High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes.
OBJECTIVE
To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy.


METHODS
In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures.


RESULTS
Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9).


CONCLUSIONS
High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.
Li
An-Fei
AF
Department of Ophthalmology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Sato
Tsuyoshi
T
Haimovici
Robert
R
Okamoto
Tamami
T
Roy
Sayon
S
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002450:Cell Communication / Q000187:drug effects*; D018929:Cell Culture Techniques; D018920:Coculture Techniques; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D020286:Pericytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein
D018031:Connexin 43; D017630:Connexins; D005947:Glucose; D012333:RNA, Messenger; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein