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Cardiovascular Complications of Non-Steroidal Anti-Inflammatory Drugs

Address correspondence to: Egil Fosslien, M.D., 502 Fairview Ave, Glen Ellyn, IL 60137, USA; tel 630 469 6824; e-mail efosslie{at}uic.edu.

	Abstract

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Coxibs, such as rofecoxib, celecoxib, and valdecoxib, selectively inhibit cyclooxygenase (COX)-2, the mainly inducible, pro-inflammatory COX isoform. Unlike traditional non-steroidal anti-inflammatory drugs (NSAIDs) most coxibs do not significantly inhibit COX-1 and are therefore less toxic to the gastrointestinal tract. Hence, coxibs widely replaced traditional NSAIDs for treatment of arthritis and other painful inflammatory conditions. In many, but not all, clinical studies, coxibs became associated with higher risks of myocardial infarction (MI) and stroke. Several mechanisms may be involved in the pathogenesis of such complications. First, selective inhibition of COX-1 lowers platelet synthesis of thromboxane (TXA₂), a thrombogenic and atherogenic eicosanoid. Selective inhibition of COX-2 limits endothelial cell synthesis of prostacyclin (PGI₂), an arachidonic acid product that opposes the effects of thromboxane. In apoE-/- mice, interruption of TXA₂ signaling by deletion of its receptor (TP) limits atherogenesis, whereas interruption of PGI2 signaling by deletion of its receptor (IP) accelerates atherogenesis. This suggests that selective inhibition of COX-2 can disrupt the physiological balance between thromboxane and prostacyclin and thus increase atherosclerosis, thrombogenesis, and the risk of cardiovascular complications. Second, COX inhibition can raise levels of arachidonic acid, which can inhibit mitochondrial oxidative phosphorylation (OXPHOS) and increase OXPHOS generation of reactive oxygen species. Several NSAIDs, including coxibs and meloxicam, directly uncouple or inhibit OXPHOS. Studies of apoE-/- mice indicate that mitochondrial dysfunction plays an early role in atherogenesis. Third, many NSAIDs exhibit COX-independent properties. For example, in animal models, short-term treatment with celecoxib reduces monocyte chemotaxis by reducing expression of monocyte chemoattractant protein (MCP)-1. However, long-term treatment results in the opposite effect and accelerates atherogenesis. In conclusion, to reduce the risk of cardiovascular complications during long-term coxib therapy, low-dose aspirin supplementation should be considered. An alternative is to use a less COX-2-selective inhibitor such as meloxicam. Genotyping of -765 alleles of the COX-2 gene promoter and examining the polymorphism of other genes involved in eicosanoid metabolism or NSAID degradation may become helpful in predicting patients who are at higher risk of cardiovascular complications during selective COX-2 inhibitor therapy.

Keywords: NSAIDs, COX-2 inhibitors, coxibs, cardiovascular disease, myocardial infarction, arthritis, neoplasia, chemotherapy

	Introduction

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Several, but not all, clinical studies have revealed an increased rate of cardiovascular complications after long-term use of coxibs such as rofecoxib, celecoxib, and valdecoxib, which are nonsteroidal anti-inflammatory drugs (NSAIDs) that selectively inhibit cyclooxygenase (COX)-2. Despite significant variability of findings in these studies, the increased rates of myocardial infarction and stroke emerging from rofecoxib studies and cardiovascular complications found in celecoxib cancer prevention studies raise concerns about the cardiovascular safety of long-term treatment with coxibs, as well as traditional NSAIDs that inhibit both COX-1 and COX-2 [1–5].

Cyclooxygenase (COX)-2 expression is elevated at sites of inflammation such as in synovial tissues in rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis [6]. Traditional, non-selective NSAIDs are effective in treating inflammation and pain. However, they can induce hemorrhage and severe gastrointestinal complications because they also inhibit COX-1, which has major roles in hemostasis and gastrointestinal cytoprotection [7].

NSAIDs that selectively inhibit COX-2, such as coxibs, were developed to retain the anti-inflammatory effects of traditional NSAIDs, but lessen the risk of adverse gastrointestinal effects. Clinical trials have shown that coxibs have analgesic profiles similar to traditional NSAIDs, but are significantly less toxic to the gastrointestinal system. They therefore widely replaced non-selective NSAIDs for treatment of inflammatory joint diseases and other painful inflammatory conditions [8,9].

The primary goal of this review is to elucidate the pathophysiology and molecular pathology of cardiovascular complications of NSAIDs, especially COX-2-selective inhibitors such as coxibs. A second goal is to highlight clinical factors and laboratory tests that may identify patients with increased risk of cardiovascular complications when using NSAIDs. A final goal is to identify potential pharmacological and other strategies for cardiovascular risk reduction.

	Cyclooxygenase

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
An analysis of effects of NSAIDs is intricate because cyclooxygenases participate in many physiological functions and human pathologies. The COX-2 isoform plays a major role in initiation of inflammatory alterations in response to injury. In addition, during later stages it may aid in repair and resolution of inflammation. It is inducible by inflammatory stimuli such as cytokines, growth factors, and bacterial endotoxin and is expressed in atherosclerotic lesions and in a variety of tumors. COX-1 is constitutively expressed in most tissues where it regulates synthesis of physiological levels of prostaglandins. COX-3 is a splicing variant of COX-1 that is unaffected by most NSAIDs [10,11].

COX-2 is expressed in various normal tissues where its expression may be important for physiological functions such as cardioprotection associated with high-density lipoprotein and apolipoprotein E (apoE) [12] (see below). Interpretation of experimental and clinical findings is complicated further by dissimilar tissue distribution and altered expression of COX-1 and COX-2 in diseased tissues and by the observation that COX expression differs in different species [13]. Because COX-1 and COX-2 have some adaptable overlapping functions, effects of their inhibition on the cardiovascular system remain complex and sometimes contentious [14].

In healthy rats, COX-2 mRNA is constitutively expressed in heart, kidney, and lung; COX-1 mRNA is expressed in kidney, lung, and liver [15]. In the monkey, COX-1 is found in the epididymis and vas deferens and COX-2 in the seminal vesicles [16]. Also important for interpretation of effects of NSAID inhibition, when immunohistochemistry is employed to determine COX-2-protein expression in normal, inflamed, or neoplastic tissues, is the fact that the results may depend on the antibody used [17].

COX-2 is induced in atherosclerotic plaques, during angiogenesis, and during wound healing [9,18]. In rabbits, experimental balloon injury and stent implantation induce expression of COX-2 in areas with atherosclerotic lesions. Normally, arteries at these anatomic locations express only COX-1 [19]. In addition, COX-2 is constitutively expressed in the macula densa and renal medullary interstitial cells [20]. Traditional NSAIDs, and coxibs as well, can decrease renal excretion of sodium and potassium. Hyperkalemia can induce cardiac arrhythmia [21]. Moreover, COX-2 inhibition can increase kidney water retention, and induce peripheral edema and weight gain. Chronic NSAID use can lead to renal failure [22,23]. In animal models, selective inhibition of COX-2 promotes hypertension, atherogenesis, and formation of thrombi, all risk factors for acute myocardial infarction. Nevertheless, the exact pathogenesis of the increased rates of cardiovascular complications caused by coxibs is unclear at this point [24].

Smoking increases COX-2 expression that induces inflammation [25]. Chronic inflammatory processes play a major role in carcinogenesis and atherogenesis. The latter is a major risk factor for myocardial infarction [26,27]. Inflammation plays a role in all stages of atherogenesis, from fatty streak development to plaque formation and plaque rupture [28]. The inflammatory features resemble the inflammatory alterations seen in rheumatoid arthritis and osteoarthritis. Rheumatoid arthritis is associated with accelerated atherosclerosis, high cardiac mortality, and shortened life expectancy. As inhibition of COX-2 reduces inflammation, it was hoped that COX-2-selective inhibitors, when used to reduce pain and inflammation in joint disease, might also inhibit atherogenesis and reduce the rates of myocardial infarction [29].

For reasons not entirely agreed upon, the opposite was observed in several, but not all, clinical studies associated with use of coxib inhibitors, particularly after long-term use, and in a few clinical studies on the use of traditional NSAIDs such as aspirin and naproxen. Several questions arise about NSAID-induced cardiovascular complications: How do NSAIDs alter lesion initiation, progression, thrombosis, and myocardial infarction? Are cardiovascular complications caused by alterations in eicosanoid synthesis? Are they due to interference with mitochondrial function? Are both mechanisms involved? What is the effect of polymorphism of genes involved in eicosanoid synthesis on responses to NSAID therapy?

	NSAIDs

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Cyclooxygenase inhibition alters metabolism of eicosanoids, which include prostaglandins, thromboxane, and leutkotrienes that are derived from arachidonic acid (AA), an omega-6 polyunsaturated acid (PUFA), and a few similar 20-carbon long-chain fatty acids (Greek, eicosa = 20) such as omega-3 PUFA found in fish oil. Cyclooxygenase is rate-limiting for conversion of arachidonic acid to prostaglandin (PG) H₂, which is converted to the important vasoactive eicosanoids thromboxane (TXA₂) and prostacyclin (PGI₂) by thromboxane synthase prostacyclin synthase respectively (Fig. 1). Furthermore, PGH₂ serves as a common substrate for specific synthases that produce other prostaglandins such as PGD₂, PGJ₂, and PGE₂. The latter plays a key role in carrageenan-induced inflammation [30].

