RICERCA ITALIANA     

Genetic and molecular determinants of the role of COX-2 in atherothrombosis.

Università degli Studi "G. d'Annunzio" Chieti-Pescara

Abstract

Cyclooxygenase (COX)-2 plays a key role both in endothelial dysfunction, "primus movens" of atherosclerosis, and in atherosclerotic plaque rupture, final step of atherosclerotic disease.
There is strong evidence that reduced endothelial nitric oxide (NO) release may play a critical role in the evolution of endothelial dysfunction. Thus, all mediators inducing NO perturbation could promote endothelial dysfunction. One of them is COX-2, which may reduce NO biosynthesis by inducing oxidative stress. This is confirmed by the observation that COX-2 blockade can restore normal endothelial function. Different enzymatic systems can modulate endothelial susceptibility to COX-2-dependent oxidative stress. In fact, p66Shc protein modulates the endothelial oxidative stress response, and mice carrying a targeted mutation of the p66Shc gene show an increased resistance to oxidative stress.
However, the role of COX-2 in vascular walls is more complex, as it can exert both anti- and pro-atherogenic effects depending on the cellular type and on the prostaglandin (PG) produced. In fact, COX-2 in endothelial cells (ECs) catalyses PGI2 production. This may be the case in the presence of high density lipoprotein (HDL)3 that increases release of PGI2, but not of the pro-atherogenic PGE2.
In addition to its role in endothelium homeostasis, COX-2 has been shown to be involved in atherosclerotic progression toward instability. In fact, in atherosclerotic plaques, COX-2 can modulate the biosynthetic activity of macrophages (Mf). Macrophages produce matrix metalloproteinases (MMP), enzymes capable of degrading plaque extracellular components. MMP release depends on the sequential activity of COX-2 and the type 1 inducible PGE synthase (mPGES-1). Consequently, systems involved in COX-2 modulation within vascular wall seem to be optimal targets for therapeutic intervention. It has been shown that angiotensin (Ang) II is a potent stimulus for COX-2 expression and is able to influence the activity of PGE2-dependent MMPs, through Ang II type 1 (AT1) receptors. In this light, Prof. Cuccurullo's team has recently reported that AT-1 receptor blockade promotes human atherosclerotic plaque stabilization. However, it is not known whether Ang II-COX-2/mPGES-1 axis is overexpressed in ECs also and, consequently, whether it may play a role in Ang II-induced endothelial dysfunction.
Moreover, COX-2 activity can be modulated by enzymatic activity of proteins that compete with COX-2 for the same substrate, arachidonic acid. In particular, overexpression of "Fatty acid CoA ligase" (FACL)4 may reduce in vitro PGE2 biosynthesis up to 55%. However, no studies have ever examined FACL4 expression in the different types of atherosclerotic plaque (stable vs unstable).
In this project, we intend to pursue four major scientific objectives:
1. To investigate the role of FACL in controlling COX-dependent events in human atherosclerotic plaques.
2. To characterize the intracellular signaling mechanisms involved in susceptibility to COX-2 dependent oxidative stress.
3. To examinate the selective influence of HDL3-induced COX-2 expression on the anti-atherogenic eicosanoids production.
4. To investigate whether COX-2 may be involved in Ang II-induced vascular dysfunction in hypertension.
Four Research Units will work together within the same Research Project.
The Project is organized through a targeted approach addressing different steps of the cascade of events leading to endothelial dysfunction, the initial event in atherosclerotic process, and to atherosclerotic plaque instability:
1.Signalling pathways upstream of COX-2 expression, developed by Research Unit (RU) #3.
2.Competitive regulation of COX-2 activity, investigated by RU #3.
3.Protection mechanisms against COX-2-induced injury, investigated by RU #2
4.Regulation pathways orienting COX-2 activity toward anti-atherogenic profile, examined by RU#1. <<<

