ROCK inhibitor fasudil attenuated high glucose-induced MCP-1 and VCAM-1 expression and monocyte-endothelial cell adhesion.
BACKGROUND
Previous studies suggested that the RhoA/ROCK pathway may contribute to vascular complications in diabetes. The present study was designed to investigate whether ROCK inhibitor fasudil could prevent high glucose-induced monocyte-endothelial cells adhesion, and whether this was related to fasudil effects on vascular endothelial cell expression of chemotactic factors, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1).


METHODS
HUVECs were stimulated with high glucose (HG) or HG + fasudil in different concentration or different time. Monocyte-endothelial cell adhesion was determined using fluorescence-labeled monocytes. The mRNA and protein expression of VCAM-1 and MCP-1 were measured using real-time PCR and western blot. The protein levels of RhoA, ROCKI and p-MYPT were determined using western blot analysis. ELISA was employed to measure the expression of soluble VCAM-1 and MCP-1 in cell supernatants and human serum samples.


RESULTS
Fasudil significantly suppressed HG-induced adhesion of THP-1 to HUVECs. Fasudil reduced Rho/ROCK activity (as indicated by lower p-MYPT/MYPT ratio), and prevented HG induced increases in VCAM-1 and MCP-1 mRNA and protein levels. Fasudil also decreased MCP-1 concentration in HUVEC supernatants, but increased sVCAM-1 shedding into the media. In human diabetic subjects, 2 weeks of fasudil treatment significantly decreased serum MCP-1 level from 27.9 ± 10.6 pg/ml to 13.8 ± 7.0 pg/ml (P < 0.05), while sVCAM-1 increased from 23.2 ± 7.5 ng/ml to 39.7 ± 5.6 ng/ml after fasudil treatment (P < 0.05).


CONCLUSIONS
Treatment with the Rho/ROCK pathway inhibitor fasudil attenuated HG-induced monocyte-endothelial cell adhesion, possibly by reducing endothelial expression of VCAM-1 and MCP-1. These results suggest inhibition of Rho/ROCK signaling may have therapeutic potential in preventing diabetes associated vascular inflammation and atherogenesis.
Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China.
Hailing
H
Li
Wenhui
W
Peng
Weixia
W
Jian
Yuanmin
Y
Li
Qi
Q
Li
Weiming
W
Li
Yawei
Y
Xu
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D000368:Aged; D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D002681:China; D018920:Coculture Techniques; D003920:Diabetes Mellitus / Q000097:blood / Q000188:drug therapy / Q000201:enzymology / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D006801:Humans; D007262:Infusions, Intravenous; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016037:Single-Blind Method; D013997:Time Factors; D019010:Vascular Cell Adhesion Molecule-1 / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000893:Anti-Inflammatory Agents; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; D005947:Glucose; C049347:fasudil