacolon due to abnormalities in the enteric nervous system  <cit> .
studies in chick embryos suggest that dfd, the hoxa4-like gene in chickens, is not critical to aortic arch artery patterning, since treatment of the cardiac neural fold with a dfd antisense oligonucleotide resulted in no apparent change in heart development at stage  <dig> to  <dig>  <cit> .
it is possible that hoxa <dig> is involved in regulating angiogenic function similar to other hox genes.
an in vitro comparison of ovarian carcinoma cell lines showed that cells exhibiting higher levels of hoxa <dig> expression have decreased motility by scratch assay  <cit> .
similarly, overexpression of hoxa <dig> further decreased cell motility and led to a corresponding increase in ß <dig> integrin  <cit> .
ß <dig> integrin has been implicated in aaa pathogenesis and is decreased in aaa tissues compared to non-aneurysmal aorta  <cit> .
sirna knockdown of hoxa <dig> does not ablate ß <dig> integrin expression, however, which is consistent with hox genes having redundant functions  <cit> .
a role for hoxa <dig> in mediating cell contacts is further supported by an increase in basal lamina thickness and collagen in the colonic mucosa of transgenic mice overexpressing hoxa <dig>  <cit> .
it is, therefore, plausible that loss of hoxa <dig> function in medial smcs contributes to adverse phenotypic changes in aneurysms, including disruption of cell-cell interactions.
hoxa <dig> can bind to its own promoter region and there is some evidence for hoxa <dig> regulating the adjacent hoxa <dig> gene under certain conditions  <cit> .
additionally, hoxa <dig> has also been implicated in the regulation of microrna genes during monocyte differentiation, suggesting micrornas as one mechanism for hox gene regulation of cell-type specific effects  <cit> .
CONCLUSIONS
our results suggest that hoxa <dig> is important for maintaining spatial identity even in the adult aorta and that downregulation of hoxa <dig> expression may increase susceptibility to aortic disease such as aaas.
to test these hypotheses embryonic targeted deletions of hoxa <dig> in animal models are needed to determine the exact contribution of this gene family to maintaining spatial identity.
similarly, targeted deletion mutants could be employed to prove that downregulation of hoxa <dig> increases susceptibility to aaa in a mouse model.
authors' contributions
jhl participated in the design of the study, analyzed microarray data, carried out western blot experiments with human tissue lysates, and drafted the manuscript.
re carried out all experiments with cultured cells and prepared illustrations.
cms and zg carried out and interpreted the immunohistochemical analyses.
ag and kd catalogued phenotypic information, processed samples, and carried out rt-pcrs.
lac provided baboon samples.
js and ll interpreted results.
rsvh and lh provided control samples and interpreted results.
gml participated in the design of the study, analyzed microarray data and interpreted results.
jre, dpf, jlg and rpg recruited aaa patients for the study and collected tissue specimens.
djc and wdl interpreted results.
gt participated in the design of the study, analyzed results, carried out statistical analyses, prepared illustrations and interpreted results.
hk obtained funding for the study, participated in the design of the study, interpreted results, and participated in drafting the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
table s <dig>  human tissue samples from non-aneurysmal aortas used in assessing protein abundance of hoxa <dig>  list of sample ids with donor age, ethnicity, sex and cause of death for human samples used to study hoxa <dig> protein levels.
click here for file
additional file 2
table s <dig>  human aortic tissue samples used in microarray and q-rt-pcr experiments.
list of sample ids with donor disease status classification, ethnicity, sex, age, and cause of death for human samples used in microarray and rt-pcr studies.
click here for file
additional file 3
figure s <dig>  specificity of hoxa <dig>  acta <dig> and actb antibodies.
images of western blots performed with antibodies used in the study.
click here for file
additional file 4
table s <dig>  commercially available human cell lines for hoxa <dig> experiments.
list of cell lines used in the study with codes, description of cell type, vendor, and catalog number.
click here for file
additional file 5
table s <dig>  mrna expression of hox genes in human aneurysmal  and non-aneurysmal  abdominal aorta based on microarray analyses.
list of differentially expressed homeodomain-containing genes in human aaa compared to age-sex-and ethnicity-matched controls.
