Atherosclerotic lesions are not evenly distributed throughout the vascular system
. For example, in humans atherosclerotic lesion development occurs predominantly in the lower limbs while the upper limbs are protected from advanced disease
[3, 6]. Location dependent hemodynamic environments and a blood pressure gradient due to upright posture and bipedal locomotion are partially responsible for this heterogeneous disease distribution in humans
[3, 7–9]. However, in swine a human-like atherosclerotic disease distribution has been observed despite the absence of the blood pressure gradient
[3–6, 10] and similar mean arterial blood pressure, mean blood velocity, and shear rate
. Therefore, hemodynamics cannot fully account for the heterogeneous disease distribution between porcine brachial and femoral arteries. We recently identified 430 differentially expressed (DE) genes associated with biological adhesion and Ca-signaling between these arteries in 13 day old healthy swine
. Therefore, we now hypothesized that these differences in healthy arteries would enable rapid disease development in femoral but not brachial arteries. This proposed divergence in atherosclerotic phenotype, classified by Sudan IV, VVG, and H/E staining, between arteries would be accompanied by an increasing number of DE genes with disease progression. In contrast to our hypothesis, the total number of differential transcripts between arteries was reduced with the heterogeneous progression of atherosclerotic disease. However, 56 genes with differential abundance throughout atherosclerotic disease progression are most likely to influence the heterogeneous lesion distribution. In agreement with our previous results
, this study also reports that these genes are associated with calcium signaling, extracellular function, and biological adhesion.
Previous research has shown that Rapacz FHC swine rapidly develop human-like peripheral atherosclerotic disease due to an allelic variant for apolipoprotein B and a low-density lipoprotein receptor dysfunction
[17–19]. Despite similar mean arterial blood pressure, mean blood velocity, and shear rate, this species develops severe atherosclerosis in femoral but not brachial arteries
. We assessed atherosclerotic disease distribution using quantitative and qualitative methods. Firstly, Sudan IV staining for lipid depositions in the endothelial and sub-endothelial layer showed no differences between brachial and femoral arteries in nine month-old animals. In two year-old swine a trend (p = 0.09) towards increased lipid depositions in femoral compared to brachial arteries was observed. VVG and H/E staining allow for Stary stage classification
 and a qualitative assessment of disease progression. The brachial arteries of the nine month-old animals were classified as healthy (Stary stage zero). Femoral arteries from this age group had healthy arteries or very mild disease (Stary stage 1) with the exception of one artery with mineral infiltration (Stary stage two). Brachial arteries from two year-old swine exhibited no (three arteries) or very mild atherosclerotic disease (two arteries). However, the rapid atherosclerotic disease progression in the femoral artery was seen in three of the five two year-old animals. Overall, quantitative and qualitative histological atherosclerotic disease classification using Sudan IV and Stary classification verified that brachial arteries are protected from advanced disease, while femoral arteries rapidly develop atherosclerotic lesions in Rapacz FHC swine. Therefore, the arterial phenotype provided a large spectrum of disease states (healthy, mild and advanced atherosclerosis) for gene expression profiling.
We hypothesized that the number of differential transcripts between arteries would increase due to the rapid disease progression in femoral but not brachial arteries. Contrary to this hypothesis, our current investigation demonstrates that the number of DE genes between arteries decreases with a diverging disease progression. With substantial differences in atherosclerotic disease between arteries of two year old swine, 370 DE genes were identified. This is a reduction of 344 probe sets compared to the number of DE genes for nine month-old animals (Figure
2B). Although 17,984 detectable transcripts did not change with age and disease progression in this study, a reduction of more than 50% of the total number of DE transcripts within the same tissue type is significant. Our findings are supported by a previous cross-sectional study which investigated gene expression patterns of human ECs from early and advanced atherosclerosis compared to healthy controls
. Specifically, this study reported a total of 713 DE genes between ECs from mild atherosclerotic disease compared to those from healthy controls. However, when ECs from advanced atherosclerosis were compared to healthy controls, only 403 transcripts reached significantly different levels of relative transcript abundance. Therefore, similar to our findings in whole arteries, the number of DE transcripts in ECs decreased despite a diverging disease classification. This is remarkable since the development of atherosclerosis is generally accompanied by an up-regulation of inflammatory genes in the diseased arteries
. In addition to the reduced number of transcripts, the volcano plot for the two year old animals shows a larger number of up-regulated genes in the healthier brachial arteries rather than the diseased femoral arteries. Overall, Volgers et al.
 and our findings suggest that differences in disease phenotype do not have to be accompanied by an increased number of DE genes.
Quantitative differences in total number of DE transcripts between samples do not compare gene expression signatures within arteries with a variety of disease states. For this purpose we analyzed the 56 genes with a significant main effect for age as well as a significant age × artery interaction using two-way hierarchical clustering. The heatmap, visualizing the clustering results, groups arteries from nine month old animals (cluster 1 and 4) furthest away from each other, even though brachial arteries in this age group are healthy and femoral arteries only show very mild signs of disease (i.e. low Stary stage and Sudan IV staining). This is in agreement with our previous study reporting 430 DE probe sets between healthy brachial and femoral arteries in 13 day old swine
. Gene expression profiling for ECs from atheroprotective (iliac) and –susceptible (coronary) arteries in the central circulation reported 51 genes with significantly different levels of transcript abundance
. Furthermore, some genes persisted to have differential expression after cell culture
. The authors concluded that these differences are due to embryonic vascular bed origin. Even though current knowledge about peripheral artery development is sparse, based on ours and previous results, one may speculate that developmental differences during angio- and vasculogenesis contribute to our observed pattern of differential gene expression between brachial and femoral arteries in Rapacz FHC swine.
