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Am J Physiol Heart Circ Physiol. Sep 2010; 299(3): H837–H846.
Published online Jun 11, 2010. doi:  10.1152/ajpheart.00002.2010
PMCID: PMC2944485

Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries

Abstract

Atherosclerotic plaques tend to form in the major arteries at certain predictable locations. As these arteries vary in atherosusceptibility, interarterial differences in endothelial cell biology are of considerable interest. To explore the origin of differences observed between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of harvested porcine coronary (CECs) and iliac artery endothelial cells (IECs) grown in static culture out to passage 4. Fewer differences were observed between the transcriptional profiles of CECs and IECs in culture compared with in vivo, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. One-class significance of microarrays revealed that most in vivo interarterial differences disappeared in culture, as fold differences after passaging were not significant for 85% of genes identified as differentially expressed in vivo at 5% false discovery rate. However, the three homeobox genes, HOXA9, HOXA10, and HOXD3, remained underexpressed in coronary endothelium for all passages by at least nine-, eight-, and twofold, respectively. Continued differential expression, despite removal from the in vivo environment, suggests that primarily heritable or epigenetic mechanism(s) influences transcription of these three genes. Quantitative real-time polymerase chain reaction confirmed expression ratios for seven genes associated with atherogenesis and over- or underexpressed by threefold in CECs relative to IECs. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial endothelium. The transcriptional differences observed here may provide new insights into pathways responsible for coronary artery susceptibility.

Keywords: vascular endothelium, atherosclerosis, pathophysiology, genomics, epigenetics

atherosclerosis is the primary cause of coronary heart disease, which is the leading cause of death in the United States and other developed countries (43, 63). Major arteries vary in their atherosusceptibility (2, 44), with plaque formation more prevalent in the coronary arteries compared with the carotid and femoral vessels (16). A recent human biopsy study suggested that the initiation, development, and composition of atherosclerotic plaques differ among arteries (16). As the endothelial lining of blood vessels is known to play an essential role in the development of atherosclerosis (44), interarterial differences in endothelial cell (EC) biology are of considerable interest. Both local environment and vascular bed origin have been implicated in atherosclerosis as plaques tend to form in the major arteries at certain predictable locations (19).

“Disturbed” flow is a commonly studied component of the local environment that is thought to cause EC dysfunction and contribute to subsequent plaque formation. Atheroprone regions exhibiting disturbed flow fields include the inner curvature of curved vessels and walls opposite flow dividers at branches and bifurcations (18). Local vessel geometry affects flow patterns, which may alter EC phenotype between arteries and even within the same artery. Indeed, microarray analysis revealed transcriptional differences between putatively disturbed and undisturbed flow regions of the porcine aorta (39).

ECs originating from different vascular beds display a remarkable heterogeneity in both structure and function under normal and pathological conditions (2, 3). The notion of phenotypic diversity is supported by both in vivo (50, 52) and in vitro (10, 13, 53) studies, which revealed distinct transcriptional profiles for ECs derived from different anatomic locations. Although the underlying causes and persistence of the observed heterogeneity are currently unclear, recent studies suggest that the developmental origin and/or differentiation pathway of ECs induce “signature” or vascular bed-specific transcriptional profiles, which are independent of local flow patterns (13, 20, 37). For example, characteristic differences between arteries and veins remain in cell culture, despite removal from the in vivo environment and growth under static conditions (13). Additionally, coronary artery and saphenous vein ECs have been shown to respond differently to both atherogenic stimuli under static conditions (20) and cytokine stimulation under vessel-specific flow patterns (37). In particular, exposing saphenous vein ECs to coronary artery flow patterns and vice versa revealed that expression of adhesion molecules involved both flow-dependent and flow-independent pathways, depending on the specific genes under investigation (37). The impact and relative influences of flow and vascular bed origin on endothelial transcriptional profiles are likely to be complex and interconnected.

