Oxidative stress mediates age-related hypertrophy of ligamentum flavum by inducing inflammation, fibrosis, and apoptosis through activating Akt and MAPK pathways.

The role of oxidative stress in ligamentum flavum (LF) hypertrophy has not been elucidated. We hypothesize that oxidative stress induces inflammatory responses and the subsequent fibrotic processes in LF, via activation of the Akt and MAPK pathways. Specimens of LFs were collected during surgeries for lumbar disc herniation (LDH) or lumbar spinal stenosis (LSS). Part of the LF specimens underwent analyses for ROS, fibrotic markers, and inflammatory mediators, with the remainder minced for cell cultures. The cell cultures were treated with H2O2, after which the cells were lysed and analyzed via western blotting. The specimens of the LSS patients showed increased infiltration of inflammatory cells and were stained positively for MMP-3, MMP-9, vimentin, and fibronectin. The LF of the LSS patients had increased oxidative stress and inflammation compared to that of the LDH patients. In vitro analyses demonstrated that oxidative stress rapidly activated the Akt and MAPK pathways. Inflammatory mediators, iNOS and NF-κB, and fibrotic markers, including TGF-ß, ß-catenin, α-SMA and vimentin, were significantly upregulated after induction of oxidative stress. Oxidative stress activated the intrinsic apoptotic pathway. These findings revealed that oxidative stress is one of the etiological factors of LF hypertrophy, which might provide new insights into treatment approaches.

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF-ß Pathway.

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor-ß (TGF-ß) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF-ß mechanism. Human LF cells were used for the experiments. First, we created TGF-ß receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF-ß1 to determine the effects of blocking the TGF-ß pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF-ß pathway. To assess the predisposition toward fibrosis, α-smooth muscle actin (αSMA), fibronectin, collagen-1, collagen-3, and CCN2 were evaluated with quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild-type (WT) cells, treatment with TGF-ß1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis-related factors. However, in KO cells, the responses to TGF-ß1 stimulation were significantly lower. In addition, CCN5 and TGF-ß1 co-treatment caused a notable reduction in mRNA expression levels compared with TGF-ß1 stimulation only. The αSMA protein expression increased with TGF-ß1 but decreased with CCN5 treatment. TGF-ß1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF-ß pathway. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634-2644, 2019.

Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway.

BACKGROUND/AIMS: Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy Methods: We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in in vivo studies. RESULTS: The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our in vitro findings were supported by our in vivo evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. CONCLUSION: LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy.

iTRAQ quantitative proteomic study in patients with thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is a unique disease with ectopic ossification, and is a major cause of thoracic spinal stenosis and myelopathy. However, the underlying etiology remains largely unknown. In this study, the ligamentum flavum was systematically analyzed in TOLF patients by using comprehensive iTRAQ labeled quantitative proteomics. Among 1285 detected proteins, there were 282 proteins identified to be differentially expressed. The Gene Ontology (GO) analysis regarding functional annotation of proteins consists of the following three aspects: the biological process, the molecular function, and the cellular components. The function clustering analysis revealed that ten of the above proteins are related to inflammation, such as tumor necrosis factor (TNF). This finding was subsequently validated by ELISA, which indicated that serum TNF-α of TOLF patients was significantly higher compared with the control group. To address the effect of TNF-α on ossification-related gene expression, we purified and cultured primary cells from thoracic ligamentum flavum of patients with TOLF. TNF-α was then used to stimulate cells. RNA was isolated and analyzed by RT-PCR. Our results showed that TNF-α was able to induce the expressions of osteoblast-specific transcription factor Osterix (Osx) in ligamentum flavum cells, suggesting that it can promote osteoblast differentiation. In addition, as the Osx downstream osteoblast genes OCN and ALP were also activated by TNF-α. This is the first proteomic study to identify inflammation factors such as TNF-α involved in ossified ligamentum flavum in TOLF, which may contribute to a better understanding of the cause of TOLF.

Dose-dependent regulation of cell proliferation and collagen degradation by estradiol on ligamentum flavum.

