Korean J Physiol Pharmacol.  2010 Dec;14(6):385-390. 10.4196/kjpp.2010.14.6.385.

Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

• Park J
• Seo JY
• Ha H

• KMID: 2071710
• DOI: http://doi.org/10.4196/kjpp.2010.14.6.385

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)-beta1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 microM 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-beta1. 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-beta1 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-beta1-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.

Keyword

Plasminogen activator inhibitor-1; Antisense oligodeoxynucleotide; Plasmin; Matrix metalloproteinase; Mesangial cells

MeSH Terms

Animals
Blotting, Western
Diabetic Nephropathies
Extracellular Matrix
Fibrinolysin
Fibronectins
Fibrosis
Glucose
Liposomes
Mesangial Cells
Oligodeoxyribonucleotides
Plasminogen
Plasminogen Activator Inhibitor 1
Plasminogen Activators
Rats
Renal Insufficiency, Chronic
Transfection
Transforming Growth Factor beta1
Transforming Growth Factors
Up-Regulation
Fibrinolysin
Fibronectins
Glucose
Liposomes
Oligodeoxyribonucleotides
Plasminogen
Plasminogen Activator Inhibitor 1
Plasminogen Activators
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  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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