Epigenetics, defined as heritable changes in gene expression without a change in DNA sequence (47), has been increasingly implicated in the control of vascular gene expression in both healthy and diseased states (36, 41, 57). Epigenetics provides a molecular basis for understanding how exposure to past and current cues can alter gene expression and thereby modify disease susceptibility. Chromatin-based epigenetic mechanisms enable so-called “cellular memory” in the form of specific marks that record the past history of cellular exposure to developmental pathways and cues, such as shear flow (6, 62). Epigenetic marks vary in their longevity from relatively stable DNA methylation to extremely dynamic and reversible histone modifications and RNA-based mechanisms (36). Wall shear stress has been shown to induce alteration of chromatin structure via histone modifications, demonstrating that flow can affect gene expression through epigenetic mechanisms (30). Although flow can alter gene expression, by itself it cannot completely account for expression of EC-specific genes, since endothelial markers remain in the absence of flow (22).

Previously, we found that in vivo the endothelial transcriptional profile of a coronary artery (the right coronary artery) is different from that of a major conduit vessel (the external iliac artery), and that this difference is consistent with the former vessel being more prone to atherosclerosis (64). However, although we demonstrated that transcriptional differences exist between these major arteries in vivo, we were not able to discern the origin of the observed differences. In this study, we compare the transcriptional profiles of the first four passages of right coronary (CECs) and external iliac artery ECs (IECs) to those observed in vivo to determine whether these differences persist in culture. Young and healthy swine were again used as the source of ECs, because swine vasculature is one of the best models of the human circulation with respect to normal and pathological physiology (4, 46). We find that local environment accounts for most differences observed in vivo, although vascular bed origin does play a role, especially for homeobox (HOX) genes.

MATERIALS AND METHODS

Animal surgery.

Animal experiments were performed in accordance with a protocol approved by the Duke University Institutional Animal Care and Use Committee. Four commercial juvenile female swine (60–70 kg) were maintained under anesthesia with inhaled isoflurane. Basic physiological parameters, including heart rate and blood pressure, were recorded for at least 20–25 min. The animal was then euthanized, and the arterial system was flushed with Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) to clear the blood from the vessels. The left and right external iliac arteries and right coronary arteries were dissected out. After the adventitial tissue was removed, the arteries were cut open and pinned out flat. A metal spatula was used to scrape ECs from the arterial wall, as described previously (34). IECs were scraped from the straight portion of the vessel between the circumflex iliac and deep femoral branches, as shown previously (64). Cells scraped from the right and left iliac arteries were combined for each animal, as no genes were previously found to be differentially expressed between the two at a false discovery rate (FDR) of 10% (64). CECs were scraped from the proximal portion, approximately 5–8 cm from the ostium.

Cell culture and RNA isolation.

Scraped cells were transferred directly into centrifuge tubes filled with media (EGM-2MV, Clonetics, Walkersville, MD). Cells were spun down (1,000 rpm for 5 min at 4°C) and seeded into tissue culture flasks. On reaching the desired confluency, cells were split 1:3 and cultured out to passage 4 (p4). As cells in the initial plating grew more slowly than those in subsequent passages, the first splits were performed at 53 ± 10% (SE) confluency after 7.4 ± 0.6 (SE) days. Subsequent splits were performed at 85 ± 3% (SE) confluency after 2.8 ± 0.4 (SE) days. Aliquots of cells from p1–p4 were spun down and resuspended in cell lysis solution, and total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA). Due to low cell yield for IECs on the first animal, RNA was not isolated from p1 IECs, as all of the cells were used to seed the flask for p2.

Microarray experiments.

The microarray experiments were performed as described earlier (27). Briefly, RNA samples were amplified and labeled with Cy5 dyes. A reference RNA sample (Cy3 labeled) was derived from cultured porcine aortic ECs (p3). Samples were hybridized to Sus Scrofa DNA microarrays (version 1.0, Operon Biotechnologies, Huntsville, AL) with 7,373 unique, known genes. The arrays were scanned, and the fluorescence intensity for each spot was quantified using GenePix (Molecular Devices, Sunnyvale, CA). Data for one CEC p1 sample was not included, as the signal-to-background ratio was low, indicating poor dye incorporation. Microarray data are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xtgltymassqgqhu&acc=GSE19593.

Flow cytometry and polymerase chain reaction (PCR) for calponin had been performed earlier (34) to confirm that no smooth muscle cells were scraped from the artery wall using this technique. The purity of the scraped ECs was further confirmed in this study by the microarray results; indeed, the expression of the smooth muscle cell-specific genes, calponin and myosin heavy chain, was lower in all samples than in a reference sample of cultured ECs.

Statistical analysis and bioinformatics for microarray.

Lowess normalization was performed on each array using GeneSpring GX (Agilent, Santa Clara, CA, version 10.0). Statistical tests were performed using significance analysis of microarrays (SAM) (58), and the FDRs were estimated based on permutation methods in SAM. The expression values for each gene were normalized within each animal by taking the ratio of CEC to IEC expression. One-class paired t-tests were used to identify the genes differentially expressed between CECs and IECs for each of the passages, p0–p4. In vivo or p0 microarray data previously reported (64) from four similar animals were reanalyzed using the same software versions and techniques as the p1–p4 data presented here. Technical limitations precluded both RNA isolation and culture of cells freshly scraped from the in vivo vessels; it was necessary to passage cells once before isolating RNA.

Two-way analysis of variance (12) on microarray expression (log2) values was performed by passage number and artery type (either CEC or IEC) over p2–p4 for all genes reported in Figs. 114 and Tables 1 and and2.2. Only passage, artery, and interaction effects with P ≤ 0.05 were considered significant.

Fig. 1.
Number of differentially expressed genes between coronary endothelial cells (CECs) and iliac artery endothelial cells (IECs) at 5–25% false discovery rate (FDRs) using one-class paired significance analysis of microarrays (SAM). For passage 0 ...
Fig. 2.
Genes identified as differentially expressed in p0 at 5% FDR using one-class paired SAM that also exhibit significant fold differences between the two arteries in cell culture. Values are means ± SE; for p0 and p2–p4, n = 4; for p1, n ...
Fig. 3.
One-class paired microarray results for genes that are putatively atheroprotective (A), associated with atherosclerosis with uncertain function (B), and atherogenic (C). Values are means ± SE; for p0 and p2–p4, n = 4; for p1, n = 2. CECs ...
Fig. 4.
PCR results for selected genes. For p0–p4, expression fold differences between CECs and IECs (A), and average ΔCT for CECs and IECs (B) are shown. C: expression ratios for HOXA9 and HOXA10. Lines represent linear least squares fits with ...
Table 1.
Differentially expressed genes identified in at least three passages at 25% false discovery rate using one-class paired significance analysis of microarrays
Table 2.
P values for two-way ANOVAs of PCR ΔCT values by passage number and arterial source (coronary or iliac artery endothelial cells)

Quantitative real-time PCR.

PCR was performed with the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA), using iQ SYBR Green (Bio-Rad) to detect double-stranded DNA, as described previously (64). Briefly, the 2−ΔΔCT method (35) was used to quantify the fold difference in expression, using ribosomal 18S as the internal control gene. Three replicate amplifications were used for each sample, and melting curves verified the quality of DNA product in each well. The RNA samples used for PCR were isolated at the same time and from the same animals as those used in the microarray measurements. cDNA was synthesized from 50 ng of total RNA using the Quantitect Reverse Transcription Kit (Qiagen, Valencia, CA), which includes a DNase treatment step. Two-way ANOVA on PCR ΔCT values was performed by passage number and artery type (either CEC or IEC) over both p0–p4 and p1–p4 for all genes analyzed by PCR.

RESULTS

Differential gene expression.

One-class SAMs (58) revealed that the number of unique, differentially expressed genes between CECs and IECs varied for each passage number (Fig. 1). As expected, for each passage, the number of differentially expressed genes increased as the FDR was relaxed from 5 to 25%. For p1, the numbers were low for all FDRs due to a small sample size of two, compared with data available from four animals in the other passages. In general, there were fewer differences between CECs and IECs in cell culture compared with in vivo. The only exceptions were for p2, in which a comparable number of differentially expressed genes were found using a 20% FDR and almost twice as many when the FDR was raised to 25%. The overall decrease in number of differentially expressed genes on culturing suggests that most differences observed in vivo were due to different environmental cues in the two arteries.

