Asymmetric dimethyl L-arginine (ADMA) is a critical regulator of myocardial reperfusion injury.

OBJECTIVE: Endothelial dysfunction by the loss of nitric oxide (NO) is a critical event during reperfusion of ischemic myocardium. Reduced NO availability signals important pathophysiological changes leading to myocardial reperfusion injury. We have recently shown that NO biosynthesis can be disturbed by the endogenous NO synthase (NOS) inhibitor ADMA and that these changes are mediated by an impairment of its metabolism by dimethylarginine dimethylaminohydrolase (DDAH). We therefore analyzed the role of ADMA and its metabolism in the setting of myocardial ischemia and reperfusion. METHODS: C57-bl6 mice underwent myocardial ischemia for exactly 30 min followed by 2, 4, 8, 12, 24, and 72 h of reperfusion achieved by occlusion and re-opening of the left coronary artery. The reperfused left ventricle was subsequently homogenized for measurements of determinants of the NO synthase pathway. Furthermore, the effects and its mechanisms of ADMA on reperfusion injury were analyzed in a genetic mouse model. RESULTS: A significant accumulation of ADMA was found in myocardial tissue when mice were subjected to 30 min of ischemia followed by reperfusion in our in vivo model. The maximum increase of tissue ADMA at 4 h of reperfusion coincided with reductions of NO tissue concentrations and DDAH activity; protein expression of NOS isoforms, however, was not changed. Furthermore, DDAH overexpression in a genetic mouse model as well as treatment with oral L-arginine markedly reduced reperfusion injury by 40-50% at 4 h of reperfusion. The effects of ADMA on reperfusion injury were shown to be mediated by reduced eNOS activity and phosphorylation, expression of adhesion molecules, and leukocyte activity. CONCLUSION: Accumulation of tissue ADMA by impairment of DDAH was found to be a significant determinant of reperfusion injury. Our results indicate that ADMA could be a potential new target for the treatment of myocardial ischemia/reperfusion injury.

Countervailing effects of rapamycin (sirolimus) on nuclear factor-kappa B activities in neointimal and medial smooth muscle cells.

OBJECTIVE: Local application of rapamycin (sirolimus) by drug-eluting stents prevents lumen obliteration after angioplasty by inhibition of neointimal hyperplasia. The effects of rapamycin on neointimal smooth muscle cells (niSMC) which are responsible for the occurrence of restenosis have not been investigated so far. METHODS AND RESULTS: Rat niSMC and medial SMC (mSMC) were obtained from balloon catheter-injured arteries. The niSMC exhibited higher basal NF-kappaB activity and TNF-alpha mRNA levels. Nuclear protein binding to NF-kappaB-DNA was attenuated in niSMC by incubation with rapamycin (0.1 and 1 microg/ml) for 24 and 48 h. In contrast in mSMC, 0.1 microg/ml rapamycin had no effect and at 1 microg/ml even increased nuclear protein binding to NF-kappaB-DNA. After 12 h incubation, rapamycin (0.001-10 microg/ml) induced IkappaB-alpha protein in niSMC, whereas in mSMC it stimulated IkappaB-alpha at much lower levels. Prolonged rapamycin treatment (1 microg/ml for 72 h) had no effect on TNF-alpha mRNA level and NF-kappaB activity in niSMC, whereas it led to their increase in mSMC. Vascular endothelial growth factor (VEGF) secretion was higher in mSMC than in niSMC; rapamycin decreased VEGF levels in both cell types. Ultrastructural analysis suggested that rapamycin caused early signs of degeneration in niSMC, but enhanced protein synthesis in mSMC. CONCLUSIONS: This study shows that rapamycin influences the inflammatory phenotypes of SMC in opposite directions: it reduces the high basal NF-kappaB activity in niSMC and enhances NF-kappaB activity and TNF-alpha expression in mSMC. In addition, rapamycin inhibits VEGF production regardless of the phenotype of SMC. These findings shed light on molecular mechanisms and structural changes underlying therapeutic applications of rapamycin in prevention of restenosis, inhibition of chronic transplant arteriosclerosis and reduction of secondary malignoma formation due to immunosuppression.

