PB0416 - Platelet polyphosphate activates NFκB signaling to induce myofibroblast differentiation

Background: Inorganic polyphosphate, composed of linear polymers of orthophosphate units, is remarkably conserved, being found in organisms from bacteria to humans. However, roles for polyphosphate in mammalian biology have only recently been identified and are not well understood. Polyphosphates ranging from ~60 to 100 phosphates in length are stored at relatively high levels in platelet dense granules and are secreted upon platelet activation. Our lab and others have shown that platelet polyphosphate is potently procoagulant and proinflammatory. We also previously reported that platelet polyphosphate is chemotactic for fibroblasts and promotes their transition into myofibroblasts, a pro-wound healing and pro-fibrotic cell type.

Aims: To determine underlying mechanisms by which platelet-derived polyphosphate induces myofibroblast differentiation.

Methods: NIH-3T3 cells and primary human fibroblasts were incubated with platelet-sized polyphosphate or platelet releasates in the presence or absence of cell-signaling inhibitors. Differentiation was assessed through the accumulation of markers of myofibroblast phenotype such as increased alpha-smooth muscle actin localized along F-actin stress fibers, and increased collagen levels. Markers were assessed by immunofluorescence.

Results: We now report that polyphosphate initiates an intracellular signaling cascade in fibroblasts modulated by the Receptor for Advanced Glycation Endproducts (RAGE), leading to nuclear translocation of NFκB and myofibroblast differentiation. Pharmacologic inhibition of RAGE or NFκB abrogated the differentiation-inducing activity of platelet-sized polyphosphate and platelet releasates. We also report that a novel, non-toxic, dendrimer-like polyphosphate inhibitor (MPI-8) potently blocked the differentiation-inducing activity of platelet polyphosphate in vitro.

Conclusion(s): This work identifies the signaling pathway utilized by polyphosphate to promote the transition of fibroblasts into myofibroblasts, a finding which may have implications for both wound healing and fibrosis. Furthermore, the availability of the novel polyphosphate inhibitor, MPI-8, now allows selective targeting of polyphosphate in the context of its biological activities.