University of New Hampshire University of New Hampshire Scholars' Repository New Hampshire Agricultural Experiment Station Publications New Hampshire Agricultural Experiment Station 7-1-2013 Atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortic cells express different genes in vivo Janet L. Anderson University of New Hampshire, [email protected] C. M. Ashwell University of New Hampshire - Main Campus S. C. Smith University of New Hampshire - Main Campus R. Shine University of New Hampshire - Main Campus E. C. Smith University of New Hampshire - Main Campus See next page for additional authors Follow this and additional works at: https://scholars.unh.edu/nhaes Part of the Poultry or Avian Science Commons Recommended Citation J. L. Anderson, C. M. Ashwell, S. C. Smith, R. Shine, E. C. Smith and R. L. Taylor, Jr. Atherosclerosis- susceptible and atherosclerosis-resistant pigeon aortic cells express different genes in vivo Poultry Science (2013) 92 (10): 2668-2680 doi:10.3382/ps.2013-03306 This Article is brought to you for free and open access by the New Hampshire Agricultural Experiment Station at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in New Hampshire Agricultural Experiment Station Publications by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. Authors Janet L. Anderson, C. M. Ashwell, S. C. Smith, R. Shine, E. C. Smith, and Robert L. Taylor Jr. This article is available at University of New Hampshire Scholars' Repository: https://scholars.unh.edu/nhaes/207 Atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortic cells express different genes in vivo J. L. Anderson ,* C. M. Ashwell ,† S. C. Smith ,* R. Shine ,* E. C. Smith ,* and R. L. Taylor Jr. *1 * Department of Animal and Nutritional Sciences, University of New Hampshire, Durham 03824; and † Department of Poultry Science, North Carolina State University, Raleigh 27695 ABSTRACT Spontaneous atherosclerosis in the White NOT were exclusive to the SR-Ar. Microarray analysis Carneau (WC-As) pigeon is inherited as a single gene revealed 48 genes with differential expression. Vascular disorder, and its progression closely mirrors the human endothelial growth factor and p53 binding protein were disease. Representational difference analysis and micro- among the 17 genes upregulated in the WC-As. Thirty- Downloaded from array were used to identify genes that were differen- one genes were upregulated in the SR-Ar including the tially expressed between the susceptible WC-As and transforming growth factor-β signaling factor SMAD2 resistant Show Racer (SR-Ar) aortic tissue. The RNA and heat shock protein 90. Genes representing several extracted from 1-d-old squab aortas was used to make biochemical pathways were distinctly different between cDNA for each experiment. Fifty-six unique genes were breeds. The most striking divergences were in cytoskel- http://ps.oxfordjournals.org/ found using representational difference analysis, with 25 etal remodeling, proteasome activity, cellular respira- exclusively expressed in the WC-As, 15 exclusive to the tion, and immune response. Actin cytoskeletal remod- SR-Ar, and 16 nonexclusive genes having copy number eling appears to be one of the first differences between variation between breeds. Caveolin and β-actin were susceptible and resistant breeds, lending support to the expressed in the WC-As, whereas the proteasome mat- smooth muscle cell phenotypic reversion hypothesis of uration protein and the transcription complex CCR4- human atherogenesis. Key words: atherosclerosis , pigeon , aorta , Show Racer , White Carneau at University of New Hampshire Library on July 17, 2014 2013 Poultry Science 92 :2668–2680 http://dx.doi.org/ 10.3382/ps.2013-03306 INTRODUCTION level of the arterial wall. The Show Racer (SR-Ar) pi- geon is resistant to the development of atherosclerosis Investigation of human disease relies on animal mod- under identical diet and housing conditions, and with els that parallel specific human pathologies. One of the similar blood cholesterol levels (Clarkson et al., 1959). significant human diseases that relies heavily on models Crossbreeding and backcross experiments demonstrat- is atherosclerosis, especially at the earliest stages of the ed aortic atherosclerosis susceptibility to be inherited disease. Diverse animal models exist, each of them hav- in a pattern consistent with an autosomal recessive ing unique features appropriate for investigating differ- Mendelian trait (Smith et al., 2001). ent stages of atherosclerosis. The susceptible-resistant Atherosclerosis continues to be a major factor affect- pigeon (Columba livia) model (Anderson et al., 2012a) ing American mortality. According to the Centers for has