BACKGROUND
mounting evidence linking environmental exposures in early life to later risk of cardiovascular disease has led to intense interest in the process of vasculature development in utero <cit> .
primarily, arteries and veins are defined by physiologic factors such as the direction and pressure of blood flow and by functional and anatomical differences such as the arrangement of smooth muscle cells around the vessels.
in general, arteries carry oxygenated blood, and have tighter endothelial junctions, whilst veins carry deoxygenated blood and have looser endothelial junctions  <cit> .
the feto-placental vascular system differs from that of most other human organs, because the arteries carry the deoxygenated blood coming from the fetus whilst the veins carry the oxygen enriched blood.
not surprisingly, given their different physiologic functions, the identity of arterial and venous endothelial cells is established before the onset of circulation  <cit>  in association with distinct gene expression signatures that define endothelial cell identity  <cit> .
this includes endothelial nitric oxide synthase  encoded by nos <dig>  von willebrand factor , vascular endothelial cadherin , intracellular adhesion molecular- <dig> , endothelial growth factor receptor tyrosine kinases vegf-r <dig>  and vegf-r <dig> , angiopoietin receptors tie- <dig> and tie <dig>  and notch <dig> .
in addition, a number of transcription factors have been shown to be preferentially expressed in endothelial progenitor cells and mature endothelial cells and have been argued to orchestrate the expression of such genes  <cit> .
despite this, the associated epigenetic regulators determining this specific gene expression profile are largely unclear.
numerous lines of evidence suggest that the endothelial compartment represents a potential target tissue for transmission of environmentally mediated risk of complex disease, potentially via a process of epigenetic disruption in early development  <cit> .
epigenetic mechanisms, including dna methylation, are now widely accepted to underpin developmentally regulated changes in cell morphology and function  <cit> , however little is known about the role of such modifications, and their relative plasticity, during endothelial cell development.
it is clear that one of the key regulators of vascular function, endothelial nitric oxide synthase  is under epigenetic control by several mechanisms, including dna methylation  <cit> .
indeed the chromatin structure of the enos promoter is transcriptionally permissive only in cells of the endothelial compartment, such as those isolated from human umbilical vein, mouse aortic, and pooled human dermal microvascular endothelial cells  <cit> .
altered chromatin has also been reported for other endothelial-specific genes including vwf <cit> , notch <dig> <cit> .
furthermore, studies have shown changes in epigenetic marks induced by oxidative stress  in vegf <cit>  and nos <dig> <cit> , supporting the potential epigenetic roles in tissue-specific regulation of those genes.
recent genome-scale comparison of dna methylation in dermal derived lymphatic and blood-derived endothelial cells has highlighted the role of differential methylation in the specification of endothelial function  <cit> , but beyond these limited data, little is known about the relationship between epigenetic modifications and gene expression underpinning endothelial phenotype or developmental stage at the genome-wide level.
as a first step towards addressing these questions, we have examined the relationship between genome-scale dna methylation and gene expression in purified human placental arterial and venous endothelial cells .
RESULTS
placenta-derived venous and arterial endothelial cells play especially important functional roles  as part of the fetal-maternal supply line  <cit> .
furthermore, several physiological differences exist between these two cell types  <cit>  and they differ in their degree of maturity.
whereas placental arterial endothelial cells have a mature arterial phenotype , venous derived cells show a juvenile  phenotype, potentially representing a pool of endothelial progenitor cells  <cit> .
we compared dna methylation in  <dig> purified cell populations each of human placental arterial endothelial cells  and venous equivalents .
after removing probes that did not pass a quality control cutoff  and probes on sex chromosomes, a total of  <dig>  probes remained that were common to all datasets for inclusion in subsequent analyses.
