The use of network analyses for elucidating mechanisms in cardiovascular disease

Systems biology offers the potential to provide new insights into our understanding of the pathogenesis of complex diseases such as atherosclerosis. It seeks to comprehend the system properties of the non-linear interactions of the multiple biomolecular components that characterize a living organism. An important component of this research approach is identifying the biological networks that connect the differing elements of a system and in the process describe the characteristics that de. ne a shift in equilibrium from a healthy to a diseased state. The utility of this method becomes clear when applied to multifactorial diseases with complex etiologies such as inflammatory-related diseases, herein exemplified by cardiovascular disease. In this study, the application of network theory to systems biology is described in detail and an example is provided using data from a clinical biobank database of carotid endarterectomies from the Karolinska University Hospital (Biobank of Karolinska Endarterectomies, BiKE). Data from 47 microarrays were examined using a combination of Bioconductor modules and the Cytoscape resource with several associated plugins to analyze the transcriptomics data and create a combined gene association and correlation network of atherosclerosis. The methodology and workflow are described in detail, with a total of 43 genes found to be differentially expressed on a gender-specific basis, of which 15 were not directly linked to the sex chromosomes. In particular, the APOC1 gene was 2.1-fold down-regulated in plaques in women relative to men and was selected for further analysis based upon a purported role in cardiovascular disease. The resulting network was identified as a scale-free network that contained specific sub-networks related to immune function and lipid biosynthesis. These sub-networks link atherosclerotic-related genes to other genes that may not have previously known roles in disease etiology and only evidence small alterations, which are challenging to find by statistical and comparison-based methods. A number of Gene Ontology (GO), BioCarta and KEGG pathways involved in the atherosclerotic process were identified in the constructed sub-network, with 19 GO pathways related to APOC1 of which 'phospholipid efflux' evidenced the strongest association. The utility and functionality of network analysis and the different Cytoscape plugins employed are discussed. Lastly, the applications of these methods to cardiovascular disease are discussed with focus on the current limitations and future visions of this emerging field.