Systems biology analyses of CVD-associated proteins were performed using the Ingenuity Pathways Knowledge Base (Qiagen, Redwood City, CA), a repository of biological interactions and functions created from millions of individually modeled relationships ranging from the molecular (proteins, genes) to organism (diseases) level. Ingenuity Pathway Analysis (IPA) uses enrichment analysis-based approaches66,67 to calculate the significance of observing a candidate protein set within the context of biological systems.

Ingenuity Pathways Analysis (IPA) has generated a knowledge database that defines and incorporates many different categories and subcategories of biological processes and disease functions based on gene/protein expression or interactions. Each function defined by IPA includes a specific set of genes/proteins that have been linked to the particular functional category based on published data. IPA then calculates the enrichment of genes/proteins from the list of genes/proteins in the particular test set to the genes/proteins included in each functional category and calculates the p-value for enrichment or overlap between the test set and the IPA knowledge base using Fisher’s Exact test.

The different functional categories are defined by IPA based on genes/proteins associated with such functions and can be displayed in a tree map which clusters related functions together, thus providing a high-level view of the function families. Consequently, there may be various related functions such as “recruitment of neutrophils” and “recruitment of granulocytes” that have many overlapping genes/proteins, but have also some that are unique to one but not the other. Since granulocytes includes basophiles, eosinophils, and neutrophils, granulocytes would be a higher-level hierarchical category than neutrophils and neutrophils would be a subcategory of granulocytes.

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