β-values  were calculated for each probe in hpaec and hpvec samples.
general hypomethylation in placental venous relative to arterial endothelial cells
as a first comparison of methylation within the two cell types, we calculated an average β-value across the entire dataset as a proxy for global methylation levels in venous and arterial cell samples as previously described  <cit> .
interestingly, we found clear evidence for a general hypomethylation in hpvec  relative to hpaec , with the latter comparable to peripheral blood average methylation, whereas hpvec levels were more similar to that of buccal cells taken at birth.
both hpaec and hpvec were generally more methylated on average than placental tissue .
widespread epigenetic differences in placental arterial and venous endothelial cell compartments
importantly, where tested, hm <dig> dna methylation levels showed strong correlations with those derived using locus-specific sequenom massarray epityper platform , validating the hm <dig> platform, in accordance with other recent studies  <cit> .
unsupervised hierarchical clustering of variable probes in this dataset  clearly discriminated arterial from venous samples  highlighting the genome-wide dna methylation differences between these endothelial compartments.
hpaec were more variable as a group than hpvec in terms of dna methylation profile.
this discriminatory capacity was maintained following unsupervised clustering of the  <dig> most highly variable probes .
further, a similar comparison with recently reported methylation datasets generated from cultured adult dermal blood and lymphatic endothelial cell populations  <cit>  with our dataset, along with unpublished human umbilical vein endothelial cell  data, highlights the distinct dna methylation profile of endothelial source from different vascular compartments.
hpvec show the most unique methylation profile, clustering separately from lymphatic and blood derived endothelial cells, huvecs and hpaec cells .
to identify sets of differentially methylated probes  between hpaec and hpvec, average β-values were calculated for each probe in hpaec and hpvec groups.
linear regression analysis revealed a large set of significantly differentially methylated probes .
a total of  <dig>  probes, associated with  <dig>  genes, showed elevated methylation in hpvec relative to hpaec  whereas  <dig>  probes associated with  <dig>  genes showed the reciprocal pattern .
these data highlight the coordinated nature of methylation change, with blocks of probes , showing a common shift in the direction and magnitude of methylation change between cell types.
in order to examine the potential for altered dna methyltransferase activity to explain the observed global dna methylation differences described above, we extracted methylation and gene expression data for found evidence for differential methylation of both the dnmt <dig> and dnmt3a  genes, with elevated average methylation in both promoter regions in hpvec relative to hpaec .
further, this was associated with a reciprocal pattern of gene expression specifically for dnmt3a consistent with methylation induced down regulation of dnmt activity in the placental venous compartment .
of particular interest to endothelial cell biology, many dmp/dmrs were identified in genes previously implicated in endothelial functioning, including nitric oxide synthase  <dig>   <cit>  and nos <dig>   <cit> , von willebrand factor   <cit> , notch <dig> <cit> , vegfa, vegfc <cit> ,sele <cit> , flt <cit> , and kdr <cit>  genes, although the number of dmps and direction of methylation difference between cell types was variable .
interestingly, many genes showed a reciprocal effect on methylation profile according to genomic location relative to the transcription start site.
for example, nos <dig>  showed two regions of highly significant differential methylation.
the first, located in the major promoter previously described to drive endothelial expression, encompassed  <dig> probes with higher mean methylation in hpvec relative to hpaec cells, while a reciprocal mean methylation pattern was observed for  <dig> probes in a downstream exon-associated region in a previously described, placenta-specific variant of this gene .
locus-specific analysis of methylation confirmed highly differential methylation across each of these regions , however the regulatory function  of this second region remains to be determined.
such complex patterns of methylation within individual genes have recently emerged as a common feature in gene regulation in humans  <cit> .
nos <dig> is constitutively expressed in the vascular endothelium and plays a critical role in cardiovascular physiology as evidenced by systemic and pulmonary hypertension, abnormal vasculature, defective angiogenesis, poor healing in response to injury and impaired mobilization of stem cells, in enos-null mice .
disruption of methylation in either of these regions early in development could be a modifiable risk factor for disease in later life.
complex interplay between endothelial dna methylation and gene expression
extensive epigenetic studies have highlighted the complex relationship between dna methylation and gene expression levels.
in addition to the well documented role of elevated methylation at promoter-associated cpg islands in down-regulation of gene expression, it is also apparent that some methylation marks at these  sites may be independent of gene expression status, or even predict active expression, as observed in several dmrs of imprinted genes or in the case of gene-body methylation  <cit> .
thus, in order to examine the relationship between dna methylation and gene expression, we integrated the methylation and gene expression data sets.
for each hm <dig> probe, differences in β-values between the two groups  were calculated and these were compared with corresponding gene expression differences .
by setting cutoffs in average methylation difference higher than 10%  and log <dig> fold change in gene expression of ~ <dig>  , we identified a subset of genes that showed a reciprocal relationship between methylation and expression level in hpvec and hpaec , consistent with a role of increasing methylation in down-regulation of gene expression.
this includes  <dig> hm <dig> probes showing lower methylation in association with elevated expression of  <dig> genes in arterial cells , and  <dig> probes covering  <dig> genes showing the same relationship in venous cells .
in order to test for any pattern regarding genomic location and likelihood of regulating gene expression, we examined the proportion of each probe type showing coordinate changes in differential methylation and gene expression, according to annotation .
this revealed that the majority of probes were located in enhancer or upstream regulatory  regions .
q <dig>  probes showing less methylation  and higher expression ; q <dig>  probes showing higher methylation  and expression; q <dig>  probes showing higher methylation and less expression ; q <dig>  probes showing less methylation and expression in hpaec relative to hpvec, genomic regions may overlap between one or more categories .
interestingly, endothelial genes likely to be directly regulated by dna methylation in our dataset included nos <dig>  vwf, apold <dig>  angptl <dig> genes.
many endothelial genes show both differential methylation at upstream regulatory regions and gene expression differences, but not always in the anticipated reciprocal direction .
* denotes number of probes located near promoter regions of each gene where highly differential dna methylation patterns were detected between two cell types out of total numbers of probes associated with each gene.
† denotes probes where strong dna methylation differences and gene expression patterns were detected but not inverse relationship.
it may indicate there exists other regulatory regions or gene expression is regulated by epigenetic mechanism other than dna methylation.
the flt <dig> and kdr genes code for two different subtypes of vegf receptors.
these genes have previously been shown to be expressed in most endothelial cells including aortic, vein, microvessel and in many tumors, albeit variably across tumors and individuals  <cit> .
flt <dig> and kdr are postulated to play a significant role in angiogenesis and/or tumor progression by influencing the activity of vegf <cit> .
these genes are shown to be hypermethylated in some cancer cell lines   <cit> .
in our study, flt <dig> showed differential promoter methylation and gene expression in hpaec and hpvec, however expression and methylation were positively correlated rather than anti-correlated .
in contrast, the kdr gene was differentially expressed between two cell types but this was independent of any dna methylation differences in the vicinity of the transcription start site.
these findings are interesting given that previous studies have shown down regulation of each of these genes in association with promoter methylation in various cell lines, while cell lines expressing kdr  appear universally unmethylated  <cit> .
the tlr <dig> gene has previously been shown to be expressed in endothelial cells in a cell-type specific manner  <cit> .
we found no significant gene expression difference in tlr <dig> in hpvec relative to hpaec  despite significant differences in promoter methylation levels, with hpvec having higher regional methylation , spanning seven hm <dig> probes, located proximal to the transcription start site.
the activity of this gene in huvecs has previously been shown to be regulated by inflammation in the absence of altered dna methylation in huvecs  <cit> .
in combination these data highlight the differential and complex role of epigenetics in the regulation of gene expression in endothelial cells of different origin, with several genes likely to be regulated by alternative epigenetic mechanisms  in a cell context-dependent manner.
differential utilization of genomic regulatory elements or expression of upstream transcription factors may also contribute to the observed pattern of expression.
epigenetic regulation of mir expression in endothelial cells?
multiple examples of mirna gene differential methylation were apparent in hpaec and hpvec cells , including mirs previously implicated in regulation of endothelial function and/or angiogenesis such as mir- <dig> and mir-130a  <cit> .
interestingly mir- <dig> family members , involved in regulation of vegf signaling in endothelial cells  <cit> , were found to be more highly methylated in hpaec than hpvec.
furthermore, mir-10a-associated probes, previously linked with arthero-function, were specifically hypermethylated in hpaec relative to hpvec  <cit> .
in contrast, the gene encoding mir-125b, a translational suppressor of ve-cadherin  <cit> , were hypermethylated in hpvec, relative to hpaec.
despite the clear evidence of differential methylation, an examination of mir expression profiles failed to reveal a corresponding difference in gene expression between the two cell types .
thus, the biological significance of the methylation differences remains.
ontology of genes associated with dmrs
in order to more fully understand any biological/cellular functions subject to coordinated regulation in endothelial cells, we performed gene ontology and ingenuity pathway analyses  on differentially methylated genes.
