Combination of cilostazol and clopidogrel attenuates rat critical limb ischemia.
OBJECTIVE
Procedural failure and untoward clinical outcomes after surgery remain problematic in critical limb ischemia (CLI) patients. This study tested a clopidogrel-cilostazol combination treatment compared with either treatment alone in attenuating CLI and improving CLI-region blood flow in rats.


METHODS
Male Sprague-Dawley rats (n = 40) were equally divided into five groups: control, CLI induction only, CL I + cilostazol (12.0 mg/day/kg), CLI + clopidogrel (8.0 mg/kg/day) and CLI + combined cilostazol-clopidogrel. After treatment for 21 days, Laser Doppler imaging was performed.


RESULTS
On day 21, the untreated CLI group had the lowest ratio of ischemic/normal blood flow (p < 0.001). Inflammation measured by VCAM-1 protein expression; oxidative stress; PAI-1, MMP-9 and TNF-α mRNA expressions; and immunofluorescence staining (IF) of CD68+ cells was lower with combined treatment than with the other treatments, and lower in the two single-treatment groups than the untreated CLI group (all p < 0.01). Anti-inflammatory mRNA expression of interleukin-10, and eNOS showed a reverse pattern among these groups. Apoptosis measured by Bax, caspase-3 and PARP; and muscle damage measured by cytosolic cytochrome-C, and serum and muscle micro-RNA-206 were all lowest with combination treatment, and the two single-treatment groups showed lower values than the untreated group (all p < 0.001). Angiogenesis measured by eNOS, IF staining of CD31+ and vWF + cells; and number of vessels in CLI region were highest with combination treatment and higher in the single-treatment groups than the untreated group (all p < 0.001).


CONCLUSIONS
Combined cilostazol-clopidogrel therapy is superior to either agent alone in improving ischemia in rodent CLI.
Division of Thoracic and Cardiovascular Surgery, Chang Gung Memorial Hospital- Kaohsiung Medical Center, Chang Gung University College of Medicine, Gueishan, Taiwan.
Jiunn-Jye
JJ
Sheu
Kun-Chen
KC
Lin
Ching-Yen
CY
Tsai
Tzu-Hsien
TH
Tsai
Steve
S
Leu
Chia-Hung
CH
Yen
Yung-Lung
YL
Chen
Hsueh-Wen
HW
Chang
Cheuk-Kwan
CK
Sun
Sarah
S
Chua
Jenq-Lin
JL
Yang
Hon-Kan
HK
Yip
D000818:Animals; D015153:Blotting, Western; D000077407:Cilostazol; D000077144:Clopidogrel; D004359:Drug Therapy, Combination; D005121:Extremities / Q000098:blood supply*; D005434:Flow Cytometry; D007150:Immunohistochemistry; D007511:Ischemia / Q000517:prevention & control*; D008297:Male; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D013777:Tetrazoles / Q000008:administration & dosage / Q000627:therapeutic use*; D013988:Ticlopidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000627:therapeutic use
D013777:Tetrazoles; D000077144:Clopidogrel; D000077407:Cilostazol; D013988:Ticlopidine