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. 2020 Aug 28;82(8):1209-1218.
doi: 10.1292/jvms.20-0091. Epub 2020 Jul 7.

Effects of pentosan polysulfate on cell proliferation, cell cycle progression and cyclin-dependent kinases expression in canine articular chondrocytes

Ekkapol Akaraphutiporn  1 , Eugene C Bwalya  2 , Sangho Kim  1 , Takafumi Sunaga  1 , Ryosuke Echigo  3 , Masahiro Okumura  1
Affiliations

Effects of pentosan polysulfate on cell proliferation, cell cycle progression and cyclin-dependent kinases expression in canine articular chondrocytes

Ekkapol Akaraphutiporn et al. J Vet Med Sci. .

Abstract

Pentosan polysulfate (PPS) is a semi-synthetic sulfated polysaccharide compound which has been shown the benefits on therapeutic treatment for osteoarthritis (OA) and has been proposed as a disease modifying osteoarthritis drugs (DMOADs). This study investigated the effects of PPS on cell proliferation, particularly in cell cycle modulation and phenotype promotion of canine articular chondrocytes (AC). Canine AC were treated with PPS (0-80 µg/ml) for 24, 48 and 72 hr. The effect of PPS on cell viability, cell proliferation and cell cycle distribution were analyzed by MTT assay, DNA quantification and flow cytometry. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) and glycosaminoglycan (GAG) quantification. PPS significantly reduced AC proliferation through cell cycle modulation particularly by maintaining a significantly higher proportion of chondrocytes in the G1 phase and a significantly lower proportion in the S phase of the cell cycle in a concentration- and time-dependent manner. While the proportion of chondrocytes in G1 phase corresponded with the significant downregulation of cyclin-dependent kinase (CDK) 1 and 4. Furthermore, the study confirms that PPS promotes a chondrogenic phenotype of AC through significant upregulation of collagen type II (Col2A1) mRNA and GAG synthesis. The effect of PPS on the inhibition of chondrocyte proliferation while promoting a chondrocyte phenotype could be beneficial in the early stages of OA treatment, which transient increase in proliferative activity of chondrocytes with subsequent phenotypic shift and less productive in an essential component of extracellular matrix (ECM) is observed.

Keywords: articular chondrocyte; cell cycle; cyclin-dependent kinases; osteoarthritis; pentosan polysulfate.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Morphological appearance and confluency condition of pentosan polysulfate (PPS) treated chondrocytes observed under a light microscope. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 10, 20, 40 and 80 µg/ml) for 72 hr. (A) Magnification: ×40, Scale bar: 500 µm. (B) Magnification: ×200, Scale bar: 100 µm.
Fig. 2.
Fig. 2.
Treatment with pentosan polysulfate (PPS) resulted in reduced chondrocyte viability. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 10, 20, 40 and 80 µg/ml) for 72 hr. The cell viability of cultured chondrocytes was analyzed by MTT assay at 24, 48 and 72 hr during PPS treatment. The data are expressed as the mean ± SEM (*P<0.05 and **P<0.01).
Fig. 3.
Fig. 3.
Treatment with pentosan polysulfate (PPS) showed no cytotoxic effect on cultured chondrocytes. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 10, 20, 40 and 80 µg/ml) for 72 hr. Cell apoptosis was evaluated by flow cytometry analysis with annexin V and propidium iodide (PI) staining at 72 hr after exposure to PPS. Flow cytometry results showed the percentage of cells binding to annexin V and PI.
Fig. 4.
Fig. 4.
Pentosan polysulfate (PPS) increases the proportion of chondrocytes distributed in the G1 phase while reducing the proportion of chondrocytes distributed in the S phase of the cell cycle. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 10, 20, 40 and 80 µg/ml) for 72 hr. Cell cycle was analyzed by flow cytometry and propidium iodide (PI) staining at 24, 48 and 72 hr during PPS treatment. A represents histogram showing the cell distribution pattern between control and treatment with PPS at 80 µg/ml. The results were analyzed by the FlowJo software program using Watson Pragmatic model.
Fig. 5.
Fig. 5.
Treatment with pentosan polysulfate (PPS) promotes glycosaminoglycan (GAG) synthesis but reduces DNA content of chondrocytes in a concentration-dependent manner. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 10, 20, 40 and 80 µg/ml) for 72 hr. Biochemical analysis was performed at 72 hr after exposure to PPS. (A) Quantification of DNA content in cell lysates by Hoechst assay. (B) Quantification of GAG content (normalized with DNA content) in cell lysates by dimethylmethylene blue (DMMB) assay. The data are expressed as the mean ± SEM (*P<0.05 and **P<0.01).
Fig. 6.
Fig. 6.
Pentosan polysulfate (PPS) downregulates cell cycle regulator genes, while promoting a chondrocyte phenotype. Chondrocytes were cultured as a monolayer for 24 hr prior to the treatment with various concentrations of PPS (0, 5, 20 and 80 µg/ml) for 72 hr. Relative mRNA expression of chondrocytes was evaluated by quantitative real-time PCR (qPCR) analysis at 24 and 72 hr during PPS treatment. The relative mRNA expression of (A) cyclin-dependent kinase (CDK) 1, (B) CDK2, (C) CDK4, (D) CDK6, (E) collagen type II (Col2A1) and (F) matrix metalloproteinase 13 (MMP13) were normalized to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data are expressed as the mean ± SEM (*P<0.05, **P<0.01 and ***P<0.001).
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References

    1. Akkiraju H., Nohe A.2015. Role of chondrocytes in cartilage formation, progression of osteoarthritis and cartilage regeneration. J. Dev. Biol. 3: 177–192. doi: 10.3390/jdb3040177 - DOI - PMC - PubMed
    1. Archer C. W., Francis-West P.2003. The chondrocyte. Int. J. Biochem. Cell Biol. 35: 401–404. doi: 10.1016/S1357-2725(02)00301-1 - DOI - PubMed
    1. Bernstein P., Sticht C., Jacobi A., Liebers C., Manthey S., Stiehler M.2010. Expression pattern differences between osteoarthritic chondrocytes and mesenchymal stem cells during chondrogenic differentiation. Osteoarthritis Cartilage 18: 1596–1607. doi: 10.1016/j.joca.2010.09.007 - DOI - PubMed
    1. Berridge M. J.2014. Cell cycle and proliferation. Cell Signal. Biol. 6: csb0001009. doi: 10.1042/csb0001009 - DOI
    1. Bwalya E. C., Kim S., Fang J., Wijekoon H. M. S., Hosoya K., Okumura M.2017. Pentosan polysulfate inhibits IL-1β-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes. PLoS One 12: e0177144. doi: 10.1371/journal.pone.0177144 - DOI - PMC - PubMed

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