Mechanisms involved in the remyelinating effect of sildenafil

Abstract

Multiple Sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system characterized by a coordinated inflammatory attack on the myelin sheaths with ensuing damage to the underlying axons. Using myelin oligodendrocyte glycoprotein (MOG)-induced chronic experimental autoimmune encephalomyelitis (EAE) as a MS model, it has been previously demonstrated that daily administration of the PDE-5 inhibitor sildenafil starting at peak disease rapidly ameliorates clinical symptoms whereas administration at the onset of symptoms prevents disease progression. These beneficial effects involved down-regulation of adaptive and innate immune responses and protection of axons and oligodendrocytes and promotion of remyelination. The aim of this work was confirm the remyelinating potential of sildenafil treatment and investigate mechanisms involved in this effect in CNS cells. Results show that sildenafil induces remyelination in EAE mice even when the administration of the drug starts during the chronic stage of the disease. Sildenafil also stimulates remyelination in cerebellar organotypic cultures demyelinated with lysophosphatidylcholine and this effect is prevented by inhibitors of nitric oxide-dependent guanylyl cyclase (NO-GC), NO synthase type 2 (NOS-2) and cGMP-dependent protein kinase (PKG), indicating the involvement of the endogenous NO-cGMP-PKG pathway. Maturation of oligodendrocytes as a potential mechanism implicated in the remyelinating effect of sildenafil was investigated by immunostaining for transcription factors involved in different stages of oligodendrocyte development. Results in the EAE model show that sildenafil treatment increases oligodendrocyte precursor cells (OPCs; Nkx2.2+ cells) and promotes the final stage of oligodendrocyte maturation (olig2-/MBP+ cells). These later result was confirmed in LPC-demyelinated cerebellar slices treated with cGMP increasing compounds. This work also shows that expression of the neurotrophic factor CNTF, that has been implicated in oligodendrocyte maturation, is increased in astroytes of sildenafil-treated EAE mice spinal cord, as well as in cerebellar slice cultures. Results also show that cGMP-increasing treatments alter expression of inflammatory phenotype markers (COX-2 and Arg-1) in microglia in demyelinated slice cultures. The potential of cGMP-increasing treatments for regulating the inflammatory phenotype of monocytes was confirmed in bone marrow derived macrophages (BMDM). In these cells sildenafil treatment induces arginase activity and potentiates the effect of IL-4 suggesting the promotion of an M2 phenotype. Analysis by flow cytometry of BMDM confirmed that cGMP augments the number of cells expressing an M2 phenotype marker (CD206). This work further demonstrates that sildenafil significantly increases the myelin phagocytic capacity of microglia/macrophages in EAE mice and in BMDM. Taken together these data suggest that promotion of oligodendrocyte maturation, growth factor expression, modulation of the inflammatory process and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue.