ENDOTHELIAL DYSFUNCTION AND DEFECTIVE SMOOTH MUSCLE CONTRACTILITY IN ANEURYSMAL FIBULIN-4 DEFICIENT MICE: 8A.05

Abstract

Objective: 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. Aortic pulse pressure was 2 to 3-fold higher in Fibulin-4R/R mice, resulting from increased aortic stiffness. Here, we analyzed the altered biological pathways in aneurismal disease at the molecular, cellular and functional level. Methods: Aorta transcriptome changes of Fibulin-4+/R and Fibulin-4R/R aortas were performed using ANOVA and Ingenuity Pathway Analysis. Histology was applied to verify cellular abnormalities. Functional analysis occurred by measuring isometric forces of thoracic aortas in vitro and aortic pressures with a pressure transducer catheter in vivo. Results: 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 á-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. In vitro, maximum contractility was determined for phenylephrine and compared to KCl (100 mM). KCl responses showed a reduced contractile capacity of ascending aortas in Fibulin-4R/R mice correlating with a reduction of SMCs in this area. In descending aortas, phenylephrine-induced contractility decreased from 54 ± 7% in wildtypes to 13 ± 2% in Fibulin-4R/R mice. Endothelium-dependent vasorelaxation by acetylcholine, following preconstruction with U46619, was strongly reduced in Fibulin-4R/R mice. Finally, with reducing expression of fibulin-4, diastolic blood pressure decreased from 63 ± 3 in Fibulin-4+/+ to 60 ± 5 in Fibulin-4+/R and 41 ± 7 in Fibulin-4R/R mice. Conclusion: Our results uncover a role for Fibulin-4 in the maintenance of SMC contractile function and endothelial responsiveness.