Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

Abstract

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5 ؊/؊ mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced ␣-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGF␤ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5 ؊/؊ cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan hdf/؉ ) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5 ؊/؊ cells resulted from increased PCM versican content, we generated Adamts5 ؊/؊ ;Vcan hdf/؉ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5 ؊/؊ mice. In Adamts5 ؊/؊ fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.In addition to the role of cells in building specialized structural extracellular matrix, they maintain a pericellular matrix (PCM) 2 that is dynamic, provisional, and intimately associated with the cell surface (1, 2). Thus, it is appropriate to view a cell and its PCM as a functional unit. Processes that could potentially be regulated by PCM include macromolecular assembly (e.g. assembly of collagen, fibronectin fibrils, or fibrillin microfibrils), cell surface receptor-ligand interactions, binding of pathogens, and cell-matrix and cell-cell adhesion. PCM is sometimes referred to as the glycocalyx because of its abundant carbohydrate content (3). The PCM in many cell types such as chondrocytes, ova, neurons (in which PCM is also termed the perineuronal net), vascular smooth muscle cells, and fibroblasts contains aggregates of hyaluronan (HA) with chondroitin sulfate proteoglycans (CSPG) (4 -7). These aggregates are formed from non-covalent association of HA with large CSPGs such as aggrecan, versican, brevican, and neurocan. In contrast to the aforementioned cell types, heparan sulfa...

Homotypic Versican G1 Domain Interactions Enhance Hyaluronan Incorporation into Fibrillin Microfibrils

“…Recently, proteolysis of versican by ADAMTS proteases has been shown to play key biological roles in ovulation (40), neointimal atrophy (41), interdigital web regression (42), and palatal mesenchyme proliferation (43). Dermal fibroblasts that are deficient in ADAMTS-5 exhibit a myofibroblastic phenotype (44), and transfection of cancer cells with the versican G1 domain exaggerates tumor progression (45). However, the mechanisms mediating these activities and whether versican cleavage products perform important roles are not fully understood.…”

The extracellular matrix in myocardial injury, repair, and remodeling

“…In vivo, loss of CD44, the main receptor for haluronan, impaired collagen synthesis in infarct fibroblasts (40). Versican loss in dermal fibroblasts attenuated myofibroblast conversion (68). Although versican is upregulated in healing myocardial infarcts (69), its in vivo role in transdifferentiation of injury-site cardiac myofibroblasts has not been directly documented.…”

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

“…Reduced Adamts5 mRNA Results in the Accumulation of Pericellular Matrix Around C2C12 Myoblasts-Using the red blood cell exclusion assay (27), a significant increase in pericellular matrix was observed after treatment with Adamts5 targeted siRNA, using either computer imaging (Fig. 7, A and B, Adamts5-1 siRNA versus control) or blinded visual analysis, A, significant knockdown of Adamts5 mRNA levels upon siRNA treatments are seen at 72 h after differentiation.…”

“…As a control, hyaluronidase digestion was done using Streptomyces hyalurolyticus hyaluronidase (Sigma) prior to the particle exclusion assay. Resultant images of controls or Adamts5-1 siRNA treatments were analyzed using custom, semiautomated scripts generated in Image-Pro version 6.2 (Media Cybernetics, Silver Springs, MD) as previously described (27). In addition, scoring of the RBC exclusion area was performed by 3 investigators blinded to the experimental groups using a scale of 0 -3, where 0 ϭ no matrix accumulation and 3 ϭ maximal matrix accumulation.…”

“…Particle Exclusion Assay and Quantitation of Pericellular Matrix Area-The assay was done essentially as previously described by Hattori et al (27). C2C12 myoblasts transfected with Adamts5 siRNA were trypsinized and re-plated at a density of ϳ2,000 cells/well into 6-well plates 24 h after siRNA transfection.…”