Principal Investigator

Franco CUCCURULLO Università degli Studi "G. d'Annunzio" CHIETI-PESCARA

Research Objectives

This Research Program is focused on different research activities in the area of cardiovascular disease.
There is strong evidence that inflammation plays a key role in the events leading to plaque rupture. In fact, inflammation is more common in symptomatic plaques, with a greater number of macrophages (Mf) and T cells infiltration, and plaque rupture is related to increased inflammation within the plaque rather than plaque morphology, or degree of vessel stenosis. Mf synthesizes metalloproteinases (MMPs) that are enzymes capable of degrading plaque constituents. Increased expression of MMPs has been reported in Mf in vulnerable regions of unstable plaques. Thus, localized MMP increase may cause acute plaque disruption, and the identification of pathways that regulate MMPs is critical to the formulation of strategies for plaque stabilization. Production of MMPs by Mf is induced through a prostaglandin (PG)E2-dependent pathway. In contrast, MMP biosynthesis may be suppressed by PGD2 metabolite via mechanisms involving both Peroxisome Proliferator Activated Receptor (PPAR)-gamma activation and NF-kB inhibition. Signaling through these pathways involves cyclooxygenase (COX), the enzyme that catalyzes the conversion of arachidonic acid (AA) to PGH2, then further metabolized by PGE synthase (PGES) or PGD synthase (PGDS) (isomerases) to opposing prostanoids (PGE2 and PGD2). It is important to note that COX-2 is expressed in Mf in human atherosclerotic plaques. Thus, COX-2 overexpression could be a key step for MMP generation in Mf in unstable plaques, and systems involved in COX-2 regulation appears as candidate targets to test "the inflammatory hypotesis" of plaque instability.
In the recent years, research has demonstrated that the final effect of COX-2 in the pathophysiology of the atherosclerotic lesion is significantly dependent on the prevalent activation of downstream isomerases. In fact, if the presence of PGES is responsible for MMPs induction in macrophages, instead the expression of the PGDS or PGIS may have strong protective effects and it is known that the HDL3 are able to selectively induce the PGIS pathway. So, the identification of the principal phases responsible of the effect of HDL3 on arachidonic acid metabolism represent the primary step in obtaining interventional strategies on atherosclerotic disease. In addition, enzymatic systems competes with COX-2 for the metabolization of arachidonic acid could represent an important control of the inflammatory output of COX-2. In this light, the enzyme fatty acid CoA ligase 4 (FACL4) converts arachidonic acid in arachidonoyl CoA ester, recycling it into membrane phospholipids, and thus making it unavailable metabolization by COX-2. Consequently, the recent demonstration that FACL4 induction during COX-2 overexpression may reduce in vitro PGE2 biosynthesis up to 55% with respect to COX-2 overexpression alone is of great interest. However, no studies have ever examined FACL4 expression in atherosclerotic plaque tissue, or the potential role of FACL-4 in the pathophysiology of COX-2-dependent atherosclerotic plaque instability. Enhanced oxygen free radical (ROS) generation is assumed to have a key role in the beginning of the atherosclerotic process by exerting several negative effects on the vascular wall, generally via the NFkB pathway. Indeed, ROS may promote monocyte adhesion and inflammation in the vessel. Endothelial vascular cells express NO synthase (eNOS) and are an active source of NO, which plays a key role in regulating vascular tone. Furthermore, NO may affect the vascular structure by also inhibiting NF-kB. Thus, we can speculate that enhanced oxidative stress and secondary NO perturbation could occur in vivo in the vessel wall, thus leading in turn to plaque progression and instability. Experimental evidence both in animal and in human models support this hypothesis enough. Interestingly, it has been demonstrated that one of the mechanisms responsible for this endothelial dysfunction is the pathological activity of COX, which, through yet to be defined mechanisms, causes oxidative stress therefore inducing NO breakdown. In line with this possibility, COX blockage can restore the normal endothelial function. Finally, it has been reported that when cells are subjected to oxidative stress, COX-2 expression is upregulated, thus suggesting the presence of a positive feedback loop.