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect of cyclooxygenase (COX) inhibition by non-steroidal anti-inflammatory drugs (NSAIDs) on main vasoactive eicosanoid and related pathways associated with cardiovascular complications (CV) during NSAID use. Platelet (PLT) COX-1 is rate-limiting for conversion of arachidonic acid (AA) to thromboxane (TXA2,)(1). Endothelial cell (EC) COX-1 and COX-2 (inducible, pro-inflammatory isoform) are rate-limiting for AA conversion to prostacyclin (PGI2) (2), an eicosanoid that counteracts TXA2 effects. Aspirin (ASA) inhibits COX-1, lowers TXA2 synthesis, reduces platelet aggregation, and inhibits atherogenesis (open arrows), but increases risk of hemorrhage and gastrointestinal (GI) complications. Traditional NSAIDs such as indomethacin (INDO) inhibit both COX-1 and COX-2. Coxibs selectively inhibit COX-2 and reduce PGI2 levels thus generating a prothrombotic state (black filled arrows). Less arachidonic acid AA is consumed. Excess AA stimulates leukotrienes synthesis via lipoxygenase (LO) (3), inhibits mitochondrial oxidative phosphorylation (OXPHOS) (4), lowers mitochondrial membrane potential (m) (5) and ATP generation, but elevates OXPHOS generation of radical oxygen species (ROS) (6). Feedback loops via prostaglandin (PG) E2 and tumor necrosis factor (TNF)- (not shown) increase COX-2 synthesis and ROS generation from the peroxidase site of COX-2 increases (not shown). NSAIDs such as INDO and some coxibs (7) inhibit or uncouple mitochondrial oxidative phosphorylation (OXPHOS). Mitochondrial dysfunction lowers cardiac contractility and is atherogenic. Some important NSAID effects are cyclooxygenase independent, such as celecoxib regulation of expression of monocyte chemoattractive protein-1 (MCP-1) that regulates monocyte chemotaxis (8). Polymorphism of COX-2 affects the risk of CV complications. Polymorphism of CYP2C9 affects the rate of celecoxib degradation (9). For details see text.

NSAIDs can induce secondary effects by altering the size of the arachidonic acid pool, which depends on a dynamic balance between supply and consumption. Supply is determined by cytosolic (c) and soluble (s) phospholipase A₂ (PLA₂), which catalyse cleavage of fatty acids from membrane phospholipids [32]. Consumption is regulated by conversion to PGH₂ by COX-1 and COX-2, depletion of arachidonic acid via the lipoxygenase pathway, and non-enzymatic conversion of arachidonic acid to isoprostanoids by ROS.

Selective inhibition of COX-2 by coxibs, or selective inhibition of COX-1 by tenoxicam, increases the cellular arachidonic acid pool, which stimulates leukotriene synthesis by lipoxygenase (LO). Because traditional NSAIDs such as acetyl-salicylic acid (ASA, aspirin) and ibuprofen inhibit both COX isoforms they can increase the arachidonic acid pool size as well. Furthermore, some NSAIDs also exert functions that are independent of cyclooxygenase inhibition, for example, many NSAIDs affect mitochondrial function, expression of cytokines, and growth factors [33–35].

A high serum level of oxidized LDL (oxLDL) is a strong predictor of myocardial infarction [36]. In vitro, rofecoxib (4-(4'-methylsulfonylphenyl-3-phenyl-2-(5H)-furanone) [37] increases, but cele-coxib (4-[[5-(p-tolyl)-3-(trifluoromethyl)-1H-pyra--zol-1-yl]]benzenesulfonamide) decreases, oxidation of low-density lipoprotein (LDL). By comparison, valdecoxib (4-[5-methyl-3-phenyl-isoxazol-4-yl]-benzene-sulfonamide) [38], meloxicam (4-hydroxy-2-methyl-N-[5-methyl-2-thiazolyl] -2H-1,2-benzothiazine-3-carboxamide-1,1 dioxide), ibuprofen (2-(4-isobtyl-phenyl)-propionic acid), naproxen (D-6-methoxy--methyl-2-naphthaleneacetic acid), or diclofenac (2-[(2,6-dichlorophenyl)-amino-benzene-acetic acid) exhibit no significant effect on LDL oxidation [34,39].

Aspirin.  Aspirin inhibits COX-1 and to a lesser extent COX-2 by acetylating single serine residues (Ser529 and Ser516, respectively) [40]. Many, but not all, clinical studies show that aspirin significantly reduces the risks of MI and stroke. Aspirin lowers cardiovascular risk by blocking platelet thromboxane synthesis and platelet aggregation. Aspirin lowers the serum levels of C-reactive protein (CRP) [41] and lipoprotein (L)a (Lp(a) [42], which are both independent risk factors for atherosclerosis and myocardial infarction.

Aspirin inhibits platelet COX-1 irreversibly, and since platelets lack nuclear DNA, neither additional COX-1 nor further thromboxane can be synthesized by affected platelets. Aspirin treatment alone lessens clot formation and mitigates rates of fatal and non-fatal myocardial infarction by 25–30% [43,44]. Cardiovascular patients who are resistant to aspirin therapy have more than twice the rate of cardiovascular events, compared to aspirin-sensitive patients [45].

However, a 2-year study of platelet sensitivity to aspirin (100–330 mg/day) in 150 patients without aspirin resistance showed progressive loss of anti-platelet effects after 6–12 months of treatment [46]. One hundred mg/day of aspirin is considered 3 times the dose needed to saturate platelet COX-1 [40]. The altered hemostasis induced by aspirin and other traditional NSAIDs increases the risk of gastrointestinal hemorrhage, intestinal perforation [3], and excessive perioperative bleeding. Moreover, the inhibition of COX-1 lowers gastrointestinal synthesis of cytoprotective prostaglandins. Aspirin treatment doubles the rate of gastrointestinal complications and raises the rate of hemorrhagic stroke by about 50% [47]. Also, in isolated guinea pig hearts, aspirin significantly depresses cardiac recovery after mild ischemia/perfusion, probably due to mitochondrial uncoupling rather than altered eicosanoid release or non-enzymatic formation of isoprostanes from arachidonic acid by radical species [48,49].

Surprisingly, in a clinical study designed to evaluate valdecoxib compared to aspirin, diclofenac, ibuprofen, and placebo, aspirin was associated with a higher risk of thrombotic events than placebo [50]. However, no increased risk of myocardial infarction was caused by simultaneous use of aspirin and ibuprofen [51].

Coxibs.  In experimental models coxibs cause significantly fewer gastrointestinal complications than traditional NSAIDs [52]. Clinical studies corroborate these advantages of coxib over traditional NSAIDs. Rofecoxib, celecoxib, and valdecoxib [5,53] (oral medication, injectable counterpart: parecoxib) differ considerably in their COX-2-selectivity. The exact degree of inhibition of COX-2 versus inhibition of COX-1 varies in different publications, and the following values are representative examples: rofecoxib 80-, etodolac 23-, meloxicam 11-, and celecoxib 9-fold [54]. Other investigators found that in a whole blood assay rofecoxib exhibited a 36-fold preference for COX-2 compared with COX-1; the corresponding values for celecoxib, diclofenac, meloxicam, and indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid) [34,55] were 6.6-, 3-, 2-, and 0.4-fold respectively [37,56].

Importantly, in vitro selectivity values are method-dependent, and may not accurately predict selectivity in clinical applications. Taken together, these sources suggest the following gradation of affinity for the COX-2 versus the COX-1 isoform: Aspirin < indomethacin < meloxicam < diclofenac < celecoxib < etodolac < rofecoxib < valdecoxib. Note that some reported selectivity values for meloxicam and indomethacin differ somewhat from those indicated in this gradation list.

Rofecoxib.  Rofecoxib is a compound with high analgesic and antiphlogistic efficacy in carrageenan- induced inflammation in rodents. It has demonstrated analgesic potency similar to ibuprofen. However, unlike ibuprofen rofecoxib does not inhibit thromboxane synthesis [37,57]. A study by the rofecoxib manufacturer of a group of patients treated with different NSAIDs for osteoarthritis found no difference in cardiovascular thrombotic events between users of rofecoxib, celecoxib, or non-selective NSAIDs such as ibuprofen, nabumetone (4-[6-methoxy-2-naphthalenyl]-2-butan-one) [34], and diclofenac [58]. A Canadian study supported this interpretation; it reported no increased risk of myocardial infarction in rofecoxib users [59]. Additionally, an extensive review of data from multiple rofecoxib studies affirmed that there was no solid evidence for increased cardiovascular risk in patients using rofecoxib [60]. This point of view was corroborated by other experts [61].

In a gastrointestinal outcomes research study of rofecoxib, which included patients with rheumatoid arthritis treated either with rofecoxib or naproxen (a non-selective cyclooxygenase inhibitor), patients in the rofecoxib treatment group had significantly fewer gastrointestinal complications than those in the naproxen-treated group [56]. However, the study showed an increased incidence of myocardial infarction and other thrombotic complications in the group of patients treated with rofecoxib. The rates of myocardial infarction were 0.1% and 0.5% in naproxen- and rofecoxib-treated groups, respectively [40].

Several clinical studies have shown that naproxen [62] may be cardioprotective [44,60,63, 64]. These findings led to a suggestion that the interpretation of an increased rate of myocardial infarction in rofecoxib users was biased because of the cardioprotective effects of naproxen. However, other studies corroborated that rofecoxib therapy in patients with rheumatoid arthritis is associated with increased risk of myocardial infarction [65,66]. Also, in a group of patients over 65 years of age, use of rofecoxib was associated with increased risk of myocardial infarction compared to use of celecoxib or other NSAIDs. The risk was higher with rofecoxib dosages >25mg/day vs <25 mg/day [67].

