Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans

Objective: Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-beta 1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factor beta 1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-beta 1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-beta 1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. Methods and results: Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-beta 1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-beta 1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-beta 1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-beta 1-mediated [S-35]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-beta 1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-beta 1 receptor signaling resulted in decreased lipid binding. Conclusions: Classic transforming growth factor-beta 1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-beta 1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease. (C) 2013 Elsevier Inc. All rights reserved.