Site-Specific Atherogenic Gene Expression Correlates With Subsequent Variable Lesion Development in Coronary and Peripheral Vasculature
Objectives— The relationship between specific gene regulation and subsequent development and progression of atherosclerosis is incompletely understood. We hypothesized that genes in the vasculature related to cholesterol metabolism, inflammation, and insulin signaling pathways are differentially regulated in a site-specific and time-dependent manner.
Methods and Results— Expression of 59 genes obtained from coronary, carotid, and thoracic aortic arteries were characterized from diabetic (DM)/hypercholesterolemic (HC) swine (n=52) 1, 3, and 6 months after induction. Lesion development in the 3 arterial beds was quantified and characterized at 1, 3, 6, and 9 months. Progressive lesion development was observed in the coronary>thoracic aorta≫carotid arteries. Genes involved in cholesterol metabolism and insulin pathways were upregulated in coronaries>thoracic aortae>carotids. Inflammatory genes were more markedly upregulated in coronary arteries than the other 2 arteries. Genes implicated in plaque instability (eg, matrix metalloproteinase-9, CCL2 and Lp-PLA2 mRNAs) were only upregulated at 6 months in coronary arteries.
Conclusions— Variable gene expression, both in regard to the arterial bed and duration of disease, was associated with variable plaque development and progression. These findings may provide further insight into the atherosclerotic process and development of potential therapeutic targets.
Atherosclerosis is a multi-factorial pathological process resulting from environmental and genetic influences involving multiple vascular territories. Patients with diabetes mellitus (DM) have an increased risk of developing accelerated cardiovascular disease which is potentiated by hypercholesterolemia (HC).1 Although gene expression of atherosclerotic genes is associated with the presence of atherosclerosis, the sequence of specific gene regulation and the relationship to subsequent development of atherosclerosis is incompletely understood. Also unclear is whether development of atherosclerosis in separate vascular beds is associated with differential gene expression.
We hypothesized that genes relating to vascular cholesterol metabolism, insulin pathways, and inflammatory responses are differentially regulated in a site-specific and time-dependent manner. We tested this hypothesis in the atherosclerotic DM/HC swine model which develops advanced coronary lesions within 6 months.2 We chose genes that had previously (1) shown differential expression in stable and unstable human atherosclerotic plaques, (2) demonstrated involvement in the atherosclerotic process, and (3) had a pig ortholog.3
Diabetes mellitus was induced in male pigs (n=52) weighing 25 to 30 kg by 125 mg/kg of intravenous streptozotocin (Sicor Pharmaceuticals). Exogenous insulin was administered to insure that glucose levels did not exceed 350 mg/dL. The animals were fed chow containing 0.5% to 2% cholesterol, 5% to 20% lard, and 1.5% sodium cholate (Animal Specialties) to achieve a cholesterol level of 250 to 1000 mg/dL. Animals were euthanized at 1, 3, 6, or 9 months after induction. The protocol followed institutional guidelines.
Please see supplemental methods, available online at http://atvb. ahajournals.org.
Please see supplemental methods.
Gene Expression Using Quantitative Real-Time Polymerase Chain Reaction
A Taqman plate was constructed based on a literature review (1990 to 2006) with a limited choice of 59 genes that showed increased pro- and antiatherosclerotic expression in human atherosclerotic plaques.3 The Taqman plate was constructed with those genes that showed high mRNA expression in human plaques by microarray and had a porcine analog. The selected genes were then annotated for various pathways using GO terms. (For a complete list of genes evaluated and their GO annotation, please see supplemental Table I). Following removal of overlying adipose and connective tissue the entire vessel was used for analysis. Total RNA was extracted using RNeasy Mini Kit (Qiagen), followed by removal of genomic DNA contamination using DNase I (Ambion). The efficiency of the DNase procedure was validated in a standard TaqMan assay using RNA samples not subjected to a reverse transcription step and GAPDH primer set. The DNA-free total RNA samples were quantified using RiboGreen RNA quantitation reagent (Molecular Probes) mRNA was converted to cDNA by reverse transcription utilizing High Capacity cDNA Archive Kit (Applied Biosystems) and the equivalent of 10 ng per well was arrayed into high-density 384-well plates using a Biomek FX robot (Beckman Coulter) allowing expression profile generation for 1 gene at a time. Quantitative RT-PCR was carried out using a 7900HT Sequence Detector System (Applied Biosystems) in 10 μL reaction volume. TaqMan Universal PCR Master Mix 2X (Applied Biosystems) and universal PCR conditions recommended by the manufacturer were followed. Copy numbers of a given mRNA detected in each sample were calculated after normalization to the average log of the expression of 3 housekeeping genes (cyclophilin, GAPDH, and B-actin). Four non-DM/non-HC age- and sex-matched animals acted as controls for comparison in the gene expression studies. In these animals for the 59 evaluated genes the group coefficient of variation was 63%, 35%, and 30% for coronary, carotid, and aortic samples, respectively.
Numeric data are expressed as mean±SD, unless otherwise noted. Comparisons of multiple groups were made using analysis of variance (ANOVA). If the results of the ANOVA were significant, posthoc analysis using the Sheffe method was performed to evaluate intergroup differences. Significance of up- and downregulation of gene expression used ANACOVA. SPSS version 12 was used for statistical analysis. A probability value <0.05 was considered significant.
DM/HC animals (n=52) were euthanized at 1 month, (n=7), 3 (n=10), 6 (n=26), or 9 months (n=9) after induction. Originally 7 animals were enrolled in the 1-month group and 10 animals in the other groups. One animal died suddenly in the 9-month group. Additional vascular tissue was available for histology, immunohistochemistry, and gene expression studies at the 6-month time point and we elected to enrich this sample. There was no difference in the care or results in the additional 16 animals compared to the original 10. After initiation of DM/HC the serum glucose levels increased to approximately 300 mg/dL and cholesterol levels increased to >700 mg/dL at 3 months. Both remained elevated for the duration of the study (P<0.0001, supplemental Table II).
Arterial samples were obtained 1, 3, 6, and 9 months to evaluate the time course of complex lesion development. The 9-month samples were obtained to document variable progression of atherosclerosis in the 3 arterial territories, specifically the continued differential lesion progression over time. There were no lesions 1 month after initiation of DM/HC in any arterial bed. By 3 months lesions were observed in all arterial beds but were more frequently observed in coronary arteries and thoracic aortae (Table 1⇓). Over the study period the intimal-medial ratio increased in the coronary artery and thoracic aorta (coronary≫aorta) and was relatively stable in the carotid arteries. Coronary lesions at 3 months generally consisted of lipid laden foam cells or smooth muscle α-actin negative cells, whereas at 6 months increased complexity was observed with the lesions consisting of smooth muscle cells (α-actin positive), extracellular matrix, and macrophages. Lipid pools and necrotic cores were also observed. By 9 months high-grade complex lesions consisting of smooth muscle cells, extracellular matrix, and macrophages with lipid pools, necrotic debris, and calcifications were commonly observed. Examples of coronary, carotid, and thoracic aortic lesions obtained from 1 animal at each time point are shown in Figure 1. Adjacent sections demonstrating macrophage accumulation in the 3 arterial beds at the 3 time points is seen in supplemental Figure II. Histological evaluation of all coronary arteries demonstrating an atherosclerotic plaque (n=78) showed that the most severe lesion was generally located in the proximal segment of the coronary artery (60/78, 77%). In 62 arteries 1 or more sections per artery contained a branching vessel. Of the 62, 55 arteries had a lesion located near the ostium of the side branch (89%). In 13 (of the 55) the lesions were only noted near the branching vessel while in 3 (of the remaining 7) a lesion was noted only in the nonbranching segment.
A progressive increase in the percentage of advanced atherosclerotic lesions was noted in left coronary arteries (Tables 2 and 3⇓). In contradistinction, the thoracic aorta demonstrated less advanced lesions at each time point, as 8 of 9 thoracic aortae had intimal xanthomas at 3 months. At 6 months 19 of 21 had intimal xanthomas, 1 had a fibrous atheroma, and 1 a thin fibrous cap atheroma. At 9 months, of the 9 arteries, 7 exhibited an intimal xanthoma and 2 a fibrous cap atheroma. Of the 10 carotid arteries, at 3 months only 2 had intimal xanthomas whereas at 6 months, 6 of 26 arteries had intimal xanthomas and 2 had fibrous atheromas. By 9 months of 6 arteries there was 1 with an intimal xanthoma and 1 with a fibrous atheroma. The remaining arteries at these time points demonstrated no lesions.