The reports of such diverse clinical findings have led to controversy, dispute, and uncertainty. Ethical stances emerged about perceived undue delay in issuing warnings about cardiovascular complications [68]. Then, based on an increase in cardiovascular complications observed in a clinical study designed to evaluate the efficacy of rofecoxib (25 mg/day) in preventing recurrence of colorectal polyps in a group of 2,600 patients, the manufacturer discontinued the study and withdrew rofecoxib from the market at the end of September, 2004 (www.vioxx.com) [39,43,69,70–72]. Public controversy over rofecoxib findings and the final decision to withdraw the drug from the market drew attention to other COX-2-selective inhibitors, eg, celecoxib [73,74] and valdecoxib [5].

Celecoxib.  Celecoxib is approved at recommended daily doses of 100–200 mg for treatment of osteoarthritis and 200–400 mg for rheumatoid arthritis (manufacturer’s news release, 17 December 2004). In a 6-mo long multicenter clinical trial involving patients with rheumatoid arthritis or osteoarthritis, celecoxib 400 mg (twice daily) was associated with significantly lower incidence of upper gastrointestinal bleeding, perforation, and obstruction compared to traditional NSAIDs (ibuprofen 800 mg (thrice daily) or diclofenac 75mg/(twice daily)). The rates of cardiovascular events were similar for celecoxib and traditional NSAIDs, and surprisingly, were unaffected by concomitant aspirin use [75].

Since celecoxib is a sulfonamide compound, structurally different from rofecoxib and less COX-2-selective, it was argued that celecoxib probably provides some inhibition of platelet COX-1 and thus diminishes thrombogenesis and cardiovascular complications by reducing thromboxane synthesis. In view of such reasoning, celecoxib was considered safer than rofecoxib. This opinion was supported by the results of several clinical studies [76,77]. A search of a large, 15-trial database covering use of celecoxib showed no evidence of undue thrombotic risk. The analysis covered 2 post-marketing trials, the Celecoxib Long-term Arthritis Safety Study (CLASS) [78] and the Successive Celecoxib Efficacy Studies (SUCCESS), and 13 new drug application studies. In all, data from >18,000 patients and almost 6000 patient-years of exposure were analyzed. Celecoxib dosages ranged from 100 mg to 400 mg twice daily [79]. Moreover, no increased risk of cardiovascular complications due to celecoxib treatment with 400 mg daily was detected in a Prevention of Spontaneous Adenomatous Polyps (PreSAP cancer trial) (manufacturer’s news release, 17 December 2004).

However, in the Adenoma Prevention with Celecoxib (APC) trial, which included 2035 patients, an increased rate of myocardial infarction was detected among patient using celecoxib. Patients who had been consuming celecoxib 200 or 400 mg twice daily had 2.5-fold and 3.4-fold increases respectively of cardiovascular events compared to the placebo group [80]. The APC trial was therefore discontinued in late December 2004. These findings raised additional concerns about the cardiovascular safety of long-term administration of coxibs.

On the other hand, some clinical studies have indicated that use of traditional NSAIDs may also be associated with increased risks of cardiovascular complications. For example, an Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT) involved a comparison of celecoxib and naproxen against placebo. It was halted because it showed a 50% increase in cardiovascular risk in patients who received naproxen compared to patients treated with celecoxib [81,82], a surprising finding in view of results of other studies discussed above that deemed naproxen to be cardioprotective. A clue to why naproxen may increase cardiovascular risk may be derived from a study of 9 healthy subjects, which showed that naproxen 500 mg (twice daily) mimicked the effect on thromboxane synthesis of 100 mg of aspirin daily, and importantly, that naproxen, but not aspirin, significantly lowered prostacyclin synthesis [40].

Valdecoxib.  Valdecoxib is a potent anti-inflammatory coxib, which is even more COX-2 selective than rofecoxib [83]. Valdecoxib at 10 mg/day has a better GI tolerance profile but similar analgesic efficacy compared to traditional NSAIDs. Like other NSAIDS it can cause fluid retention and hypertension [84]. An analysis of data from about 8000 patients with osteoarthritis or rheumatoid arthritis treated with daily doses of 10–20 mg, and even at doses up to 80 mg, showed that valdecoxib was not associated with higher rates of thrombotic events than naproxen (500 mg/twice daily, ibuprofen (800 mg thrice daily), or diclofenac (75 mg twice daily) [50].

On the other hand, short-term (10 day) treatment with valdecoxib after cardiac surgery led to increased rates of myocardial infarction, cardiac arrest, pulmonary embolism, and stroke [85]. Valdecoxib was removed from the market in early 2005 [54]. Interestingly, removal of the sulfonamide group of valdecoxib yields compounds that are COX-1-selective inhibitors [86].

Other COX-2-selective inhibitors.  Meloxicam inhibits COX-2 about 12 times more potently than COX-1 [87]. Pooled clinical data from >13,000 patients who received meloxicam and were followed for >60 days showed that it had good GI and cardiovascular safety profiles compared to patients treated with diclofenac, naproxen, or piroxicam (4-hydroxy-2-methyl-3-[pyrid-2-y1-carbamoyl]-2H-1, 2-benzothiazin-1,1-dioxide) [34,88]. In a recent 1-yr clinical trial involving >200 subjects, its efficacy and safety were comparable to naproxen [89]. Etoricoxib, lumiracoxib, and parecoxib [90] are newer NSAIDs that are highly selective for COX-2 [91,92]. Etoricoxib (MK-0663) is more COX-2-selective than rofecoxib [93–95]. In whole blood assays etoricoxib showed >100-fold selectivity for COX-2 compared to COX-1 [96]. Clinical trials indicated that etoricoxib is well tolerated; at 60 mg or 90 mg dosages it is effective for lower back pain [97] and at 120 mg dosage for post-operative dental pain [98]. Studies to evaluate the effects of these drugs on cardiovascular risk are pending.

	Pathophysiology

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Therapeutic doses of coxibs can increase the risk of thrombosis by reducing endothelial cell synthesis of prostacyclin (PGI₂), a vital vasodilator and platelet inhibitor. This disrupts the normal balance between prostacyclin and COX-1-derived thromboxane (TXA₂), a vasoconstrictive eicosanoid that promotes platelet aggregation and thrombus formation. Furthermore, inhibition or gene knockout of thromboxane receptors (TP) and inhibition or gene knockout of prostacyclin receptors (IP) significantly inhibits and accelerates atherogenesis, respectively, showing the importance of proper balance of thromboxane and prostacyclin signaling. Aspirin lowers thromboxane synthesis by inhibiting platelet COX-1 irreversibly, but too much inhibition of COX-1 increases the risk of hemorrhage (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Illustration shows effects of non-steroidal anti-inflammatory drugs (NSAIDs) on thromboxane (TXA2) synthesis. NSAIDs inhibit platelet (PLT) cyclooxygenase (COX)-1, which is rate-limiting for PLT conversion of arachidonic acid (AA) to prostaglandin (PG) H2, the substrate for synthesis of TXA2 by PLT thromboxane synthase. Platelets lack nuclear genes; consequently levels of enzymes in platelets are determined by expression of genes of the megakaryocytes that formed the platelets. The gene for TXA2 synthase, TBXAS1, is located on megakaryocyte chromosome region 7q34. Its expression determines the initial platelet thromboxane synthesis level, which controls platelet conversion of prostaglandin (PG) H2 to thromboxane, which promotes atherosclerosis by activating platelets and increasing leukocyte interaction with endothelial cells. Additionally, TXA2 contracts vascular (v) smooth muscle cells (vSMCs) via their thromboxane-prostanoid (TP) receptors, which are coded for by the TBX2R gene on chromosome region 19p13.3. Contraction of vSMCs can lead to hypertension, another risk factor for atherogenesis. Gene induction, polymorphism and pharmacological inhibition of enzymes regulate platelet thromboxane synthesis. Dexamethasone (DEX) significantly increases TXA2 synthase levels, lipopolysaccharide (LPS) less so. NSAIDs such as aspirin (ASA), naproxen (NAP), and indomethacin (INDO), inhibit COX-1 at the cyclooxygenase site and reduce TXA2 synthesis; resveratrol (RESV) in addition inhibits the peroxidase site of COX-1; sulindac (SUL) inhibits cytosolic phospholipase A2 (cPLA2) Deleting the gene for the TP receptors (TP-/-) in apoE null mice significantly hinders atherogenesis compared to apoE null mice that express thromboxane receptors. Similarly, inhibition of TP receptors with the receptor antagonist S18886 slows atherosclerotic lesion formation in such mice. BM-613 is an antagonist for the TP receptor and also inhibits thromboxane synthase. For details see text.

Endothelial cells are a major source of prostacyclin. Inhibition of endothelial cell COX-2 but not platelet COX-1 by coxibs can therefore interfere with the proper balance, on the one hand, of platelet synthesis of prothrombotic thromboxane A2, which is unaffected by coxibs (with the possible exception of celecoxib), and, on the other hand, reduced synthesis of antithrombotic prostacyclin. Selective inhibition of COX-2 may alter the balance of prostacyclin and thromboxane synthesis. This is the most frequently proposed mechanism to explain the adverse prothrombotic effects of coxibs: they inhibit vascular synthesis of prostacyclin and thereby remove the platelet-inhibitory effect of prostacyclin [100].

This scenario is supported by clinical findings; administration of rofecoxib to patients reduced systemic prostacyclin synthesis to one-half or less of normal levels. This mechanism may, at least in part, explain the hypertension and increased rates of thromboembolic events observed in rofecoxib clinical studies [60]. The imbalance promotes a prothrombotic state, which implies a class effect that may apply to all coxibs [101,102]. Others opine that existence of such a class effect cannot be supported by currently available clinical data. They consider that the degree of imbalance differs for each coxib, and is probably related to the COX-2 selectivity of the coxib administered [103].

