Abstract 2293: Endothelial Dysfunction and Defective Smooth Muscle Contractility in Aneurysmal Fibulin-4 Deficient Mice
Using Fibulin-4 knockdown mice (Fibulin-4R/R), we previously showed that the dosage of Fibulin-4 can determine the severity of aneurysm formation. Strikingly, even a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4+/R mice occasionally resulted in small aneurysm formation. To get insight into underlying molecular pathways involved in aneurysm formation we rigorously analyzed the aorta transcriptome changes of Fibulin-4+/R and Fibulin-4R/R aortas. To examine the quality of the various arrays, raw intensities of all samples were normalized and visualized by Principal Component Analysis. Cut-off values for significantly expressed genes were set at a false discovery rate of ≤0.5 and a fold change of 1.5. Ingenuity pathway analysis identified three major dysregulated pathways including TGFβ signaling, immune response and specifically Ca2+ signaling genes involved in the maintenance of contractile function. Histological analysis and α-smooth muscle actin immunostaining showed loss of smooth muscle cells (SMCs), which coincided with cartilage bone formation, already in Fibulin-4+/R animals. Severe loss of SMCs and increase of extracellular matrix depositions resulted in overall thickening of the aortic wall in Fibulin-4R/R mice. In addition, Fibulin-4R/R mice showed increased endothelial damage, endothelial cellular proliferation as evidenced by BrdU labeling and thrombus formation, indicative for endothelial dysfunction. Subsequently, functional analysis of the contractile capacity was performed by isometric force measurements of ascending and descending aortas. Maximum contractile responses were determined for phenylephrine and compared to KCl (100 mM). KCl contraction measurements showed a significantly reduced contractile capacity of ascending aortas in Fibulin-4R/R mice correlating with a reduction of SMCs in this area. In descending aortas, contractility decreased from 54±7% in wildtypes to 2±2% in Fibulin-4R/R mice. Endothelium-dependent vasorelaxation by acetylcholine, following preconstruction with U46619, was strongly reduced in Fibulin-4R/R mice. In summary, our results uncover a role for Fibulin-4 in the maintenance of SMC contractile function and endothelial responsiveness.