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European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society

Myofibroblast in the ligamentum flavum hypertrophic activity.


PMID 28180980

Abstract

Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study. Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using α-smooth muscle actin (αSMA) as a marker. To study the myofibroblast in TGF-β pathway, LF tissues were analyzed for protein levels of αSMA/TGF-β1 by Western blot. In addition, from LF cells cultured with exogenous TGF-β1 conditioned medium, expression of αSMA/collagen-1 was assessed and the cell morphology was identified. The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-β1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-β1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p < 0.05). We detected myofibroblast in the patient samples by αSMA staining, and the protein levels of αSMA were positively correlated with TGF-β1. In LF cell culture, exogenous TGF-β1 upregulated αSMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast. We conclude that the transition of fibroblast to myofibroblasts via TGF-β pathway is a key linker between inflammation and fibrosis in LFH mechanism.