Mechanisms of Aortic Aneurysm Formation
The effects of IL-1β on smooth muscle cell phenotypic during experimental aneurysm formation
Abstract:
With no proven medical therapy to treat or prevent aortic aneurysms, understanding molecular mechanisms of aneurysm formation are critical to developing novel treatment strategies. Although the etiology of formation of aneurysms is poorly understood, there is evidence that local inciting events of unknown nature lead to upregulation of inflammatory cytokines, leukocyte infiltration into the aortic wall, smooth muscle cell (SMC) proliferation, upregulation of matrix metalloproteinases (MMPs) by leukocytes and SMCs, and subsequent destruction of the aortic wall. MMP-2 produced by mesenchymal SMCs and MMP-9 produced by macrophages have both been demonstrated to be critical to experimental aneurysm formation. In late stages of aneurysm progression, SMCs undergo apoptosis, thereby eliminating the cells that are the primary source of extracellular matrix proteins necessary for mechanical stabilization of the blood vessel, thus increasing the risk of aneurysm rupture. To date, studies have primarily focused on the role of leukocytes in aneurysm development, while little is known of the role of SMCs.
Vascular SMCs have the ability to undergo rather profound changes in phenotype in response to pathological stimuli and in experimental models of atherosclerosis. The nature of changes in SMC phenotype can vary over a wide range, but typically involves coordinated repression of SMC marker genes and production of MMPs and their inhibitors. Of major relevance to the present application, recent studies in the Owens Lab and work from the Principal Investigator (PI) have independently observed that the pro-inflammatory cytokine IL-1β (more than any other cytokine tested) profoundly increases the expression of MMP-2,-9 in vitro while simultaneously suppressing the expression of all SMC marker genes including SM α-actin. However, little is known regarding the effect of local inflammatory cytokines on SMC phenotypic switching and their role in aneurysm formation in vivo.
The focus of this proposal is to test the hypothesis that IL-1β within aortic aneurysms, induces an inflammatory phenotype in smooth muscle cells characterized by marked suppression of SMC marker gene, activation of MMPs and inflammatory mediators, and that this process plays a critical role in mediating degradation of the aortic wall matrix and aneurysm formation. Our rationale for focusing on IL-1β is as follows: 1) Tissue from human aortic aneurysms has demonstrated a profound upregulation of IL-1β. 2) Preliminary data from the Owens' laboratory and from prior experience of the PI has suggested that IL-1β, compared to other cytokines, induces the most profound phenotypic switching in SMCs as noted above. 3) The availability of IL-1β and IL-1 R knockout (KO) mice in the Owens laboratory will allow this hypothesis to be tested. We recognize that cytokines other than IL-1β may be relevant to SMC phenotypic switching during aneurysm formation but believe that testing these is beyond the scope of this proposal. However, the studies described will provide a template for potential future studies testing other cytokines present in human aortic aneurysm tissue.
Smooth muscle cell phenotypic switching during experimental aneurysm formation is KLF4 dependent
Abstract:
Although the etiology of aneurysms are poorly understood, there is evidence of upregulation of inflammatory cytokines, leukocyte infiltration into the wall, smooth muscle cell (SMC) apoptosis, and production of matrix metalloproteinases by leukocytes and SMCs leading to aortic wall destruction. Recent evidence has suggested an important role for transforming growth factor-beta (TGF-β) during aneurysm development. Mutations of the TGF-β receptors, TGFBR1 and TGFBR2, have been causally linked to familial aneurysm syndromes. Furthermore, mouse models of Marfan's syndrome deficient in fibrillin are characterized by increased TGF-β and can be inhibited by specific antibodies to TGF-β.
To date, studies have primarily focused on the role of leukocytes in aneurysm development, while little is known of the role of SMCs. Vascular SMCs have the ability to undergo profound changes in phenotype, defined by coordinated repression of SMC marker genes and production of MMPs, in models of atherosclerosis. Of major relevance to the present application, studies in the Owens Lab have identified and cloned a novel protein, Kruppel Like Factor-4 (KLF4) to be a key effector that binds to the TGF-β control element and represses TGF-β dependent increases in SMC marker genes in vivo. Recent studies using a conditional knockout KLF4 mouse created in the Owens Lab have shown that wire injury of the carotid artery results in delayed repression of SMC marker genes and enhanced neointimal formation. Moreover, evidence from the Owens Lab suggests that KLF4 plays a key role in activating expression of inflammatory genes through profound myocardin-dependent inhibition of NFkB. Thus, KLF4 represses SMC marker genes and may alter both SMC and leukocyte responses to vascular injury/ inflammation as related to atherosclerosis. However, nothing is known regarding the role of KLF4 in regulating SMC phenotypic switching in vivo during aneurysm formation.
The focus of this proposal is to test the hypothesis that SMCs within aortic aneurysms undergo phenotypic switching to an inflammatory phenotype characterized by marked suppression of SMC marker genes and activation of MMPs, and that this process is dependent on KLF4 which plays a critical role in mediating matrix degradation in aneurysm formation. Our rationale for focusing on KLF4 is as follows: 1) TGF-β is complex and a difficult molecule to target; KLF4 appears to be a key effector of TGF-β signaling and may be a potential therapeutic target. 2) Because conventional KLF4 null mice are lethal, the recent availability of conditional KLF4 deletion mice in the Owens Lab (via a tamoxifen inducible Cre recombinase and floxed KLF4 gene) will allow this hypothesis to be tested. While other effectors of TGF-β signaling may be relevant to SMC phenotypic switching during aneurysm formation, examining these are beyond the scope of this proposal.