AngII infusion into hypercholesterolemic mice has been used in many studies to induce AAAs and augment the development of atherosclerosis.4,8
These diseases are frequently co-morbid, and therefore the model permits simultaneous evaluation of both vascular pathologies. Previous studies have demonstrated distinctions between mechanisms that influence the processes of AAAs versus atherosclerosis.31–34
In the present study, MyD88 and TLR4 deficiencies had equivalent effects on AngII-induced AAAs and atherosclerosis. However, while TLR2 deficiency reduced AngII-induced atherosclerosis, we were not able to discern any effect on the development of AAAs.
Deficiency of MyD88 was studied on the effects of AngII-induced AAAs in both apoE−/−
mice fed a normal laboratory diet and LDLR−/−
mice fed a saturated fat-enriched diet. There are marked differences in plasma cholesterol concentrations and lipoprotein characteristics between the two strains. There is also evidence that apoE has direct effects on innate and adaptive immune responses independent of lipoproteins, which has the potential to lead to different responses between apoE−/−
Despite these differences, aneurysms that form during AngII infusion in the two strains are similar in size and characteristics. Consistently as demonstrated in this study, deficiency of MyD88 had a similar effect in attenuating AngII-induced AAAs in both strains.
Infusion of AngII leads to medial accumulation of macrophages in regions that are prone to aneurysmal formation.35
Following luminal dilation, there is enhanced macrophage accumulation and the presence of other leukocyte types, such as lymphocytes.35
Macrophages are the most abundant leukocyte type that infiltrates aortic tissue at all stages of aneurysmal formation.35,36
Despite this abundance, the function of macrophages in aneurysmal pathology has not been determined. Our previous study using osteopetrotic mice that have marked reductions in circulating monocytes37
was confounded by several defects in this strain and a low incidence of AngII-induced AAAs in the genetic background-matched wild type mice. A later study demonstrated that substantial reductions of circulating monocytes by clodronate-lipoproteins decreased severity of AngII-induced AAAs.6
In the present study, we found that the striking effect of MyD88 on AAA formation was attenuated by selective deletion of MyD88 in hematopoietic cells, strongly suggesting a role for MyD88 signaling in leukocytes in promoting AAAs. Interestingly, the same effect did not occur for TLR4 deficiency in hematopoietic cells, despite the pronounced attenuation of AAA formation in mice with whole body TLR4 deficiency.
monocytes are the predominant population of macrophages accumulating in AngII-induced AAAs.28
We demonstrated that MyD88 deficiency ablated AngII-induced Ly-6Clow
monocyte switching in both peripheral blood and spleens. These results are consistent with the previous report that AngII mediates monocyte recruitment from the spleen to peripheral tissues after injury.38
It is possible that MyD88 deficiency blunts macrophage infiltration into the aortic wall via abolishing the ability of AngII-induced Ly-6C monocyte switching.
AngII exerts its bioactive effects predominantly through binding AT1a receptors. AT1a receptors are ubiquitously present on many cell types including macrophages and resident cell types of the aorta. Comparable to whole body deficiency of AT1a receptors,39
whole body deficiency of MyD88 strikingly reduced AngII-induced AAAs. However, while MyD88 deficiency in bone marrow-derived cells also profoundly reduced AngII-induced AAAs, AT1a receptor deficiency on bone marrow-derived cells failed to influence AngII-induced AAAs.39
These findings infer that AngII induces changes in cells of non-hematopioetic origins that subsequently promote AAAs through a mechanism based on MyD88 of hematopoietic origin.
The role of MyD88 and its link to TLRs in the development of atherosclerosis have been previously studied in apoE−/−
Consistent with the previous studies, we demonstrated that deficiency of MyD88 also decreased atherosclerotic lesions that were augmented by AngII infusion in apoE−/−
mice. We also demonstrated that deficiency of MyD88 had a comparable effect on the reduction of atherosclerosis in LDLR−/−
mice. The effect of MyD88 deficiency on AngII-induced atherosclerosis occurred in both mouse strains, despite profound differences in both concentrations and characteristics of plasma lipoprotein cholesterol distributions. Our study advances the previous understanding of the role of MyD88 in atherosclerosis by demonstrating that MyD88 in bone marrow-derived cells accounts for the predominant effect of this adaptor protein in the development of atherosclerosis. Deficiency of TLR2 or TLR4 decreases atherosclerosis in both apoE−/−
The present study also demonstrated that deficiency of these TLRs decreased atherosclerosis in AngII-infused LDLR−/−
mice. There is compelling evidence for the similarities of the effects of MyD88, TLR2, and TLR4 in the development of atherosclerosis. However, it was unclear whether MyD88 served as a required adaptor signaling molecule for TLR2 or TLR4 to promote atherosclerosis. Our findings using bone marrow transplantation in the present study clearly demonstrate that reductions in atherosclerosis in mice with complete deficiency of TLR4 were attributable to MyD88-independent mechanisms. This conclusion is consistent with the recent demonstration that TRIF deficiency reduced atherosclerosis by a mechanism not involving hematopoietic cells.40
In conclusion, this study demonstrates that MyD88 deficiency in hematopoietic cells has a profound effect in reducing AngII-induced AAAs and atherosclerosis. While whole body deficiency of TLR4 had a similar ability to reduce AngII-induced vascular pathologies, the use of bone marrow transplantation clearly demonstrated that these effects were unrelated to MyD88 mediated signaling in hematopoietic cells. Future studies will systemically evaluate other MyD88-linked receptors, such as IL-1β and IL-18, in AngII-induced AAA formation and atherosclerosis.41–44