prior to the analysis, the list of dmps was selectively culled to focus on those probes predicted to play a role in gene regulation according to genomic location .
ingenuity pathway analysis revealed an enrichment of genes involved in biological functions such as “tissue development”, “cardiovascular system development and function”, and “tissue morphology” as differentially methylated in hpaec and hpvec .
many of these genes warrant further investigation in the development of arterial or venous endothelial cell phenotype.
gene ontology analysis performed by david  highlighted related biological processes associated with the “extracellular matrix”, previously shown to be critical for proper vascular development  <cit> , and “plasma membrane” and “vasculature development” .
importantly, coordinated regulation of genes involved in specific cellular pathways such as “cardiovascular pathway genes” was apparent .
CONCLUSIONS
placenta-derived arterial and venous endothelial cells differ in both their functional characteristics  <cit>  and differentiation state  <cit> .
whereas arterial cells are mature and fully differentiated, their venous counterparts have been regarded as immature, representing a juvenile phenotype with a high degree of plasticity.
we speculate that differential phenotype of these cells is largely driven by distinct gene expression changes, many of which are mediated by promoter methylation differences in key genes identified here.
at present it is not possible to distinguish which epigenetic changes are associated with the degree of maturity of the cells, and which drive more general distinct differences in venous vs arterial characteristics.
this will require further investigation.
the two cell types examined show a distinct difference in global dna methylation level, with the hpvec hypomethylated relative to hpaec, and other endothelial cells from a variety of tissues.
the low average methylation seen in the venous compartment is reminiscent of the hypomethylation seen in placental tissue and may be one of the underlying mechanisms by which environmental cues modulate their phenotype to adapt to the microenvironment.
indeed, the hpvecs are more sensitive to changes in their local environment in vitro relative to the hpaec counterparts  <cit> .
to the best of our knowledge this is the first study comparing global gene expression and dna methylation of primary arterial and venous endothelial cells isolated from the same organ.
in general our data have revealed a highly coordinated series of dna methylation events, many of which are directly implicated in regulating underlying gene expression levels, while the functional significance of others is less apparent.
included in our dataset are numerous genes previously not studied in relation to endothelial function, that warrant such an investigation in future studies.
the combination of dna methylation and gene expression profiling of early life endothelial cells represents a powerful approach to identify candidate loci potentially subject to environmentally mediated epigenetic disruption, in association with modified risk of later complex diseases involving cardiovascular dysfunction.

 mounting evidence linking environmental exposures in early life to later risk of cardiovascular disease has led to intense interest in the process of vasculature development in utero. primarily, arteries and veins are defined by physiologic factors such as the direction and pressure of blood flow and by functional and anatomical differences such as the arrangement of smooth muscle cells around the vessels. in general, arteries carry oxygenated blood, and have tighter endothelial junctions, whilst veins carry deoxygenated blood and have looser endothelial junctions. the feto-placental vascular system differs from that of most other human organs, because the arteries carry the deoxygenated blood coming from the fetus whilst the veins carry the oxygen enriched blood. not surprisingly, given their different physiologic functions, the identity of arterial and venous endothelial cells is established before the onset of circulation in association with distinct gene expression signatures that define endothelial cell identity. this includes endothelial nitric oxide synthase encoded by nos von willebrand factor, vascular endothelial cadherin, intracellular adhesion molecular-, endothelial growth factor receptor tyrosine kinases vegf-r and vegf-r, angiopoietin receptors tie- and tie and notch. in addition, a number of transcription factors have been shown to be preferentially expressed in endothelial progenitor cells and mature endothelial cells and have been argued to orchestrate the expression of such genes. despite this, the associated epigenetic regulators determining this specific gene expression profile are largely unclear. numerous lines of evidence suggest that the endothelial compartment represents a potential target tissue for transmission of environmentally mediated risk of complex disease, potentially via a process of epigenetic disruption in early development. epigenetic mechanisms, including dna methylation, are now widely accepted to underpin developmentally regulated changes in cell morphology and function, however little is known about the role of such modifications, and their relative plasticity, during endothelial cell development. it is clear that one of the key regulators of vascular function, endothelial nitric oxide synthase is under epigenetic control by several mechanisms, including dna methylation. indeed the chromatin structure of the enos promoter is transcriptionally permissive only in cells of the endothelial compartment, such as those isolated from human umbilical vein, mouse aortic, and pooled human dermal microvascular endothelial cells. altered chromatin has also been