This involvement of oxidative stress in the evolution of atherosclerosis suggests a critical role for the endogenous antioxidant systems in plaque stabilization.
Indeed, in the rabbit aorta the impaired relaxations are restored by non-selective COX blockade, prostanoid receptor antagonists as well as SOD, suggesting that vasocostrictor prostanoids and ROS are the underlying cause of endothelial dysfunction. In patients with NIDDM and IDDM vitamin C increases endothelium-dependent vasodilation in the forearm circulation. We have evidence that, in spite of a paradoxical upregulation of eNOS, NO release is reduced and coincubation with antioxidants restores the balance between NO and ROS formation.
It is well established that the modulation of the cell redox state and its ability to induce tissue injuries occurs through the expression of regulatory proteins promoting or counterbalancing the activity of ROS. Furthermore, it was recently reported that p66Shc protein modulates the oxidative stress response. Mice carrying a targeted mutation of the p66Shc gene show an increased resistance to oxidative stress which is coupled to a 30% increase in total life span compared with wild-type mice.
In conclusion, there is strong evidence that a number of mechanisms regulating critical genes are implicated in the shift towards atherosclerotic plaque destabilization. These processes include the regulation of enzymatic systems that utilize arachidonic acid, modulation of COX-2 and of the downstream enzymes PGES/PGIS, the homeostasis of the intravascular oxidant/antioxidant system and the balance between PGI2 and NO.
In the present research proposal, we intend to pursue four major scientific objectives:
1.To identify novel molecular targets for pharmacological modulation of the inflammatory, gelatinolytic and proliferative response with improved safety profile vis-à-vis currently available COX-inhibitors, in the following cellular and clinical paradigms:
The role of FACL4 in controlling COX-dependent events related to the balance of inflammation/anti-inflammation, extracellular matrix degradation/production, and cell proliferation/apoptosis in human models of atherosclerotic cardiovascular disease.
2.To characterize the genetic and molecular determinants of individual physiological and pathological vascular effects of cyclooxygenase activity, by integrating the assessment of functional end-points with the study of genetic polymorphisms of the COX-1 and COX-2 genes, and measurement of COX-1 and COX-2 activity, oxidant and inflammatory status, and endothelial dysfunction in the following experimental paradigms:
a. selective inhibition of COX-2.
b. selective inhibition of PGES.
c. reduction of COX-2 expression in presence of the polymorphism -765 G>C.
3.To explore alternative therapeutic strategies to prevent plaque destabilization, based on mechanisms inhibiting the degradation and/or stimulating the generation of GSH in clinical and experimental paradigms characterized by the interplay of oxidative stress, COX-2 induction and chronic inflammation.
4.To identify new molecular targets for the pharmacologic modulation of the inflammatory response in the artery wall investigating the role of the HDL3 and the correlated pathways in the control of the events related to the inflammatory balance of COX-2, to the stable production of PGI2 and ROS, to cellular proliferation and apoptosis in cellular and murine models of endothelial dysfunction. <<<

