Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Jehyun Park; Ji Yeon Seo; Hunjoo Ha

doi:10.4196/kjpp.2010.14.6.385

Journal List > Korean J Physiol Pharmacol > v.14(6) > 1025708

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Park, Seo, and Ha: Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Original Article

Korean J Physiol Pharmacol 2010;14(6):385-390.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.6.385

Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Jehyun Park, Ji Yeon Seo, Hunjoo Ha

Department of Bioinspired Science, Division of Life and Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-752, Korea

Corresponding to: Hunjoo Ha, College of Pharmacy, Ewha Womans University, 11-1, Daehyun-dong, Seodaemun-gu, Seoul 120-752, Korea. (Tel) 82-2-3277-4075, (Fax) 82-2-3277-2851, (E-mail) hha@ewha.ac.kr

Received 10 October 2010 Revised 30 October 2010 Accepted 6 November 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1μM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

Keywords: Plasminogen activator inhibitor-1, Antisense oligodeoxynucleotide, Plasmin, Matrix metalloproteinase, Mesangial cells

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Fig. 1.

Effect of PAI-1 antisense ODN on HG- (A) and TGF-β 1- (B) induced PAI-1 protein expression in mesangial cells. Growth arrested and synchronized primary rat mesangial cells were stimulated with 30 mM high D-glucose (HG) (A) or 2 ng/ml of TGF-β 1 (B) for 48 hours after PAI-1 antisense or mis-match sense ODN transfection. PAI-1 protein was measured by Western blot analysis as described in the Methods. Data are presented as means±SE of 4 experiments. ^∗p<0.05 vs control, ^†p<0.05 vs HG or TGF-β1, C: control without HG or TGF-β1, AS: PAI-1 antisense ODN, MS: PAI-1 mis-match sense ODN.

Fig. 2.

Effect of PAI-1 antisense ODN on HG- (A) and TGF-β1- (B) suppressed plasmin activity in mesangial cells. Growth arrested and synchronized primary rat mesangial cells were stimulated with 30 mM high D-glucose (HG) (A) or 2 ng/ml of TGF-β 1 (B) for 48 hours after PAI-1 antisense or mis-match ODN transfection. Plasmin activity in the media was measured as described in the Methods. Data are presented as means±SE of 4 experiments. ^∗p<0.05 vs control without HG or TGF-β 1, p<0.05 vs HG or TGF-β 1, C: control without HG or TGF-β1, AS: PAI-1 antisense ODN, MS: PAI-1 mis-match sense ODN.

Fig. 3.

Effect of PAI-1 antisense ODN on HG- (A) and TGF-β1- (B) suppressed MMP-2 activity in mesangial cells. Growth arrested and synchronized primary rat mesangial cells were stimulated with 30 mM high D-glucose (HG) (A) or 2 ng/ml of TGF-β1 (B) for 48 hours after PAI-1 antisense or mis-match ODN transfection. MMP-2 activity was detected by gelatin zymography as described in the Methods. Data are presented as means±SE of 4 experiments. ^∗p<0.05 vs control without HG or TGF-β1, ^†p<0.05 vs HG or TGF-β1, C: control without HG or TGF-β1, AS: PAI-1 antisense ODN, MS: PAI-1 mis-match sense ODN.

Fig. 4.

Effect of PAI-1 antisense ODN on HG- (A) and TGF-β1- (B) increased fibronectin secretion in mesangial cells. Growth arrested and synchronized primary rat mesangial cells were stimulated with 30 mM high D-glucose (HG) (A) or 2 ng/ml of TGF-β1 (B) for 48 hours after PAI-1 antisense or mis-match sense ODN transfection. Fibronectin protein was detected by Western blot analysis as described in the Methods. Data are presented as means±SE of 4 experiments. ^∗p<0.05 vs control without HG or TGF-β1, ^†p<0.05 vs HG or TGF-β1, C: control without HG or TGF-β1, AS: PAI-1 antisense ODN, MS: PAI-1 mis-match sense ODN.

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