WISP-1 Induced by Mechanical Stress Contributes to the Fibrosis and Hypertrophy of Ligamentum Flavum Through the Hedgehog-Gli1 Signaling

Abstract

Background: The chronic fibrosis and hypertrophy of ligamentum flavum (LF) is an important cause of lumbar spinal canal stenosis (LSCS). Our previous work showed WNT1 inducible signaling pathway protein 1 (WISP-1) is a critical driver of LF fibrosis. However, its potential mechanism has not been explored. Methods: We studied the expression of Hedgehog-related proteins in human LF tissues. Cell viability, cell cycle, apoptosis rate and molecular mechanisms were evaluated in LF cells with lentivirus-mediated knockdown or over-expression of WISP-1 and Gli1. Finally, the molecular mechanism was confirmed in in vivo studies. Findings: The results showed Gli1 was upregulated in hypertrophic LF tissues and required for fibroblast proliferation, apoptosis inhibition and collagen expression in fibroblasts. Moreover, mechanical stretching increased the expression of WISP-1 in LF fibroblasts. Furthermore, WISP-1 induced fibrogenesis in vitro through Hedgehog-Gli1 pathway. This conclusion was supported by the fact that WISP-1 activated Hedgehog-Gli1 pathway in LF fibroblasts and cyclopamine attenuated the effect of WISP-1-induced fibrogenesis. WISP-1 also promoted the transition of fibroblasts into myofibroblasts via Hedgehog pathway. Importantly, hypertrophic LF rabbit model induced by mechanical stress also showed pathological change of fibrosis and more higher expression of WISP-1, Gli1 and ɑ-SMA. Therapeutic administration of cyclopamine reduced collagen expression, fibroblast proliferation, myofibroblast differentiation and ameliorated fibrosis in rabbit model of mechanical stress-induced LF fibrosis. Interpretation: Our study showed the mechanical stress/WISP-1/Hedgehog signaling as a new fibrotic axis contributing to the LF hypertrophy and identified Hedgehog signaling as a therapeutic target for the prevention and treatment of LF fibrosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant No. 81802198) and Natural Science Foundation of Jiangsu Province (Grant No. SBK2019022658). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: An informed consent form was signed by the all patients and the study was approved by the Ethics Committee of Nanjing Medical University (Approval No.20180104).