First Results

Studies on endothelial dysfunction.
It is expected that this 12-months period of study will allow the identification and the recruitment of a number of patients with different cardiovascular risk profiles adequate to obtain a statistical power. Sample size of the different research activities was usually determined by power analyses as follows: power = 0.95 and alpha= 0.05. In the different statistical calculations, we have estimated the means value of analyzed parameters based on previously published data by Unit #4, and assuming differences of 25% between groups of patients with different risk profile with respect to diverse molecular markers of endothelial function. The supposed difference in molecular marker was always a conservative estimate.

Studies on plaque instability.
It is expected that this 12-months period of study will permit the identification and the recruitment of a number of patients with symptomatic and asymptomatic carotid atherosclerotic plaques adequate to obtain a statistical power.
Sample size of the different research activities was usually determined by power analyses as follows: power = 0.95 and alpha= 0.05. In the different statistical calculations, we have estimated the means value of analyzed parameters based on previously published data, and assuming differences ranging from 30% to 60% between symptomatic and asymptomatic groups with respect to diverse molecular markers, the required sample of patients per group of study was determined. The supposed difference in molecular marker was always a conservative estimate.

Studies on animals.
In the animal model of hyperglycaemia, a single high-dose streptozotocin (STZ) regimen will be used to induce pancreatic islet beta-cell destruction and consequently persistent hyperglycaemia, both in p66Shc knock-out mice (p66Shc-/-) and in wild-type littermates male 12- to 16 weeks-old.
In the protocol study regarding Ang II and vascular injury, chronic treatment with Ang II will allow the development of vascular injury typical of arterial hypertension, and of minor lesions (according to the hypothesis of the study) in the genetic mouse model with homozygous deficiency for mPGES-1.
After 4 weeks, in all protocols, mice will be anaesthetized by intraperitoneal administration of 50 mg/kg sodium pentobarbital and then sacrificed. Aortas and small resistance arteries will be excised and snap frozen, embedded in paraffin or immediately used according to the study protocol.It is expected that research activities implemented under the first objective will allow the identification of novel molecular drug targets, alternative to COX-2, that could affect fundamental inflammatory processes related to plaque progression towards instability, without altering the potential anti-inflammatory effects due to COX-2 induction in presence of PGDS overexpression.
It is expected that research activities implemented under the second objective will allow to identify the control mechanism involved in the vascular reactivity regulation in patients with a different cardiovascular risk profile. These activities will lead to an improved benefit/risk profile of existing COX-inhibitors. In particular, clarification of the mechanism(s) underlying the regulation of vascular reactivity by arachidonate bio-products might have a significant impact on the Italian National Health System, given the wide number of subjects in treatment with conventional NSAIDs and with coxibs. On the other hand, characterization of the genetic and molecular determinants of the individual therapeutic and untoward responses to coxibs will able to lead to improved safety, optimizing this novel therapeutic strategy in areas such as prevention of coronary artery disease.
It is expected that research activities implemented under the third objective will allow to identify the molecular mechanisms underlying endothelial dysfunction in diabetes and to explore alternative molecular targets to prevent its onset. The study of gene-redox environment interactions as determinants of the individual response to these molecular mediators will be the start points for the design of effective drugs with minimal side effects. In particular, the studies on hyperglycaemia-induced endothelial dysfunction in diabetic p66Shc knock-out mice could lead to consider the pharmacological block of the p66Shc protein as an attractive therapeutical option against diabetic cardiovascular complications.
It is expected that research activities implemented under the fourth objective will allow to identify the mechanism by which HDL-induced COX-2 may affect the production and the activities of eicosanoids in vitro and in vivo. This will allow to generate reconstituted lipoproteins that can mime the HDL activity on COX-2/PGIS pathway. Results of this study could open new perspectives in the pharmacological treatment of atherosclerosis via selective interventions on circulating lipoproteins profiles. <<<

Timescale

24 months

National and international background

1.Arachidonic acid metabolism via the PGH-synthase pathway.
Arachidonic acid (AA; 20:4, n-6) is an essential polyunsaturated fatty acid; its oxidation generates prostanoids and leukotrienes, collectively designated as "eicosanoids". When tissues are exposed to diverse physiological and pathological stimuli, AA is liberated from membrane phospholipids and converted to prostanoids like the prostaglandins and thromboxane (TX) A2, by the action of PGH-synthase (PGHS), known as cyclooxygenase as well. The cyclooxygenase and hydroperoxidase catalytic activities of this bifunctional enzyme result in the sequential formation of unstable endoperoxide intermediates, PGG2 and PGH2, which in turn is metabolized to PGD2, PGE2, PGF2alpha, PGI2 and TXA2 by cell-specific isomerases and synthases.
Two distinct isoforms of PGHS have been recognized since 1991, also designated as COX-1 and COX-2. Both COXs are membrane-anchored proteins existing as dimers and have remarkable structural similarity. The AA gains access to the active site via a hydrophobic channel, but this access can be blocked by drugs, which reversibly anchor to strategically located residues or irreversibly modify them. COX-1 is expressed constitutively in many tissues of the body and plays a central role in platelet aggregation and gastric cytoprotection. COX-2 is predominantly induced during inflammation, wound healing and neoplastic transformation, although it is expressed constitutively in the human kidney and brain. Prostanoids coordinate signaling to both the cell of origin (autocrine effect) and neighbouring cells (paracrine effect) by binding to transmembrane G protein-coupled rhodopsin-type receptors. Nuclear actions of prostanoids have also been reported and putative nuclear prostanoid receptors have been proposed, particularly for cyclopentenone PGs (CyPGs). Organs and tissues vary in the concentration of specific isomerases that convert PGH2 into different prostanoids. A further complication of the downstream effects and potential drug targets of AA metabolism is that there may be more than one isoform of upstream and downstream enzymes, such as phospolipases (PLA2) and PGE-synthases. During the past two years, the concept of functional coupling among the PLA2-COX-PGH isomerase enzymes has gained experimental support. This model implies that basal formation of PGE2 involves preferential coupling between cPLA2 and COX-1 and a cytosolic isoform of PGES (cPGES), all of which are expressed constitutively. As conditions favour the induction of COX-2 and of a membrane-bound PGES (mPGES), terminal formation of PGE2 involves coupling between cPLA2 and these latter two enzymes. Finally, when exposure to receptor ligands is enduring, the inducible sPLA2 isoenzyme begins to participate, creating an amplification loop to align AA availability with the sustained capacity for biosynthesis by inducible COX-2 and inducible mPGES.

