**4. Relevance to atherogenic theories**

The idea that susceptible and resistant populations exhibit differences in VSMC differentia‐ tion is vigorously supported by our data. Both experiments showed SR-Ar VSMC to be in the contractile phase, while this was clearly not the case for WC-As. The WC-As did not ex‐ press myosin-related genes or proteins, and the presence of beta actin correlates with a syn‐ thetic phenotype. Contractility was a major difference in our previous gene analysis [70], so finding related proteins strengthens this pathway's relevance to the resistant phenotype. Contraction depends on the ATP produced by oxidative phosphorylation. Although mito‐ chondrial respiration was a significant biological process in this analysis, monosaccharide catabolism was upregulated in the WC-As.

Glycolytic enzymes ENO1 and lactate dehydrogenase A (LDHA), both found in the WC-As, contain hypoxia response elements [78]. This may indicate that during oxygen deficit, VSMC shift their energy production from oxidative phosphorylation, which has an absolute oxygen requirement, to glycolysis, which is anaerobic. Glycolysis does not produce enough ATP to support a contractile phenotype, and the upregulation of ENO1 to support glycoly‐ sis would be problematic, given its dual role as a transcription factor (Figure 1). In the ab‐ sence of oxygen, lipids cannot be fully oxidized which could have a two-fold effect. First, more substrate is available for cholesterol and TAG synthesis, an idea supported by DGAT2 expression in the WC-As. Second, the increased ROS generation from partially-oxidized lip‐ ids could be triggering the observed WC-As inflammatory pathway.

The theory that atherosclerosis is a chronic inflammatory disease is supported by the data sets, although the chemokines expressed in the WC-As are secondary indicators of inflam‐ mation, and the primary mediator remains unclear. In addition to the TNF alpha pathway uncovered in the WC-As proteomics experiment (Figure 3), CXCL12 was significantly upre‐ gulated in the RDA experiments. This is relevant to the human disease as it was recently identified as a CVD- susceptibility locus in a genome wide association study [79, 80]. Al‐ though newly implicated in atherosclerosis, CXCL12 is thought to exacerbate the probability of plaque rupture during the advanced stages of the disease [81]. The over-expression of CXCL12 in the earliest stages of atherogenesis is an important observation in the WC-As, es‐ pecially given that it activates c-src (Figure 1). As described earlier in this review, c-src acti‐ vates beta actin (synthetic phenotype) and enolase (glycolytic enzyme), and, like ENO1, CXCL12 has a hypoxia response element in its promoter [82]. CXCL12 has been implicated in the metastasis and angiogenesis of some cancers, and is an "independent predictor" of ovarian cancer survival rates [83]. In this capacity, it may contribute to the migration and proliferation of VSMC that occur early in atherogenesis.

induces JAK1 gene expression via two receptors, TNFR1 and TNFR2. JAK1 turns on STAT1 and this pathway is known to regulate VSMC inflammatory processes [76]. TNFα also exerts its biological effect on apoptotic peptidase activating factor 1 (Apaf-1), which induces apop‐ tosis via TRADD and the cellular caspases. This would be a simple story of inflammation and apoptosis, if it wasn't for the concomitant HSP70 expression in the WC-As. This heat shock protein also works through JAK1, but has an inhibitory effect on Apaf-1 [77]. There‐ fore, although Apaf-1 expression is stimulated by c-Jun, both via TNFα and cytochrome c, HSP70 simultaneously blocks Apaf-1, possibly causing problematic cells to resist apoptosis

The idea that susceptible and resistant populations exhibit differences in VSMC differentia‐ tion is vigorously supported by our data. Both experiments showed SR-Ar VSMC to be in the contractile phase, while this was clearly not the case for WC-As. The WC-As did not ex‐ press myosin-related genes or proteins, and the presence of beta actin correlates with a syn‐ thetic phenotype. Contractility was a major difference in our previous gene analysis [70], so finding related proteins strengthens this pathway's relevance to the resistant phenotype. Contraction depends on the ATP produced by oxidative phosphorylation. Although mito‐ chondrial respiration was a significant biological process in this analysis, monosaccharide

Glycolytic enzymes ENO1 and lactate dehydrogenase A (LDHA), both found in the WC-As, contain hypoxia response elements [78]. This may indicate that during oxygen deficit, VSMC shift their energy production from oxidative phosphorylation, which has an absolute oxygen requirement, to glycolysis, which is anaerobic. Glycolysis does not produce enough ATP to support a contractile phenotype, and the upregulation of ENO1 to support glycoly‐ sis would be problematic, given its dual role as a transcription factor (Figure 1). In the ab‐ sence of oxygen, lipids cannot be fully oxidized which could have a two-fold effect. First, more substrate is available for cholesterol and TAG synthesis, an idea supported by DGAT2 expression in the WC-As. Second, the increased ROS generation from partially-oxidized lip‐

The theory that atherosclerosis is a chronic inflammatory disease is supported by the data sets, although the chemokines expressed in the WC-As are secondary indicators of inflam‐ mation, and the primary mediator remains unclear. In addition to the TNF alpha pathway uncovered in the WC-As proteomics experiment (Figure 3), CXCL12 was significantly upre‐ gulated in the RDA experiments. This is relevant to the human disease as it was recently identified as a CVD- susceptibility locus in a genome wide association study [79, 80]. Al‐ though newly implicated in atherosclerosis, CXCL12 is thought to exacerbate the probability of plaque rupture during the advanced stages of the disease [81]. The over-expression of CXCL12 in the earliest stages of atherogenesis is an important observation in the WC-As, es‐ pecially given that it activates c-src (Figure 1). As described earlier in this review, c-src acti‐

and continue to proliferate. This pathway is not present in the SR-Ar.

ids could be triggering the observed WC-As inflammatory pathway.

**4. Relevance to atherogenic theories**

178 Current Trends in Atherogenesis

catabolism was upregulated in the WC-As.

**Figure 3.** Network analysis (GeneGo, Carlsbad, CA) incorporating annotated differentially-expressed soluble proteins extracted from vascular smooth muscle cells (VSMC) of White Carneau (WC-As) and Show Racer (SR-Ar) pigeons. Ab‐ breviations listed in [72].

The response to retention theory is marginally supported by the data. Ribophorin and man‐ nosidase expression in both experiments suggests increased glycosylation in the WC-As, a prerequisite of lipid retention. Lipid retention was further suggested by the expression of CAV1 in the WC-As. This finding is important because APOE knockout mice studies have shown that the loss of CAV1 is actually protective against atherosclerosis [84]. Therefore, its differential expression in the pigeon model may play an important role in the susceptible/ resistant phenotypes. Finally, the response to injury, monoclonal nature, and the effect of he‐ modynamic stress were not tested in either experiment, so their contribution to atherosclero‐ sis in the pigeon model could not be determined.

Gene and protein expression in susceptible and resistant pigeon VSMC support current the‐ ories of human atherogenesis. Many genetic factors have been identified that contribute to plaque progression, but the gene or genes responsible for initiation of the disease remain un‐ clear. The autosomal inheritance of spontaneous atherosclerosis in the White Carneau sug‐ gests that the affected gene is one having broad effects, such as a transcription factor. Because pigeon VSMC genes and proteins are differentially expressed prior to foam cell for‐ mation, the pigeon is a valuable model for studying the earliest events of atherogenesis.

## **Acknowledgements**

Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2490.

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