columns give the full protein name, gene symbol, geneid, expression levels in control group and aaa group, fold-change in expression, false discovery rate and illumina detection score in control and aaa group.
click here for file
additional file 6
figure s <dig>  localization of hoxa <dig> protein in cultured human aortic ecs  and smcs  using immunofluorescence.
images of stained cultured cells.
click here for file
acknowledgements and funding
we thank dr. susan land, daniel lott and sara mcnorton at the applied genomics technology center of wayne state university for their work on the microarray experiments.
this project was funded in part by a grant from the national heart, lung, and blood institute , as well as by funds from the geisinger clinic.
j.h.l.
is a recipient of a predoctoral fellowship from the national institute on aging, nih , and in 2005- <dig> g.m.l.
was a recipient of a predoctoral fellowship from the american heart association .
we acknowledge use of human aortic tissues provided by the national disease research interchange , with support from nih grant  <dig> u <dig> rr006042- <dig>

 many vascular diseases have the tendency to manifest at specific sites in the vasculature. aortic aneurysms are one such disease with approximately 90% developing between the renal arteries and iliac bifurcation. aneurysms develop less frequently in the ascending and descending thoracic aorta, and are distinct from abdominal aortic aneurysms in prevalence, risk factors, genetics and histology. several intrinsic structural differences between the abdominal and thoracic aorta may contribute to aneurysm susceptibility. the abdominal aorta has a narrower diameter and contains several major branch points, both of which result in turbulent blood flow and decreased shear stress. areas of low shear stress are more susceptible to atherosclerotic lesions. in addition, the abdominal aortic wall is thinner than that of the thoracic aorta, contains fewer elastic lamellae, as well as decreased amounts of the structural proteins, elastin and collagen. these structural differences may confer greater susceptibility to injury and dilatation to the abdominal aorta. there are also important embryological differences between the thoracic and abdominal aorta. neural crest-derived smooth muscle cells populate the ascending thoracic aorta, while mesoderm-derived cells are found in the abdominal aorta. these distinct cell populations respond differently to growth factors such as tgf-β. one group of genes that may contribute to regional heterogeneity in the vasculature is the homeobox transcription factors, which are known to control embryonic spatial patterning. in multiple adult cell types microarray analyses found a correlation between hox gene expression and anatomic site of origin. animal experiments have demonstrated distinct hox gene expression domains within the vasculature. finally, hox genes regulate proliferation and differentiation of vascular cells. characterization of regional heterogeneity in the aorta may help identify novel mechanisms of aaa formation. in the current study we used microarray-based expression profiling to identify genes whose expression differed along the aorta in baboons. we identified several members of the hox family of transcription factors with spatial expression changes, as well as reduced mrna levels in samples from human aaa. further studies on hoxa with human aortic samples confirmed its spatial expression pattern and identified it as a novel gene of interest for human aaa pathogenesis. methods baboon samples full thickness aortic wall tissue specimens were collected from baboons euthanized for old age at the southwest national primate research center, san antonio, texas, usa. the aortas were divided into 10- segments, placed in rnalater solution and processed for rna isolation. aortic segments were assigned similar numbers based on anatomical landmarks including the aortic arch, renal arteries and iliac bifurcation. human samples full thickness aortic wall tissue specimens were collected from patients undergoing aaa repair operations at the harper university hospital, detroit, michigan or at geisinger medical center, danville, pennsylvania, usa, and from controls without aneurysms at autopsies or through the national disease research interchange. samples were stored in rnalater for rna isolations, fixed in phosphate-buffered formalin for histological and immunohistochemical analyses, snap-frozen in liquid nitrogen for protein analyses, or embedded in o.c.t.