Physiological mechanical loading reduces inflammatory signalling in numerous cell types including articular chondrocytes however the mechanism responsible remains unclear. This study investigates the role of chondrocyte primary cilia and associated intraflagellar transport (IFT) in the mechanical regulation of interleukin-1β (IL-1β) signalling. Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1β. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1β signalling whilst Sulphated glycosaminoglycan (sGAG) release provided measurement of cartilage degradation. Measurements were made of HDAC6 activity and tubulin polymerisation and acetylation. Effects on primary cilia were monitored by confocal and super resolution microscopy. Involvement of IFT was analysed using ORPK cells with hypomorphic mutation of IFT88. Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1β. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1β signalling and abolished the anti-inflammatory effects of loading indicating the mechanism is IFT-dependent. Loading reduced the pool of non-polymerised tubulin which was replicated by taxol which also mimicked the anti-inflammatory effects of mechanical loading and prevented cilia elongation. This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.

Cartilage health is maintained in response to a range of mechanical stimuli including compressive, shear and tensile strains and associated alterations in osmolality. The osmotic-sensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) is required for mechanotransduction. Mechanical stimuli inhibit interleukin-1β (IL-1β) mediated inflammatory signalling, however the mechanism is unclear. This study aims to clarify the role of TRPV4 in this response. TRPV4 activity was modulated glycogen synthase kinase (GSK205 antagonist or GSK1016790 A (GSK101) agonist) in articular chondrocytes and cartilage explants in the presence or absence of IL-1β, mechanical (10% cyclic tensile strain (CTS), 0.33 Hz, 24hrs) or osmotic loading (200mOsm, 24hrs). Nitric oxide (NO), prostaglandin E2 (PGE2) and sulphated glycosaminoglycan (sGAG) release and cartilage biomechanics were analysed. Alterations in post-translational tubulin modifications and primary cilia length regulation were examined. In isolated chondrocytes, mechanical loading inhibited IL-1β mediated NO and PGE2 release. This response was inhibited by GSK205. Similarly, osmotic loading was anti-inflammatory in cells and explants, this response was abrogated by TRPV4 inhibition. In explants, GSK101 inhibited IL-1β mediated NO release and prevented cartilage degradation and loss of mechanical properties. Upon activation, TRPV4 cilia localisation was increased resulting in histone deacetylase 6 (HDAC6)-dependent modulation of soluble tubulin and altered cilia length regulation. Mechanical, osmotic or pharmaceutical activation of TRPV4 regulates HDAC6-dependent modulation of ciliary tubulin and is anti-inflammatory. This study reveals for the first time, the potential of TRPV4 manipulation as a novel therapeutic mechanism to supress pro-inflammatory signalling and cartilage degradation.

To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146⁺ cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146⁺ cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and −150, and inhibition of ZEB1 was required for miRNA-200C or −150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and −150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or −150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1. Stem Cells 2013;31:1749-1762

The endothelial glycocalyx is a thin layer of polysaccharide matrix on the luminal surface of endothelial cells (ECs), which contains sulphated proteoglycans and glycoproteins. It is a mechanotransducer and functions as an amplifier of the shear stress on ECs. It controls the vessel permeability and mediates the blood–endothelium interaction. This study investigates the spatial distribution and temporal development of the glycocalyx on cultured ECs, and evaluates mechanical properties of the glycocalyx using atomic force microscopy (AFM) nano-indentation. The glycocalyx on human umbilical vein endothelial cells (HUVECs) is observed under a confocal microscope. Manipulation of the glycocalyx is achieved using heparanase or neuraminidase. The Young's modulus of the cell membrane is calculated from the force–distance curve during AFM indentation. Results show that the glycocalyx appears predominantly on the edge of cells in the early days in culture, e.g. up to day 5 after seeding. On day 7, the glycocalyx is also seen in the apical area of the cell membrane. The thickness of the glycocalyx is approximately 300 nm–1 μm. AFM indentation reveals the Young's modulus of the cell membrane decreases from day 3 (2.93 ± 1.16 kPa) to day 14 (0.35 ± 0.15 kPa) and remains unchanged to day 21 (0.33 ± 0.19 kPa). Significant difference in the Young's modulus is also seen between the apical (1.54 ± 0.58 kPa) and the edge (0.69 ± 0.55 kPa) of cells at day 7. By contrast, neuraminidase-treated cells (i.e. without the glycocalyx) have similar values between day 3 (3.18 ± 0.88 kPa), day 14 (2.12 ± 0.78 kPa) and day 21 (2.15 ± 0.48 kPa). The endothelial glycocalyx in vitro shows temporal development in the early days in culture. It covers predominantly the edge of cells initially and appears on the apical membrane of cells as time progresses. The Young's modulus of the glycocalyx is deduced from Young's moduli of cell membranes with and without the glycocalyx layer. Our results show the glycocalyx on cultured HUVECs has a Young's modulus of approximately 0.39 kPa.