Thirty-nine genes were identified as differentially expressed in CECs relative to IECs in vivo at 5% FDR using one-class SAM (58). Most arterial differences disappeared immediately in culture, as only six of these genes displayed significant (P ≤ 0.05) fold differences at any passage in culture (Fig. 2). Three of the six were HOX genes with all passages exhibiting significant fold differences, except for p2 of HOXD3, for which P = 0.1. Differences between the two arteries decreased slightly for both HOXA10 and HOXD3 in the last two passages compared with the difference in vivo. Significant overexpression in CECs was found in p0 and p2 for ectodermal-neural cortex 1 (ENC1), which is also known as NRP/B (nuclear restricted protein in brain). ENC1 binds actin and also interacts with the transcription factor NRF2 to promote cellular defenses against reactive oxygen species (49). Protocadherin 7 (PCDH7), a calcium-dependent cell-cell adhesion glycoprotein, was significantly overexpressed in CECs for p2–p3. The only gene with a significant fold difference that changed sign with passage was MPPED2, a metallophosphoesterase that may be a tumor suppressor (59).

Overall, interarterial fold differences for 17 of the 39 genes differed significantly (P ≤ 0.05) from those in vivo for all passages; for 19 genes, significant differences from the in vivo fold difference were seen for at least two passages. The only genes whose fold difference did not change significantly compared with in vivo for any passage were HOXA9, ENC1, and MEOX2 (mesenchyme homeobox 2). MEOX2, also known as GAX (growth arrest-specific homeobox), is a key regulator of vascular cell function with either a pro- or anti-angiogenic effect, depending on concentration and EC origin (21).

Twenty-two genes were differentially expressed in at least three out of five passages at 25% FDR (Table 1). For all passages, HOXA9, HOXA10, and HOXD3 were underexpressed in coronary endothelium by at least nine-, eight-, and twofold, respectively. Although no significant fold differences were found for HOXB5, it was underexpressed in CECs by at least 1.4-fold in all passages and appeared on the p0, p2, and p4 lists. Calmodulin 4 (CALML4) and carboxypeptidase E (CPE) were also underexpressed in CECs for all passages by at least 1.2- and 1-fold, respectively. CALML4 binds calcium, and CPE is involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. Two genes underexpressed in CECs in cell culture were the oxidative stress mediators sestrin 1 (SESN1) and thioredoxin interacting protein (TXNIP). Another gene that experienced a decrease in CEC transcript levels in culture, SLC7A7 (solute carrier family 7, member 7), plays a role in nitric oxide synthesis via transport of l-arginine. Other genes differentially expressed between the two arteries in three passages code for proteins involved in cell adhesion [COL1A1 (collagen type I α1), EMILIN1 (elastin microfibril interface 1), and PCDH7], cell cycle regulation [BTG2 (B-cell translocator gene) and KLHL13 (kelch-like 13)], cell proliferation [IGFBP3 (insulin-like growth factor binding protein 3)], inflammation [IL17B (interleukin 17B) and HLA-DRB1 (major histocompatibility complex, class II, DR β1)], transcriptional regulation [GTF2IRD2B (general transcription factor IIi repeat domain containing 2B)], intracellular trafficking [CPNE8 (copine VIII) and SNX10 (sorting nexin 10)], cytoskeletal rearrangement [PLEK2 (pleckstrin 2)], and sulfation [ARSK (arylsulfatase family, member K)]. As expected, ANOVA of microarray expression values revealed that most of the genes identified as differentially expressed in three or more passages display artery and/or passage effects in p2–p4 (Table 1).

Microarray results for atherosclerosis-related genes.

In a previous in vivo study with similar swine (64), 17 genes that are known or have been suggested to be involved in atherosclerosis were found to be differentially expressed in CECs relative to IECs at 5% FDR. Figure 3 shows the fold differences between arteries observed for these genes from p0 to p4. Of the 17 genes, only HOXA9 and HOXA10 remained differentially expressed (P ≤ 0.05) for all passages out to p4. HOX genes are transcription factors that serve as mediators in vascular remodeling and angiogenesis and are known to be essential for both the development of the vascular system and its maintenance in the adult (21, 24). Continued differential expression of HOXA9 and HOXA10, despite removal of the cells from the in vivo environment, suggests that heritable factor(s) influences the observed interarterial differences. For these two genes, the only significant changes in fold difference compared with in vivo were for the p3 and p4 of HOXA10.