-media for immunohistochemical analyses. the collection of the human tissues was approved by the institutional review boards of wayne state university, detroit, michigan, usa, and geisinger clinic, danville, pennsylvania, usa. rna isolation, microarray studies, and rt-pcr we isolated total rna from baboon and human blood vessels as described previously. quality of rna was assessed by bioanalyzer and quantified using a nanodrop instrument. our laboratory used two microarray platforms to generate global mrna expression profiles for both aneurysmal and non-aneurysmal human abdominal aorta. the details on these studies have been described previously, and the microarray data can be obtained at the gene expression omnibus database. details on the samples used for the microarray studies can be found in additional file table s the aaa case and control groups were matched for age, sex and ethnicity. for the baboon experiments ng of total rna was reverse transcribed and μg of crna was hybridized to either sentrix human wg- version or humanref- version microarrays. qc procedures included examination of raw and adjusted intensity histograms and principal component analysis for systematic bias. we used the "detection score" to determine expression in the illumina platform. the illumina data were adjusted using a cubic spline function. to identify genes with relative increases or decreases in expression along the length of the aorta, linear regression was performed using the r statistical package. 99% confidence intervals were constructed for the calculated slope and those probes with a ci not crossing zero were considered to have increasing or decreasing expression. real-time quantitative rt-pcr for hoxa on rna isolated from human aaa samples and control abdominal aorta samples was performed using a commercially available assay according to the manufacturer's instructions. aaa case and aortic control groups did not differ significantly by age. levels of 18s rna were also determined to standardize the results. all experiments were run in triplicate. after pcr, amplification plots were inspected and baselines and threshold values were set for signals using sequence detection system software according to the manufacturer's recommendations. the threshold cycle numbers were computed for each well. for calculating the Δct values for each well and processing the results further, the raw data were transferred into a microsoft access database. briefly, 18s amplification ct values were subtracted from the hoxa ct values for each sample to calculate the Δct. the p-values were calculated using the wilcoxon rank-sum test. the results of the statistical analysis were summarized using the program r and are presented as a box-and-whiskers plot. western blot analysis of aortic proteins between and mg of flash-frozen aortic tissue was homogenized in buffer consisting of mm tris, mm nacl, mm edta, mm egta, 1% triton x- % sds, and 1x mammalian protease inhibitor cocktail, ph. additionally, either 1x halt phosphatase inhibitor or phosphatase inhibitor cocktail ii was included in the homogenization buffer. homogenized samples were centrifuged and supernatants were used for analysis. protein concentrations were determined using the bicinchoninic bicinchoninic acid assay from pierce following the manufacturer's instructions. a total of μg of protein was used for hoxa and actb, and μg for acta analyses. samples were loaded onto precast 4-15% gradient polyacrylamide gels and electrophoresed for minutes at v. proteins were transferred to nitrocellulose membranes in a buffer containing 20% methanol, m glycine, and mm tris in a bio-rad mini trans-blot cell at v for hour. after protein transfer, blots were fixed at room temperature for minutes in a solution containing 25% isopropanol and 10% acetic acid, rinsed briefly with m tris, m nacl, ph with % tween and blocked for minutes at room temperature in 5% skim milk in tbst. blots were then transferred to heat-sealable bags and incubated overnight at 4°c with primary antibody diluted in blocking solution. following washes the blots were incubated with hrp-conjugated igg preabsorbed species-specific secondary antibody diluted to 1: in tbst containing 4% serum of the secondary antibody species. after a final washing in tbst, blots were visualized using pierce supersignal west pico enhanced chemiluminescent substrate according to manufacturer's instructions and a bio-rad chemidoc imaging system. autoradiography film exposures were also obtained for each blot. the hoxa antibody was produced against a synthetic peptide derived from residues - of human hoxa the immunogen is specific for hoxa in ncbi blast search and shows no similarity to other hox proteins. the same antibody has been used previously by ota et al.. blots were quantified using quantity one® software. an intensity-per-area value was obtained for each sample band as well as for a control sample loaded on all blots. band intensities were adjusted between blots using control sample intensities. intensity values adjusted for expression of either acta or actb were input into the r statistical package. a wilcoxon signed rank test was used to compare the intensity values of protein bands from thoracic and abdominal aortic wall samples. linear regression with stepwise backwards elimination was used to evaluate age, race and sex as predictors of atcb-adjusted hoxa protein levels. immunohistochemical staining of aortic tissues immunohistochemical staining of o.c.t-embedded frozen tissues was carried out using the vectastain