J Menopausal Med.  2014 Aug;20(2):57-68. 10.6118/jmm.2014.20.2.57.

Sildenafil Inhibits Advanced Glycation End Products-induced sFlt-1 Release Through Upregulation of Heme Oxygenase-1

• Jeong JH
• Kim HG
• Choi OH

• KMID: 2325443
• DOI: http://doi.org/10.6118/jmm.2014.20.2.57

Abstract

OBJECTIVES
We examined the effect of sildenafil citrate on advanced glycation end products (AGEs)-induced soluble fms-like tyrosine kinase 1 (sFlt-1) release in JEG-3 choriocarcinoma cells.
METHODS
Cells were incubated with control bovine serum albumin (BSA) or AGEs-BSA, and expression of sFlt-1 mRNA and protein release was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. AGEs-BSA increased sFlt-1 mRNA expression and protein release in a dose-dependent manner.
RESULTS
Sildenafil citrate suppressed sFlt-1 mRNA expression and protein release in cells treated with AGEs-BSA in a dose-dependent manner. Likewise, it inhibited the increase of reactive oxygen species (ROS) production and NF-kappaB activity in these cells. Cobalt protoporphyrin (CoPP) and bilirubin also inhibited sFlt-1 release and ROS production in cells treated with AGEs-BSA, whereas zinc protoporphyrin IX (ZnPP IX) antagonized the effect of sildenafil citrate. In cells transfected with the heme oxygenase-1 (HO-1) siRNA, sildenafil citrate failed to inhibit the sFlt-1 release and ROS production.
CONCLUSION
These results strongly suggest that sildenafil citrate inhibits sFlt-1 release and ROS production in cells treated with AGEs-BSA through upregulation of the HO-1 expression in JEG-3 cells.

Keyword

Advanced glycoxylation end products; sFLt-1; Sildenafil

MeSH Terms

Bilirubin
Choriocarcinoma
Citric Acid
Cobalt
Enzyme-Linked Immunosorbent Assay
Female
Glycosylation End Products, Advanced
Heme Oxygenase-1*
NF-kappa B
Pregnancy
Reactive Oxygen Species
RNA, Messenger
RNA, Small Interfering
Serum Albumin, Bovine
Up-Regulation*
Vascular Endothelial Growth Factor Receptor-1
Zinc
Sildenafil Citrate
Bilirubin
Citric Acid
Cobalt
Glycosylation End Products, Advanced
Heme Oxygenase-1
NF-kappa B
RNA, Messenger
RNA, Small Interfering
Reactive Oxygen Species
Serum Albumin, Bovine
Vascular Endothelial Growth Factor Receptor-1
Zinc

Figure

• 

  Fig. 1 Upregulated expression and release of soluble fms-like tyrosine kinase-1 (sFlt-1) in JEG-3 cells treated with advanced glycation end-products-vine serum albumin (AGEs-BSA). Cells were incubated in the absence of BSA (Con), in the presence of unmodified BSA (BSA, 200 g/mL) or indicated concentrations of AGEs-BSA (AGE) for 24 h. (A) sFlt-1 mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (B) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. BSA control. sFlt-1: soluble fms-like tyrosine kinase-1, AGE: advanced glycation end-products.

• 

  Fig. 2 Effect of anti-receptors for advanced glycation end-product (RAGE) antibody on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-induced stimulation of soluble fms-like tyrosine kinase-1 (sFlt-1) expression and release in JEG-3 cells. Cells were incubated in the presence of each 200 µg/mL of unmodified BSA or AGEs-BSA (AGE) for 24 h. (A) sFlt-1 mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (B) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. BSA control, #P < 0.01 vs. without anti-RAGE. sFlt-1: soluble fms-like tyrosine kinase-1, BSA: bovine serum albumin, AGE: advanced glycation end-products.

• 

  Fig. 3 Effect of anti-receptors for advanced glycation end-product (RAGE) antibody on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-induced stimulation of reactive oxygen species (ROS) production and NF-κB activity in JEG-3 cells. Cells were incubated in the presence of each 200 µg/mL of unmodified BSA or AGEs-BSA (AGE) for 24 h. (A) ROS production was determined by 2',7'-dichlorodihydrofluorescein (DCFH) fluorescence assay. (B) NF-κB activity was determined by reporter gene assay. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. BSA control, #P < 0.01 vs. without anti-RAGE. ROS: reactive oxygen species, BSA: bovine serum albumin, AGE: advanced glycation end-products.