been employed to understand genetic components Disease Control (Heron, 2012), diseases of the heart of this disease. White Carneau (WC-As) pigeons de- (#1) account for 25% of the total number of deaths, velop spontaneous atherosclerosis without known risk and cerebrovascular accidents (#4) account for ap- factors (Clarkson et al., 1959; Santerre et al., 1972). proximately 5%. Atherosclerosis contributes to both of The pigeon lesions (St. Clair, 1998; Moghadasian et these leading causes of death in the United States be- al., 2001) have greater similarities to human athero- cause, depending on the location of the affected artery, sclerosis than any other animal model of heart disease. the condition can promote heart disease, stroke, and St. Clair (1983) has reviewed multiple studies clearly kidney failure. Atherosclerosis most commonly occurs demonstrating that WC-As susceptibility resides at the along branch points of the arterial tree, such as the cor- onary and carotid arteries, and the celiac bifurcation of the aorta (Bassiouny et al., 1994; Kjaernes et al., 1981). © 2013 Poultry Science Association Inc. There is a strong familial component to the disease, Received May 10, 2013. Accepted July 1, 2013. and many risk factors that contribute to lesion progres- 1 Corresponding author: [email protected] sion and plaque stability have been identified. However, 2668 IN VIVO PIGEON ATHEROSCLEROSIS GENES 2669 the earliest events of atherogenesis remain unclear, and balanced salt solution and placed on an ice-cooled nu- approximately 50% of heart attacks occur in individu- clease-free surface where extraneous lipid, blood, and als displaying none of the classical risk factors (Ridker, connective tissue were removed. After blotting on filter 2000). Until the genetic predisposition to the disease is paper, the aorta was flash-frozen in liquid nitrogen and fully understood, prevention efforts will remain limited. placed in a −20°C freezer. Multiple hypotheses have been proposed to explain The aortas from each breed were pooled and placed atherosclerotic lesion initiation. Some of these hypoth- in a liquid-nitrogen chilled mortar. They were crushed eses have been based on work in animal models. Lipid with a pestle to a fine powder and homogenized with infiltration, response to retention, response to injury, TRIzol reagent (catalog 15596–026, Life Technolo- inflammation, monoclonal origin, smooth muscle cell gies, Grand Island, NY). The resulting slurries were (SMC) phenotypic reversion, and hemodynamics have transferred into 1.7 mL of nuclease-free microcentrifuge all been explored (Anderson et al., 2013). Many of tubes and centrifuged for 10 min at 16,000 × g at room these explain the preferred site of fatty streak forma- temperature. tion, some explain the accumulation of lipid in the arte- The supernatants were transferred to 1.5 mL of rial wall and the appearance of macrophage cells, but heavy phase Lock Gels (catalog 955154151, Eppendorf, Downloaded from none of the current hypotheses describe the complete Hauppauge, NY) followed by chloroform extraction. set of events that occurs in atherogenesis. Genetic fac- The RNA was mixed with cold isopropanol and incu- tors clearly influence cell proliferation rates (Lichter, bated overnight at −20°C. Following incubation, the 2000), the mitochondrial oxidative capacity for cellular tubes were centrifuged for 15 min at 16,000 × g at room lipids (Scheckhuber, 2005; Yu et al., 2012), cholesterol temperature to sediment the RNA. The supernatant http://ps.oxfordjournals.org/ metabolism (Ordovas and Shen, 2002), and the immune was decanted and the pellets rinsed with 1 mL of cold response (VanderLaan and Reardon, 2005; Hansson 70% ethanol. After decanting, the pellets received an and Libby, 2006), thereby manifesting an underlying additional rinse with 95% ethanol at −20°C. The etha- influence on all aspects of atherogenesis that warrants nol was again decanted, the pellets dried under vacuum additional investigation. and dissolved in 20 μL of distilled, deionized water. Genetic differences between aortic SMC in pigeon Total RNA was assessed in a 3.0 mM sodium phos- breeds were previously characterized in vitro and cat- phate, monobasic buffer at wavelengths ranging from egorized into 6 general metabolic themes (Anderson 220 to 320 nm. Ratios calculated from spectrophoto- at University of New Hampshire Library on July 17, 2014 et al., 2012b). Disparities in energy metabolism and metric readings revealed no protein (OD260/OD280) SMC phenotypic markers were the most remarkable. or polysaccharide (OD230/OD260) contamination. The However, due to the compressed time frame of cell de- RNA concentration was calculated