elite avidin:biotinylated enzyme complex kit. air-dried slides with 8-μm sections were fixed in methanol with 3% hydrogen peroxide to quench endogenous peroxidases and then blocked with serum for minutes. incubation with hoxa primary antibody, secondary antibody, abc reagent, and diaminobenzidine substrate were performed according to the manufacturer's instructions. each treatment was preceded by a 10-minute pbs wash. immunostained slides were counterstained with hematoxylin and mounted with a coverslip. non-specific igg antibody in lieu of primary antibody served as a negative control. when using formalin-fixed paraffin embedded aortic tissue, 5-μm sections were used in a protocol described previously. the slides were incubated with hoxa primary antibody on an automatic immunostainer. a secondary antibody with avidin-biotin peroxidase amplification from dako was used and the signal detected using diaminobenzidine as a chromogen. images were obtained using either a leica dm4000b or nikon microscope. western blot analysis of protein lysates from cultured cells human aortic smcs, human aortic endothelial cells, and monocyte/macrophage cells listed in additional file table s were cultured in appropriate medium according to suppliers' recommendations. aortic ecs and smcs were stimulated for hours using ng/ml ifn-γ. monocyte/macrophage cell lines were stimulated hours using ng/ml lps, and ng/ml ifn-γ. during stimulation, serum concentrations were half the concentrations used for maintenance culture. this stimulation was considered relevant to aaa, since mice lacking ifn-γ are resistant to aaa formation in the cacl model and ifn-γ-producing t cells are present in the blood and aortic wall of most aaa patients. cells were washed twice with cold pbs and lysed in ripa buffer with included protease inhibitors. cytoplasmic and nuclear extracts were also prepared using ne-per nuclear and cytoplasmic extraction reagents according to the manufacturer's recommendations. cell lysates were subjected to sds-page, transferred to pvdf membrane and immunoblotted with anti-hoxa rabbit polyclonal antibody or anti-actb mouse monoclonal antibody. blots were quantified to obtain an intensity-per-area value for each band. intensity values were adjusted for actb protein levels. actb-adjusted band intensity values were input into the r statistical package. repeated measures mixed effects linear model, was used to evaluate the effect of inflammatory stimulation on hoxa levels. fluorescence microscopy on cultured cells human aortic smcs and human aortic ecs were grown on 2-well permanox chamber slides, fixed with 3% paraformaldehyde and permeabilized with % triton x- in pbs, and then incubated with rhodamine-phalloidin at 1: dilution, and anti-hoxa with 1: dilution, followed by incubation with fluorescein-conjugated affinity-purified donkey anti-rabbit igg. the nuclei were stained with dapi. the stained cells were examined using a zeiss observer z inverted light microscope equipped for epifluorescence.  regional differences in the expression of hox genes in the baboon aorta to identify novel candidate genes with spatial expression differences in the aorta, genome-wide expression profiling was undertaken using segments along the entire aorta from baboons, which have been used as a model organism for vascular disease. aortic tissue from the baboons was preserved immediately following euthanasia and high-quality rna was subsequently extracted. as no baboon-specific microarrays were available, rna was hybridized to human arrays. without a completed baboon genome the full extent of sequence conservation is not known, but over 95% conservation would be expected based on sequence similarity between humans and other primate species. for highly conserved genes such as the hox family of genes, this degree of conservation is expected; hoxa coding region sequence identity is % and protein sequence identity is % between humans and baboons. genes with increasing or decreasing trends in expression along the baboon aorta included several members of the hox gene family. figure shows the regional expression of hoxa in two baboon aortas separately. differences in the expression of hox genes in human abdominal aortic aneurysms compared to matched control aortas the regional expression of hox genes in the baboon aorta led us to hypothesize that hox gene expression might be altered in human aaa. indeed, using microarray data generated previously, we found homeodomain-containing genes which were expressed in the human aorta; of these had significantly different mrna levels in aaa compared to control abdominal aortic tissues from an age-, sex-, and ethnicity-matched group of individuals. ten of the differentially expressed homeodomain-containing genes were classic hox-genes; the mrna levels of all of them were significantly decreased in aaa. hoxa was the most significantly decreased hox gene in human aaa tissue based on microarray results. in a different set of aaa cases and similarly-aged controls real-time quantitative rt-pcr was