Another concern is the observation that myocardial COX-2 expression may be important in limiting damage caused by cardiac ischemia/reperfusion. Selective inhibition of COX-2 may directly interfere with cardiac synthesis of prostacyclin and PGE₂, both cardioprotective eicosanoids [104]. As an example, treatment of rats with atorvastatin, which induces an increase in cardiac expression of cPLA₂, COX-2, prostacyclin synthase and PGE₂ synthase, reduces murine experimental infarct size. Simultaneous inhibition of COX-2 with valdecoxib eliminates this cardioprotective effect [105].

Vasoactive products.  Arachidonic acid can be metabolized to vasoactive products via COX-1, COX-2, lipoxygenase, and cytochrome P-450 (cP450) mono-oxygenase [106]. The most important vasoactive eicosanoids are thromboxane and prostacyclin, generated via the platelet COX-1 pathway and the endothelial cell COX-1 and COX-2 pathways, respectively. Thromboxane contracts and prostacyclin relaxes vascular mural smooth muscle cells (SMCs). In addition, thromboxane promotes and prostacyclin prevents and reverses thromboxane-induced platelet aggregation [107]. Conversely, thromboxane up-regulates COX-2 expression in endothelial cells and enhances endothelial synthesis of prostacyclin [108].

Some have suggested that, in evaluating effects of NSAIDs on vasoactive eicosanoids, species differences must be considered. Thus, in a canine model of coronary vasodilation, naproxen (but not the COX-2 selective inhibitor SC-58236) significantly reduced prostacyclin-mediated vasodilation induced by arachidonic acid. It was suggested that, in the canine model, vasodilation is mostly COX-1-dependent [62]. Other investigators corroborate that naproxen significantly lowers prostacyclin synthesis [40], and some have indicated that systemic prostacyclin synthesis is mainly COX-2-dependent (see below) [109].

Thromboxane.  Platelet COX-1 is rate-limiting for conversion of arachidonic acid to prostaglandin H₂, the substrate for synthesis of thromboxane A₂ by thromboxane synthase. Thromboxane causes platelet aggregation and contraction of mural smooth muscle cells, which constricts vessels. It also increases leukocyte interaction with endothelial cells. All are important steps in the development of atherosclerosis. Thromboxane promotes atherosclerosis by activating platelets and contracting arteries (Fig. 2). Megakaryocyte gene polymorphism regulates platelet thromboxane synthesis. Aspirin inhibits platelet COX-1, lowers platelet synthesis of thromboxane (reducing prothrombotic activity and vasoconstriction), and reduces leukocyte interaction with endothelial cells [110].

Prostacyclin.  Prostacyclin participates in cardio-protective functions in various ways. It inhibits platelet aggregation on the endothelium, reduces leukocyte interaction with endothelial cells, and hinders atherogenesis. Prostacyclin also protects mitochondrial function (see below). Cyclooxygenases in endothelial cells and activated macrophages convert arachidonic acid to PGH₂, which is then converted by prostacyclin synthase (PGIS) to prostacyclin, which dilates vessels by relaxing vascular smooth muscle cells (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Illustration showing effects of non-steroidal anti-inflammatory drugs (NSAIDs) on prostacyclin synthesis. Inhibition of cyclooxygenase (COX)-2 by indomethacin (INDO) or coxibs reduces endothelial cell conversion of arachidonic acid (AA) to prostaglandin (PG) H2, the substrate for synthesis of prostacyclin (PGI2) by prostacyclin synthase (PGIS). The gene for PGIS, PTGIS, is located on chromosome region 20q13.11–q13.13. Prostacyclin exerts its vascular dilator and antihypertensive effects by relaxing vascular smooth muscle cells via prostaglandin (inducible protein, IP) receptors, which are coded for by the PTGIR gene located on chromosome region 19q13.3. Flow shear stress and mechanical stress induce PTGS2 and PGIS respectively. In addition, prostacyclin counteracts prothrombotic and atherogenic effects of thromboxane. Targeted deletion (TD, intron 9) of PTGES causes renal pathology and hypertension. Complete deletion (IP–/–) of PTGIR accelerates atherogenesis. IP receptor function requires isoprenylation (ISOPR), which is inhibited by statins. C-reactive protein (CRP) and 15-HPAA inhibit PGIS and reduce PGI2 synthesis, which can also be reduced by inhibition of cPLA2 by quinacrine or hypoxia. Also, sulindac inhibits transcription of the cPLA2 gene and has shown anticancer effects. Whereas prostacyclin is cardioprotective, other eicosanoid metabolites downstream of COX-2 are pro-inflammatory and this may explain why C-alleles at -765 in PTGS2, which reduce expression of COX-2 and CRP, are associated with reduced risk of myocardial infarction and stroke. For details see text.

1

Lesion-free healthy endothelium expresses only COX-1, whereas both COX-1 and COX-2 are expressed in atherosclerotic lesions; the 2 isoforms are found in macrophages, endothelium, and to a lesser extent smooth muscle cells [112,113].

In vitro studies demonstrate that estrogen can up-regulate endothelial cell COX-1 expression [114]. Thus, COX-1-dependent prostacyclin synthesis may protect against atherosclerosis in premenopausal women. However, local differences in the response of artery types to relaxation signals were found in studies on the effect of 17ß– estradiol (ES2) in sheep: Exogenous ES2 increased PGIS proteins in renal and uterine arteries but not in coronary arteries [115].

In rat coronary arteries, mild physical trauma induces endothelial cell COX-2 expression and COX-2 is expressed in cultured endothelial cells [116]. Prostacyclin (like nitric oxide) is produced by endothelial cells in response to shear stress or other physical forces [117]. For example, in cultured human umbilical vein endothelial cells (HUVEC), laminar fluid shear stress induces COX-2 (mainly via the promoter CRE-responsive element) and stabilizes COX-2 mRNA. However, COX-1 expression is unaltered [118].

No COX-2 expression was detected in freshly removed internal mammary artery segments removed during coronary artery bypass grafting. However, when such segments were placed in organ culture for 2 days they expressed COX-2 [116]. This was a small study of only 3 male and 1 female patients, age 59–68 yr. By comparison, a study of 22 males and 15 females with a median age 28 yr indicated that prostacyclin synthesis in healthy humans is mostly COX-2-dependent [109].

In pigs, COX-1 and COX-2 inhibition by indo-methacin (a non-selective NSAID) results in coronary vasoconstriction at rest and during exercise [117]. Moreover, prothrombotic effects of COX-2-selective inhibition were demonstrated in a hamster model using NS-398, a COX-2 selective inhibitor; it resulted in abridged release of endothelial PGF₁, the stable urinary metabolite of prostacyclin, augmented platelet adhesion to arteries, and increased the pace of occlusion of damaged micro-vessels [119]. Taken together, these reports suggest that both COX-1 and COX-2 contribute to prostacyclin synthesis, and if that is so, then the amounts and ratio concentration of the two isoforms in endothelial cell may significantly affect the extent to which NSAIDs can lower prostacyclin synthesis. Also, as vascular inflammatory changes increase with age, one might expect endothelial cell COX-2 expression to increase.

As noted above, selective inhibition of COX-2 alters the balance of prostacyclin and thromboxane synthesis. This is the most frequently proposed mechanism to explain the adverse prothrombotic effects of coxibs: they inhibit vascular synthesis of prostacyclin and thereby remove the platelet-inhibitory effect of prostacyclin [100]. Rofecoxib administration to patients reduced systemic prostacyclin synthesis to one-half or less of normal levels. This mechanism may, at least in part, explain the hypertension and increased frequency of thromboembolic events observed in clinical studies of rofecoxib [60].

Treatment with coxibs can cause endothelial dysfunction [120] because they inhibit COX-2-dependent synthesis of prostacyclin, the vasodilator eicosanoid. Also, LDL, an independent risk factor for atherogenesis, dose-dependently reduces COX-2 transcription and prostacyclin synthesis in cultured human umbilical vein endothelial cells (HUVECs) [121]. Ex vivo, prostacyclin relaxes coronary artery strips. Conversely, the non-selective NSAID indomethacin, and 15-hydro-peroxyarachidonic acid (15-HPAA), a prostacyclin synthase inhibitor, increase strip resting tone [122].

Such experimental findings raise concerns that selective COX-2 inhibition may cause human hypertension by reducing prostacyclin synthesis, for example, through therapeutic doses of rofecoxib. However, a 7-day study of 35 healthy volunteers revealed no significant differences of vasodilatation in response to acetylcholine or sodium nitroprusside when rofecoxib (25 mg/day) or naproxen (750 mg/ day) were administered. Vascular response was determined by forearm strain-gauge plethysmography [123]. Again, as mentioned above, it is possible that naproxen reduced COX-2-dependent prostacyclin synthesis to a similar extent as rofecoxib in this study. Or, more likely, as the study group consisted of healthy subjects, it is possible that a major part of endothelial cell prostacyclin synthesis was COX-1-dependent, there being no significant shear-stress to induce endothelial cells COX-2 [116].

Contractile dysfunction.  Altered prostaglandin synthesis can significantly affect cardiac mitochondria and cardiomyocyte contraction. In rats, PGE₂ enhances cardiac contractile function and increases cardiac output [124]. Piroxicam, indomethacin, naproxen, nabumetone, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), and meloxicam, but not diclofenac, induce a negative ionotropic effect on perfused isolated rat heart contractility. In vitro, in isolated rat liver mitochondria all of these NSAIDs except naproxen and nabumetone stimulate rates of basic respiration, reduce membrane potential, and reduce the rate of oxidative phosphorylation (OXPHOS) [34].

These findings support the concept that NSAIDs can affect cardiac contractility via effects on mitochondrial function and through cyclooxygenase inhibition that lowers synthesis of cardioprotective eicosanoids. As the heart needs to generate sufficient energy to allow it to pump efficiently, cardiac mitochondrial ATP production must rapidly adjust to a variety of energy demands and dietary supply conditions [125]. However, little is known about eicosanoid involvement in feedback mechanisms that enable OXPHOS to regulate coronary blood flow to its oxygen requirements. It has been suggested that prostaglandins are unimportant for the feed-back [126,127], and that coronary blood flow is normally adjusted to myocardial demands by neural control circuits [128].

