Integrin - Dependent Mechanotransduction in Mechanically Stimulated Human Annulus Fibrosus Cells: Evidence for an Alternative Mechanotransduction Pathway Operating with Degeneration

Intervertebral disc (IVD) cells derived from degenerate tissue respond aberrantly to mechanical stimuli, potentially due to altered mechanotransduction pathways. Elucidation of the altered, or alternative, mechanotransduction pathways operating with degeneration could yield novel targets for the treatment of IVD disease. Our aim here was to investigate the involvement of RGD-recognising integrins and associated signalling molecules in the response to cyclic tensile strain (CTS) of human annulus fibrosus (AF) cells derived from non-degenerate and degenerate IVDs. AF cells from non-degenerate and degenerate human IVDs were cyclically strained with and without function blocking RGD – peptides with 10% strain, 1.0 Hz for 20 minutes using a Flexercell® strain device. QRT-PCR and Western blotting were performed to analyse gene expression of type I collagen and ADAMTS -4, and phosphorylation of focal adhesion kinase (FAK), respectively. The response to 1.0 Hz CTS differed between the two groups of AF cells, with decreased ADAMTS -4 gene expression and decreased type I collagen gene expression post load in AF cells derived from non-degenerate and degenerate IVDs, respectively. Pre-treatment of non-degenerate AF cells with RGD peptides prevented the CTS-induced decrease in ADAMTS -4 gene expression, but caused an increase in expression at 24 hours, a response not observed in degenerate AF cells where RGD pre-treatment failed to inhibit the mechano-response. In addition, FAK phosphorylation increased in CTS stimulated AF cells derived from non-degenerate, but not degenerate IVDs, with RGD pre-treatment inhibiting the CTS – dependent increase in phosphorylated FAK. Our findings suggest that RGD -integrins are involved in the 1.0 Hz CTS – induced mechano-response observed in AF cells derived from non-degenerate, but not degenerate IVDs. This data supports our previous work, suggesting an alternative mechanotransduction pathway may be operating in degenerate AF cells

Effect of CTS on the gene expression of AF cells from non-degenerate and degenerate IVDs+/− peptides.

<p>Cells derived from non-degenerate and degenerate IVDs were treated+/− RAD (50 µg/ml) or RGD (50 µg/ml) -peptides 30 minutes prior to mechanical stimulation with CTS at 10% strain, 1.0 Hz, for 20 minutes, then incubated for up to 24 hours prior to analysis. QRT-PCR was used to analyse the gene expression of A) ADAMTS -4 or B) type I collagen, relative to the housekeeping gene GAPDH and normalised to the corresponding unloaded baseline control in non-degenerate (n = 3) and degenerate (n = 3) AF cells, respectively. Black represents AF cells cyclically strained without peptide treatment, while speckles and stripes represent cells cyclically strained after treatment with RAD or RGD –peptides, respectively. Values are mean of 3 donors+/− standard error mean. *denote a significant change (<i>p</i>≤0.05) in gene expression between mechanically stimulated and unstimulated baseline control.</p

Phosphorylation of FAK following treatment of AF cells derived from non-degenerate and degenerate IVDs with 1.0 Hz CTS.

<p>AF cells derived from non-degenerate (n = 4) and degenerate (n = 3) IVDs were treated+/−1.0 Hz CTS in serum-free media and total protein extracted at timepoints of up to 20 minutes. Mechanically stimulated and unstimulated A) non-degenerate and B) degenerate protein samples (5 µg/well) were separated using 10% SDS-PAGE and probed using primary antibodies against phosphorylated FAK. Blots were then stripped using a stripping buffer, re-blocked and probed using an antibody against total FAK protein. C) The density of bands was quantified using a Syngene imaging system and the ratio of phosphorylated: total FAK protein normalised to timepoint controls and plotted as % change. *denotes a significant change (<i>p</i>≤0.05) between treatment groups.</p

Nuclear factor-κB1 (p50) limits the inflammatory and fibrogenic responses to chronic injury

In this study we addressed the role of the nuclear factor (NF)-κB1/p50 subunit in chronic injury of the liver by determining the inflammatory and fibrotic responses of nfκb1-null mice in an experimental model that mimics chronic liver disease. Mice received repeated hepatic injuries throughout 12 weeks by intraperitoneal injection of the hepatotoxin carbon tetrachloride. In response nfκb1−/− mice developed more severe neutrophilic inflammation and fibrosis compared to nfκb1+/+ mice. This phenotype was associated with elevated hepatic expression of tumor necrosis factor (TNF)-α, which was localized to regions of the liver associated with inflammation and fibrosis. Hepatic stellate cells are important regulators of hepatic inflammatory and fibrogenic events but normally do not express TNF-α. Hepatic stellate cells derived from nfκb1−/− mice expressed TNF-α promoter activity, mRNA, and protein. By contrast the expression of other NF-κB-responsive genes (ICAM1 and interleukin-6) was similar between nfκb1−/− and nfκb1+/+ cells. We provide experimental evidence that the inappropriate expression of TNF-α by nfκb1−/− cells is because of lack of a p50-dependent histone deacetylase 1 (HDAC1)-mediated repression of TNF-α gene transcription. Taken together these data indicate that the p50 NF-κB subunit plays a critical protective role in the injured liver by limiting the expression of TNF-α and its recruitment of inflammatory cells

Tissue section AFM: In situ ultrastructural imaging of native biomolecules

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae