Genetic Determinants of Aortic CD47 Expression

The Hybrid Mouse Diversity Panel (HMDP), which includes a quantitative analysis of 109 classical and recombinant inbred mouse strains^{41 (link)}, was used to identify factors associated with vascular CD47 expression, in vivo. Briefly, whole aorta from the arch to the mid-abdomen was snap-frozen at the time of euthanasia and total RNA was isolated using the RNeasy kit (Qiagen), as described^{42 (link)}. Genome wide expression profiles were determined by hybridization to Affymetrix HT-MG_430 PM microarrays on a subset of female mice from 104 strains (N = 2 aorta per strain). Quantification of plasma cytokines was carried out in a multiplexed immune-capture microbead system (Milliplex Mouse Cytokine / Chemokine Magnetic Bead Panel MCYTOMAG-70K, EMD Millipore) as per manufacturer’s instructions. Cytokines profiled were: G-CSF, GM-CSF, IFNr, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IP-10, KC, MCP-1, MIP-1α, MIP-1β, M-CSF, MIP-2, MIG, RANTES, TNF-α. Plasma insulin was measured using the mouse insulin ELISA kit (80-INSMS-E01, Alpco) as per manufacturer’s instructions. Pearson’s correlations were generated to calculate transcript-transcript and transcript-trait correlations. Using these methods, the genes and plasma cytokines which were significantly associated with aortic CD47 expression levels were identified.