Myofibroblasts are increased in the dorsal layer of the hypertrophic ligamentum flavum in lumbar spinal canal stenosis

Abstract

BACKGROUND CONTEXTHypertrophy of the ligamentum flavum (LF) is a major contributor to the development of lumbar spinal canal stenosis (LSS). Although previous studies have identified some factors related to hypertrophy of the LF, the etiology remains unclear. It is well known that myofibroblasts have a key role in the pathology of fibrosis in other tissues, including the skin, liver, kidney, and lung. We hypothesized that myofibroblasts were also important players in the pathology of fibrosis in the LF. PURPOSETo elucidate the distribution and role of myofibroblasts in the hypertrophic LF. STUDY DESIGNA histological, immunohistochemical, and gene expression analysis of the LF in the human lumbar spine. PATIENT SAMPLEHypertrophic LF tissue samples were collected from patients with LSS. OUTCOME MEASURESHistology, immunohistochemistry, microarray, reverse transcription-quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. METHODSThe degree of fibrosis in the dural and dorsal layers of the LF was evaluated by Masson's trichrome tissue staining. Collagen gene expression was evaluated by quantitative reverse transcription polymerase chain reaction. Immunostaining of αSMA was performed to evaluate localization of myofibroblasts in LF tissue. The association between gene expression of alpha-smooth muscle actin (αSMA) and that of several types of collagen was investigated. The signal activated on the dorsal side of LF was examined by gene set enrichment analysis using microarray data. Expression levels of αSMA and several types of collagen in LF fibroblasts were investigated under hypoxic conditions. RESULTSIn the histological study using Masson's trichrome staining, the fibrosis score was significantly higher in the dorsal layer than in the dural layer. Gene expression levels for several types of collagen (COL1A1, COL1A2, COL3A1, COL5A1, COL6A1, and COL11A1) and heat shock protein 47 (a collagen-specific chaperone) were significantly higher in the dorsal layer. Furthermore, immunohistochemistry revealed a significantly greater number of αSMA-stained cells in the dorsal layer. There was a strong correlation of αSMA mRNA expression with COL1A-1 in LF fibroblasts. Gene set enrichment analysis showed that the set of fibrosis-related gene signals, including those for epithelial-mesenchymal transition, hypoxia, and inflammation, were significantly upregulated in the dorsal layer compared with the dural layer. Under hypoxic stimulation, expression of αSMA and several types of collagen was increased in LF fibroblasts. CONCLUSIONSThis study is the first to reveal that myofibroblast expression levels are higher in the dorsal layer of the LF than in the dural layer. We confirmed that hypertrophy of the LF in LSS is associated with increased expression of myofibroblasts in the dorsal layer. Hypoxia could be a cause of expression of myofibroblasts leading to fibrosis and finally to hypertrophy of the LF. CLINICAL SIGNIFICANCEThe results of this study partially elucidate the molecular mechanisms of LF hypertrophy and suggest that myofibroblasts may be involved in age-related degeneration of the LF.