Additional support for this speculation can be derived from arteries of the two year-old swine which are grouped close together in clusters 2 and 3, despite brachial arteries being healthy or having mild disease and femoral arteries showing severe atherosclerotic lesions. Specifically, brachial arteries were characterized with very low Stary stages (0 to 2), which are generally considered reversible, while femoral arteries had lymphocyte infiltration, calcification, and fibrous caps. Considering the large number of DE genes in healthy arteries from young animals, the divergent disease pattern despite a converging gene expression signature in two year old animals and the reported persistent number of DE genes from cultured ECs of porcine iliac and coronary arteries, allows for the speculation of an artery specific developmentally programmed atherosusceptibility. This is also supported by aortic homograft transplantation experiments in which segments from atheroresistant parts of the aorta were exchanged for atherosusceptible segments
. The atherosusceptible segments developed severe atherosclerosis even when exposed to an atheroprotective hemodynamic environment while the atheroprotective segments did not develop the disease even when placed in previously identified atherosusceptible regions of the aorta
. Therefore, the underlying genetic predisposition determined atherosusceptibility rather than the hemodynamic environment
. The current findings of a larger number of DE genes in nine month- compared to two year-old animals and the previous identification of 430 DE genes in healthy 13 day old swine
 supports the hypothesis of developmentally programmed atherosusceptibility.
Investigating gene networks in atherosclerosis provide a tool to explore the differential disease progression between brachial and femoral arteries. Hierarchical clustering groups genes according to similarity in abundance to identify enriched biological signaling pathways which may contribute to the heterogeneous progression of PAD. We identified a total of 13 gene expression clusters using the left dendogram (Figure
4). Five clusters (A, D, F, J, and L) are down-regulated, while three clusters (B, G, M) are up-regulated with severe disease (horizontal cluster 2). However, in addition to being down-regulated in arteries with very advanced disease, clusters F and J are also down-regulated in two year old arteries with mild disease (red color in vertical cluster 3). Therefore, whether the expression levels of these genes changed in response to disease or age cannot be determined. Nonetheless, these clusters contain five down- (TRAF3IP2, CST3, CD97, DBNL, MYD88, and CCL28) and one up-regulated gene (THBS1) which are associated with immune response, while biological adhesion was enhanced with CD97 and NID1 being down- and NPNT, KITLG, and THBS1 being up-regulated. This is consistent with previous reports about an involvement of inflammatory genes in atherosclerotic porcine coronary arteries
 as well as atherosclerotic disease progression in mice and humans
. Interestingly, our data also indicates alterations in vascular smooth muscle contraction (up-regulation of PPP1R12A and PPP1R14A) in arteries with very advanced atherosclerotic disease. This demonstrates that in addition to previously identified biological signaling pathways, which play a role in atherosclerotic disease progression, differences in vascular smooth muscle contraction, mediated by alterations in calcium signaling, may also be related to the heterogeneous progression of PAD.
Gene expression differences between healthy and atherosclerotic arteries are important to understand cellular processes involved in the progression and development of atherosclerotic disease. However, the comparison of transcript abundance in nine month-old animals, when atherosclerotic disease differences are minimal, may identify genes predisposing arteries to rapid disease development. Our results identify cluster C (PAN3, NCOA7, NPHP1, RAB18, SCUBE3) and three genes (PPP1R14A, THBS1, SH2D4F) to be up-regulated in femoral as well as DBNL and ITPR1 up-regulated in brachial arteries of nine month-old swine, respectively. We previously identified SCUBE3 to have differential abundance in healthy arteries from 13 day old swine
. Furthermore, THBS1 is known to influence atherosusceptibility. THBS1 is an antagonist of nitric oxide (NO) signaling
. Recent evidence of cultured human vascular smooth muscle cells identified THBS1 as an important pro-atherosclerotic factor that augments atherosclerotic disease progression
. Hence, THBS1 may sufficiently decrease NO-signaling in femoral arteries and thereby increase its susceptibility to atherosclerotic disease. However, molecular pathways during the early stages of atherosclerosis are not very well understood. This is exemplified by a functional comparison of porcine brachial and femoral artery endothelium-dependent and –independent vasorelaxation that reported high functional correlation between arteries, but no correlation in a variety of pro- and anti-atherogenic proteins
. Even though, our descriptive study does not allow for a definite identification of potentially atheroprotective or -susceptible genes, we demonstrate that there are substantial differences in gene expression between brachial and femoral arteries in 13 day
 and nine month-old animals prior to the onset of disease which could affect arterial atherosusceptibility later in life.
A limitation of the current investigation is that we did not measure the hemodynamic stimuli in this set of nine month- and two-year old Rapacz FHC swine. However, previous studies have reported a similar mean arterial blood pressure, mean blood velocity, and shear rate in brachial and femoral arteries of Yucatan miniature swine
. Even though Rapacz FHC swine are larger compared to Yucatan swine, we assume that the observed hemodynamic similarities between arteries are not affected by size since local artery geometry is unlikely to vary within a species. Nonetheless, our assumption needs to be verified by future research. Our approach of using whole arterial samples implies that a variety of different cell types throughout the progression of atherosclerotic disease may be responsible for our observations. Thus, we cannot determine which type of cell is responsible for the reported differential gene expression. Furthermore, we are aware of the fact that a limitation of this study is the lack of protein data possibly identifying posttranscriptional factors influencing artery specific disease progression. Lastly, an inherent limitation of DAVID analyses is their bias towards previously identified biological signaling pathways.