Mitochondrial dysfunction.  Almost one-third of all proteins in the heart are mitochondrial proteins, which emphasizes the extraordinary role of OXPHOS in converting energy from food metabolites into adenosine triphosphate (ATP) to adapt to myocardial energy demands [129]. Inhibition of cyclooxygenase may be detrimental to cardiac recovery, as it can lead to accumulation of arachidonic acid, which increases cardiac mitochondrial dysfunction. In isolated bovine heart mitochondria, arachidonic acid inhibits OXPHOS by inhibiting complex I and III of the mitochondrial electron transfer chain (ETC). Mitochondria exposed to arachidonic acid in vitro generate increased amounts of ROS [130] and mitochondrial release of cytochrome c [131] (Fig. 4).

View larger version (31K): [in this window] [in a new window]   Fig. 4. Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is associated with atherogenesis and cardiac failure. Many non-steroidal anti-inflammatory drugs (NSAIDs), including some coxibs, inhibit or uncouple OXPHOS, a part of which is illustrated above. Electrons from food metabolites travel via complexes I and II (succinate dehydrogenase, SDH), coenzyme Q10 (CoQ10), and complexes III and IV (not shown) to oxygen. Complexes I, III, and IV pump protons and build the transmembrane potential that is used by complex V (not shown) to generate adenosine triphosphate (ATP). Statins, often consumed by older NSAID users, inhibit CoQ10 synthesis, block vital electron transfer, and inhibit OXPHOS. Aspirin (ASA) inhibits Krebs cycle -ketoglutarate dehydrogenase (-KGDH). Like complexes I and II, -KGDH is a major source of reactive oxygen species (ROS), which, like salicylate (SAL), opens mitochondrial pores, uncouples OXPHOS, releases cytochrome c (cyt c), and induces apoptosis. Inhibition of cyclooxygenase by NSAIDs elevates arachidonic acid (AA) levels, which inhibits pumps I and III. PGJ2 inhibits complex I. Aconitase (ACO2) declines with age. Proper OXPHOS is also required for maintaining tissue architecture. Mutations of SDH subunit genes (B, C, D) are associated with neoplasia (paraganglioma, pheochromocytoma). For details see text.

Dysfunctional mitochondria present an important source of ROS, which can contribute significantly to atherogenesis and cardiac dysfunction (for a review, see [134]). Cardiac mitochondria produce excessive amounts of ROS during ischemia/ reperfusion [135]. The heart is especially susceptible to defective OXPHOS, which leads to oxidative stress that induces cardiac dysfunction–for example, via generation of malondialdehyde [136]. Inherited or acquired mitochondrial dysfunction can cause severe mitochondrial cardiomyopathy and fatal cardiac failure, even at a very young age, before the onset of significant coronary atherosclerosis. And rare cases of massive, premature atherosclerosis in young subjects with inherited mitochondriopathies have been reported, demonstrating that mitochondrial dysfunction can lead to atherosclerosis and cardiac failure [134].

The classical view of atherogenesis is that slowly developing coronary atherosclerosis and myocardial ischemia is the major cause of mitochondrial dysfunction, cardiac contractile failure, myocardial infarction, and death. In acute myocardial infarction (AMI) an occlusive thrombus at the site of coronary artery atherosclerotic plaques that may have ruptured causes instant downstream anoxia.

A more recent view is that accumulated products of ROS damage mtDNA; this may represent the initial common mechanism by which classical risk factors contribute to atherogenesis. In experiments that employ apoE-/- mice with defective manganese superoxide dismutase (SOD2), mitochondrial damage induced by ROS and reactive nitrogen species preceded the formation of atherosclerotic lesions. These experiments show that SOD2 can inhibit atherosclerotic lesion formation by protecting the mitochondrial genome [137]. Importantly, therapeutic drugs that increase ROS formation or cause mitochondrial dysfunction through OXPHOS inhibition or uncoupling may increase the risk of cardiovascular complications.

In mitochondria isolated from rat hearts, aspirin and salicylic acid both inhibit state 3 (ADP-dependent) respiration through inhibition of -ketoglutarate dehydrogenase (-KGDH), a vital enzyme of the Krebs cycle. However, only salicylic acid induces mitochondrial uncoupling. It inhibits -KGDH competitively, whereas aspirin causes irreversible inhibition via acetylation of the enzyme. Inhibition of the Krebs cycle reduces NADH generation and electron transfer to Complex I of the respiratory chain. This reduces ROS generation and ATP synthesis [138]. Experiments using plant mitochondria indicate that salicylic acid also inhibits electron transfer from Complex I to the coenzyme Q10 (CoQ10, Q10) pool, but only at very high concentrations [139].

In vitro experiments indicate that inhibition of the respiratory chain increases but uncoupling reduces mitochondrial generation of ROS that play important parts in the pathogenesis of inflammation. As examples, ROS production induced by hyperglycemia increases COX-2 mRNA and protein levels via activation of NF-kappaB (NF-B) [140]. Aspirin inhibits NF-B activation and reduces ROS generation [141]. Nimesulide, a sulfonanilide compound and selective COX-2 inhibitor, has shown antioxidant properties [142]. It and its metabolite, 4-OH nimesulide, subdue generation of, and scavenge, ROS released by cultured human chondrocytes [143]. Nimesulide reduces NF-B activation and protects against cardiovascular complications of endotoxemia [144]. In a mouse model of skin carcinogenesis, nimesulide and celecoxib significantly curtailed chemically induced H₂O₂ generation and lipid peroxidation [145].

Whereas some NSAIDs reduce the rate of ROS formation, others raise it. In rats, indomethacin exposure leads to renal mitochondrial damage and abnormal tubular morphology. Pretreatment with arginine, a nitric oxide donor, suppresses tubular injury, which points to ROS participation in the pathogenesis of the tubular destruction induced by indomethacin [146]. Pretreatment with melatonin, a pineal sleep hormone and strong antioxidant, lessens renal lipid peroxidation and renal damage induced by indomethacin, which corroborates that ROS is involved in the pathogenesis [147].

Results of in vitro studies may be cell-dependent. Celecoxib (but not rofecoxib) inhibits proliferation of HUVECs, but neither affected the growth of vascular smooth muscle cells. At high concentrations, celecoxib (but not rofecoxib) induced HUVEC apoptosis, suggesting that the antiproliferative effects may not be linked to COX-2 inhibition, since both drugs inhibit COX-2 [29].

Atherogenesis.  Atherosclerosis in an age-related inflammatory disease and aging may alter the effects of cyclooxygenase inhibition on atherogenesis. In apoE^–/– mice an increase of thromboxane synthesis or a decrease in prostacyclin synthesis contributes to atherogenesis by inducing platelet activation and enhancing interaction of leukocytes with endothelial cells. In an apoE-/- knockout mouse model of atherosclerosis, selective inhibition of COX-1 with SC-560 abrogates gross lesion formation even in the presence of vascular injury and inflammation, probably by lowering thromboxane synthesis By contrast, inhibition of COX-2 with SC-236 had no effect on lesion development. SC-236 inhibited prostacyclin synthesis, but did not reduce platelet-endothelial cell interaction [148]. No benefit was evident in older apoE^–/– mice with established atherosclerotic lesions treated with celecoxib compared with controls [149]. In rat aorta, prostacyclin and thromboxane levels increase with age. Interestingly, calorie restriction lessens the increase [150]. Also, the relative lack of COX-1 inhibition by coxibs may be especially important in aging patients with atherosclerosis, who synthesize increased amounts of thromboxane and prostacyclin compared to subjects without the disease [151].

Paradoxically, regional hypoxia may markedly increase COX-2 expression in response to NSAID treatment. As mentioned above, a problem with high level expression of COX-2 is that it may increase ROS generation at the enzyme peroxidase site. Whereas NSAID treatment can lower PGE₂ synthesis, simultaneous exposure to hypoxia during NSAID treatment may disrupt a negative feed-back loop in atherosclerotic lesions that normally limits macrophage synthesis of tumor necrosis factor- (TNF-), an inducer of COX-2. This insight is based upon experiments that employed cultured monocytes exposed to hypoxia, which induced expression of TNF- that elevated COX-2 mRNA and protein expression. Exogenous PGE2 dose-dependently reduced TNF synthesis. Indomethacin and NS-398 markedly reduced PGE2 and significantly increased TNF- synthesis. Since hypoxia also decreased phosphorylated cPLA₂ levels, conversion of membrane phospholipids to arachidonic acid was lowered, so that despite elevated COX-2 expression, the substrate shortage caused by hypoxia further diminished the synthesis of PGE₂ [152].

Vascular inflammation.  Histological and biochemical analyses of human intima show accumulation of inflammatory proteins not only in atherosclerotic lesions, but also in areas without local lesions in vessels with lesions elsewhere, which indicates that atherosclerotic lesions develop in a background of vascular inflammation [153–155]. Intimal inflammatory alterations are more pronounced in older subjects, even in those that lack atheromas. By comparison, intimae of young subjects lack inflammatory alterations [156]. Such age-related factors may be relevant to the clinical response to NSAID treatments, since the risk of cardiovascular complications is higher in older patients.

Elevated plasma levels of C-reactive protein (CRP), intracellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 are biomarkers of inflammation that can predict cardiovascular risk [27,28,41,157]. CRP belongs to the pentraxin (PTX) family of acute-phase proteins and is a marker for disease activity in patients with rheumatoid arthritis [158].