In general, removal from the in vivo environment reduced arterial differences for most genes, suggesting that variations in environmental factors between the two arteries contributed to the differential expression observed in vivo. Transcript ratios of DUSP1 (dual specificity phosphatase 1) and IGF1R (insulin-like growth factor I receptor) appear to be affected mostly by environmental differences between the arteries, as fold differences decreased significantly compared with in vivo for all passages. For S100B (S100 calcium binding protein B), expression was initially higher in CECs, but became higher in IECs with culture, resulting in a significant fold difference between the two arteries for p4. Arterial differences in S100B expression may thus be affected by both environmental and heritable factor(s). For FABP5 (fatty acid binding protein 5), RICS (Rho GTPase-activating protein), MAOA (monoamine oxidase A), FOS (FBJ osteosarcoma oncogene), IGFBP5, and EGR1 (early growth response 1), p1 fold differences were not significantly different from those in vivo, whereas p2 fold differences were, suggesting there may be heritable influence(s) on these genes that disappears rapidly in culture. However, due to the small sample size for p1, further measurements would be necessary to confirm that the in vivo ratio persists through this passage. Although the fold differences switched signs from p0 to p1 for C1R (complement component 1, r subcomponent), CLDN1 (claudin 1), and GADD45B (growth arrest and DNA-damage-inducible, β), arterial differences generally dissipated in subsequent passages. No significant differences compared with in vivo were found for CFI (complement factor I), DCN (decorin), or RAB34. For RAB34, significant arterial differences were found for p2 and p3.

To further explore the effects of artery type and passage, two-way ANOVA of microarray expression data was performed over p2–p4 to identify significant (P ≤ 0.05) effects. Artery effects were found for HOXA9 and HOXA10, thus confirming differential expression of these two genes in CECs and IECs. Passage effects were found for S100B, RICS, MAOA, IGFBP5, and CLDN1, indicating variation in expression with continued culture. Both artery and passage effects were found for the Ras oncogene family member RAB34.

Quantitative real-time PCR confirmation of microarray results.

To confirm the differences in gene expression observed via microarray, PCR was performed (Fig. 4) for the following seven genes: IGF1R, MAOA, EGR1, KLF2 (Kruppel-like factor 2), FABP5, HOXA9, and HOXA10. While fold difference plots (Fig. 4A) provide details on expression in CECs relative to IECs, plots of PCR ΔCT values (Fig. 4B) allow further exploration of differences both between the arteries and with passage. PCR ΔCT values quantify the difference in transcript copy numbers between a gene of interest and a housekeeping gene in the same sample. Detection after fewer PCR cycles translates to lower ΔCT values, which indicate higher concentrations of the transcript of interest and vice versa.

HOXA9 and HOXA10 expression increased in CECs on cell culture; in the previous study (64), neither were reliably detected at p0 after 50 PCR cycles, while both were consistently detected in the present study for p1–p4. PCR results (Fig. 4A) confirm differential expression of HOXA9 and HOXA10 between the two arteries for all passages. Strong linear correlations were observed for both HOXs when log fold difference was plotted vs. passage number for p1–p4 (Fig. 4C). These correlations show that expression differences between the two arteries decrease steadily for p1–p4.

For the remaining five genes, PCR indicated that expression became more similar in the two arteries with passage. KLF2, MAOA, and IGF1R were not found to be differentially expressed between CECs and IECs in cell culture (Fig. 4A). Differential expression was detected for FABP5 and EGR1 at p1. Significant changes in fold difference compared with in vivo were detected in all passages for both FABP5 and MAOA and in all but one passage for KLF2 and EGR1. IGF1R fold difference only changed significantly compared with in vivo for p4.

