الفهرس 
Introduction and aim of the workOnly 14 pages are availabe for public view
 |  | from | 203 |  |  |
| | | from | 203 | | |
Abstract
Atherosclerosis is a progressive disease that starts early in life and is manifested clinically as coronary heart disease (CHD), cerebrovascular disorders or peripheral arterial disease. Atherosclerosis and related disorders are the major causes of mortality in the western world and also becoming common in the developing countries. Endothelial dysfunction plays an important role in the pathogenesis of atherosclerosis. It is one of the main pathologic mechanisms by which different cardiovascular risk factors cause atherosclerotic vascular damage, predisposing patients to cardiovascular events. Endothelial dysfunction has been proposed to be a useful marker of early cardiovascular diseases and may result from increased production of reactive oxygen species (ROS) and decreased synthesis, release, or activity of endothelial-derived nitric oxide.
Recently, HO-l has emerged as an important mediator of antioxidant and tissue protective actions. Studies that modulate HO-l activities have clearly shown that HO-I assumes a central role in cellular antioxidant defense especially in vascular protection suggesting a significant anti-atherogenic effect of this enzyme. While many previous studies demonstrated that NO is a determinant in the modulation of the activity ofHOOI to a major resistance of the endothelium to oxidative stress, others reported that NO inhibits HO-I activity.
Statins, HMG-CoA reductase inhibitors, are now widely prescribed to patients with ischemic heart diseases. Besides the therapeutic use in hyperlipidemia, the pleiotropic effect of statins in ameliorating endothelial dysfunction is not yet completely defined. Since the understanding and evaluation of the pharmacological
176
effects of statins are increasing and accelerating their clinical importance and validity, in this study we intended to identify novel mechanisms that could modulate their pleiotropic effects. Therefore, in this work both in vitro and ex vivo studies are conducted by using two different kinds of statins (atorvastatin and simvastatin) as we are looking for a class effect of statins rather than drug-specific action.
For studying in vitro effect of statins on NO/ONOO- balance in normal and dysfunctional endothelium, a set of electrochemical nanosensors was applied for simultaneous measurements of minute concentrations of NO and ONOo- produced by cultured endothelial cells (HUVECs) treated with statins (5 Ilmol/L atorvastatin or 10 IlmollL simvastatin) for 24 h in the presence or absence of 50 Ilglml ox-LDL. An imbalance between NO/ONOO- concentrations was used as an indicator of endothelial dysfunctional and correlated with a level of endothelial nitric oxide synthase (eNOS) protein expression. The essential findings from this part are as follows:
1- Incubation of endothelial cells with ox-LDL greatly reduced the expression of eNOS protein level and also the release of NO while it produced a parallel increase in ONOOOproduction which in turn, reflects an increase in the nitroxidative stress. The increase in nitroxidative stress is of high importance in cardiovascular disorders that involve oxxLDL deposition on the endothelial wall.
2- Treatment with statins can favorably shift NO/ONOO- balance in normal, functional HUVECs, as well as, in highly dysfunctional cells (ox-LDL-treated HUVECs).
3- Statins increased bioavailable NO and reduced ONOO- production, the main component of nitroxidative stress. Therefore, a treatment with statins can preserve and extend bioavailable NO in endothelium and significantly reduce the level of cytotoxic
effect of ONOO-. Both of these processes are crucial in maintaining endothelial function.
4- Both atorvastatin and simvastatin show beneficial effects in normal endothelium and to higher extent in the dysfunctional endothelium.
The possible molecular mechanisms for pleiotropic effects of statins in this study include their effect on the induction of the antioxidant enzyme, HO-l. Since statins were reported to increase NO concentration, we investigated the role of NO in this induction as a signaling molecule. Therefore, this part was designed to study first, if HO-I is a molecular target for statins through NO pathway and second, the possible regulatory role of HO-I in modulating pleiotropic effects of statins, especially, on NO/ONOO- balance and eNOS functional activity. To perform these goals, atherosclerotic animal models were used and treated with statins (atorvastatin or simvastatin). Other atherosclerotic models were co-treated with statins and either LLNAME, or SnPP. Finally, some models were treated with either L-arginine or hemin. Plasma lipid profile (TC, TO, LDL and HDL), MDA (as an indicator for lipid peroxides) and aortic endothelial release of NO/ONOO- were estimated. In addition, aortic endothelial eNOS and HO-l protein expression (by Western blotting) and total HO activity were measured. Finally, histopathological study of hematoxylin and eosinnstained sections of the aortic arches was performed.
The essential findings from this part are as follows:
1- High cholesterol diet for two months produced marked atherosclerotic changes, which is evaluated by histopathological examination in the form of marked fatty streak with multiple plaque lesions and increasing in the thickness of the vessel wall.
2- As a function of hypercholesterolemia, there was a dramatic reduction in eNOS protein expression and NO/ONOO· ratio (K value was about three fold lower than control). Low K value indicates eNOS uncoupling and dysfunction of endothelium.
3- In atherosclerotic animals, the decrease in NO level with parallel increase in ONOO’ production is one of the candidate mechanisms that are involved in the oxidative metabolism of LDL and generating higher level of lipids peroxides.
4- Treatments with statins reduced deposited fatty streak and the thickness ofthe intima with significant decrease in the vessel wall thickness.
5- Plasma lipids significantly decreased with statins treatment while, it was not changed by treatment with either eNOS or HO-I modulators, suggesting that the antiatherogenic properties of NO and HO-I couldn’t be conducted through direct changes of plasma lipids.
6- Statins appreciably increased eNOS protein expression and restored NO-generating capacity of the enzyme in atherosclerotic animals. Accordingly, statins were capable of reversing endothelial dysfunction associated with hypercholesterolemia.
7- Treatment with statins produced appreciable increases in vascular HO-I expression and activity, which were higher than that obtained for non-treated, atherosclerotic animals. Therefore, the antioxidant defense enzyme, HO-I, is an intracellular site of action for statins.
8- The increase in HO-I expression was mediated by NO pathway. NO, being an HOOI-inducing signaling molecule, was responsible for statin-dependent HO-I induction and this might explain some pleiotropic effects of these drugs.
9- Inhibition of either eNOS or HO-I (during statins treatment) abridged the regression of atherosclerotic plaques produced by statins. While, either L-arginine or hemin
treatment produced significant decrease In vessel wall thickness compared to atherosclerotic group.
10- Inhibition of HO-I, during treatment with statins, abolished the restoration in NO/ONOO· balance and eNOS status produced by statins while, its stimulation had the reverse effect. As a result, HO-I plays an important role in mediating the antioxidative effect of statins.
11- Treatment with statins significantly decreased lipid peroxides level while, inhibition of either eNOS or HO-I abolished this effect, and also abolished the decrease in the vessel wall thickness produced by statins.
12- The beneficial effects of statins in lowering lipid peroxidation and improving dysfunctional endothelium involved the increase in eNOS expression with restoring its functional activity, increasing NO/ONOO· balapce, and also, augmentation of the expression and activity of the antioxidant enzyme, HO-I, in the vasculature.
• A real-time (onset time) measurement of NO and ONOO· release, with nanosensors, in single HUVEC provides an evidence that a decrease in the [NO]/[ONOO·] balance accompanied ox-LDL-induced endothelial dysfunction.
• The shift of [NO]/[ONOO·] balance towards high production of cytotoxic ONOO· and diminished NO generation could be of high importance in cardiovascular disorders that involve ox-LDL deposition on the vascular wall.
• The results of this study shed new light on the interaction of statinslNO/HO-l pathways and emphasize a synergistic regulatory interaction between NO and HO-I in mediating the vasculoprotective actions of statins during atherosclerosis.
• HO-l and its enzymatic products are of functional relevance and could be responsible, at least in part, for the observed antioxidant and anti-inflammatory effects (pleiotropic effects) of statins.
• Despite the clinical efficacy of cholesterol reduction in decreasing cardiovascular events, novel therapeutic approaches are still needed. Reduction of cholesterol level is still the best guide to the success of statin therapy. Greater understanding of non-lipid mechanisms of statins may help in the development of new therapies that could provide additional benefits in the form of risk reduction.
• Restoration of eNOS functional activity and activation of HO-l pathway may be one of the important mechanisms by which statins inhibit pro-oxidant and inflammatory disorders and protect the endothelium from oxidative damage. Therefore, treatment with statins could be considered in diseases other than hypercholesterolemia in which endothelial dysfunction is involved; such as hypertension, angina or ischemic stroke. However, further clinical studies are still needed to evaluate the vasculoprotective effects of statins in such diseases.
• Although statins share a common mechanism of action, there are differences in their relative efficacy for improving the lipid profile, as well as in their non-lipid (pleiotropic) effects. Consideration of these differences helps to provide a rational basis for their use in clinical practice.