2.Role of COX-2 and PGH-isomerases in cardiovascular diseases.
The relative abundance of a specific prostanoid is the result of the expression and activity of its specific isomerase, and only the concomitant expression of COX-2 and PGES may lead to an increased inflammatory/proliferative state potentially leading to atherosclerotic plaque disruption. There are two isoforms of PGES: cPGES is a constitutively expressed housekeeping gene, important for physiologic cell regulation; in contrast, mPGES shows a low basal expression that is rapidly induced by a wide range of pro-inflammatory and mitogenic stimuli in nucleated cells, suggesting that this enzyme is involved in the generation of PGE2 and PGE2-dependent MMPs in inflammatory vascular syndromes.

3.Role of the COX pathways in plaque stability.
Current understanding of the mechanisms underlying plaque development assigns a key role to oxidative modifications of specific phospholipids that are carried into the subendothelial space with low-density lipoproteins (LDL). These variably oxidized lipids in turn trigger a chronic inflammatory response, largely orchestrated by monocytes/macrophages. Thrombotic occlusion of a major coronary or cerebral vessel can complicate the sudden fissuring or rupture of a plaque and lead to myocardial infarction or ischemic stroke, respectively. There is strong evidence that inflammation plays a key role in the events leading to atherosclerotic plaque rupture. In fact, inflammation is more common in symptomatic plaques, with greater number of macrophage and T cells infiltration, and plaque rupture is related to increased inflammation within the plaque rather than plaque morphology or degree of vessel stenosis. Macrophages synthesize MMPs, enzymes capable of degrading plaque constituents, and an increased expression of MMPs has been reported in macrophages in vulnerable regions of unstable plaques. Thus, since localized MMP increase may cause acute plaque disruption, the identification of pathways that regulate MMPs is critical to the formulation of strategies for plaque stabilization. Release of MMPs by macrophages is induced through a PGE2-dependent pathway. In contrast, MMP release may be suppressed by PGD2 metabolite through mechanisms involving both PPARgamma and NF-kB. Interestingly, it has been demonstrated that COX-2 is expressed in macrophages in human atherosclerotic plaques. Thus, COX-2 overexpression could play a key role for MMP generation in macrophages in unstable plaques, and selective COX-2 inhibitors appear as interesting candidates to test "the inflammatory hypothesis" of plaque instability despite their controversial cardiovascular effects. Consistent with this hypothesis, Prof. Cuccurullo's team demonstrated the concomitant overexpression of COX-2/PGES and MMPs in "symptomatic" carotid plaques. However, PGDS may also be critically involved in the modulation of plaque stability (Cipollone et al, ATVB, under revision). In fact, 15d-PGJ2, one of PGD2 metabolites and a potent agonist of PPAR-gamma, can exert anti-inflammatory effects such as COX-2 and MMP-9 suppression. Thus, identification of the precise stimuli capable of influencing the PGE2/PGD2 ratio in plaque macrophages could greatly influence plaque stability and may be critical to develop optimal preventive strategies.

4.Role of other enzymes transforming AA in the control of the pro-inflammatory activity of COX-2 in the atherosclerotic plaque.
In addition to cyclooxygenase and other AA-oxidizing enzymes (lipoxygenase and cytochrome P450), AA can be the substrate of another enzyme, FACL4, a member of the family of acyl-CoA synthetases (or ligases). This enzyme plays a key role in fatty acid metabolism, by catalyzing the formation of fatty acyl-CoA-thio-esters, which are substrates for beta-oxidation, phospholipid biosynthesis and cellular signaling. Five isoforms of FACL have been cloned in mammals with different substrate specificity and tissue distribution. FACL4 is the isoform with the highest preference for AA; it converts AA into arachidonoyl CoA ester, recycles it into membrane phospholipids, and thus makes it unavailable for metabolization by other pathways. In this light, the recent demonstration that FACL4 induction during COX-2 overexpression may reduce in vitro PGE2 biosynthesis up to 55% with respect to COX-2 overexpression alone is of great interest. However, no studies have ever examined FACL4 expression in atherosclerotic plaque tissue, or the potential role of FACL-4 in the pathophysiology of COX-2-dependent atherosclerotic plaque instability.