Although significant differences in arterial ΔCT values compared with in vivo (Fig. 4B) were not always found for all passages, the direction of changes was consistent when they occurred in more than one passage. In general, in both CECs and IECs, expression of MAOA and IGF1R increased, while KLF2 decreased with passaging. FABP5 expression decreased in IECs and increased in CECs, while the opposite was true for EGR1.

To further explore the effects of artery type and passage, two-way ANOVAs of PCR data were performed to identify significant (P ≤ 0.05) effects (Table 2). Two ANOVAs covering both p0–p4 and p1–p4 allowed insight into possible effect(s) of the p0–p1 transition, over which the largest changes in fold difference tended to occur for most genes. For HOXA9 and HOXA10, very strong artery effects were found over p1–p4. ANOVA, including p0, was not possible for the HOX genes, as they were not reliably detected in p0 CECs. The passage number effects over p0–p4 for KLF2 and MAOA, with no artery effects, were mostly due to the large p0 to p1 changes, suggesting primarily an environmental origin. However, the passage number effect remained over p1–p4 for MAOA. For p0–p4, EGR1 and IGF1R display both artery and passage effects. Both effects remain in p1–p4 for IGF1R, suggesting that they are not entirely due to the p0 to p1 change. For EGR1, the artery effect remains in p1–p4, but the passage number effect disappears, suggesting it to be primarily due to the p0–p1 change. Both artery and interaction effects were observed for FABP5 over p0–p4. The differences in expression between CECs and IECs in vivo caused the trends upon passaging to be different in the two arteries, presumably leading to the interaction term. This is supported by loss of the artery and interaction effects in p1–p4, suggesting they were due primarily to the large p0–p1 change. As differential expression was lost quickly in culture, the interarterial difference in expression observed in vivo for FABP5 was mostly due to environmental factors.

DISCUSSION

Our results demonstrate that both environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries in vivo. Fewer differences were seen between CECs and IECs in cell culture, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. Indeed, most genes that were differentially expressed between the two arteries in vivo did not display any significant fold differences in culture. However, the three HOX genes, HOXA9, HOXA10, and HOXD3, remained underexpressed in CECs out to p4, despite removal from the in vivo environment and exposure to identical static culture conditions. Continued differential expression suggests that primarily heritable or epigenetic mechanism(s) influences transcription of these three genes. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial ECs.

As atherosclerotic plaques are most prevalent in the coronary arteries, knowledge of factors that affect transcriptional differences between the relatively atheroprotected IECs and the atheroprone CECs may yield insight into molecules and pathways that influence atherosusceptibility. The transcriptional differences observed in this study were found in cells isolated from young, healthy swine maintained on a normal diet. Our results are thus relevant to the initiation and early stage of atherosclerosis. Lesions were not observed during any of the postmortem procedures. For consistency, previous in vivo data (64) were reanalyzed with the same software versions used for the p1–p4 data presented here. Differences in normalization caused slight variations in gene expression ratios. In the present analysis, 39 genes were found to be differentially expressed in p0 compared with 47 detected previously. Nonetheless, 20 genes were identified in both analyses. Technical limitations precluded both RNA isolation and culture of freshly scraped cells; it was necessary to passage cells once before isolating RNA. Eight total animals were thus used, four each for the p0 and p1–p4 data. This is not expected to have a large impact on the analysis, as the biological variation among individual animals was previously found to be smaller than the differences between CECs and IECs in vivo using unpaired one-class SAM (64).

Distinct environmental cues in the two arteries appear to account for the majority of in vivo transcriptional differences observed between CECs and IECs. Most in vivo arterial differences disappeared in culture, as fold differences were not significant for 85% of genes differentially expressed at p0. Furthermore, expression changed significantly compared with in vivo for 92% of these genes in at least one-half of the passages. Mechanical coupling to the heart and conduit geometry distinguish the coronary arteries from the iliac arteries and affect the mechanical cues that ECs are exposed to in their local in vivo environments. Mechanical cues that vary among arteries include phasic differences in hydrostatic pressure, wall shear stress, and circumferential strain (18). Whereas the proximal iliac arteries are essentially straight tubes downstream of the aortic bifurcation, the coronary arteries are highly curved and tethered to the beating heart. The curvature of the coronary arteries may induce more complex flow patterns than those found in the proximal iliac vessels (28). Indeed, wall shear stress distributions in proximal human coronary arteries are highly variable, with low shear stress occurring in several regions throughout the cardiac cycle (32). The coronary arteries exhibit a unique asynchrony between shear stress and circumferential strain (29, 42) that has been claimed to be responsible for differences in gene expression between the coronary arteries and the aorta (17).

