Thoracic aortic aneurysms (TAAs), occurring at the aortic root, are cardinal features of Marfan Syndrome (MFS) and Loeys-Dietz Syndrome (LDS) that significantly shorten the lifespans of affected individuals because they dissect and rupture. Causative mutations for MFS and LDS have implicated perturbed TGF? signaling in TAA pathogenesis. However, considerable uncertainty and controversy abound regarding whether high and/or low TGF? signaling drives aneurysm formation. Also unclear is whether the nearly universal increases in TGF? effector phosphorylation (i.e. pSmad2/3 and pErk1/2), observed in advanced disease, are a primary cause or secondary consequence of aortic distention. Lastly, the cellular mechanism by which perturbed TGF? signaling might promote aneurysm remains undefined. Unfortunately, a small molecule that showed promise in treating aneurysm in MFS mice recently failed in a clinical trial. We have isolated and begun characterizing a novel genetic model of TAA in zebrafish. Animals deficient in Latent-TGF? binding proteins (Ltbps) 1 and 3 (ltbp1-/-; ltbp3-/-), molecules that sequester TGF? ligands in the extra cellular matrix (ECM), rapidly develop impressive aneurysm of the cardiac outflow tract (OFT), a structure homologous to the aortic root in mammals, over a two-day period following grossly unperturbed OFT morphogenesis. We present preliminary data documenting several similarities between zebrafish and mammalian aneurysm. The shared features include: 1) greater than 50% increases in aortic diameter; 2) significantly increased phosphorylation of Smad2/3 and Erk1/2 in the aneurysm wall, indicative of high canonical and non-canonical TGF? signaling, respectively; 3) disorganization of smooth muscle cells which contain prominent stress fibers; 4) a propensity to dissect and/or rupture; and 5) overlapping molecular signatures revealed through a bioinformatics comparison of our recently-acquired RNA-sequencing dataset with a published microarray dataset from MFS mice. Interestingly, we have discovered elevated expression of markers for highly differentiated ?contractile? smooth muscle in the OFTs of mutant zebrafish prior to any visible evidence of aneurysm. To my knowledge, few laboratories have investigated gene expression changes in the aortic root prior to aneurysm emergence in mouse models of MFS and LDS. Therefore, this aspect of the phenotype is highly novel. I propose several innovative and hypothesis-driven experimental approaches designed to provide impactful mechanistic insights into TAA pathogenesis. The knowledge obtained will inform ongoing debates over disease mechanisms and bolster efforts to identify novel therapeutic modalities for preventing and treating aneurysmal disease. I propose two Specific Aims: 1) to test the hypothesis that elevated TGF? signaling is necessary and sufficient to cause aortic aneurysm in zebrafish; and 2) to test the hypothesis that aberrant phenotypic modulation of smooth muscle cells undermines OFT maturation and stability in ltbp1-/-; ltpb3-/- animals.
Localized dilation of the aorta, the large blood vessel that connects the heart with the body, occurs frequently in people with connective tissue disorders. As aneurysms grow, they become increasingly susceptible to rupture, which is an often-fatal medical emergency. The experiments proposed in this grant application are designed to understand why the aorta becomes susceptible to aneurysm in the context of connective tissue disorders with the long-term goal being improved prevention and/or therapies for aneurysmal disease.