• 

  Fig. 4 Effect of sildenafil citrate on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-induced stimulation of soluble fms-like tyrosine kinase-1 (sFlt-1) expression and release in JEG-3 cells. Cells were incubated in the presence of 200 µg/mL AGEs-BSA alone or with indicated concentrations of sildenafil citrate (SC) for 24 h. (A) sFlt-1 mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and presented as % of BSA control. (B) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants and presented as % of BSA control. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.05, #P < 0.01 vs. BSA control. sFlt-1: soluble fms-like tyrosine kinase-1, BSA: bovine serum albumin, SC: sildenafil citrate.

• 

  Fig. 5 Effect of sildenafil citrate on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-induced stimulation of reactive oxygen species (ROS) production, NF-κB activity and receptors for advanced glycation end-product (RAGE) mRNA expression in JEG-3 cells. Cells were incubated in the presence of each 200 µg/mL of unmodified BSA or AGEs-BSA (AGE) with or without 20 µM sildenafil citrate (SC) for 24 h. (A) ROS production was determined by 2',7'-dichlorodihydrofluorescein (DCFH) fluorescence assay. (B) NF-κB activity was determined by reporter gene assay. (C) RAGE mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. BSA control, #P < 0.01 vs. without anti-RAGE. ROS: reactive oxygen species, BSA: bovine serum albumin, AGE: advanced glycation end-products, RAGE: receptors for advanced glycation end-product.

• 

  Fig. 6 Upregulation of heme oxygenase-1 (HO-1) expression by sildenafil citrate. Cells were incubated for 24 h in the presence of each 200 µg/mL of unmodified bovine serum albumin (BSA) or advanced glycation end products (AGEs)-BSA (AGE) with vehicle (DMSO, Veh), 20 µM sildenafil citrate (SC) and 20 µM cobalt protoporphyrin (CoPP). (A) HO-1 mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (B) HO-1 protein expression was determined by Western blot analysis of the cell extracts. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. Veh. HO-1: heme oxygenase-1, BSA: bovine serum albumin, AGE: advanced glycation end-product, Veh: vehicle, SC: sildenafil citrate, CoPP: Cobalt protoporphyrin.

• 

  Fig. 7 Effects of heme oxygenase-1 (HO-1) related agents on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-stimulated soluble fms-like tyrosine kinase-1 (sFlt-1) release and reactive oxygen species (ROS) production. Cells were incubated for 24 h in the presence of each 200 µg/mL of unmodified BSA or AGEs-BSA (AGE) with or without each 20 µM of sildenafil citrate (SC), cobalt protoporphyrin (CoPP), bilirubin (Bili), and zinc protoporphyrin IX (ZnPP). (A) sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants and presented as % of BSA control. (B) Reactive oxygen species (ROS) production was determined by 2',7'-dichlorodihydrofluorescein (DCFH) fluorescence assay and presented as % of BSA control. Mean ± S.E. of 3 independent duplicate experiments. *P < 0.01 vs. AGE alone, #P < 0.05 vs. AGE+SC without ZnPP. sFlt-1: soluble fms-like tyrosine kinase-1, BSA: bovine serum albumin, AGE: advanced glycation end-product, SC: sildenafil citrate, CoPP: cobalt protoporphyrin, Bili: bilirubin, ZnPP: zinc protoporphyrin, ROS: reactive oxygen species.

• 

  Fig. 8 Effect of heme oxygenase-1 (HO-1) downregulation on advanced glycation end-products-bovine serum albumin (AGEs-BSA)-stimulated soluble fms-like tyrosine kinase-1 (sFlt-1) release and reactive oxygen species (ROS) production. (A) Down-regulation of HO-1 protein expression by siRNA transfection. Cells transfected with 30 picomoles of HO-1 siRNA or scrambled siRNA (siRNA control) were incubated 24 h with vehicle (Veh) or each 20 µM of sildenafil citrate (SC) and cobalt protoporphyrin (CoPP). HO-1 protein expression was determined by Western blot analysis of the cell extracts. (B) Cells were incubated in the presence of each 200 µg/mL of unmodified BSA or AGEs-BSA (AGE) with or without each 20 µM of sildenafil citrate (SC). sFlt-1 protein release was determined by enzyme-linked immunosorbent assay (ELISA) analysis of the culture supernatants. (C) ROS formation was determined by 2',7'-dichlorodihydrofluorescein (DCFH) fluorescence assay. Data were presented as % of BSA control. Mean ± S.E. of 4 independent duplicate experiments. *P < 0.01 vs. BSA control, #P < 0.01 vs. AGE alone, @P < 0.01 vs. siRNA control. HO-1: heme oxygenase-1, Veh: vehicle, SC: sildenafil citrate, CoPP: Cobalt protoporphyrin, sFlt-1: soluble fms-like tyrosine kinase-1, BSA: bovine serum albumin, AGE: advanced glycation end-product, ROS: reactive oxygen species.

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