Strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way.
OBJECTIVE
To determine the effects of pericytes and angiopoietin-1 on the expression of occludin and zonula occludens-1 (ZO-1) in retinal endothelial cells (ECs) under both normoxic and hypoxic conditions.


METHODS
Rat primary retinal microvascular ECs were cultured under normoxia or hypoxia in either absence or presence of pericytes conditioned medium (PCM). PCM was pretreated with or without angiopoietin-1 neutralizing antibody. Immuofluorescent staining, Western blot and RT-PCR were used to detect the alterations of occludin and ZO-1 expression.


RESULTS
Under normoxia, PCM strengthened occludin and ZO-1 immunofluorescent staining at cytomembrane as well as increased their expression at both protein and mRNA level. When pretreated with angiopoietin-1 neutralizing antibody, occludin upregulation induced by PCM was significantly blocked at protein level (62%) and mRNA level (34%). Under hypoxia, the continuity of occludin and ZO-1 staining at cell boundaries was disrupted consistent with a decrease of their protein level by 31 and 27%, respectively. Also occludin and ZO-1 mRNA level decreased by 46 and 57%, respectively. PCM was observed to partially increase expression of occludin at protein and mRNA level. Angiopoietin-1 antibody slightly inhibited (16%) PCM induced occludin mRNA increase under hypoxia.


CONCLUSIONS
Pericytes improved the integrity of endothelial barrier through inducing occludin and ZO-1 expression at protein and mRNA level under normoxia. Under hypoxia, pericytes could partially reverse occludin decrease. These protecting effects of pericytes on endothelial barrier were at least in part mediated by angiopoietin-1.
Wang
Y L
YL
Department of Ophthalmology, Xijing Hospital, the Fourth Military Medical University, Xi'an, China. wangylnancy@163.com
Hui
Y N
YN
Guo
B
B
Ma
J X
JX
D042683:Angiopoietin-1 / Q000031:analogs & derivatives / Q000502:physiology; D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D042783:Endothelial Cells / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D062793:Occludin; D020286:Pericytes / Q000502:physiology*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000166:cytology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000502:physiology; D019108:Tight Junctions / Q000502:physiology*; D062826:Zonula Occludens-1 Protein
D042683:Angiopoietin-1; C467629:Angpt1 protein, rat; D017077:Culture Media, Conditioned; D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein