S.M. Richardson’s research while affiliated with University of Manchester and other places

What is this page?

This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Publications (7)

Investigation of the chondrocyte response to varying mechanical load: assessment of impact stress
  • Article

April 2020

·

13 Reads

·

1 Citation

Osteoarthritis and Cartilage

S.M. Higgins

·

·

·

[...]

·

Urocortin expression in cartilage is attenuated in a non-invasive mouse model of post traumatic osteoarthritis
  • Article
  • Full-text available

April 2018

·

54 Reads

Osteoarthritis and Cartilage

·

·

·

[...]

·

Download
Figure 2. Response of C-20/A4 cells and AC to Ucn1 blockade. Cells were treated for 8 h with CP-154526, astressin 2B or in combination at indicated concentrations. (A) The effects on morphology of incubating C-20/ A4 cells and AC with CP-154526 and/or astressin 2B (1-50 μM) was assessed using phase contrast microscopy. (B) Dose dependant effects of CP-154526 and/or astressin 2B on C-20/A4 cells. (C) AC cell death as determined by Annexin V/PI staining and flow cytometry. n = 3, error bars = SD *p < 0.05 and **p < 0.005 compared with untreated control cells. 
Figure 3. Western immunoblot analysis of C-20/A4 cells treated with CP-154526 and/or astressin 2B. Protein extracts were electrophoresed and probed with the indicated primary antibodies. Expected molecular sizes are indicated. p53 was upregulated in response to CP-154526 (50 μM) but not astressin 2B (50 μM). Cleaved caspases 3 and 9 were detected in the presence of CP-154526 but not in the presence of astressin 2B or nontreated cells. The images have been cropped but gels were run under the same experimental conditions. Representative images of 3 independent experiments. 
Figure 4. Antagonist induced calcium influx is inhibited by the non-selective cation channel blocker gadolinium. (A) C-20/A4 cells were treated at indicated time points CP-154526 (50 μM) alone or in the presence of Gd 3+ (100 µM). Intracellular Ca 2+ levels were visualised by fluorescent microscopy and Fluo-4 AM staining. Representative images of 3 independent experiments are shown. (B) Fluo-4 signal quantification using ImageJ software and graphical representation of Fluo-4 staining intensity for C-20/A4 cells treated with CP-154526 (50 μM) alone or in the presence of Gd 3+ (100 µM). Mean fluorescent intensities are reported, n = 3, error bars = SD, *p < 0.05. (C) The ability of Gd 3+ treatment to protect C-20/A4 cells against CP-154526 induced cell death was determined by Annexin V/PI staining and flow cytometry, n = 3, error bars = SD, *p < 0.05. (D) AC cells were treated with CP-154526 (50 μM) alone or in the presence of Gd 3+ (100 µM). Intracellular Ca 2+ levels were visualised by fluorescent microscopy and Fluo-4 AM staining. Representative images of 3 independent experiments are shown. (E) Fluo-4 signal was quantified using ImageJ software and graphical representation of Fluo-4 staining intensity for AC cells treated with CP-154526 alone or in the presence of Gd 3+ (100 µM). Mean fluorescent intensities are reported, n = 3, error bars = SD, *p < 0.05. (F) The ability of Gd 3+ treatment to protect primary AC cells against CP-154526 induced cell death was determined by Annexin V/PI staining and flow cytometry, n = 3, error bars = SD, *p < 0.05. 
Figure 5. Inhibition of mechanosensitive Piezo1 can protect C-20/A4 cells from CP-154526 induced apoptosis and calcium influx (A). C-20/A4 cells were transfected with siRNA (20 nM) against Piezo1, Piezo2 and a Scrambled control in the presence of HiPerFect, and cultured for 72 h before treatment with CP-154526 (50 µM). Phase contrast images were collected at 5 h post-CP treatment to record changes to cell morphology. Images of the centre of each well are presented. Representative images of 3 independent experiments are shown. (B) Cell death in transfected and control cells was assessed 5 h post CP treatment by AnnexinV/PI staining and flow cytometry, n = 5, error bars = SEM, *p < 0.05. (C) Cell death measured by LDH (absorbance at 490 nm) release into the media. n = 5, error bars = SEM, *p < 0.05. (D) Intracellular Ca 2+ levels were visualised in control and transfected C-20/A4 cells by fluorescent microscopy following staining with Fluo-4 AM and Hoescht-33342. Cells were treated for 3 h with CP-154526 (50 µM). Representative images of 3 independent experiments are shown. (E) Fluo-4 AM intensity was quantified on ImageJ software to assess calcium influx in all cells. Mean fluorescent intensities are reported, n = 3, error bars = SEM, *p < 0.05. 
Figure 6. Identification of downstream signalling associated with CRF-R1. (A) The effect of the adenylate cyclase activator forskolin (0.1 μM) and the PLC activator m3M3FBS (0.1 μM) on CP-154526 (50 μM) induced C-20/A4 cell death as determined by Annexin V/PI staining and flow cytometry. n = 3, error bars = SD **p < 0.005 compared with control. (B) The effect of the PLA 2 inhibitor OBAA (0.1 μM) on CP-154526 (50 μM) induced C-20/A4 cell death as determined by Annexin V/PI staining and flow cytometry. n = 3, error bars = SD **p < 0.005 compared with control (untreated). 