used to confirm the decrease in hoxa mrna expression. we also detected hoxa mrna in cultured human aortic smcs and aortic ecs. regional differences in the abundance of hoxa protein in the human aorta western blot analysis using paired human thoracic and abdominal aortic samples was performed to determine whether the observed spatial differences in baboon mrna expression were reflected in differences in protein levels in human aorta. in non-aneurysmal aortic tissue hoxa was detected using a specific antibody as a single band at ~ kda. after adjusting for β-actin, the level of hoxa protein was significantly higher in thoracic than the abdominal aorta, consistent with the baboon microarray analysis. smooth muscle α-actin protein levels were also investigated to assess whether the difference in the abundance of hoxa was a result of higher smc density in the thoracic aorta. after adjusting for α-actin levels, hoxa remained significantly higher in the thoracic aorta than in the abdominal aorta. the potential effects of age, race and sex on hoxa protein levels were also investigated. linear regression with stepwise backwards elimination showed age to be the only significant predictor of hoxa protein abundance, with evidence that protein levels decrease with age. immunohistochemical localization of hoxa in aortic tissue samples formalin-fixed paraffin-embedded samples of human aortic tissues were used for immunohistochemical staining. hoxa staining was observed in the nuclei and perinuclear regions of several cell types in all three layers of the aorta. staining patterns appeared to change with age. in samples from both young and old individuals staining was observed in and around the nuclei, but staining tended to concentrate more in the nuclei of samples from young individuals whereas samples from older individuals showed more diffuse perinuclear staining. additionally, an intense punctate staining pattern was observed in samples from older individuals that was not present in younger samples. similar staining patterns, including the punctate staining, were also observed with frozen tissue sections, demonstrating that it was not an artifact related to tissue preparation. characterization of hoxa expression in cultured vascular cells since inflammation is a hallmark of aaa pathogenesis, we examined whether inflammatory stimuli altered levels of hoxa in cultured human vascular cells. human macrophage/monocytes were stimulated using both ifn-γ and lps, whereas human aortic ecs, and human aortic smcs were stimulated with ifn-γ. hoxa protein levels were analyzed by western blots and compared with unstimulated control cells. this stimulation was considered relevant to aaa, since mice lacking ifn-γ are resistant to aaa formation in the cacl model and ifn-γ-producing t cells are present in the blood and aortic wall of most aaa patients. all but one of the cell lines exhibited downregulation of hoxa levels within hours of exposure to inflammatory stimuli. the decrease in hoxa levels was statistically significant. when investigating the cytoplasmic and nuclear fractions of proteins separately, the relative amount of hoxa protein in the nucleus decreased with inflammatory stimulation in all cell types. immunofluorescent staining for hoxa was also performed using cultured human aortic ec and human aortic smcs. in both cell types the strongest signal was observed in the perinuclear region with a small amount of nuclear staining. consistent with previously published results, stimulated smcs had fewer large actin stress fibers visualized by co-staining with rhodamine-phalloidin, whereas stress fibers in ecs did not decrease with stimulation. discussion the results of the current study provide evidence of differences in gene expression between the descending thoracic and abdominal aorta, particularly among the hox family of transcription factors. additionally, several hox transcription factors demonstrated altered expression in human aaa compared to a group of age-, race- and sex-matched control aorta, with hoxa being the most downregulated. quantitative rt-pcr using a separate set of samples confirmed that hoxa mrna levels were decreased in aaa and western blots demonstrated that protein levels were lower in the human abdominal than thoracic aorta. immunohistochemical staining suggested expression of hoxa in smcs and ecs in aortic tissue sections. finally, hoxa was observed in the nuclear and perinuclear regions of cultured aortic ecs and smcs with decreased protein levels after stimulation with inflammatory cytokines. hox genes were originally identified for their functions in embryonic patterning. increasing evidence suggests, however, that these genes also have a role in maintaining spatial identity of tissues, including vasculature, as demonstrated by microarray analyses and animal models. the results of our microarray analyses using the baboon aortas were consistent with these studies, demonstrating that the expression of hox genes varies spatially, in particular between the more aneurysm-susceptible