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Lovastatin protects chondrocytes derived from Wharton’s jelly of human cord against hydrogen-peroxide-induced in vitro injury

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Abstract

Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton’s jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 μM hydrogen peroxide, either in the presence or absence of Lovastatin (2 μM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.

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Authors and Affiliations

  1. National Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Lahore, Pakistan

    Nadia Wajid, Azra Mehmood, Fazal-ur-Rehman Bhatti, Shaheen N. Khan & Sheikh Riazuddin

  2. Allama Iqbal Medical College/Jinnah Hospital Complex, University of Health Sciences, Lahore, Pakistan

    Sheikh Riazuddin

  3. University of Lahore, 1-km Raiwind Road, Lahore, Pakistan

    Sheikh Riazuddin

Authors
  1. Nadia Wajid
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  2. Azra Mehmood
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  3. Fazal-ur-Rehman Bhatti
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  4. Shaheen N. Khan
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  5. Sheikh Riazuddin
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Corresponding author

Correspondence to Sheikh Riazuddin.

Additional information

Nadia Wajid and Azra Mehmood contributed equally to this work.

This work was supported by research grants from Higher Education Commission (HEC) of Pakistan.

The authors declare no conflicts of interest.

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Table S1

List of antibodies used (JPEG 44 kb)

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Fig. S1

Optimization of hydrogen-peroxide-induced in vitro injury conditions in WJMSC-derived chondrocytes. a Phase contrast images of WJMSC-derived chondrocytes showing the morphology of the cells subjected to 200 μM hydrogen peroxide for various time periods, i.e., C (0 h), H1 (1 h), H2.5 (2.5 h) and H5 (5 h). 200x, Bars ∼100 μm. b Trypan blue exclusion assay with maximum non-viable cells in the H5 group. c Gel bands for RT-PCR analysis of mRNA of FADD, BAX and BCL2L1. d Quantitative expression of FADD, BAX and BCL2L1 in various groups as determined by Image J software. *Significant difference between control (C) and 5-h (H5) groups (P<0.05). Data are expressed as means ± SD (JPEG 82 kb)

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Fig. S2

Dose optimization of Lovastatin during hydrogen-peroxide-induced in vitro injury in WJMSC-derived chondrocytes. a Phase contrast images of WJMSC-derived chondrocytes showing the morphology of the cells kept as C (control), H (subjected to 200 μM hydrogen peroxide alone), L0.5 (subjected to 0.5 μM Lovastatin together with 200 μM hydrogen peroxide for 5 h), L1 (1 μM Lovastatin together with 200 μM hydrogen peroxide for 5 h), or L2 (2 μM Lovastatin together with 200 μM hydrogen peroxide for 5 h). 200x, Bars ∼100 μm. b Trypan blue exclusion assay with maximum viable cells in the L2 group (JPEG 60 kb)

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Wajid, N., Mehmood, A., Bhatti, FuR. et al. Lovastatin protects chondrocytes derived from Wharton’s jelly of human cord against hydrogen-peroxide-induced in vitro injury. Cell Tissue Res 351, 433–443 (2013). https://doi.org/10.1007/s00441-012-1540-3

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  • DOI: https://doi.org/10.1007/s00441-012-1540-3

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