BACKGROUND: Estradiol plays an important role in the regulation of collagen metabolism. Deficiency of estradiol has been reported to be associated with the degeneration of many connective tissues. However, the association of estradiol and hypertrophy of the ligamentum flavum was seldom explored. Therefore, we studied the effects of estradiol on cultured cells from the ligamentum flavum. METHODS: Primary cultures of human ligamentum flavum cells obtained from surgical specimens of 14 patients undergoing spinal surgery were used to investigate the effect of estradiol on cell proliferation and the expression of collagen, elastin, and matrix metalloproteinases. Downstream pathways of estrogen receptor underlying the regulation of metalloproteinases were also investigated. RESULTS: In our study, we revealed the existence of estrogen receptors on both female and male ligamentum flavum cells with a gender difference. 17ß-estradiol increased early (24 hours) proliferation of ligamentum flavum cells in a dose dependent manner and the effect could not be seen when the cell density increased. Estradiol with a concentration of 10(-9) M decreased collagen levels and increased the expression of MMP-13. Adding an antagonist of PI3K downstream pathway could reverse the expression of MMP-13 caused by estradiol. CONCLUSIONS: The results implied estradiol regulated the expression of MMP-13 via PI3K pathway and contributed to the homeostasis of extracellular matrix in the ligamentum flavum.

Elastin-derived peptides induce inflammatory responses through the activation of NF-κB in human ligamentum flavum cells.

The formation of fibrotic tissue in the ligamentum flavum (LF) is usually preceded by breakdown of elastic fibers. Elastin-derived peptides (EDPs) from breakdown of elastic fibers display a wide range of biological activities in a variety of cells, but there is minimal information regarding the involvement in the processes of LF hypertrophy. The aim of this study is to elucidate the effects of EDPs on cultured human LF cells and to investigate their molecular and biochemical mechanisms. Human LF cells were obtained from 18 patients who underwent lumbar spine surgery. After treatment with different concentrations of EDPs with or without specific inhibitors in culture medium, the viability and proliferation of LF cells, genes expression, and the signaling pathways were evaluated and analyzed. It was found that 50 µg/ml EDPs significantly increased cell proliferation and synthesis of prostaglandin E(2). The gene expression and protein production of proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1ß, and IL-6, were also upregulated. The levels of p-ERK (extracellular signal-regulated kinase) and NF-κB increased immediately following EDP treatment and sustained up to 90 min. It was also found that NF-κB inhibitor, but not ERK1/2 inhibitor, attenuated EDP-dependent induction of IL-1α, IL-1ß, and IL-6 expression, indicating that NF-κB pathways are required for EDP-induced IL-1α, IL-1ß, and IL-6 gene expression in human LF cells. The results of this in vitro experiment suggest that EDPs do induce inflammatory responses in human LF cells and plays the key role in the development of LF hypertrophy.

Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.

To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.

Fibroblasts of spinal ligaments pathologically differentiate into chondrocytes induced by recombinant human bone morphogenetic protein-2: morphological examinations for ossification of spinal ligaments.

To elucidate the process of ossification in spinal ligaments, an aqueous solution containing recombinant human bone morphogenetic protein (BMP)-2 (40 micrograms/100 microL) was injected into murine ligamenta flava, and the ossification process was analyzed morphologically. In the control group, the solution administered lacked the protein; these flattened ligamentous fibroblasts possessing BMP receptors type IA and type II existed among type I collagen bundles. In the week immediately following the injection of BMP-2, ligamentous fibroblasts began to proliferate, differentiating into alkaline phosphatase-positive chondrocytes surrounded by an extracellular matrix composed of type I and II collagen. By the second week, differentiated chondrocytes of various stages were observed in type II collagen-rich matrix. These chondrocytes showed an abundance of BMP receptors type IA and II. The pathologically induced cartilage was resorbed by chondroclasts, permitting migration of blood vessels and osteogenic cells, as well as providing a site for endochondral ossification. By the third week, BMP-induced ossification had compressed the spinal cord, and by the sixth week, the ligamentous tissue had been almost completely replaced by bone. Ligamentous fibroblasts appeared to possess BMP receptors, as well as the potentiality to differentiate into chondrocytes. BMP receptors were upregulated during chondrification of ligamentous fibroblasts induced by exogenous BMP-2, suggesting that BMPs may play an important role in ossification of spinal ligaments.