23. Mechanical stress induces degeneration and hypertrophy of ligamentum flavum in lumbar spinal canal stenosis

BACKGROUND CONTEXT

Ligamentum flavum (LF) hypertrophy is characterized histologically by LF degeneration, including the loss of elastic fibers and the increase collagen. In patients with lumbar spinal canal stenosis (LSCS), chronic inflammation and subsequent fibrosis induced by mechanical stress play an important role in LF hypertrophy and degeneration. Several molecules, such as transforming growth factor (TGF)-β1, inflammatory cytokines, and matrix metalloproteinase (MMPs) participate in the pathological processes; however, the mechanisms underlying the induction of these molecules and process of degeneration have not been fully elucidated. Angiopoietin-like protein 2 (Angptl2), a tissue remodeling factor, is induced by various stress conditions and regulates TGF-β1, inflammatory cytokines such as interleukin (IL)-6, and MMPs. Therefore, Angptl2 induced by mechanical stress may contribute to the pathogenesis of LSCS.

PURPOSE

Analyzing the role of Angptl2 and elucidating the mechanism of degenerative processes of ligamentum flavum caused by mechanical stress.

STUDY DESIGN/SETTING

N/A

PATIENT SAMPLE

This study was conducted after approval was obtained from the Kumamoto University Ethics Committee and written informed consent was received from each patient. LF samples for this study were provided by patients who underwent lumbar surgery with LSCS or non-LSCS such as disc herniation and cauda equina tumors. For in vitro experiments, LF fibroblasts were isolated from LF tissue and cultured.

OUTCOME MEASURES

N/A

METHODS

Total RNA was extracted from LF tissue. Angptl2, TGF-β1, IL-6, MMP-2, collagen, and elastin mRNA expression was evaluated by real-time polymerase chain reaction (PCR). Anti-human Angptl2 TGF-β1, IL-6 and MMP-2 antibody was used for immunohistochemistry. For in vitro experiments, LF fibroblasts were isolated from LF tissue and cultured. LF fibroblasts were applied to stretching stimulation (10% elongation, 10 cycles/min) for 12 h, and Angptl2 expression was investigated by PCR and ELISA. Recombinant Angptl2 protein (5 μg/ml) was added to the cells, followed by 6 h incubation, after which the RNA was extracted, and TGF-β1, IL-6, MMP-2 and collagen mRNA expression was evaluated by PCR. To evaluate activating Smad signaling, p-Smad3 was analyzed by western blot after Angptl2 stimulation. MMP-2 expression was evaluated by Recombinant IL-6 protein(300ng/ml). The concentration of elastin in the supernatant was measured using a competitive ELISA kit, after 300ng/ml IL-6 protein stimulation adding 1mg elastin protein.

RESULTS

Angptl2, TGF-β1, IL-6, MMP-2 and collagen mRNA expression in hypertrophied LF tissue from the LSCS group were significantly increased relative to that in LF tissue from the non-LSCS group. On the other hand, there was no significance in elastin mRNA expression between both groups. In immunohistochemistry, we found a markedly increased number of Angptl2, TGF-β1, IL-6, MMP-2 expressing fibroblasts in hypertrophied LF tissue. In vitro experiments, Angptl2 mRNA and Angptl2 protein expression in LF fibroblasts were elevated after stretching stimulation, suggesting that mechanical stress induced Angptl2 expression in LF tissue. Angptl2 stimulation induced TGF-β1 expression, leading to phosphorylation Smad3 and elevation of collagen production. Angptl2 also induced IL-6 expression, and IL-6 promoted MMP-2 expression and activation. Actually, MMP-2 induced by IL-6 decreased elastin protein.

CONCLUSIONS

The degeneration and hypertrophy of LF is considered to be increased collagen production, and promoted degradation elastic fiber rather than decreased elastin production. In the mechanism of LF degeneration, Angptl2 could be induced mechanical stress, and promoting TGF-β1/Smad signaling, leading to increasing collagen expression. Angptl2 also could induce IL-6 expression, leading to activation of MMP-2 and degradation of elastic fiber.

FDA Device/Drug Status

This abstract does not discuss or include any applicable devices or drugs.