reported for other endothelial-specific genes including vwf, notch. furthermore, studies have shown changes in epigenetic marks induced by oxidative stress in vegf and nos, supporting the potential epigenetic roles in tissue-specific regulation of those genes. recent genome-scale comparison of dna methylation in dermal derived lymphatic and blood-derived endothelial cells has highlighted the role of differential methylation in the specification of endothelial function, but beyond these limited data, little is known about the relationship between epigenetic modifications and gene expression underpinning endothelial phenotype or developmental stage at the genome-wide level. as a first step towards addressing these questions, we have examined the relationship between genome-scale dna methylation and gene expression in purified human placental arterial and venous endothelial cells.  placenta-derived venous and arterial endothelial cells play especially important functional roles as part of the fetal-maternal supply line. furthermore, several physiological differences exist between these two cell types and they differ in their degree of maturity. whereas placental arterial endothelial cells have a mature arterial phenotype, venous derived cells show a juvenile phenotype, potentially representing a pool of endothelial progenitor cells. we compared dna methylation in purified cell populations each of human placental arterial endothelial cells and venous equivalents. after removing probes that did not pass a quality control cutoff and probes on sex chromosomes, a total of probes remained that were common to all datasets for inclusion in subsequent analyses. β-values were calculated for each probe in hpaec and hpvec samples. general hypomethylation in placental venous relative to arterial endothelial cells as a first comparison of methylation within the two cell types, we calculated an average β-value across the entire dataset as a proxy for global methylation levels in venous and arterial cell samples as previously described. interestingly, we found clear evidence for a general hypomethylation in hpvec relative to hpaec, with the latter comparable to peripheral blood average methylation, whereas hpvec levels were more similar to that of buccal cells taken at birth. both hpaec and hpvec were generally more methylated on average than placental tissue. widespread epigenetic differences in placental arterial and venous endothelial cell compartments importantly, where tested, hm dna methylation levels showed strong correlations with those derived using locus-specific sequenom massarray epityper platform, validating the hm platform, in accordance with other recent studies. unsupervised hierarchical clustering of variable probes in this dataset clearly discriminated arterial from venous samples highlighting the genome-wide dna methylation differences between these endothelial compartments. hpaec were more variable as a group than hpvec in terms of dna methylation profile. this discriminatory capacity was maintained following unsupervised clustering of the most highly variable probes. further, a similar comparison with recently reported methylation datasets generated from cultured adult dermal blood and lymphatic endothelial cell populations with our dataset, along with unpublished human umbilical vein endothelial cell data, highlights the distinct dna methylation profile of endothelial source from different vascular compartments. hpvec show the most unique methylation profile, clustering separately from lymphatic and blood derived endothelial cells, huvecs and hpaec cells. to identify sets of differentially methylated probes between hpaec and hpvec, average β-values were calculated for each probe in hpaec and hpvec groups. linear regression analysis revealed a large set of significantly differentially methylated probes. a total of probes, associated with genes, showed elevated methylation in hpvec relative to hpaec whereas probes associated with genes showed the reciprocal pattern. these data highlight the coordinated nature of methylation change, with blocks of probes, showing a common shift in the direction and magnitude of methylation change between cell types. in order to examine the potential for altered dna methyltransferase activity to explain the observed global dna methylation differences described above, we extracted methylation and gene expression data for found evidence for differential methylation of both the dnmt and dnmt3a genes, with elevated average methylation in both promoter regions in hpvec relative to hpaec. further, this was associated with a reciprocal pattern of gene expression specifically for dnmt3a consistent with methylation induced down regulation of dnmt activity in the placental venous compartment. of particular interest to endothelial cell biology, many dmp/dmrs were identified in genes previously implicated in endothelial functioning, including nitric oxide synthase and nos, von willebrand factor, notch, vegfa, vegfc,sele, flt, and kdr genes, although the number of dmps and direction of methylation difference between cell types was variable. interestingly, many genes showed a reciprocal effect on methylation profile according to genomic location relative to the transcription start site. for example, nos showed two regions of highly significant differential methylation. the first, located in the major promoter previously described to drive endothelial expression, encompassed probes with higher mean methylation in hpvec relative to hpaec cells, while a reciprocal mean methylation pattern was observed for probes in a downstream exon-associated region in a previously described, placenta-specific variant of this gene. locus-specific analysis of methylation confirmed highly differential methylation across each of these