title: Arginase blockade lessens endothelial dysfunction after thrombosis.
abstract: BACKGROUND
  Acute arterial thrombosis causes endothelial dysfunction due to decreased nitric oxide bioactivity. Increased arginase activity may modulate intracellular L-arginine levels, the substrate for nitric oxide. The purpose of this study was to identify the role of arginase in endothelial dysfunction in cell culture and in the vasomotor response of arteries exposed to thrombus.
  
  
  METHODS
  Rat aortic endothelial cells were exposed to thrombin at different time points. The cell extract was analyzed by immunoblotting and real-time polymerase chain reaction. Adult male rats underwent infrarenal aortic thrombosis by clip ligature for 1 hour. Infrarenal aortic ring segments were harvested and placed in physiologic buffer baths, and a force transducer was used to measure endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR). Arginase blockade was performed by incubating infrarenal aortic ring segments with arginase inhibitors for 1 hour before measuring EDR. Whole tissue extracts also underwent immunoblot analysis. The EDR and EIR curves were compared with analyses of variance.
  
  
  RESULTS
  A 6.76 +/- 1.4-fold induction in arginase I message levels (P = .001) was found in rat aortic endothelial cells exposed to thrombin (30 U/mL), and arginase I protein levels increased 2.1 times. The eight infrarenal aortic ring segments exposed to thrombosis for 1 hour had diminished EDR curves compared with 14 nonthrombosed normal segments (controls). The maximum (+/- SEM) EDR (acetylcholine 10(-5)M dose) in control infrarenal aortic ring segments was 108% +/- 4.3% compared with 63% +/- 6.2% for thrombosed infrarenal aortic ring segments (P < .001). Exposure to arterial thrombosis resulted in a 3.8-times increase in arginase I protein levels in infrarenal aortic ring segments. Preincubation of nine infrarenal aortic ring segments with the nonspecific (difluoromethylornithine) and six with specific ([S]-[2-boronoethyl]-L-Cysteine-HCl [BEC]) arginase inhibitor for 1 hour significantly increased the maximum EDR compared with untreated thrombosed segments (104 +/- 5.2, 108 +/- 7.6 vs 63% +/- 6.2, P < .001). EDR curves for difluoromethylornithine- and BEC-treated infrarenal aortic ring segments were superimposed on control EDR curves. The EIR and the vasoconstriction with norepinephrine for all groups were similar.
  
  
  CONCLUSIONS
  Endothelial cells exposed to thrombin have increased arginase I messenger RNA and protein levels. Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Arginase blockade can lead to normalization of arterial vasomotor function.
authors.affiliation: Department of Cell Biology, the Lerner Research Institute, Cleveland, Ohio; Department of Vascular Surgery, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Chandani
authors.initials: C
authors.lastname: Lewis
authors.forename: Weifei
authors.initials: W
authors.lastname: Zhu
authors.forename: Mircea L
authors.initials: ML
authors.lastname: Pavkov
authors.forename: Corttrell M
authors.initials: CM
authors.lastname: Kinney
authors.forename: Paul E
authors.initials: PE
authors.lastname: Dicorleto
authors.forename: Vikram S
authors.initials: VS
authors.lastname: Kashyap
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001157:Arterial Occlusive Diseases / Q000473:pathology* / Q000503:physiopathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000503:physiopathology*; D015151:Immunoblotting; D008297:Male; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D011336:Probability; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000494:pharmacology*; D014661:Vasoconstriction / Q000502:physiology
chemical_list: D012333:RNA, Messenger; D013917:Thrombin; D001119:Arginase