Of the 17 atherosclerosis-related genes previously (64) identified as differently expressed in p0, only HOXA9 and HOXA10 remained differentially expressed for all passages out to p4. However, the more sensitive PCR technique detected significant fold differences for both EGR1 and FABP5 in p1, indicating that, for these genes, arterial differences dissipated quickly, but not immediately, upon cell culture. This suggests that primarily environmental differences in vivo affect differential expression of these genes, although heritable influence(s) may initially affect FABP5 and EGR1 expression in culture. It should be noted that, because the sample size for p1 was only n = 2, it is possible that other arterial differences in p1 exist, but were not detected by microarray analysis. Both artery and passage effects were found for RAB34, while passage effects were found for S100B, RICS, MAOA, IGFBP5, and CLDN1. Variation in expression with continued culture suggests that heritable factor(s) may be dissipating with passage, and/or cells are adapting to their new in vitro, static environment.

Epigenetic or heritable factor(s) arising from vascular bed origin and/or exposure to distinct environmental cues in vivo appear to account for a minority of observed arterial differences, as only the three HOX genes retained significant arterial differences out to p4. Continued differential expression, despite removal from the in vivo environment and culture under identical conditions, suggests that epigenetic mechanisms may contribute to the arterial differences observed for HOXA9, HOXA10, and HOXD3. HOX genes are known to be regulated by epigenetic mechanisms during development and are among the best studied genes that connect epigenetic modification, chromatin structure, and genome organization (62). Environmental factor(s), such as wall shear stress, may affect maintenance of epigenetic mark(s), as fold differences measured using PCR decreased linearly over p1–p4 for HOXA9 and HOXA10.

HOX transcription factors play important roles not only during embryonic development of the cardiovascular system, but also in adults during the vessel remodeling that occurs with angiogenesis and atherosclerosis (24). In vertebrates, the highly conserved HOX genes encode transcription factors located in four distinct clusters, labeled A through D, which are characterized by a common 61 amino acid DNA-binding motif (23). Although HOXs are involved in specifying positional identity along the anterior-posterior axis during embryogenesis, less is known about the roles and downstream targets of HOX genes in adults. Several HOX transcription factors, including HOXA10, HOXD3, HOXC6, and HOXC8, regulate the expression of integrins, adhesion molecules, and/or extracellular matrix proteins in mature ECs (8, 33).

HOXA9 is a potential regulator of EC homeostasis (35) and plays a key role in both the endothelial commitment of adult progenitor cells (45) and inflammation (5, 40, 55, 56). HOXA9 mediates the shear-stress induced maturation of ECs and the expression of prototypical endothelial-committed genes (45), such as ENOS (endothelial nitric oxide synthase) (1, 38), VEGFR2 (vascular endothelial growth factor receptor 2) (51), and VE-cadherin (11). The latter two genes have been identified as part of a mechanosensory complex that confers responsiveness to flow in heterologous cells (60).

