RESUMO
BACKGROUND: Fibrotic scar formation and inflammation are characteristic pathologies of spinal cord injury (SCI) in the injured core, which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure. Pericytes were shown to be the main source of fibroblasts that form fibrotic scar. However, the mechanism of pericyte-fibroblast transition after SCI remains elusive. METHODS: Fibrotic scarring and microvessels were assessed using immunofluorescence staining after establishing a crush SCI model. To study the process of pericyte-fibroblast transition, we analyzed pericyte marker and fibroblast marker expression using immunofluorescence. The distribution and cellular origin of platelet-derived growth factor (PDGF)-BB were examined with immunofluorescence. Pericyte-fibroblast transition was detected with immunohistochemistry and Western blot assays after PDGF-BB knockdown and blocking PDGF-BB/PDGFRß signaling in vitro. Intrathecal injection of imatinib was used to selectively inhibit PDGF-BB/PDGFRß signaling. The Basso mouse scale score and footprint analysis were performed to assess functional recovery. Subsequently, axonal regeneration, fibrotic scarring, fibroblast population, proliferation and apoptosis of PDGFRß+ cells, microvessel leakage, and the inflammatory response were assessed with immunofluorescence. RESULTS: PDGFRß+ pericytes detached from the blood vessel wall and transitioned into fibroblasts to form fibrotic scar after SCI. PDGF-BB was mainly distributed in the periphery of the injured core, and microvascular endothelial cells were one of the sources of PDGF-BB in the acute phase. Microvascular endothelial cells induced pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway in vitro. Pharmacologically blocking the PDGF-BB/PDGFRß pathway promoted motor function recovery and axonal regeneration and inhibited fibrotic scar formation. After fibrotic scar formation, blocking the PDGFRß receptor inhibited proliferation and promoted apoptosis of PDGFRß+ cells. Imatinib did not alter pericyte coverage on microvessels, while microvessel leakage and inflammation were significantly decreased after imatinib treatment. CONCLUSIONS: We reveal that the crosstalk between microvascular endothelial cells and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway. Our finding suggests that blocking the PDGF-BB/PDGFRß signaling pathway with imatinib contributes to functional recovery, fibrotic scarring, and inflammatory attenuation after SCI and provides a potential target for the treatment of SCI.
RESUMO
After spinal cord injury (SCI), astrocytes gradually migrate to and surround the lesion, depositing chondroitin sulfate proteoglycan-rich extracellular matrix and forming astrocytic scar, which limits the spread of inflammation but hinders axon regeneration. Meanwhile, microglia gradually accumulate at the lesion border to form microglial scar and can polarize to generate a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. However, the effect of microglia polarization on astrocytes is unclear. Here, we found that both microglia (CX3CR1+) and astrocytes (GFAP+) gathered at the lesion border at 14 days post-injury (dpi). The microglia accumulated along the inner border of and in direct contact with the astrocytes. M1-type microglia (iNOS+CX3CR1+) were primarily observed at 3 and 7 dpi, while M2-type microglia (Arg1+CX3CR1+) were present at larger numbers at 7 and 14 dpi. Transforming growth factor-ß1 (TGFß1) was highly expressed in M1 microglia in vitro, consistent with strong expression of TGFß1 by microglia in vivo at 3 and 7 dpi, when they primarily exhibited an M1 phenotype. Furthermore, conditioned media from M1-type microglia induced astrocytes to secrete chondroitin sulfate proteoglycan in vitro. This effect was eliminated by knocking down sex-determining region Y-box 9 (SOX9) in astrocytes and could not be reversed by treatment with TGFß1. Taken together, our results suggest that microglia undergo M1 polarization and express high levels of TGFß1 at 3 and 7 dpi, and that M1-type microglia induce astrocytes to deposit chondroitin sulfate proteoglycan via the TGFß1/SOX9 pathway. The study was approved by the Institutional Animal Care and Use Committee of Anhui Medical University, China (approval No. LLSC20160052) on March 1, 2016.