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Optimized alkylated cyclodextrin polysulphates with reduced risks on thromboembolic accidents improve osteoarthritic chondrocyte metabolism.

Groeneboer S, Lambrecht S, Dhollander A, Jacques P, Vander Cruyssen B, Lories RJ, Devreese K, Chiers K, Elewaut D, Verbruggen G - Rheumatology (Oxford) (2011)

Bottom Line: The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes.Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT.CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Laboratory of Connective Tissue Biology, Ghent University Hospital, Ghent, Belgium.

ABSTRACT

Objectives: To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro.

Methods: OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) or β-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumulation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies.

Results: The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 µg/ml. Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo.

Conclusions: CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

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Related in: MedlinePlus

In vitro activation of platelets by HIT ab-positive plasma with heparin. Percentages of cell staining with CD62p after addition of 5 µl heparin (A1 and A2: 100 IU/ml; B1 and B2: 0.3 IU/ml; C1 and C2: 0 IU/ml) in the absence (A1, B1 and C1) or presence (A2, B2 and C2) of HIT ab-positive plasma. Platelets gated on side scatter (SS log) and CD41 activity (D). The interface channel for positivity was set at the point where <2% of the control fluorescence was positive.
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Figure 1: In vitro activation of platelets by HIT ab-positive plasma with heparin. Percentages of cell staining with CD62p after addition of 5 µl heparin (A1 and A2: 100 IU/ml; B1 and B2: 0.3 IU/ml; C1 and C2: 0 IU/ml) in the absence (A1, B1 and C1) or presence (A2, B2 and C2) of HIT ab-positive plasma. Platelets gated on side scatter (SS log) and CD41 activity (D). The interface channel for positivity was set at the point where <2% of the control fluorescence was positive.

Mentions: After 20 min of incubation at room temperature, the total volume of the platelet suspension was adjusted to 600 µl buffered solution and evaluated without delay by flow cytometry. Twenty thousand events per sample were analysed and the platelet populations were gated by side scatter characteristics and the platelet marker CD41. Activated platelets (CD62p+) were distinguished from resting platelets by the CD62p-PE expression (Fig. 1). The fraction of activated platelets among the total platelet population was determined and relative CD62p ratios to the internal controls were calculated. [ratio: %CD62p+ (0.3 IU heparin or 5 µg/ml β-cyclodextrin derivate)/%CD62p+ (0 IU heparin or 0 µg/ml β-cyclodextrin derivate)]. Test results for the heparin-treated platelets were evaluated by a scoring system adapted from Jy et al. [14]. Control tests were scored positive when the calculated ratios of at least two donors were >2, which is based on previously obtained results with normal individuals.Fig. 1


Optimized alkylated cyclodextrin polysulphates with reduced risks on thromboembolic accidents improve osteoarthritic chondrocyte metabolism.

Groeneboer S, Lambrecht S, Dhollander A, Jacques P, Vander Cruyssen B, Lories RJ, Devreese K, Chiers K, Elewaut D, Verbruggen G - Rheumatology (Oxford) (2011)

In vitro activation of platelets by HIT ab-positive plasma with heparin. Percentages of cell staining with CD62p after addition of 5 µl heparin (A1 and A2: 100 IU/ml; B1 and B2: 0.3 IU/ml; C1 and C2: 0 IU/ml) in the absence (A1, B1 and C1) or presence (A2, B2 and C2) of HIT ab-positive plasma. Platelets gated on side scatter (SS log) and CD41 activity (D). The interface channel for positivity was set at the point where <2% of the control fluorescence was positive.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116210&req=5

Figure 1: In vitro activation of platelets by HIT ab-positive plasma with heparin. Percentages of cell staining with CD62p after addition of 5 µl heparin (A1 and A2: 100 IU/ml; B1 and B2: 0.3 IU/ml; C1 and C2: 0 IU/ml) in the absence (A1, B1 and C1) or presence (A2, B2 and C2) of HIT ab-positive plasma. Platelets gated on side scatter (SS log) and CD41 activity (D). The interface channel for positivity was set at the point where <2% of the control fluorescence was positive.
Mentions: After 20 min of incubation at room temperature, the total volume of the platelet suspension was adjusted to 600 µl buffered solution and evaluated without delay by flow cytometry. Twenty thousand events per sample were analysed and the platelet populations were gated by side scatter characteristics and the platelet marker CD41. Activated platelets (CD62p+) were distinguished from resting platelets by the CD62p-PE expression (Fig. 1). The fraction of activated platelets among the total platelet population was determined and relative CD62p ratios to the internal controls were calculated. [ratio: %CD62p+ (0.3 IU heparin or 5 µg/ml β-cyclodextrin derivate)/%CD62p+ (0 IU heparin or 0 µg/ml β-cyclodextrin derivate)]. Test results for the heparin-treated platelets were evaluated by a scoring system adapted from Jy et al. [14]. Control tests were scored positive when the calculated ratios of at least two donors were >2, which is based on previously obtained results with normal individuals.Fig. 1

Bottom Line: The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes.Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT.CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Laboratory of Connective Tissue Biology, Ghent University Hospital, Ghent, Belgium.

ABSTRACT

Objectives: To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro.

Methods: OA chondrocytes were cultured in alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-CDPS (CE-CDPS), (2-hydroxypropyl)-β-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-β-CDPS (MA-CDPS) or β-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumulation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies.

Results: The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 µg/ml. Five micrograms per millilitre of β-CDPS concentrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo.

Conclusions: CE-CDPS is a new, structurally adjusted, sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

Show MeSH
Related in: MedlinePlus