Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes.

Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening and macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In this study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in rat ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5-fold. Gene ontology analysis showed that components of the extracellular matrix were the most significantly affected by V3 expression. In addition, genes regulating the formation of the cytoskeleton, which also serve as markers of contractile smooth muscle cells (SMCs), were significantly up-regulated. In contrast, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most down-regulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile SMC phenotype, was greatly increased, and the proinflammatory transcription factors NFκB1 and CCAAT/enhancer-binding protein ß were significantly attenuated in V3-expressing SMCs. Overall, these findings demonstrate that V3 expression reprograms ASMCs promoting differentiated and anti-inflammatory phenotypes.

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-ß1 Induction of Lung Myofibroblasts.

The contribution of hyaluronan-dependent pericellular matrix to TGF-ß1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-ß1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-ß1. Inhibition of hyaluronan synthesis in TGF-ß1-induced lung myofibroblasts over a 4day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and ß1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through ß1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with ß1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.