The present study unexpectedly demonstrated that whole body RAP deficiency reduced atherosclerosis in hypercholesterolemic mice that were infused with AngII, despite increased plasma cholesterol concentrations. Furthermore, RAP deficiency led to a manifest reduction of LRP1 protein abundance in macrophages, but the absence of RAP in bone marrow-derived cells had no effect on the development of atherosclerosis. Also unexpected was the lack of effect of whole body RAP deficiency on AngII-induced AAA formation.
Much of the interest in RAP stems from its role in inhibiting the effects of all known LRP1 ligands and its function as a chaperone for this molecule [5
]. As a consequence, a mechanistic interpretation of the effects of RAP deficiency on atherosclerosis needs to consider the contribution of LRP1. No studies have been performed on the effects of LRP1 deficiency in atherosclerosis, since whole body depletion of LRP1 results in embryonic death [25
]. Also, there are no reports of LRP1 floxed mice being used with Cre expression that is ubiquitously expressed in an inducible manner in mature mice. However, atherosclerotic development has been determined in cell-specific LRP1 depletion in macrophages, SMCs, and hepatocytes. Each of these cell-specific deficiencies of LRP1 increased lesion size in hypercholesterolemic mice [9
]. Despite the many associations of RAP with LRP1 expression, the effects of RAP deficiency on atherosclerosis contrast those of LRP1 deficiency.
In LDL receptor -/- mice that were infused with AngII and fed the saturated fat-enriched diet, the most abundant cell type in atherosclerotic lesions was lipid-laden macrophages [14
], whereas the presence of smooth muscle cells in lesions was not readily distinguishable. LRP1 protein abundance was equivalent in aortic media between mice of the two RAP genotypes. In addition, we did not detect any difference of LRP1 protein abundance in cultured aortic SMCs from RAP +/+ and -/- mice that were incubated with AngII. The inability of RAP deficiency to mimic the effects of SMC-specific LRP1 deficiency might be explained by the findings in the present study that deficiency of RAP does not lead to a LRP1 hypomorphic phenotype in SMCs. In contrast, RAP deficiency promoted a pronounced reduction of LRP1 protein abundance in macrophages. Since macrophage-specific deletion of LRP1 increases atherosclerosis [10
], the lack of effect of bone marrow cell-derived RAP deficiency appears to contradict the findings in these studies. However, it should be noted that RAP has regulatory functions that extend to many molecules beyond LRP1. It is possible that RAP deficiency leads to compensatory increases or contemporary defects of other members in the LDL receptor family or other classes of proteins that are associated with RAP. Of the molecules that interact with RAP, LPL deficiency in macrophages has been demonstrated to reduce atherosclerosis in mice [26
]. RAP deficiency did not change mRNA abundance of LPL in macrophages in the present study. Since RAP regulates many proteins [27
], it is likely that reduction of atherosclerosis in RAP -/- mice is mediated through a complex mechanism involving multiple proteins.
Whole body RAP deficiency increased plasma cholesterol concentrations in LDL receptor -/- mice. This has been demonstrated previously and was attributed to inhibition of chylomicron uptake in liver [4
]. RAP deficiency diminishes protein abundance of LRP1 in mouse liver [4
]. As a consequence, in mice with double deficiencies of RAP and LDL receptor, clearance of large size lipoproteins in plasma such as chylomicron, VLDL and LDL were impaired, thereby resulting in increased plasma cholesterol concentrations of these lipoproteins [4
]. It is unclear whether chylomicron-sized lipoprotein particles play a role in the development of atherosclerosis [29
Chronic AngII infusion augments atherosclerosis and promotes AAAs in hypercholesterolemic mice. Although both are influenced by AngII, these two diseases are distinct in both pathologies and mechanisms. Previously, we demonstrated region-specific effects of AngII on aortic contractile responses and morphological heterogeneity [22
]. Chronic AngII infusion promotes hyperplasia of SMCs in ascending aortas but hypertrophy of SMCs in thoracic and abdominal aortas [23
]. In contrast, AngII produces hardly discernable contraction of ascending, descending, and suprarenal aortas, but striking contraction of infrarenal aortas [22
]. Although the mechanism is not clear, the phenotypic diversity of aortic SMCs may contribute to the differential effects of RAP deficiency on AngII-induced atherosclerosis and AAAs.
SMC-specific LRP1 deficiency led to development of severe medial thickening and aneurysmal formation in hypercholesterolemic mice [12
]. We considered whether the similar phenotype of SMC-specific LRP1 deficiency and AngII-induced AAAs were ascribed to AngII decreasing LRP1 abundance in SMCs. We were unable to determine any change of LRP1 abundance in SMCs isolated from RAP -/- mice. In addition, unlike a study that has reported increased LRP1 protein abundance in human vascular SMCs by AngII [30
], we did not identify any contribution of AngII in cultured mouse aortic SMCs to the protein abundance of LRP1. Furthermore, whole body RAP deficiency had no effect on AngII-induced AAA formation. Therefore, the lack of effect of RAP deficiency on AngII-induced AAA formation is consistent with the lack of a hypomorphic LRP1 phenotype in SMCs of RAP -/- mice.
In conclusion, whole body RAP deficiency reduced atherosclerosis, but had no effect on AAA formation in LDL receptor -/- mice infused with AngII. The effects on atherosclerosis were not seen in mice with RAP deficiency in bone marrow-derived cells, despite a profound reduction of LRP1 in macrophages. This study provides strong evidence that RAP contributes to atherosclerosis in an LRP1-independent manner in AngII-infused mice that is not attributable to its absence in bone marrow-derived cells.