regions, however the regulatory function of this second region remains to be determined. such complex patterns of methylation within individual genes have recently emerged as a common feature in gene regulation in humans. nos is constitutively expressed in the vascular endothelium and plays a critical role in cardiovascular physiology as evidenced by systemic and pulmonary hypertension, abnormal vasculature, defective angiogenesis, poor healing in response to injury and impaired mobilization of stem cells, in enos-null mice. disruption of methylation in either of these regions early in development could be a modifiable risk factor for disease in later life. complex interplay between endothelial dna methylation and gene expression extensive epigenetic studies have highlighted the complex relationship between dna methylation and gene expression levels. in addition to the well documented role of elevated methylation at promoter-associated cpg islands in down-regulation of gene expression, it is also apparent that some methylation marks at these sites may be independent of gene expression status, or even predict active expression, as observed in several dmrs of imprinted genes or in the case of gene-body methylation. thus, in order to examine the relationship between dna methylation and gene expression, we integrated the methylation and gene expression data sets. for each hm probe, differences in β-values between the two groups were calculated and these were compared with corresponding gene expression differences. by setting cutoffs in average methylation difference higher than 10% and log fold change in gene expression of ~, we identified a subset of genes that showed a reciprocal relationship between methylation and expression level in hpvec and hpaec, consistent with a role of increasing methylation in down-regulation of gene expression. this includes hm probes showing lower methylation in association with elevated expression of genes in arterial cells, and probes covering genes showing the same relationship in venous cells. in order to test for any pattern regarding genomic location and likelihood of regulating gene expression, we examined the proportion of each probe type showing coordinate changes in differential methylation and gene expression, according to annotation. this revealed that the majority of probes were located in enhancer or upstream regulatory regions. q probes showing less methylation and higher expression; q probes showing higher methylation and expression; q probes showing higher methylation and less expression; q probes showing less methylation and expression in hpaec relative to hpvec, genomic regions may overlap between one or more categories. interestingly, endothelial genes likely to be directly regulated by dna methylation in our dataset included nos vwf, apold angptl genes. many endothelial genes show both differential methylation at upstream regulatory regions and gene expression differences, but not always in the anticipated reciprocal direction. * denotes number of probes located near promoter regions of each gene where highly differential dna methylation patterns were detected between two cell types out of total numbers of probes associated with each gene. † denotes probes where strong dna methylation differences and gene expression patterns were detected but not inverse relationship. it may indicate there exists other regulatory regions or gene expression is regulated by epigenetic mechanism other than dna methylation. the flt and kdr genes code for two different subtypes of vegf receptors. these genes have previously been shown to be expressed in most endothelial cells including aortic, vein, microvessel and in many tumors, albeit variably across tumors and individuals. flt and kdr are postulated to play a significant role in angiogenesis and/or tumor progression by influencing the activity of vegf. these genes are shown to be hypermethylated in some cancer cell lines. in our study, flt showed differential promoter methylation and gene expression in hpaec and hpvec, however expression and methylation were positively correlated rather than anti-correlated. in contrast, the kdr gene was differentially expressed between two cell types but this was independent of any dna methylation differences in the vicinity of the transcription start site. these findings are interesting given that previous studies have shown down regulation of each of these genes in association with promoter methylation in various cell lines, while cell lines expressing kdr appear universally unmethylated. the tlr gene has previously been shown to be expressed in endothelial cells in a cell-type specific manner. we found no significant gene expression difference in tlr in hpvec relative to hpaec despite significant differences in promoter methylation levels, with hpvec having higher regional methylation, spanning seven hm probes, located proximal to the transcription start site. the activity of this gene in huvecs has previously been shown to be regulated by inflammation in the absence of altered dna methylation in huvecs. in combination these data highlight the differential and complex role of epigenetics in the regulation of gene expression in endothelial cells of different origin, with several genes likely to be regulated by alternative epigenetic mechanisms in a cell context-dependent manner. differential utilization of genomic regulatory elements or expression of upstream transcription factors may also contribute to the observed pattern of expression. epigenetic regulation of mir expression in endothelial cells? multiple examples of mirna gene differential methylation were apparent in hpaec and hpvec cells, including mirs previously implicated in regulation of endothelial function and/or angiogenesis such as mir- and mir-130a. interestingly