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PURPOSE: Osteopontin (OPN) is a secreted phospho-protein that interacts with cell surface receptor, α_vβ₃-integrin and CD44, as well as extracellular matrix elements, fibronectin and vitronectin. The interaction of OPN with its cell surface receptors induces cellular signaling responses which contribute to several physiological and pathological processes including bone remodeling, cancer metastasis and invasion, inflammation, and inhibition of apoptosis. OPN also has pro-fibrotic effects. It is highly expressed in idiopathic pulmonary fibrosis and renal fibrosis. OPN is involved in dermal scarring. Previous in vivo studies have demonstrated that knockdown of OPN with siRNA reduces dermal fibrosis. However, it is unknown how OPN exerts its pro-scarring affects. RNA aptamers are short (12-30nt) single-stranded RNA sequences with stable three-dimensional conformations that specifically bind target proteins with high affinity. They are attractive therapeutic biologics because they are stable, highly specific, and have minimal toxicity. An aptamer, OPN-R3, blocks OPN signaling, and inhibits cancer growth and metastasis. The role of OPN-R3 in preventing dermal fibrosis has not been investigated.
METHODS: Human scar tissue was stained for OPN and CD44. OPN-R3 was evaluated in vivo for its inhibitory effects on an excisional mouse wound healing model. Human dermal fibroblasts (HDF) were explanted from four different donors undergoing elective procedures. Fibroblast migration was analyzed with the use of boyden chambers and HEMA-3 staining. Inverted confocal microscopy was used to assess fibroblast focal adhesion length. Adhesion was measured by incubating fluorescently stained fibroblasts on OPN coated 96-well plates. CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay was utilized to investigate the proliferative activity of fibroblasts. Free floating collagen lattices were utilized to assess fibroblast contractility.
RESULTS: Immunohistological data of human tissue that shows OPN expression was increased in scar tissue as compared to normal surrounding dermis. This expression was temporal, with OPN being expressed in the inflammatory cells of wound healing. The expression of CD44 was increased during the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Preliminary data shows that OPN-R3 attenuates wound healing in vivo. HDF migrated significantly in response to OPN and this effect was suppressed by the addition of OPN-R3 (p<0.05). OPN did not induce a significant increase in focal adhesion length compared to controls. Adhesion studies demonstrated that OPN increased fibroblast adhesion (p<0.05). OPN-mediated adhesion is inhibited by addition of OPN-R3. Proliferation assays indicate that OPN increased fibroblast growth (p<0.05). OPN-mediated increase in proliferation was inhibited by the addition of OPN-R3. OPN increased fibroblast contractility of collagen lattices (p<0.05). OPN-mediated contractility is inhibited in the presence of OPN-R3.
CONCLUSION: OPN exerts pro-scarring effects by increasing cellular migration, adhesion, proliferation, and contractility of human dermal fibroblasts. OPN-R3 prevents OPN mediated activity. OPN may be useful for promoting closure of non-healing wounds and the OPN specific aptamer, OPN-R3, may be useful for preventing fibrosis.