Vascular Smooth Muscle Cell Phenotypic Changes in Patients With Marfan Syndrome

Abstract

Objective— Marfan’s syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan’s syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results— Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. Conclusions— In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan’s syndrome aneurysm formation.

Killing Me Unsoftly

“…27 Mechanistically, recent data published by Crosas-Molist et al indicate that in Marfan syndrome chronic TGF-β signaling is associated with increased expression of vascular smooth muscle cell (VSMC) contractile markers and actin stress fiber formation, as well as collagen I secretion, ultimately leading to increased VSMC and extracellular matrix (ECM) stiffness. 28 On the other hand, active VSMC contraction may serve as a crucial protective mechanism against aortic dissection, as recently suggested by Ferruzzi and colleagues following elegant ex vivo experiments in dissection prone murine aortas. 29 …”

“…In this regard, pressure myography systems may be better suited to obtain vascular pressure-diameter or force-length relations as a direct measure of vascular circumferential or axial stiffness. 47, 49 For dissection of vascular mechanical properties at the cellular or even sub-cellular level, established methods such as atomic force microscopy (AFM), 28 nanoindentation, or micropipette aspiration are available. While these have proven to be a powerful tools to assess individual cell stiffness, it remains to be determined if changes in cell stiffness in vitro directly correlate with changes in vessel stiffness in vivo .…”

Hereditary Influence in Thoracic Aortic Aneurysm and Dissection

“…In contrast TGF-β is a signal associated with transcriptional activation of contractile gene expression through SMAD3 and myocardin 80 . Although increased contractile protein expression has recently been described in aneurysm samples taken from patients with MFS 81 , the exact opposite has been observed in patients with mutations in TGFBR2 82 and sporadic TAA 83 . These conflicting observations may indicate that relative “upregulation” or “downregulation” is less important than disruption, per se , of proper contractile protein homeostasis.…”

Vascular Smooth Muscle Cells

“…For example, dilated aortas from Marfan patients showed increased expression of SMC differentiation markers including ActA2, SM22α, calponin-1, and smoothelin along with nuclear accumulation of phospho-Smad2 to levels greater than healthy aortas 53 . Elevated expression of these SMC differentiation markers was maintained in aortic medial explant-derived cell cultures along with increased expression of myocardin.…”

“…Further analysis showed that SMCs obtained from Marfan patients displayed more robust actin stress fibers, increased rhoA-GTP levels, nuclear accumulation of MRTF-A, greater numbers of FAs at the cell surface, and increased levels of nuclear pSmad2 and pSmad3 compared to similar SMC cultures from healthy aortas. Pharmacological inhibition of TGFβ signaling in Marfan SMCs with the TβR1 inhibitor LY364947 (LY) strongly reduced the high pSmad2/pSmad3 levels, SMC differentiation marker expression, and mRNA levels for myocardin and SRF 53 . Atomic force microscopy was used to show that aortic SMCs from Marfan patients were stiffer than counterparts from healthy aortas, and similar results were found for the secreted ECM from these SMC cultures.…”