abdominal aorta and the less aneurysm-susceptible thoracic aorta. several of these hox genes have been implicated in vascular function and disease, supporting the hypothesis that regional gene expression could alter disease susceptibility. a recent study supports this hypothesis, with hoxa exhibiting higher expression at atherosusceptible sites in the porcine aorta. to assess the potential role of hox genes in aaa pathogenesis, their expression profiles from human aaa and control aorta were compared, demonstrating significant downregulation of classic hox genes in aaa tissue. these findings are consistent with previous array-based studies, which demonstrated downregulation of hox genes in atherosclerosis and ulcerative colitis compared with non-diseased aorta and colon, respectively. protein analyses described here using cultured human vascular cells demonstrated a decrease in hoxa protein levels after in vitro stimulation with ifn-γ or lps and show that inflammatory stimuli alter hox gene expression. previously, treatment with interleukin 1ß, an inflammatory cytokine implicated in aaa pathogenesis, was shown to downregulate several hox genes. downregulation of hox genes could have several detrimental effects. hox genes have well-established roles in regulating cell proliferation and differentiation; downregulation of hox genes could alter cell phenotype in aaa. decreased hox gene expression could alter transcriptional regulation; hox gene targets include spp and the matrix metallopeptidases, which have been implicated in aaa pathogenesis. in the current study we concentrated on hoxa since it exhibited the most significant downregulation in human aaa tissue, had a distinct regional expression pattern along the aorta, and a specific antibody against it was available. western blots confirmed regional variation in hoxa protein abundance, with significantly higher hoxa levels in human thoracic aorta compared with abdominal aorta. immunohistochemical staining suggested that multiple cell types in the aorta express hoxa primarily ecs, and smcs, as well as some adventitial fibroblasts. immunofluorescent staining of cultured ecs and smcs showed nuclear and perinuclear protein localization of hoxa hoxa expression in both ecs and smcs is interesting, as human hoxa and murine hoxa are also expressed in both endothelium and smooth muscle, whereas murine hoxc is expressed in the smc only, suggesting that expression in both ec and smc is characteristic of the hoxa gene cluster. similar to other hox genes, hoxa is involved in embryonic patterning. its function in embryogenesis does not appear to be critical, since targeted disruption of hoxa results in a relatively mild phenotype; hoxa knockout mice are viable and exhibit only some deformations of the cervical spine. conversely, transgenic mice overexpressing the hoxa gene develop congenital megacolon due to abnormalities in the enteric nervous system. studies in chick embryos suggest that dfd, the hoxa4-like gene in chickens, is not critical to aortic arch artery patterning, since treatment of the cardiac neural fold with a dfd antisense oligonucleotide resulted in no apparent change in heart development at stage to. it is possible that hoxa is involved in regulating angiogenic function similar to other hox genes. an in vitro comparison of ovarian carcinoma cell lines showed that cells exhibiting higher levels of hoxa expression have decreased motility by scratch assay. similarly, overexpression of hoxa further decreased cell motility and led to a corresponding increase in ß integrin. ß integrin has been implicated in aaa pathogenesis and is decreased in aaa tissues compared to non-aneurysmal aorta. sirna knockdown of hoxa does not ablate ß integrin expression, however, which is consistent with hox genes having redundant functions. a role for hoxa in mediating cell contacts is further supported by an increase in basal lamina thickness and collagen in the colonic mucosa of transgenic mice overexpressing hoxa. it is, therefore, plausible that loss of hoxa function in medial smcs contributes to adverse phenotypic changes in aneurysms, including disruption of cell-cell interactions. hoxa can bind to its own promoter region and there is some evidence for hoxa regulating the adjacent hoxa gene under certain conditions. additionally, hoxa has also been implicated in the regulation of microrna genes during monocyte differentiation, suggesting micrornas as one mechanism for hox gene regulation of cell-type specific effects.  