The name for CRP was derived from its ability to precipitate pneumococcal C-polysaccharide. Pneumoccal vaccination ameliorates atherogenesis in homozygous low density lipoprotein (LDL)-receptor deficient mice. Plasma from such mice inhibits binding of oxidized LDL to macrophages, suggesting molecular mimicry between epitopes of Streptococcus pneumoniae and oxLDL [159]. In cultured vascular smooth muscle cells, atherogenic lipoproteins can also significantly induce expression of another pentraxin, 3 (PTX-3), which is associated with foam cell formation via rapid cholesterol uptake by macrophages [160].

Monocytes/macrophages.  Monocyte chemotaxis plays a central role in atherogenesis (Fig. 5). Increased expression of CCR2, the most important monocyte chemotaxis receptor, increases monocyte binding to monocyte chemoattractant protein-1 (MCP)-1 and facilitates the entry of monocytes into the subendothelial space, where they become macrophages and may develop into lipid-laden foam cells.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Illustration showing effects of non-steroidal anti-inflammatory drugs (NSAIDs) on monocyte/endothelial cell interaction in atherogenesis. Monocyte/endothelial cell interaction guides monocytes to enter into the subendothelial space, where they become macrophages and can develop into lipid-laden foam cells. Increased monocyte chemotaxis receptor 2 (CCR2) expression increases monocyte binding to monocyte chemoattractant protein-1 (MCP)-1, which is produced by endothelial cells (EC), macrophages (M), and vascular smooth muscle cells (vSMCs), and is particularly abundant in macrophage-rich shoulder areas of human atherosclerotic lesions. CCR2 knockout in apoE–/– mice lowers chemotaxis, reduces the number of macrophages in lesions, and slows down atherogenesis. By contrast, common atherogenesis risk factors such as C-reactive protein (CRP), free cholesterol (CHOL), and low density lipoprotein (LDL) increase CCR2 levels and promote atherogenesis by enhancing monocyte chemotaxis. Simvastatin reduces CCR2 expression. Estrogen (ES) and ibuprofen reduce MCP-1 expression. In animal models, effects of celecoxib on MCP-1 expression depend upon treatment duration; short-term treatment inhibits MCP-1 expression, whereas long-term treatment significantly increases MCP-1 mRNA levels, possibly caused by reduced synthesis of PGE2, a potent suppressor of MCP-1 synthesis. Polymorphism of the CCR2 gene may also influence the rate of atherogenesis. For further details see text.

Several atherogenic risk factors increase chemotaxis by stimulating expression of CCR2 or MCP-1. For example, CRP in addition to its indirect inhibition of prostacyclin synthase noted above may enhance atherogenesis by increasing chemotaxis: it up-regulates monocyte CCR2 levels in cultured monocytes. Patients with acute inflammatory conditions may have CRP levels of 10 mg/ L or higher, and one-half of that concentration can activate cells in atherosclerotic lesions. Furthermore, in vitro, co-incubation with free cholesterol or LDL enhances CCR2 expression, suggesting that CRP, free cholesterol, and LDL may synergistically contribute to atherogenesis by enhancing monocyte chemotaxis [164,165].

C-reactive protein promotes monocyte chemotaxis with abluminal transmigration of monocytes during atherogenesis by inducing IL-8. In cultured human aortic endothelial cells, CRP activates NF-B, which induces expression of interleukin (IL)-8, a chemokine that mediates monocyte chemotaxis. IL-8 is present in large quantities in human atherosclerotic lesions. Hyperglycemia, oxidized LDL, and TNF- also induce IL-8 expression and augment monocyte-endothelial interaction [166].

The importance of monocyte chemotaxis receptors for monocyte/endothelial cell interaction in atherogenesis has been demonstrated in apoE^–/– mice lacking such receptors. The number of macrophages and atherosclerotic lesions in aortas from apoE-null mice lacking CCR2 expression was significantly reduced compared to apoE^–/– mice that expressed CCR2 [162].

Activated macrophages in human atherosclerotic lesions express COX-2. But in mice models of atherogenesis, COX-2 inhibition has led to mixed results ranging from inhibition to acceleration to lack of effects. It has been suggested that such divergent results may depend upon the stage of disease development: it seems beneficial during early stages whereas during later stages COX-2 inhibition may have less impact on the disease [167]. For example, rofecoxib and indomethacin administered for 6 wk to LDL-receptor deficient mice (LDLR null mice) fed a Western diet significantly reduced atherosclerotic lesion formation in the proximal aorta [168]. Transplantation of COX-2^–/– and COX-2^+/+ fetal liver cells into LDLR null mice that were fed a Western diet for 8 wk showed that macrophage COX-2 expression plays an important role in the initial steps of atherogenesis by enhancing monocyte attachment and mural entry. Reconstituting LDLR null mice with macrophages lacking the COX-2 gene significantly reduced lesion areas compared to such mice with COX-2-expressing macrophages [167].

Different durations of celecoxib inhibition of COX-2 result in divergent experimental findings. For example, atherogenesis studies using 2 animal models, rabbits and apoE^–/–-mice, suggest that the effect on MCP-1 expression and mural entry of monocytes depends upon the duration of celecoxib treatment. In the rabbit, short-term (21 day) celecoxib treatment significantly decreased MCP-1 expression, macrophages infiltration, and intimal hyperplasia associated with balloon injury and also reduced post-injury neo-intimal proliferation after stent placement [19]. In contrast, in older (26 wk) apoE-/- mice with established atherosclerotic lesions, 15 wk of celecoxib treatment markedly increased MCP-1 mRNA levels, but did not affect serum cholesterol level, atheroma size, or atheroma composition, compared to non-treated mice. The MCP-1 increase may have been mediated by reduced synthesis of PGE₂, a potent suppressor of MCP-1 synthesis [149]. Whereas these experimental observations involve 2 different animal models, they may help to explain why some long-term clinical studies of selective COX-2 inhibition have revealed an increased risk of cardiovascular complications that was not evident after initial short-term trials. They call attention to the need for further studies using the same animal species in order to compare the effects of short-term and long-term treatment.

By comparison, another study, using rabbits that were fed a cholesterol-rich diet, showed that supplementation with ibuprofen for 2 mo lowered MCP-1 expression, but did not alter the extent of atherosclerosis in aortas compared to rabbits not supplemented with ibuprofen [169]. Regrettably, no comparison was conducted of long-term versus short-term ibuprofen treatment. Also, aspirin and evening primrose oil inhibit, but advanced glycation end products and TNF- stimulate, endothelial cell MCP-1 expression in vitro [170–172].

In 2 standard murine models of acute inflammation, the carrageenan-induced pleurisy and the paw edema models, rofecoxib and celecoxib reduced PGE₂ expression, pleural exudation, and paw edema, but less than dexamethasone or indomethacin, but surprisingly, had little effect on inflammatory cell influx, suggesting that in these models coxibs have only incomplete anti-inflammatory properties [173]. By contrast, in acute myocardial infarction induced by coronary ligation in rats, NS-398 [174] significantly reduced inflammatory influx into the myocardium [175].

Infectious agents.  Coxib inhibition may be beneficial in patients with arthrosclerosis who are infected with Chlamydia pneumoniae. This intra-cellular pathogen has been detected in atherosclerotic lesions and monocytes in up to a quarter of patients with atherosclerosis. Chlamydia induces expression of monocyte COX-2, PGE₂ synthase, and mRNA of the proteolytic enzyme matrix metalloproteinase (MMP)-1. NS-398 blocks the increase in PGE₂ synthesis and matrix induction [176]. Studies show that polymorphism of the MMP1 gene significantly affects lesion development (see below).

A study of >5,000 subjects showed that gingivitis is an independent risk factor for atherosclerotic cardiovascular disease [177]. Systemic treatment with ibuprofen or flurbiprofen reduces gingivitis [178]. Daily use of dental floss reduces bacterial gum infection [179]. However, whereas an association between gingivites and atherosclerosis has been found, there is at present no evidence that dental flossing prevents or retards the development of atherosclerosis.

Serum levels of IL-1ß and TNF- are elevated in patients with periodontitis caused by agents such as Porphyromonas gingivalis [180]. Also, examination of gingival plaques shows a close relationship between bacterial DNA and carotid sub-clinical atherosclerosis, which is independent of serum CRP levels [181]. Moreover, structures consistent with nanobacteria have been detected in calcified atherosclerotic plaques [182].

Studies of atherosclerotic plaques removed during endarterectomy suggest that thrombosis and plaque hemorrhage may contribute to lesion calcification [183]. Some studies show that eicosanoids contribute to the development of calcifications, but the exact pathophysiology remains debatable. As examples, IL-1ß induces PLA₂, COX-2, and PGES, and marked increases in PGE₂ levels in the mineralizing phase of cultured osteoblasts [184]. In rats, ibuprofen inhibits new bone formation and calcification of bone [185]. On the other hand, indomethacin increases alkaline phosphatase activity and mineralization in an osteoblastic cell line [186]. An effect of NSAIDs on atherosclerotic calcification has not been established.

Cytomegalovirus (CMV) has been detected in atherosclerotic lesions and may promote atherogenesis by inducing inflammation. Infection of cultured coronary artery smooth muscle cells with CMV causes generation of ROS, which induces COX-2 via NF-B and promotes expression of inflammatory response genes such as the CRP gene and COX-2 gene (prostaglandin-endoperoxidase synthase 2, PTGS2). NSAIDs such as aspirin and indomethacin, as well as ROS scavengers inhibit CMV-induced NF-B activation [187].

Statins.  Many elderly patients who use NSAIDs also are treated with statins, which inhibit hydroxyl-methylglutaryl-coenzyme A (HMG-CoA) reductase. Statins lower cholesterol, CRP and oxLDL levels. They improve coronary endothelial function, induce vasodilation, lessen rates of coronary events, and reduce cardiovascular mortality [39,188,189].