Gene Expression in Cholesterol Metabolism and Insulin Related Gene Pathways
A complete listing of gene expression changes over time are presented in supplemental Table III. There was persistent upregulation in the ATP-binding cassette transporter gene, ABCA 1, and consistent downregulation of fatty acid synthase in all arterial beds at all time points except at 6 months in carotids for ABCA 1 and 1 month for aortic fatty acid synthase. In coronary arteries only there was late upregulation of apoE and the mitochondrial gene UCP2 and downregulation of PPARα gene expression at 6 months. Elevation in insulin related pathway gene expression (adiponectin, leptin, PPARγ, and preproadipsin) was only noted early (generally at 1 month) and only in coronary arteries, whereas IRS1 (insulin receptor substrate 1) was downregulated only in the thoracic aorta at 3 and 6 months.
Gene Expression in Inflammatory Signaling Related Gene Pathways
With the exception of intercellular adhesion molecule-1 (ICAM-1), early increased inflammatory gene expression was noted in the coronary arteries only. ICAM-1 upregulation was also observed at all time points in carotid arteries but not the thoracic aortae. At 1 month, genes involved with cell-cell adhesion (ICAM-1, vascular cell adhesion molecule [VCAM]-1), regulation of an inflammatory-immune response (PTGS2 or COX-2, NOS-2A), and macrophage internalization of hemoglobin/haptoglobin (CD163),7 were only upregulated in coronary arteries. At 3 months only ICAM-1 and NOS2A continued to be significantly upregulated in the coronary arteries and ICAM-1 in the carotid arteries. At 6 months, increased expression of multiple genes was observed. Several were preferentially upregulated in coronary arteries, including those involved with monocyte chemotaxis (CCL2 or Monocyte chemotactic protein-1), progression of macrophages to foam cells (CD68),8 cell growth and maintenance (EV12B), and genes associated with increased lesion complexity such as MMP-9 and the urokinase plasminogen activator (uPA) receptor (PLAUR) which degrade extracellular matrix proteins and increase cellular migration. Multiple genes were upregulated both in the coronary arteries and thoracic aorta (but not carotid arteries). These included genes involved in cell-cell signaling and immune response (CCR1, interleukin [IL]-8), neutrophil chemotaxis (ITGB2, IL-8), and activation (IL-8), Lp-PLA2, and NOS2A. Finally, CD163 was upregulated at 6 months in the thoracic aorta whereas it was upregulated at 1 month in the coronary artery. EV12A expression (cell proliferation and growth) was increased in coronary arteries and thoracic aortae at 1 month and increased in carotid arteries at 3 months. The cytoprotective gene HMOX-1 was upregulated at 1 month only in coronary arteries.
Genes With Unknown Function
We added genes that were previously shown to be highly expressed within inflamed areas of human plaque tissue but do not have as yet have a published known function.3 The majority of genes upregulated were noted in the coronary artery. FLJ22457 was increased in coronary arteries at all time points but only at 6 months in carotid arteries.
To further investigate the correlation between expression of activated monocyte markers and the high-fat diet associations between several prominently upregulated inflammatory genes were determined. There were significant associations between gene expression of CD68, Lp-PLA2, and MMP9 (supplemental Figure III); genes involved in macrophage activation and function. A significant correlation was noted between these genes and the total cholesterol exposure.
The induction of Type I diabetes mellitus and hypercholesterolemia led to progressive yet variable development of atherosclerosis with a majority of coronary vessels developing advanced lesions whereas a minority of thoracic aortae and carotid arteries demonstrated complex atheroma development. The observation that gene expression of inflammatory mediators was most pronounced in the coronary arteries suggests a particularly important role for vascular inflammation in the development of advanced atherosclerosis in this arterial bed compared to the thoracic aortae and carotid arteries.