+1

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

July 2017

·

140 Reads

·

42 Citations

·

·

·

[...]

·

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca²⁺) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd³⁺). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

Download

Citations (3)

... The lack of significant changes in TGFBR2 suggests that this pathway has not been fully activated, implying that the cell seems to be exploring how to keep certain regulatory pathways relatively silent under different stress loads, thereby achieving a more flexible and targeted response strategy. Mechanical stimulation is not a simple additive process; rather, it involves discrimination and selection, akin to a systematic inputoutput module that only opens or strengthens certain signaling pathways at the appropriate time to ensure that the ultimately manifested biological effects of the cell align with actual needs [22]. This selective amplification and stability maintenance pattern provides a basis for explaining how articular cartilage maintains function and homeostasis under long-term stress environments. ...

Reference:

In vitro study on the effects of mechanical load on the behavior and signal pathway regulation of pediatric hip cartilage cells
Investigation of the chondrocyte response to varying mechanical load: assessment of impact stress
  • Citing Article

  • April 2020

Osteoarthritis and Cartilage

S.M. Higgins

·

·

·

[...]

·

... These insights can help reconcile reports of TRPV4 modulation failing as a therapy in animal models of post-traumatic OA despite multiple studies providing evidence of a role for TRPV4 in mechanotransduction in loading in normal articular cartilage 53,69,70 . It will be critical to identify the molecules responsible for the persistently high calcium levels in OA chondrocytes and the effects of modulating their activities [71][72][73] . It will also be important to determine the timing and mechanism of the molecular switch in OA chondrocytes that impairs their ability to respond to ECM viscoelasticity. ...

Reference:

A dysfunctional TRPV4–GSK3β pathway prevents osteoarthritic chondrocytes from sensing changes in extracellular matrix viscoelasticity
Piezo1 expression is increased in response to non-invasive impact of mouse knee joint

Osteoarthritis and Cartilage

·

·

·

[...]

·

... Mechanical loading enhances the ability of chondrocytes to uptake Ca 2+ and induces chondrocyte apoptosis; however, the addition of specific small interfering (si)RNA to reduce the expression of Piezo1 protein can reduce the mechanical stress-induced apoptosis of chondrocytes. In addition, GsMTx4, a pharmacological inhibitor of Piezo1, can also reduce the mechanical stress-induced apoptosis of chondrocytes (Lawrence et al. 2017). In recent years, research on Piezo1 protein has increased, highlighting its important role in promoting the apoptosis of articular chondrocytes and aggravating joint degeneration. ...

Reference:

Piezo1 promotes intervertebral disc degeneration through the Ca2+/F-actin/Yap signaling axis
Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

·

·

·

[...]

·