| Research Abstract |
Several lines of evidence indicate that inflammation plays a pivotal role in the process of atherosclerosis. Although local inflammation of vulnerable plaque is responsible for the development of acute coronary syndrome, systemic inflammatory process as estimated by high sensitive C-reactive protein is closely associated with later cardiovascular events in subjects with hypercholesterolemia. Although precise mechanisms which link systemic inflammation to vascular diseases remain to be elucidated, reactive oxygen species (ROS) play a pivotal role in inflammation-mediated vascular injury. Polymorphonuclear leukocytes (PMN) are ubiquitous effector cells in numerous inflammatory conditions. Relevance of PMN function is usually thought in terms of host-defense in bacterial infections. PMN produce a large amount of ROS through the respiratory burst and cause lipid peroxidation. In addition, upon stimulation, PMN release cytokines which promote endothelial cell activation and hence cell-to-ce
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ll adhesion. Collectively, these findings suggest a possible role of PMN in the development of vascular diseases via impairment of endothelial function. Indeed, widespread activation of PMN was demonstrated in subjects with hypercholesterolemia. Three-hydroxyl-3-methylglutanyl coenzyme A (HMG-CoA) reductase inhibitors (statins) significantly decrease cardiovascular mortality associated with hypercholesterolemia. In vitro studies have shown that stating exert beneficial effects on vascular diseases by inhibiting leukocyte rolling and adhesion and by stabilizing endothelial NO synthase (eNOS) posttranslationally. Several clinical trials revealed that statins decrease markers of inflammation/oxidative stress and restore endothelium-dependent vasodilation in the placebo controlled design. However, whether the restorations of endothelial function by statin are attributable to LDL reduction or to its pleiotropic effects remain to be tested by the alternative active treatment arm. Accordingly, we hypothesized that i) circulating PMN in hypercholesterolemia are activated, leading to oxidative stress in vivo and hence impair endothelial function, and that if so ii) statins may restore the PMN-mediated endothelial dysfunction independently of LDL reduction. To test these hypotheses, we measured PMN activity, oxidative stress, and endothelial function in subjects hypercholesterolemia and their age-matched controls and we conducted a prospective randomized study using two different classes of lipid lowering agents, fluvastatin and colestimide, in a cross-over design. Moreover, to investigate the direct interrelation between PMN and endothelia, cell adhesion and eNOS phosphorylation as a process of eNOS activation in cultured human endothelial cells were investigated before and after ex vivo exposure of PMN obtained from hypercholesterolemic subjects with or without fluvastatin treatment. Three months administration of fluvastatin or colestimide lowered LDL to the same levels. Endothelial function was restored and the suscepitibility of LDL to oxidation was diminished only in fluvastation-treated subjects. Similarly, fluvastatin attenuated ROS release from PMN, whereas colestimide had no effects on either lipid peroxidation or ROS release. Furthermore, co-incubation with PMN obtained from HC significantly impaired eNOS Ser1177 phosphorylation of HUVEC comared to controls. Fluvastin treatment attenuated cell adhesion, and restored eNOS Ser1177 phosphorylation in HC. Thus, our results raise a novel concept that circulating PMN may directly attack endothelia and play a pivotal role in the pathogenesis of atherosclerosis.
Next, we investigated PMN activity in general population. Myeloperoxidase (MPO), a hemoprotein abunbantly expressed by polymorphonuclear leukocytes and secreted during activation, possesses potent proinflammatory properties. It has been shown that serum MPO levels are elevated in patients with acute coronary syndrome and that its levels predict subsequent cardiovascular events. Although its precise mechanism remains uknown, in vitro MPO catalytically consumes endothelium-derived nitric oxide. In apparently healthy subjects, coronary risk factors were evaluated. Endothelial function and arterial compliance were estimated by flow-mediated vasodilation of the brachial artery and stiffness beta of the common carotid artery, by vascular ultrasonography, respectively. Serum MPO levels were determined by ELISA. Univariate analyses revealed that MPO was significantly and positively correlated with age and high-sensitivity C-creative protein (hsCRP), but not associated with gender, lipid profile, plasma glucose or smoking habit. There was significant positive relationship between MPO and stiffness beta. MPO was also inversely correlated with flow-mediated vasodilation. Multiple regression analysis revealed that MPO was an independent determinant of flow-mediated vasodilation, irrespective of classical risk and hsCRP. Thus, MPO was closely associated with aging and systemic inflammation and correlated with indices of arterial compliance. Furthermore, MPO was an independent risk factor of endothelial function. Our results suggest that MPO, neutrophil-derived peroxidase, may promote atherosclerosis via endothelial dysfunction.
Peroxisome proliferator activated receptor (PPAR) gamma plays a pivotal role in vascular inflammatory process. Indeed, stimulation of the PPAR ligand improved endothelial function partly mediated by renin-angiotensin (RA) axis. Recently, a number of study demonstrated that blocking AT(1)R and activating PPAR gamma signaling attenuate asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system, an endogenous inhibitor of nitric oxide. Therefore, we investigated the regulatory mechanisms of ADMA-DDAH system in the model of chronic kidney disease. Plasma ADMA was elevated and DDHA activity was reduced in the CKD model. Chronic administration of AT(1)R blocher restored DDAH activity and decreased ADMA. Thus, the crosstalk among PPAR-ADMA-DDAH system-RA axis may play a role in the progression of vascular disease.
As a future prospect, we are now developing our projects for clinical application. For this purpose, we have organized the committee on the standardized-measurement of endothelial function by the ultrasonography-guided flow mediated vasodilation, and published the statement. The multi-center clinical trial using FMD, as well as simultenous several workshops for the methods will be conducted next year. Less
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