Effect of atorvastatin on circulating proinflammatory T-lymphocyte subsets and soluble CD40 ligand in patients with stable coronary artery disease--a randomized, placebo-controlled study.

BACKGROUND: Coronary atherosclerosis includes an activation of circulating T lymphocytes. Statins exert anti-inflammatory effects beyond lipid lowering. Whether these properties influence systemic T lymphocytes is unclear. METHODS: To investigate the effect of atorvastatin on circulating T-lymphocyte subsets producing proinflammatory and anti-inflammatory cytokines (interferon gamma [IFN-gamma(+)], interleukin 2 [IL-2(+)], IL-4(+), and IL-10(+)) and on the T-cell-activating soluble CD40 ligand (sCD40L), 30 hypercholesterolemic patients with angiographically documented stable coronary artery disease (CAD) were randomized to placebo or atorvastatin (20 mg/d) for 3 months. Eight healthy volunteers served as controls. Levels of peripheral cytokine-producing CD4+ and CD8+ T cells and their CD28- subsets were determined by FACS. Serum soluble CD40L was measured with ELISA. RESULTS: IL-2(+) T lymphocytes and sCD40L levels were higher in patients with CAD compared with controls, whereas IFN-gamma(+) and anti-inflammatory IL-4(+) and IL-10(+) T lymphocytes were similar. Levels of IL-2(+), IFN-gamma(+), IL-4(+), and IL-10(+) T-cell subsets as well as CD28- T lymphocytes were neither changed by atorvastatin nor by placebo, whereas sCD40L was lowered only in atorvastatin-treated patients (P < .01). CONCLUSION: Circulating IL-2(+) T lymphocytes are increased in patients with stable CAD reflecting an activation of the global immune system, but are not influenced by atorvastatin therapy. The elevated levels of platelet-derived T-lymphocyte-stimulating sCD40L are decreased by atorvastatin probably reflecting an atheroprotective effect. Hence, sCD40L may be an additional biomarker to be considered when evaluating the treatment effects of statins in patients with stable CAD.

Effect of atorvastatin on peripheral endothelial function and systemic inflammatory markers in patients with stable coronary artery disease.

BACKGROUND: Endothelial dysfunction, detectable by an impaired flow-mediated vasodilation (FMD) of the brachial artery, has been shown to be associated with increased levels of circulating proinflammatory markers. Therapeutic interventions such as lipid-lowering with statins increase FMD and decrease inflammatory markers, like soluble (s) E-selectin, soluble intercellular adhesion molecule-1 (sICAM-1) or high-sensitivity Creactive protein (hsCRP). The effect of atorvastatin therapy on both FMD and inflammatory markers in patients with stable coronary artery disease (CAD) has not been investigated. METHODS: Thirty hypercholesterolemic patients with angiographically documented stable coronary artery disease (CAD) were randomized to placebo or atorvastatin (20 mg/d) for 3 months. FMD was assessed using highresolution ultrasound (13 MHz, Acuson Sequoia, C256). High-sensitivity CRP was measured with Latex agglutination assay, sE-selectin and sICAM-1 were determined with ELISA. RESULTS: Baseline characteristics were not different between groups. FMD improved in patients on atorvastatin (6.7+/-3.8% to 8.5+/-4.4%; p<0.01), but remained unchanged in placebo-treated patients (8.2+/-3.3% to 8.9+/-5.1%; p=NS). Atorvastatin treatment was associated with decreases of sICAM-1 (from 274.2+/-92.2 to 197.9+/-70.0 ng/ml; p<0.01) and hsCRP (from 0.57+/-0.45 to 0.18+/-0.15 mg/dl; p<0.01), whereas placebo treatment had no effect on these markers. sE-selectin levels were not influenced by either treatment. No correlations were found between changes in FMD, lipids and inflammatory markers. CONCLUSIONS: Treatment with atorvastatin leads to an improvement in endothelial function and a reduction in inflammatory markers in patients with stable CAD. The lack of correlation between changes in FMD and inflammatory markers may support the concept of pleiotropic effects of statins in humans.