Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro

Abstract

Versican is an extracellular matrix proteoglycan produced by many cells. Although versican is generally known as a large chondroitin sulfate proteoglycan (CSPG), the smallest splice variant, V3, consists only of the amino- and carboxy-terminal globular domains and is therefore predicted to be a small glycoprotein, lacking CS chains. The large size, negative charge, and ability of versican variants to form pericellular coats with hyaluronan are responsible for many of its effects. V3, lacking the large size and high charge density, but retaining the hyaluronan-binding domain of the larger isoforms, may have different effects on cell phenotype. To determine whether V3 alters cell phenotype, Fisher rat arterial smooth muscle cells (ASMCs), which express the larger CSPG versican splice forms (V0 and V1) were retrovirally transduced with the rat V3 cDNA. Northern analysis for versican RNAs confirmed that cells transduced with V3 retrovirus, but not cells tranduced with the empty vector, expressed RNA of the size expected for V3/neo(r) bicistronic RNA. V3 overexpressing cells were more spread on tissue culture plastic, had a smaller length-to-breadth ratio and were more resistant to release from the culture dish by trypsin. Interference reflection microscopy of sparsely plated cells showed larger areas of close contact between the V3 expressing cells and the coverslip, in comparison to control cells. Focal contacts in the periphery of V3 expressing cells were larger. Growth and migration studies revealed that V3 transduced cells grow slower and migrate a shorter distance in a scratch wound assay. The increased adhesion and the inhibition of migration and proliferation resulting from V3 overexpression are the opposites of the known and predicted effects of the other variants of versican. V3 may exert these effects through changes in pericellular coat formation, either by competing with larger isoforms for hyaluronan-binding, or by altering other components of the pericellular matrix.

Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury

“…In our previous study, 11 we reported on the reduced pericellular coat in cultured V3-transduced ASMCs and hypothesized that this phenotypic feature might arise through properties associated with the G1 domain of V3 that contains the HAbinding region. The larger versican variants V0 and V1, with their attached chondroitin sulfate chains, bind to HA to form pericellular coats that are antiadhesive and necessary for both proliferation and migration.…”

“…11 Compared with vector-alone cells, V3 cells were more spread, flatter, and less spindle-shaped (Figure 1). The most notable difference, however, and one not seen in short-term cultures, was the presence of an irregular network of fibers, especially prominent around the periphery of cells adherent to the culture dish (Figure 1).…”

“…11 Briefly, the rat V3 cDNA was inserted into the BamHI site of the retroviral vector LXSN (courtesy of Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash). 15 Orientation was confirmed by PCR.…”

“…9 Recently, we reported that retroviral insertion and overexpression of the gene for V3 into cultured Fischer rat ASMCs induces a number of phenotypic changes. 11 Compared with vector-alone-transduced cells, V3 ASMCs are more flattened and spread, show a large increase in area of close contacts and in the size of peripheral focal contacts, increased resistance to trypsin detachment, reduced pericellular coats, and decreased rates of growth and migration. The mechanism by which V3 effects these changes is not clear, although the morphological features, the reduced growth and migration, and the reduced cell coat, raise the possibility that V3 may affect the accumulation of components of the extracellular matrix.…”

Expression of Versican V3 by Arterial Smooth Muscle Cells Alters Tumor Growth Factor β (TGFβ)-, Epidermal Growth Factor (EGF)-, and Nuclear Factor κB (NFκB)-dependent Signaling Pathways, Creating a Microenvironment That Resists Monocyte Adhesion

“…Expression of V3 in ASMCs-The V3 splice variant of versican was expressed in Fischer rat ASMCs using retroviral vectors, as described previously (17)(18)(19). Briefly, the rat V3 sequence was inserted into the BamHI site of the retroviral vector LXSN (courtesy of Dr. A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA).…”