our results suggest that hoxa is important for maintaining spatial identity even in the adult aorta and that downregulation of hoxa expression may increase susceptibility to aortic disease such as aaas. to test these hypotheses embryonic targeted deletions of hoxa in animal models are needed to determine the exact contribution of this gene family to maintaining spatial identity. similarly, targeted deletion mutants could be employed to prove that downregulation of hoxa increases susceptibility to aaa in a mouse model. authors' contributions jhl participated in the design of the study, analyzed microarray data, carried out western blot experiments with human tissue lysates, and drafted the manuscript. re carried out all experiments with cultured cells and prepared illustrations. cms and zg carried out and interpreted the immunohistochemical analyses. ag and kd catalogued phenotypic information, processed samples, and carried out rt-pcrs. lac provided baboon samples. js and ll interpreted results. rsvh and lh provided control samples and interpreted results. gml participated in the design of the study, analyzed microarray data and interpreted results. jre, dpf, jlg and rpg recruited aaa patients for the study and collected tissue specimens. djc and wdl interpreted results. gt participated in the design of the study, analyzed results, carried out statistical analyses, prepared illustrations and interpreted results. hk obtained funding for the study, participated in the design of the study, interpreted results, and participated in drafting the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 table s human tissue samples from non-aneurysmal aortas used in assessing protein abundance of hoxa list of sample ids with donor age, ethnicity, sex and cause of death for human samples used to study hoxa protein levels. click here for file additional file 2 table s human aortic tissue samples used in microarray and q-rt-pcr experiments. list of sample ids with donor disease status classification, ethnicity, sex, age, and cause of death for human samples used in microarray and rt-pcr studies. click here for file additional file 3 figure s specificity of hoxa acta and actb antibodies. images of western blots performed with antibodies used in the study. click here for file additional file 4 table s commercially available human cell lines for hoxa experiments. list of cell lines used in the study with codes, description of cell type, vendor, and catalog number. click here for file additional file 5 table s mrna expression of hox genes in human aneurysmal and non-aneurysmal abdominal aorta based on microarray analyses. list of differentially expressed homeodomain-containing genes in human aaa compared to age-sex-and ethnicity-matched controls. columns give the full protein name, gene symbol, geneid, expression levels in control group and aaa group, fold-change in expression, false discovery rate and illumina detection score in control and aaa group. click here for file additional file 6 figure s localization of hoxa protein in cultured human aortic ecs and smcs using immunofluorescence. images of stained cultured cells. click here for file acknowledgements and funding we thank dr. susan land, daniel lott and sara mcnorton at the applied genomics technology center of wayne state university for their work on the microarray experiments. this project was funded in part by a grant from the national heart, lung, and blood institute, as well as by funds from the geisinger clinic. j.h.l. is a recipient of a predoctoral fellowship from the national institute on aging, nih, and in 2005- g.m.l. was a recipient of a predoctoral fellowship from the american heart association. we acknowledge use of human aortic tissues provided by the national disease research interchange, with support from nih grant u rr006042- 
we analyzed gene expression with microarrays in baboon aortas, and found that members of the hox gene family exhibited spatial expression differences. western blot analysis from human aortas demonstrated significantly higher hoxa protein levels in thoracic compared to abdominal tissues. our results demonstrated spatial variation in expression of hoxa in human aortas that persisted into adulthood and that downregulation of hoxa expression was associated with aaas, an important aortic disease of the ageing population. hoxa was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression. the hoxa transcript levels were significantly decreased in human abdominal aortic aneurysms compared to age-matched non-aneurysmal controls.
 the infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.  we analyzed gene expression with microarrays in baboon aortas, and found that members of the hox gene family exhibited spatial expression differences. hoxa was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. western blot analysis from human aortas demonstrated significantly higher hoxa protein levels in thoracic compared to abdominal tissues. immunohistochemical staining for hoxa showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. the hoxa transcript levels were significantly decreased in human abdominal aortic aneurysms compared to age-matched non-aneurysmal controls. cultured human aortic endothelial and smooth muscle cells stimulated with inf-γ showed decreased levels of hoxa protein.  our results demonstrated spatial variation in expression of hoxa in human aortas that persisted into adulthood and that downregulation of hoxa expression was associated with aaas, an important aortic disease of the ageing population. 