Although seemingly contradictory, findings from recent reports identifying HOXA9 as both an inhibitor (55, 56) and activator (5) of inflammation in ECs are consistent with a more complex interplay between HOXA9 and NF-κB. Trivedi et al. (56) recently demonstrated the potential role of HOXA9 in maintaining ECs in a “basal” state by negatively regulating EC activation through the NF-κB pathway. HOXA9 overexpression inhibited the NF-κB-dependent induction of ICAM1, VCAM1, and E-selectin by proinflammatory cytokines downstream of NF-κB nuclear localization by interfering with NF-κB DNA binding (56). Bandyopadhyay et al. (5) found that HOXA9 protein is required for transcriptional induction of E-selectin by the upstream activator of NF-κB, TNF-α, which they interpreted as a proinflammatory role for HOXA9. However, sustained overexpression of HOXA9 did not negate cessation of E-selectin expression at later time points following TNF-α stimulation (5). This is consistent with the findings of Trivedi et al. (56) that, by inhibiting NF-κB DNA binding, overexpression of HOXA9 may turn off inflammatory adhesion molecule expression, which requires NF-κB. Due to a known association between radiotherapy and cardiovascular disease years after treatment, a recent study compared gene expression in human conduit arteries and identified HOXA9 as the most underexpressed gene in irradiated compared with nonirradiated arteries (25). Significant differences in HOXA9 expression between arteries and dysregulation of genes related to the NF-κB pathway persisted in patients from 4 wk to almost 10 yr after radiation treatment (25). The findings of Halle et al. (25) support the notion of HOXA9 as anti-inflammatory because of its strong downregulation in irradiated arteries with sustained inflammation due to NF-κB activation.

Expression of HOXA9 was not detectable in CECs in vivo (64), and, as found in this study, was at least ninefold higher in IECs out to p4. In IECs in vivo, high HOXA9 expression levels may increase the inflammatory activation set point by interfering with NF-κB DNA binding and thus buffer against inflammation (55, 56). In CECs in vivo, NF-κB-promoted transcription of adhesion molecules may be induced at lower levels of cytokine stimulation compared with IECs due to lack of HOXA9 buffering. However, E-selectin expression may yet be blocked or reduced because, unlike ICAM1, its induction requires HOXA9 protein (5). As inflammation is thought to play a major role in the development of atherosclerosis (44), and coronary arteries are generally more atheroprone than systemic arteries, the relationship between HOXA9 expression and endothelial sensitivity to inflammatory cytokines in healthy and diseased systemic and coronary arteries deserves further study.

Endothelial proliferation and integrin signaling, two important processes in atherosclerosis, are affected by HOXA10 targeting of P21 (cyclin-dependent kinase inhibitor 1A, CIP1) (14) and β3-integrin (15). HOXA10 directly targeted P21 (9) to either promote proliferation (48, 54) or induce cell cycle arrest (9), depending on the cellular context. HOXA10 also directly upregulated β3-integrin (15), which is a subunit in one of the major integrins in vascular ECs, αvβ3. Shear stress induced association between αvβ3 and SHC resulted in activation of the RAS pathway, demonstrating that integrins play a significant role in the initiation of signaling in response to shear stress (31).

HOXD3 promotes EC invasion and migration (7) by inducing proangiogenic genes, including the integrin subunits α5, αv, β1, and β3 (8, 65). Previous studies have shown that increased HOXD3 expression in ECs after wounding led to an upregulation of COL1A1 and β3-integrin, which improved wound healing (26, 61). In this study, HOXD3 expression was at least twofold higher in IECs out to p4, and COL1A1 expression was one- to threefold higher in IECs, with a significant fold difference between the two arteries observed for p3–p4. Our microarray did not contain a spot for β3-integrin. Based on increased HOXD3 expression in IECs, one may speculate that IECs may demonstrate improved wound healing compared with CECs. Whether wounding responses vary among artery types deserves further study, as it may impact atherosusceptibility, as well as the tendency to develop vulnerable plaque morphology.

Endothelial properties are context dependent, with transcriptional profiles affected by cues arising from both the local environment and the vascular bed of origin. Although the interplay between the two sources of cues is likely to be complex, the lists of differentially expressed genes presented here may suggest potential avenues for further study. Caution should be exercised when interpreting results from in vitro studies of endothelial response, as the present study demonstrates that arterial cells in vivo exhibit transcriptional differences that persist to varying degrees upon passaging. Cells from different arterial beds may possess epigenetic marks that alter the expression of certain genes, leading to the speculation that cells in vivo may be “primed” or exhibit different levels of preparation for handling stimuli and stress. Some of the genes identified here may prove to be essential regulators of atherogenesis and could serve as therapeutic targets.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-050442.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

The authors thank Ellen Dixon-Tulloch, Ji Zhang, and Amanda Basciano for surgical assistance.

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