Gene expression profiling has previously been used to identify genes and pathways relevant to atherosclerosis and has identified differentially expressed genes involved in lipid metabolism, inflammation, cell turnover, matrix degradation, and coding for matrix proteins.9 In the present study a dynamic interplay of pro- and antiatherosclerotic gene profile expression was noted with upregulation of some genes preceeding atherosclerotic plaque development whereas some lipid metabolism genes, which may play a role in preventing lesion development, were also expressed (Figure 2). Persistent upregulation of ABCA1 may indicate a defensive mechanism to offload cholesterol from macrophages onto HDL in the setting of the increased serum LDL levels. The persistent downregulation of fatty acid synthase (FAS), the major enzyme involved with fatty acid synthesis, likely reflects the perturbation in the insulin and glucagon ratio.
Early increases in vascular inflammation gene expression in coronary arteries, specifically genes for ICAM-1 (also observed in carotid arteries), VCAM-1, IL-1, the nitric oxide isoform NOS2A, as well as the cytoprotective gene HMox1 were noted. Early and persistent upregulation of ICAM-1 supports previous data showing increases within circulating peripheral blood mononuclear cells and coronary tissue.10 However, given the distinct difference in lesion development in coronary and carotid arteries ICAM-1 may not play a major role in subsequent plaque progression. Later, upregulation was observed for CCR-1, produced by macrophages, T cells, neutrophils and dendritic cells, CCL2; genes associated with a vascular inflammatory-immune response. Upregulation of the Lp-PLA2 gene indicates the potential role that this molecule plays in the development and progression of atherosclerosis and supports previous data showing upregulation of Lp-PLA2 mRNA in peripheral blood mononuclear cells at 3 months and in coronary artery tissue at 6 months.10 Lp-PLA2 acts on the truncated phospholipid of oxidized LDL resulting in generation of 2 important mediators of inflammatory cell homing: nonesterified fatty acids (NEFA) and lysoPC. Other genes also upregulated at 6 months, such as MMP-9 and PLAUR, have been postulated to major roles in lesion instability deemed vulnerable plaques. Importantly there was a lack of significant change in gene expression in the thoracic aorta of MMP-9 and PLAUR paralleling the relative lack of development of high-grade complex lesions at this location.
Increased inflammatory and insulin signaling pathway gene expression suggests a mechanism of the observed increased susceptibility to lesion development and increased complexity observed in coronary arteries compared to carotid arteries and thoracic aortae. Indeed, the differential development of lesions in the 3 vascular beds supports previous observations that atherosclerosis is unevenly distributed. Passarini et al11 has shown the presence of heterogeneous populations of endothelial phenotypes from hemodynamically distinct regions of adult nonatherosclerotic porcine aortae, indicating the potential for atherogenic vulnerability in the setting of additional risk factors. Chatzizisis et al12 have shown using a DM/HC model that areas of low endothelial shear stress in coronary arteries predicted future development of atherosclerotic plaque development and progression to high-risk lesions with increased lipid accumulation, inflammation, and expansive remodeling, hallmarks of vulnerable lesions with increased risk of causing clinical instability. There are several theories on the origin of the differential vascular gene expression which include (1) that additional or different risk factors are required, eg, hypertension or smoking for complex lesion development in carotids or thoracic aortae, (2) variable deposition of LDL particles,13 (3) shear stress dependent accumulation of inflammatory cells in specific vascular regions,14 (4) differential shear stress dependent endothelial gene expression resulting from direct biomechanical effects,15 or (5) blood vessel geometry. Curved and branching arteries, such as the coronaries, possess areas of disturbed blood flow, whereas a higher shear stress and a notable lack of atherosclerosis is noted in arteries with unidirectional laminar blood flow, such as the carotid artery.11
This is the first study to evaluate gene expression over a 6-month time period in a large atherosclerotic animal model. Mouse models, developed through genetic manipulation, have proven useful in elucidating underlying mechanisms of disease development; however, they are limited by their varying lipid profiles, lack of spontaneous coronary artery disease, and nonhuman like atherosclerotic disease.16–18 For example, the apoE knock-out and LDL receptor negative model generally develop atherosclerosis in the proximal aorta, aortic arch, and the brachiocephalic arteries19–21 beds dissimilar from human atherosclerosis. In mice DM has little effect on atherosclerosis development,19 and their small size makes tissue acquisition and physiological evaluation difficult. A distinct advantage of the porcine model is the complex human-like appearance of the lesions and abundant tissue available for genetic, molecular, and cellular studies. Prolonged DM/HC results in increased arterial stiffness, reduced vascular compliance, and compromised cardiac functional reserve; characteristics of diabetic heart disease.22 Early coronary lesions, however, are intimal xanthomas rather than intimal thickening, as often noted in humans.6 Nonetheless, the location and composition of later atherosclerotic lesions, metabolism, diets, and cardiovascular physiology are similar to that of humans, and their size makes them amenable to both cellular and preclinical testing. One difference is that after cholesterol feeding pigs exhibit high HDL and low triglyceride levels in the setting of very high LDL levels (supplemental Table II), whereas humans often exhibit low HDL and high triglyceride levels. Specifically, clinically relevant studies designed to evaluate treatment aimed at preventing or regressing complex atherosclerotic complex lesions and the subsequent genetic response can be studied in this model.
