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

Abstract

Abstract-Versican is an extracellular matrix (ECM) proteoglycan that is synthesized as multiple splice variants. In a recent study, we demonstrated that retroviral-mediated overexpression of the variant V3, which lacks chondroitin sulfate (CS) chains, altered arterial smooth muscle cell (ASMC) phenotype in short-term cell culture. We now report that V3-overexpressing ASMCs exhibit significantly increased expression of tropoelastin and increased formation of elastic fibers in long-term cell cultures. In addition, V3-overexpressing ASMCs seeded into ballooned rat carotid arteries continued to overexpress V3 and, at 4 weeks after seeding, produced a highly structured neointima significantly enriched in elastic fiber lamellae. In contrast to the hydrated, myxoid neointima produced by rounded or stellate vector-alonetransduced cells, V3-expressing cells produced a compact and highly ordered neointima, which contained elongated ASMCs that were arranged in parallel arrays and separated by densely packed collagen bundles and elastic fibers. These results indicate that a variant of versican is involved in elastic fiber assembly and may represent a novel therapeutic approach to facilitate the formation of elastic fibers. T he extracellular matrix (ECM) proteoglycan versican, the major chondroitin sulfate proteoglycan of vessel wall, 1 is distinguished by a central extended region with attached glycosaminoglycan (GAG) chains by an amino-terminal globular domain (G1) that binds hyaluronan (HA) and by a carboxy-terminal selectin-like domain (G3) that binds to other matrix components including tenascin-R and fibulin-1. [2][3][4][5][6] The mRNA that codes the GAG attachment region, which in the full-length form has 2 domains, can undergo differential splicing to produce 4 variants: V0, with 2 (␣ and ␤) GAG binding regions; V1 (with the ␤ GAG exon); V2 (with the ␣ GAG exon); and V3 with neither GAG exon and formed from the G1 and G3 domains only. 7-9 V3 is thus predicted to be a glycoprotein and not a proteoglycan. V3 mRNA has been demonstrated in aortic tissue as well as cultures of ASMCs by Northern blot 10 and has been detected, by polymerase chain reaction (PCR), from cDNA libraries for various tissues including brain, stomach, and liver. 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. For example, Evanko et al 12 showed recently that the V1 isoform of versican in ...

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

“…Elastic fiber deposition was examined by immunofluorescence using anti-tropoelastin antibodies after 3 weeks of culture. Consistent with our previous studies (19,28), V3 expression by ASMCs significantly increased mRNA levels of tropoelastin as well as fibulin 5, a component of microfibrils critical in elastic fiber formation (Fig. 1, A and B) (22)(23)(24).…”

“…Deficiency in fibulin 2 and fibulin 5 in animals results in disrupted elastic laminae accompanied by an increase in vascular adhesion molecules and tissue factor expression as well as thrombus formation after carotid artery ligation injury (23), suggesting that the elastin-enriched ECM present in healthy aortas protects against vascular injury. Previously, we have demonstrated that expression of the V3 isoform of versican in ASMCs generates an ECM enriched in elastic fibers and that this ECM had reduced capacity to bind monocytes (4,18,19,28). We now show a causal relationship between changes in elastin accumulation and monocyte adhesion such that knocking down tropoelastin or fibulin 5, a component of microfibrils critical for elastic fiber formation (22)(23)(24), partially reverses the inhibition of monocyte adhesion caused by V3 expression.…”

“…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).…”

Tissue elasticity and the ageing elastic fibre

“…The repair of damaged elastic fibres within these tissues remains a difficult challenge for biomedicine, but in tissues such as photoaged skin, topical treatment with all-trans retinoic acid has been shown to promote the deposition of fibrillin-1 but not of type I collagen (Watson et al 2008). In the vasculature, whilst there is a great deal of interest in the potential of in vitro tissue engineering approaches (for a review see Kielty et al 2007), Merrilees and coworkers have demonstrated the ability of an ECM glycoprotein (verscian) to stimulate elastic fibre deposition in vivo (Merrilees et al 2002). Perhaps the best hope, however, lies with preventing the damage from occurring; elastic fibres have a widespread tissue distribution, where they fulfil vital mechanical and biochemical roles and are uniquely long-lived and hence susceptible the accumulation of age-related damage.…”

Hyaluronan-dependent pericellular matrix☆

“…Elastic fiber formation may also be regulated by the pericellular matrix. For example, retrovirally mediated overexpression of versican splice variant V3 by arterial smooth muscle cells induced tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury [53]. This was proposed to be due to removal of the chondroitin sulfate containing forms of versican, VO and V1, by competition for hyaluronan in the pericellular matrix.…”