Abdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta with over 80% mortality after rupture. The current therapy is restricted to surgical repair, highlighting the need to explore mechanic insights into the development of effective, non-surgical therapeutics. In AAA patients, structural integrity of the aortic wall is disrupted due to dissociation of smooth muscle cell (SMC) contractile filaments from extracellular matrix by proteases. This association is mediated by cytoskeletal linker proteins. However, the mechanism underlying loss of vessel wall structural integrity in AAA formation is not understood. Recently, using Angiotensin II (AngII)-induced animal model of AAA, we found a profound increase in protein and activity of aortic calpain, a calcium dependent cysteine protease in aneurysmal tissue. In addition, calpain inhibition significantly attenuated AngII-induced AAA formation. Calpains are the only known proteases targeting an array of cytoskeletal proteins that maintain structural integrity of the aorta, including filamin. Using calpain-1 or -2, the two major ubiquitous isoform-specific deficient mice, we determined that genetic deficiency of calpain-2, but not calpain-1, completely blunted AngII-induced AAA formation. Calpain-2 deficiency attenuated AngII- induced fragmentation of cytoskeletal proteins, filamin A, and talin. Immunostaining of human and mouse AAA revealed expression of calpain-2 protein by infiltrated macrophages and aortic adventitial fibroblasts (AoAFs). Macrophage specific calpain-2 deficiency in mice had no influence on AngII-induced AAA formation, which suggest a critical role for vessel wall mainly AoAFs derived-calpain-2. In cultured AoAFs, AngII promotes ASK-1/ NF-kB mediated inflammatory cytokine induction via activation of calpain-2. Further, calpain-2 silencing suppressed (myo)fibroblast differentiation and migration via TGF-? / Rho kinase signaling. Based on the described background, we will test the hypothesis that calpain-2 activation promotes AngII-induced AAAs by accelerating adventitial fibroblast-derived NF-kB/ASK-1 mediated adventitial inflammation, and (myo)fibroblasts migration to aortic media, which in turn causes medial destruction by upregulating cytoskeletal filament fragmentation in SMCs. To test this hypothesis, the following aims are proposed:
Aim 1 : Determine the contribution of fibroblasts derived-calpain-2 in AngII-induced AAA. A. Does calpain-2 deficiency in fibroblasts attenuate AngII-induced AAA formation? B. Does calpain-2 regulate TGF-?/Rho A in promoting fibroblasts differentiation via LTBP3/RhoGDI1? C. Does calpain-2 depletion in AoAFs attenuate AngII-induced cytoskeletal protein fragmentation in aortic SMCs in vitro? Aim 2: Define the mechanism by which calpain-2 promotes AngII-induced adventitial inflammation? A. Does calpain-2 contribute to ASK-1/ NF-kB activation and correlate with adventitial inflammation during AAA development? B. Does calpain-2 regulate AngII-induced ASK-1/NF-kB activation via TRX/IkB in AoAFs? C. Does cytoskeletal protein fragmentation in human AAA tissue associate with calpain? Aim 3: Determine the effect of calpain-2 deficiency on progression of established AAAs.

Public Health Relevance

Abdominal Aortic Aneurysm (AAA) is a permanent dilation of the abdominal aorta. AAAs are the 13th leading cause of morbidity and mortality in the US population due to aortic rupture (>80%), resulting in an estimated 12,000 deaths per year. The purpose of this application is to elucidate the mechanisms responsible for AAA and thereby identify new therapeutic targets to treat and prevent the devastating AAA disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Reid, Diane M
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University of Kentucky
Internal Medicine/Medicine
Schools of Medicine
United States
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Muniappan, Latha; Javidan, Aida; Jiang, Weihua et al. (2017) Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice. Sci Rep 7:14398