There are potential limitations in this study. Gene expression was determined in the right coronary and carotid arteries, and lesion complexity was determined in the left coronary and carotid arteries. Although we have previously observed no differences in the incidence or composition of atherosclerotic lesions in the right (supplemental Figure I) and left coronary arteries, given the potential variability in lesion development it is possible that a variation in gene expression or inflammation was present in the 2 arterial beds. Insofar as the entire vessel was sampled, gene expression would differ in the presence of an atherosclerotic plaque. Hence, cellular heterogeneity at the different time points and different arterial beds may limit interpretation of the results. We have attempted to reduce this potential variation by sampling many animals and using sufficient quantities of tissue to reduce sampling error. The limited list of genes selected for Taqman analysis was constructed from published human plaque microarray data and not from a porcine microarray. Other potentially important porcine pathways may have been missed. However, the purpose of this study was to focus on a human plaque profile to understand the similarities in gene expression between humans and pigs. The diet used contained 0.5 to 1.5% sodium cholate to increase cholesterol levels and development of atherosclerotic lesions. In murine models of atherosclerosis the addition of sodium cholate has been associated with pleitropic effects which may confound interpretation of chronic inflammation.23 No such effects have been reported in swine models; nevertheless, it is possible that some of the effects observed in inflammatory pathway gene expression may have reflected the use of sodium cholate rather than atherosclerosis per se. However, all 3 arterial beds were subjected to the same cholate dose and the noted heterogeneity in inflammation would not be present if cholate was the sole instigator of vascular inflammation. The pigs used in this study were “farm raised” and not genetic clones and thus there are some individual genetic differences. This may, however, be an advantage as humans also exhibit genetic heterogeneity, and this model mimics some of the challenges in studying a complex environmental and genetic disease such as atherosclerosis. It should be noted that an association of increased or decreased gene expression with increased development and complexity of coronary artery and thoracic aortic atherosclerosis does not indicate causality. Finally, this is a model of type I diabetes mellitus and although the addition of hypercholesterolemia results in aspects of metabolic syndrome, there is no evidence of insulin resistance. As such it should be viewed purely as a model of coronary and peripheral atherosclerosis.
In summary, atherosclerosis in the DM/HC porcine model is phenotypically similar to human atherosclerosis. Variability in the extent and complexity of the subsequent lesions observed in the 3 arterial beds was associated with differential gene expression and provides insight into the use of gene expression to predict arterial response to DM and HC. Several genes with as of yet unknown function, when understood, may provide further insights into the atherosclerotic process and possibly novel targets for therapy.
We gratefully acknowledge the assistance of Harrilla Profka in the care of the animals and the performance of the procedures.
Sources of Funding
This work was supported by the Juvenile Diabetes Foundation and GlaxoSmithKline.
Drs Mohler, Shi, and Wilensky are recipients of research grants from GlaxoSmithKline. Drs Sarov-Blat, Zalewski, MacPhee, and Steplewski are employees of GlaxoSmithKline.
E.R.M. and L.S.-B. contributed equally to this study.
Original received August 27, 2007; final version accepted January 30, 2008.
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