In the present study, we first examined the gene expression profiles of PAD. Data analysis identified a number of genes that might be significantly correlated with different levels of PAD severity. The list of differentially expressed genes in intermediate and advanced lesions contains many genes which can be important for atherosclerosis. Most of these genes have not been reported to be related to atherosclerosis before. For example, MAP4K4 is a member of the serine/threonine protein kinase family. It has been shown to specifically activate MAPK8/JNK and mediate the TNF-alpha signaling pathway [31, 32]. In this study, it was significantly and consistently up-regulated in both intermediate and advanced lesions.
A large multidisciplinary study is currently underway to comprehensively assess PAD at multiple levels , The goal of that study is to investigate 300 symptomatic patients with PAD undergoing medical management with or without vascular intervention by lower extremity angioplasty/stenting or vein graft bypass, and to test the hypothesis that the systemic inflammatory response after vascular intervention influences the local milieu responsible for vascular repair and adaptation . Identification of genes through the work may be significant in the selection of candidate genes that can be investigated through these cases-control genetic epidemiology studies. Our research supports the idea that immune responses play a key role in the development of PAD.
In this report, immune related genes were shown to be significantly expressed during the development of PAD. Gene functional analysis further revealed that immune related categories and pathways were significant enriched in the different stages of PAD. In these immune related genes, several genes have been shown to modulate the development of atherosclerosis in mice models. For example, IgG Fc receptors (FcgammaRs) play a role in activating the immune system and in maintaining peripheral tolerance. Previous research suggested that Fcγ receptor deficiency protects against atherosclerosis in Apolipoprotein-E knockout mice . The results suggest that broad-range inhibitors of immune and inflammatory responses can be considered as potential targets for the treatment of PAD. However, gene expression patterns of immune related genes can be different in different stages of PAD. For example, in intermediate lesions, MHC class II molecules were significantly up-regulated including HLA-DMA, HLA-DMB, HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DRB1 and HLA-DRB5. MHC class II molecules are normally restricted to a subset of antigen presenting dendritic cells, B cells, macrophages, and thymic epithelium cells . These cells can be detected close to CD4+ T cells and present peptides to the T cells. The results suggest that there can be an ongoing immune activation in the intermediate lesions. However, MHC class II molecules were not differentially expressed in advanced lesions, even with a higher false discovery rate, which may suggest that the HLA-mediated immune activation may occur mainly in the progression stages of PAD. In addition, complement molecules were also significantly up-regulated in intermediate lesions, not in advanced lesions. Previous studies have implicated that activation of the complement system is probably associated with the initiation and progression of atherosclerosis [36, 37]. Our data thus provide direct evidence from clinical samples demonstrating that complement system mainly play a role in the development stages of PAD. It is therefore conceivable that different and complex immune/inflammatory responses may take place at different stages of PAD.
Atherosclerosis is a systemic, multifocal disease leading to various symptoms and clinical events including cardiovascular disease, cerebrovascular disease, and peripheral arterial disease. Our results reveal that many genes identified in the report are also expressed in coronary or carotid atherosclerotic lesions. For example, C3AR1 and C5R1 are receptors of C3 (C3a) and C5a respectively. A recent study shows that C3AR1 and C5R1 are expressed in human atherosclerotic coronary plaques . Double immunofluorescence staining has shown that the plaque of cells that express both C3aR and C5aR are macrophages, T cells, endothelial cells, and sub-endothelial smooth muscle cells. In addition, gene expression changes between atherosclerosis from coronary and carotid artery samples have been measured by microarray technology in recent years. One study using microarray found that 82 genes were differentially expressed in both animal model and human coronary artery atherosclerosis disease . Our data confirmed 29 genes and 18 genes had significantly different expression in intermediate lesions and advanced lesions, respectively. Moreover, these genes had expression trends similar to the ones found in our data, but our data showed higher fold-changes. In these overlapping genes, 14 were reported to be involved in immune response. Another microarray study found that 206 genes were differentially expressed in aortic atherosclerosis samples . Our data confirms 43 genes and 32 genes had significantly different expression in intermediate and advanced lesions (FDR<1%), respectively. Importantly, in these overlapping genes, 15 were reported to be involved in immune response. Taken together, the results suggested that immune response is a common feature in atherosclerosis-related diseases. Our microarray study differs from prior microarray studies in the array type, sample type, sample classification, and analytical techniques. Nevertheless, the high level of overlapping genes suggests that there are similar molecular mechanisms in the development of peripheral arterial disease and other atherosclerosis-related diseases.
Several limitations of our approach should be noted. First, hybridization-based microarrays, despite their immense potential, have inherent shortcomings related to deficient standardization of methods employed in normalization, statistical analysis, and so on [41, 42]. In this study, we have attempted to limit these shortcomings by selecting subjects who were phenotypically similar to each other except for hypertension. In addition, the initial phases of data analysis, we used different normalization and statistical methods to identify differentially expressed genes. After choosing SAM, we used a rigorous false discovery rate to minimize false positive results. Expression patterns were validated by confirming mRNA expression patterns with conventional molecular techniques. We attempted, based on current literature, to suggest a potential functional role for genes whose expression was markedly altered. Second, atherosclerosis is a slow, progressive disease that may start in childhood; entirely normal arteries can only be obtained from young donors, a factor that can affect gene expression measurements. Although previous research and our data analysis suggest that age had very little effect on genes, further work is needed to identify age-related genes. Third, the relatively small number of patients did not allow us to assess serial changes in the disease development in more detail as would have been possible in animal models . Furthermore, we do not know to what extent the observed changes in gene expression translate into protein synthesis and function, and which genes cause atherosclerosis. Future studies are needed to address these issues.