5.COX-inhibitors and endothelial function: transducing biochemical selectivity into clinical read-outs.
Both animal models of genetic hypertension and essential hypertensive patients are characterized by the presence of endothelial dysfunction and reduced nitric oxide (NO) availability. This vascular alteration causes an early aging of the vascular wall, promoting the pathogenesis of atherosclerosis. It has been shown that one of the mechanisms responsible for this endothelial dysfunction is a pathological activity of COX-2, which, via yet to be defined mechanisms, causes oxidative stress and therefore induces NO breakdown. In line with this possibility, a normal endothelial function can be restored by blocking COX-2. Worth consideration is the fact that when cells are subjected to oxidative stress, COX-2 expression is upregulated, thus suggesting the presence of a positive feedback loop. In contrast, it is still unknown whether oxidative stress produced by COX-2 is caused by the intrinsic COX-2 activity or mediated by prostanoids acting on specific targets. Because a strong correlation between COX-2 up-regulation and PGI2 production by measuring PGI2 metabolites has been shown, it has been hypothesized that selective blockade of endothelial COX-2 can be potentially dangerous in patients at cardiovascular risk, since COX-2 would be the main source of endothelial prostacyclin. However, other results suggest that COX-2 induction in activated endothelial cells may contribute to aspirin-insensitive TXA2 biosynthesis in patients with unstable angina. Thus, the role of COX-2 enzymes in endothelial cells might be diverse with respect to the different phases of atherosclerosis.

6.Molecular mechanism of COX-2 control.
The molecular mechanisms by which different stimuli, like the high density lipoprotein (HDL) 3, induce COX-2 are poorly understood. COX-2 expression is modulated by multiple growth factors and cytokines via mitogen-activated protein kinase (MAPKs) cascade. Once activated, the MAPKs modulate the activity of several transcription factors such as CREB, NFAT, AP-1, NF-kB, which are involved in COX-2 expression. HDL3 activate two of the major kinases pathways involved in COX-2 gene transcription: ERK1/2 and p38 MAPK. HDL can activate ERK1/2 via cell surface S1P receptor in astroglial cells, but it is even possible that MAPK activation results from plasma membrane cholesterol depletion. In support of this hypothesis, Smith et al. showed that increased concentration of LDL or free cholesterol decreases COX-2 expression and PGI2 synthesis. As HDL triggers the release of cholesterol from cells, cellular cholesterol balance may play an important role in determining COX-2 levels. HDL3 also activate CREB in a time dependent fashion; CREB binds to CRE and serves as an anchor for P300 interaction with both upstream transactivators and downstream transcription machinery, suggesting that CRE plays a relevant role in COX-2 induction by a number of stimuli. Using transient transfection experiments, Prof. Catapano's team has shown that mutation in CRE abrogated the luciferase activity induced by HDL3 confirming the role of CRE in HDL3 induced COX-2 gene transcription. Moreover, inhibition of the p38 MAPK pathway is responsible for the induction of COX-2 by HDL3. As p38 MAPK plays a housekeeping role in maintaining COX-2 mRNA stability, Prof. Catapano's team studied Cox-2 mRNA stability in cells stimulated with HDL3. Simultaneous addition of actinomycin D and SB 203580 to the cells resulted in a more rapid decrease in COX-2 mRNA compared to actinomycin D alone, suggesting a role for HDL3 in mRNA stabilization.

7.Genetic determinant of vascular damage induced by COX-2.
The COX-2 activity can be modulated by functional mutation of the gene responsible for its expression and activity, other than the transcriptional and post-transcriptional genetic expression control mechanism. Recently, the identification of a new variant in the COX-2 promoter, called -765G>C, was reported. Patients carrying the -765C allele had significantly lower promoter activity and lower plasma levels of C reactive protein, a sensitive marker of low-grade inflammation, compared to patients homozygous for -765G. Because lower COX-2 activity could have, as direct consequence, the reduction in the generation of MMPs in atherosclerotic plaque, the possibility that -765G>C might protect persons against plaque instability was considered. In press data (Cipollone et al, JAMA 2004) indicate that patients carrying the -765C allele have a reduced risk of myocardial infarction and stroke. At the present time, whether this genetic polymorphism plays a role in modulating endothelial function and weather it is able to influence the effect of acute and chronic administration of COX-2 inhibitors on endothelial function still needs to be evaluated.
Other than the direct impact of COX-2 gene mutation on the pro-inflammatory and pro-oxidant output, it is well established that the modulation of the cell redox state and its ability to induce tissue injuries is occurring through the expression of regulatory proteins promoting or counterbalancing the activity of ROS. Furthermore, it was recently reported that p66Shc protein modulates the oxidative stress response. Mice carrying a targeted mutation of the p66Shc gene show an increased resistance to oxidative stress, which is coupled to a 30% increase in total life span compared with wild-type mice. Nevertheless, it is still unknown whether the inactivation of the p66Shc protein is protective against hyperglycaemia-induced endothelial dysfunction. <<<