mir- family members, involved in regulation of vegf signaling in endothelial cells, were found to be more highly methylated in hpaec than hpvec. furthermore, mir-10a-associated probes, previously linked with arthero-function, were specifically hypermethylated in hpaec relative to hpvec. in contrast, the gene encoding mir-125b, a translational suppressor of ve-cadherin, were hypermethylated in hpvec, relative to hpaec. despite the clear evidence of differential methylation, an examination of mir expression profiles failed to reveal a corresponding difference in gene expression between the two cell types. thus, the biological significance of the methylation differences remains. ontology of genes associated with dmrs in order to more fully understand any biological/cellular functions subject to coordinated regulation in endothelial cells, we performed gene ontology and ingenuity pathway analyses on differentially methylated genes. prior to the analysis, the list of dmps was selectively culled to focus on those probes predicted to play a role in gene regulation according to genomic location. ingenuity pathway analysis revealed an enrichment of genes involved in biological functions such as “tissue development”, “cardiovascular system development and function”, and “tissue morphology” as differentially methylated in hpaec and hpvec. many of these genes warrant further investigation in the development of arterial or venous endothelial cell phenotype. gene ontology analysis performed by david highlighted related biological processes associated with the “extracellular matrix”, previously shown to be critical for proper vascular development, and “plasma membrane” and “vasculature development”. importantly, coordinated regulation of genes involved in specific cellular pathways such as “cardiovascular pathway genes” was apparent.  placenta-derived arterial and venous endothelial cells differ in both their functional characteristics and differentiation state. whereas arterial cells are mature and fully differentiated, their venous counterparts have been regarded as immature, representing a juvenile phenotype with a high degree of plasticity. we speculate that differential phenotype of these cells is largely driven by distinct gene expression changes, many of which are mediated by promoter methylation differences in key genes identified here. at present it is not possible to distinguish which epigenetic changes are associated with the degree of maturity of the cells, and which drive more general distinct differences in venous vs arterial characteristics. this will require further investigation. the two cell types examined show a distinct difference in global dna methylation level, with the hpvec hypomethylated relative to hpaec, and other endothelial cells from a variety of tissues. the low average methylation seen in the venous compartment is reminiscent of the hypomethylation seen in placental tissue and may be one of the underlying mechanisms by which environmental cues modulate their phenotype to adapt to the microenvironment. indeed, the hpvecs are more sensitive to changes in their local environment in vitro relative to the hpaec counterparts. to the best of our knowledge this is the first study comparing global gene expression and dna methylation of primary arterial and venous endothelial cells isolated from the same organ. in general our data have revealed a highly coordinated series of dna methylation events, many of which are directly implicated in regulating underlying gene expression levels, while the functional significance of others is less apparent. included in our dataset are numerous genes previously not studied in relation to endothelial function, that warrant such an investigation in future studies. the combination of dna methylation and gene expression profiling of early life endothelial cells represents a powerful approach to identify candidate loci potentially subject to environmentally mediated epigenetic disruption, in association with modified risk of later complex diseases involving cardiovascular dysfunction. 
the aim of this study was to investigate the potential for dna methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. dna methylation was measured at > cpg sites in parallel with expression measurements taken from annotated genes. endothelial cellsdna methylationepigeneticsplacentanos3gene expressionhpaechpvecreprogramming differentially methylated region this included many loci previously not investigated in relation to endothelial function. an inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including nos encoding endothelial nitric oxide synthase. these are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life. a consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. the endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes.
 the endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. the molecular mechanisms underpinning this process in utero have yet to be fully elucidated. the aim of this study was to investigate the potential for dna methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells.  placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. dna methylation was measured at > cpg sites in parallel with expression measurements taken from annotated genes. a consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. this included many loci previously not investigated in relation to endothelial function. an inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including nos encoding endothelial nitric oxide synthase.  endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. these are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life. endothelial cellsdna methylationepigeneticsplacentanos3gene expressionhpaechpvecreprogramming differentially methylated region 