Inhibitory mechanism of vascular endothelial growth factor (VEGF) by bucillamine.
1. Vascular endothelial growth factor (VEGF) plays an important role in the neovascularization of ischaemic retinal diseases such as proliferative diabetic retinopathy. We determined that bucillamine, an anti-rheumatic drug, inhibits the VEGF production induced by hypoxia in bovine retinal microcapillary endothelial cells (BREC). To further clarify the inhibitory mechanism, we investigated the possible mechanism by which bucillamine exerts this inhibitory effect. 2. Bucillamine (100 micro M) decreased the hypoxia-induced increase of VEGF mRNA by 54.5% (P<0.001). Bucillamine (100 micro M) reduced the hypoxia-induced VEGF content in culture media by 29.0% (P<0.001), while monosulfydryl drugs, N-acetylcysteine and D-penicillamine, did not. 3. Bucillamine (100 micro M) did not affect VEGF mRNA half-life (hypoxia, 4.3 h; hypoxia+bucillamine, 3.9 h; normoxia, 2.7 h; normoxia+bucillamine, 2.7 h). 4. Reporter gene studies revealed that bucillamine reduced transcriptional activity in the 5'-flanking region of the VEGF gene by 74.0%. Hypoxia stimulated binding activity of BREC nuclear protein to a hypoxia responsive element (HRE), which was decreased by bucillamine. 5. Bucillamine inhibited hypoxic-induction of HIF-1alpha mRNA by 73.1% (P<0.001). Bucillamine also inhibited spontaneous VEGF mRNA expression by 26.6%. Furthermore, it inhibited activity of VEGF promoter and decreased binding activity to Sp1 and HRE, but did not alter AP1 and AP2 activity in normoxia. 6. These data suggest that bucillamine inhibits hypoxic induction of VEGF through inhibition of HIF-1 induction and binding activity in BREC. Bucillamine also inhibits the spontaneous expression of VEGF mRNA by its effect on Sp1 and HRE binding.
Koyama
Shinji
S
Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8397, Japan.
Takagi
Hitoshi
H
Otani
Atsushi
A
Oh
Hideyasu
H
Nishimura
Kazuo
K
Honda
Yoshihito
Y
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008222:Lymphokines / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D008156:Luciferases; D003545:Cysteine; C026535:bucillamine