On the other hand, because statins inhibit HMG-CoA reductase and block the mevalonate pathway of cholesterol synthesis, they simultaneously limit the endogenous synthesis of CoQ₁₀, which shares part of the same pathway for synthesis of its long side chain [190] (Fig. 4). CoQ₁₀ is a potent free radical scavenger. In the blood it is carried in the low-density-lipoprotein (LDL) fraction and protects LDL against oxidation. Moreover, CoQ₁₀ is an indispensable electron carrier as part of the mitochondrial electron transport chain and for that reason is vital for OXPHOS and ATP generation.

Inhibition of prostacyclin synthesis interferes with valuable cardioprotection by CoQ₁₀. A study of aging in rats showed that beneficial effects of CoQ₁₀ on arterial relaxation are largely mediated by prostacyclin [191]. Furthermore, in the isolated guinea-pig heart, the mitochondrial OXPHOS inhibitor 2,4-dinitrophenol depresses contractile function, which is restored by CoQ₁₀ or prostacyclin. Pretreatment with indomethacin (which inhibits both COX-1 and COX-2) or with 15-HPAA, which inhibits prostacyclin synthase, abolishes CoQ₁₀ restorative action but not that of prostacyclin [192]. CoQ₁₀ enhances prostacyclin synthesis, probably by reducing mitochondrial ROS generation and scavenging free radicals that may otherwise inactivate prostacyclin synthase (see below).

	Molecular Pathology

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Genotyping may help identify patients at high risk of developing myocardial infarction and stroke and reduce rates of cardiovascular complications caused by NSAIDs. For example, a polymorphism located in the 5' transcriptional regulatory promoter region of the COX-2 gene, PTGS2, (region 1q24.2–1q24.3) may be cardioprotective because it lowers promoter response. In a British study, >25% of healthy Caucasians carried one -765G>C allele in the promoter (Fig. 3). The study included 173 patients who underwent first-time coronary bypass surgery and developed significant post-operative acute phase reactions with increased COX-2 activity. Plasma levels of C-reactive protein and of IL-6 rose, the latter in response to rising PGE₂ levels. Patients carrying one or more -765C alleles had significantly lower post-operative plasma CRP levels than individuals with two -765G alleles, indicating that presence of one or more C alleles within the promoter renders it less responsive to inflammatory stimuli compared to promoters having two G alleles [193].

An Italian study supports this interpretation. It found that -765G>C was associated with decreased risk of myocardial infarction and stroke, and with superior prospects of healthy aging. The study included an analysis of internal carotid endarterectomy samples from 864 patients who had had first-event myocardial infarction or stroke, and 864 controls (high-risk patients; no history of MI or stroke). Immunohistochemical staining for COX-2 and MMP-9 in plaques was strongly positive in -765GG patients, intermediate in patients with the -765GC genotype, and minimal in the -765CC genotype. Cultured macrophages exposed to LPS, oxLDL, or angiotensin II expressed high, medium, and low COX-2 levels in -765GG, -765GC, and -765CC phenotypes respectively. Presence of the -765C allele was associated with significantly lower serum CRP levels. Conspicuously, the reduced cardiovascular risk was not associated with changes in prostacyclin synthesis or altered in vivo vasodilation [194].

Taken together, these findings suggest that genotyping of alleles at -765 of the COX-2 promoter may predict patient responses to NSAID treatment. Further clinical studies are needed to verify that such genotyping can help to reduce cardiovascular complications associated with selective inhibition of COX-2.

Serum concentration of active transforming growth factor (TGF)-ß 1 is markedly depressed in patients with advanced atherosclerosis compared to subjects with normal coronary arteries, suggesting that TGF-ß 1 plays a role in atherogenesis [195]. TGF-ß 1 can induce COX-2 and IL-8 via NF-B activation [196] but can also directly induce COX-2 transcription (see below). In coronary endarterectomy atherosclerotic plaque samples TGF-ß 1 expression is higher in stable than in unstable lesions and correlates negatively with plaque expression of MMP-9. High expression of this metalloproteinase is associated with lesion progression [197]. On the other hand, TGF-ß up-regulates expression of MMP-1 [198], which slows down atherogenesis by degrading type III and type I collagen. The latter stimulates differentiation of monocytes into lipid-laden foam cells [198]. Inhibition of TGF-ß signaling by employing TGF-ß antisense-oligonucleotides reduces MMP-1 expression, corroborating that TGF-ß induces MMP-1 expression [199].

MMP-1 is expressed in atherosclerotic lesions but lacking in normal blood vessels. Transgenic mice expressing MMP-1 develop less advanced atherosclerotic lesion with reduced collagen content, which deters lesion progression. Moreover, recent studies of 471 human subjects indicate that single nucleotide polymorphism located at -1607bp of the MMP1 promoter affects expression of the gene [200]. Subjects homozygous for a transcriptionally more active 2G allele had less atherosclerosis than patients homozygous for the 1G allele [201].

Aspirin, which inhibits iNOS, IL-4, and NF-kB [202], also induces TGF-ß, which stops cells in G0 of the cell cycle and may help restrain atherosclerotic plaque growth [203]. Lowering TGF-ß signaling using anti-TGF-ß antibody treatment in apoE-/- mice accelerates development of atherosclerotic lesions. Lesions show increased infiltration by T-lymphocytes and many macrophages [204]. Moreover, abrogating TGF-ß signaling via its TGF-ß type II receptors in apoE^–/– mice causes massive increase in plaque size [205].

The COX-2 gene promoter contains a TGF-ß responsive element. Thus TGF-ßcan induce COX-2 expression [206] and stimulate endocardial endothelial cells (EECs) to synthesize prostacyclin, which may be important in preventing intracardiac thrombus formation and coronary artery thrombosis. In cultured porcine EECs, TGF-ß 1 rapidly induces prostacyclin synthesis, as evidenced by a rise in release of 6-keto-PGF₁, a stable prostacyclin metabolite. Exposure of platelets for 2 min to cultured EECs or coronary endothelial cells inhibits platelet aggregation induced by thrombin. Pretreatment with indomethacin, but not with a nitric oxide inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), restores platelet aggregation, which shows that prostacyclin and not nitric oxide is involved in the inhibition of platelet aggregation [207].

The rates of degradation of NSAIDs may modulate their effects. For example, cytochrome P450 2C (CYP2C8) polymorphism can reduce ibuprofen clearance [208]. CYP2C8 is involved in metabolism of cerivastatin and several other drugs [209]. The leukotriene receptor antagonist, montelukast, is a strong, selective inhibitor of CYP2C8, but other therapeutic drugs such as lovastatin, simvastatin, and levothyroxine also inhibit CYP2C8, but to a lesser degree [210]. No report is presently available on the effect of CYP2C8 polymorphism on the metabolism of coxibs.

Several NSAIDs are metabolized by CYP2C9 and drugs that modify CYP2C9 expression may affect their blood and tissue levels [211]. As examples, CYP2C9 is involved in the degradation of ibuprofen, diclofenac, naproxen, and celecoxib. A polymorphism that occurs in ~0.5% of Caucasians significantly lowers celecoxib disposition: homozygous carriers of the CYP2C9*3 phenotype (particularly elderly patients), may be prone to accumulate coxib, leading to adverse effects [212].

Thromboxane signaling.  Platelets lack nuclear genes; consequently levels of enzymes in platelets are determined by expression of genes of the megakaryocytes that formed the platelets. The gene for thromboxane synthase, TBXAS1, is located on chromosome region 7q34 (Fig. 2). Its expression controls platelet conversion of PGH2 to thromboxane. Dexamethasone (DEX) increases TXA2 mRNA and protein levels, lipopolysaccharide (LPS) much less so [213,214]. Several polymorphisms of the gene have been identified, but their effects on enzyme activity need clarification [215].

Thromboxane exerts its effects on platelets, endothelial cells, and vascular SMCs via binding to G-protein-coupled thromboxane-prostanoid (TP) receptors, which are coded for by the TBXA2R gene located (OMIM # 188070 [OMIM] ) on chromosome region 19p13.3. A gene polymorphism T924C is associated with bronchial constriction and severity of asthma [216]. There are no data available on its effect, if any, on arteries and cardiovascular risk, but as the bronchial constriction involves smooth muscle cell contraction, further studies would be of interest to clarify this question. Deleting TBXA2R in apoE null mice significantly hinders atherogenesis compared to apoE null mice that express thromboxane receptors [4], [151].

An alternative to use of NSAIDs to reduce thromboxane synthesis is to inhibit downstream signaling, by inhibiting thromboxane synthase, or the TP receptor, or both. Thus, inhibition of TP receptors with the receptor antagonist S18886 (3–6-(4-chlorophenylsulfonylamido)-2-methyl-5,6,7,8 tetrahydronaphtyl-propanoic acid, Na salt) slows atherosclerotic lesion formation in apoE-/- mice. Interestingly, it has no effect on established lesions, but significantly reduces early atherogenesis. In pigs, S18886 displays potent, dose-dependent anti-aggregant effects at both low and high shear stress conditions [217,218]. Another compound, BM-613 (N-n-pentyl-N’-[2-(4'-methyl-phenylamino)-5-nitrobenzenesulfonyl]urea) inhibits thromboxane synthase and is also a TP-receptor antagonist: It inhibits human platelet aggregation in vitro and reduces thrombus size in the rat [219].

Chromosome region 19p13.3, in addition to containing the thromboxane receptor gene, contains a cluster of genes associated with increased risk of atherosclerosis, such as the atherosclerosis susceptibility gene (ATHS), which is associated with a significant risk of myocardial infarction, adhesion molecule genes (ICAM1 and ICAM3), and several mitochondrial genes such as translocases of inner mitochondrial membrane (TIMM 13 and TIMM 44), and nearby ATP5J (OMIM), which codes for a subunit of the F₀ moiety of mitochondrial ATPase. (See below for a discussion of the important role of mitochondria in atherogenesis.)

Prostacyclin signaling.  As noted above, both COX-1 and COX-2 can contribute to prostacylin synthesis by endothelial cellls (EC, Fig. 3). In the chain of synthetic steps for prostacyclin, prostacyclin synthase is located downstream of COX-1 and COX-2. The latter is therefore rate-limiting for shear-stress-induced prostacyclin synthesis, which is induced via COX-2. Selective inhibition of COX-2, which reduces substrate availability for prostacyclin synthesis by endothelial cells, can therefore reduce the atheroprotective effect of PGI₂-induced vascular dilatation (Fig. 3).

PTGIS (CYP8A1, OMIM), the gene for prostacyclin synthase, is located on chromosome region 20q13.11–q13.13. Its promoter contains several transcription factor recognition sites such as a shear-stress responsive element, NF-B, Sp-1, AP-2, and glucocorticoid response elements [220]. Studies employing cultured human myometrial cells indicated that cyclic mechanical stretch can significantly augment PTGIS expression via an AP-1 promoter binding site [221]. Thus, mechanical stress can apparently augment both COX-2 [117] and PGIS expression. It follows that vascular shear stress associated with vessel contraction may up-regulate both genes and increase prostacyclin release, which would tend to induce vascular dilatation, reduce shear stress, lower blood pressure, and lessen atherogenesis.

Prostacyclin relaxes vascular smooth muscle cells (SMC) via binding to their PGI₂ receptors (inducible protein, IP), coded by the PTGIR gene of chromosome region 19q13.3. SMC relaxation dilates vessels and lowers blood pressure. Targeted deletion (intron 9) of the prostacyclin synthase gene that produces a truncated protein has been associated with essential hypertension [222]. Mice with genetic disruption of PGIS developed mural thickening of the aorta and of renal arteries, with arteriosclerosis, renal cysts, and progressive renal fibrosis. Moreover, blood pressure and thromboxane synthesis increased with age [223]. Signal transmission via the IP receptor required receptor isoprenylation, which is inhibited by statin therapy. This finding may raise concerns about long-term statin use [224].

Disruption of the prostacyclin receptor gene in mice significantly accelerated arterial thrombus formation in response to local injury, compared to wild mice [225]. Moreover, experiments using apoE^–/– mice corroborate that altered vasoactive eicosanoid signaling caused by cyclooxygenase inhibition can promote atherogenesis; complete deletion of the prostacyclin receptor gene accelerates atherosclerosis, but retention of one allele is atheroprotective [226].

Reducing PGI₂ synthesis by combined inhibition of COX-2 and COX-1 increases the rate of atherogenesis. By comparison, a murine model suggests that even a modest amount of PGI₂ may be atheroprotective. Remarkably, in mice, selective COX-2 inhibition alone fails to accelerate atherogenesis, even if prostacyclin levels are significantly reduced. Reducing prostacyclin synthesis in mice by up to 60% by inhibition of COX-2 failed to generate atherosclerosis [226]. These findings support the notion that EC COX-1-induced prostacyclin synthesis in mice may be sufficient to inhibit atherogenesis, even if COX-2 is inhibited. However, as noted above, thromboxane synthsis tends to increase with age. Thus, such lowered prostacyclin synthesis may become insufficient to counteract the prothrombotic atherogenic effects of thromboxane.

Evidence that C-reactive protein can contribute to atherogenesis by reducing prostacyclin synthesis was obtained by supplementing cultured human aortic endothelial cells with CRP, which decreased prostacyclin release by the cells. CRP induced superoxide and nitric oxide production, which increased nitration of prostacyclin synthase (PGIS) and inactivated the enzyme, but did not alter PGIS mass [227]. Conversely, in HUVECs, - high-density lipoprotein (HDL), which is deemed cardioprotective, induced COX-2 expression and increased synthesis of prostacyclin [228].

Interestingly, prostacyclin expression may also protect against neoplasia. In mice with lung-specific PTGIS over-expression, after exposure to mainstream cigarette smoke, the lung tumor incidence and multiplicity were significantly reduced, compared to wild-type littermates similarly exposed. PTGIS overexpression enhanced PGI₂ levels and reduced PGE₂ levels. Expression of CYP2E1 (ethanol-inducible P450) in type II pneumocytes was significantly reduced [229]. CYP2E1 catalyzes N-nitrosoamines and other procarcinogens [230]. Polymorphism of CYP2E1 has been associated with increased risks of cancers of the esophagus and gastric cardia in smokers [230,231].

Prostacyclin may be an important regulator of renin release, acting directly on juxtaglomerular (JG) cells to stimulate secretion. In a mouse hypertension model, administration of SC-58125, a COX-2 selective inhibitor, or deletion of IP (IP- null mouse), significantly reduced JG cell renin mRNA levels and the IP-deficiency suppressed renovascular hypertension. Surprisingly, SC-58125 did not lower blood pressure in this model, and it is unclear why. The effect of prostacyclin signaling on renin secretion in humans is unclear and needs further study [232].

Even after inhibition of cyclooxygenase and nitric oxide synthesis, significant vasodilator effects remain that are provided by metabolites of arachidonic acid derived via cytochrome P450 2C (CYP2C8) located at chromosome region 10q24 [233]. The cP450 pathway generates regional synthesis of hydroxyl-eicosatetraenoic acids (HETEs), epoxides, and epoxyeicosatrienoic acids (EETs). The latter has been identified as an endothelium-derived hyperpolarizing factor (EDHF) that is a potent vasodilator of coronary arteries [106]. In response to bradykinin, a vasodilator, porcine and bovine coronary endothelial cells release EDHF, which diffuses and relaxes mural smooth muscle cells by activating their calcium-dependent potassium channels [234,235].

	Neoplasia

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Chemotherapy.  COX-2 is over-expressed in a variety of pre-malignant lesions and malignant tumors [236]. Importantly, experimental over-expression of COX-2 alone can transform cells. Tumor-induced COX-2 expression promotes angiogenesis, stimulates tumor cell invasion, diminishes apoptosis, and suppresses anti-tumor immunity.

Many in vitro and in vivo studies show that inhibition of COX-2 can inhibit cancer cell growth and metastases. For example, in vitro, celecoxib inhibits growth of cultured lymphoma cells via a Bcl-2-independent pathway [237]. Celecoxib, but not rofecoxib, inhibits growth of cultured cholangiocarcinoma cells by inactivating Akt and translocating Bax to mitochondria [238]. Furthermore, celecoxib (but not rofecoxib) induces apoptosis in a line of transformed cells derived from normal rat intestinal cells [239].

COX-2 inhibition with NS-398 reduces growth of cultured human endometrial cancer cells. In this type of tumor, mutated phosphatase tensin homolog (PTEN), a tumor suppressor and negative regulator of Akt/protein kinase B (Akt/PKB), fails to suppress Akt, an inducer of COX-2 expression. Akt phosphorylation levels increase, causing elevation of COX-2 expression via NF-B, and increases of tumor PGE₂ levels. Blocking COX-2 catalytic activity induces cancer cell apoptosis [240].

In vivo, etodolac, a highly selective COX-2 inhibitor, reduces cancer-induced neovascularization [241]. The selective COX-2 inhibitor JTE-522 restrains metastatic spread to the lung in a rat model of colorectal cancer [242]. Topical treatment with celecoxib suppresses ultra-violet (UV)-induced inflammation of the skin and restrains skin cancer growth in mice [243,244]. Also, celecoxib induces apoptosis and reduces cell proliferation and tumor burden in a murine mammary tumor model [245]. Clinical studies that employ celecoxib to treat breast cancer are presently ongoing [246]. Clinical trials are also being conducted to investigate the efficacy of selective COX-2 inhibitors for the treatment of lung cancer [247].

On the other hand, NSAIDs may inhibit neoplastic growth via COX-2-independent pathways. Various NSAIDs may differ in the ways they inhibit cancer cell growth. For example, indomethacin and aspirin caused cellular release of cytochrome c and apoptosis and inhibited growth of several cultured endometrial cancer cell lines. Both NSAIDs increased COX-2 expression and PGE₂ synthesis. By comparison, NS-398 failed to alter COX-2 expression or PGE₂ synthesis and induced weak apoptosis in only one cell line. Nevertheless, all 3 NSAIDs exhibited similar anti-proliferative effects [248]. Sulindac inhibits COX-2 enzymatic activity, stimulates COX-2 mRNA expression, and markedly reduces cPLA₂ transcription. It has been suggested that the anti-cancer effect of sulindac may be due to reduction in cPLA₂ synthesis [249]. Steps distal to cyclooxygenase may also be interesting targets for cancer chemotherapy and chemoprevention.

Chemoprevention.  Chronic inflammation is a risk factor for neoplasia. There is therefore great interest in identifying NSAIDs that may be suitable for chemoprevention to lower cancer risk. By reducing inflammation and sparing the gastrointestinal system, selective COX-2 inhibitors are potentially significant anticancer drugs. For instance, colonic polyposis is a risk factor for colon cancer. Celecoxib treatment attenuates polyposis in patients with Peutz-Jegher syndrome (PJS) and in mouse models of the disease [250].

However, chemoprevention trials show that patients differ significantly in their response to therapeutic COX-2 inhibition, leading to searches for laboratory methods to discriminate between responders and non-responders to enhance the benefit-to-risk ratio. In one clinical cancer prevention trial, proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) analysis was successful in identifying familial adenomatous polyposis (FAP) patients who were celecoxib responders [251]. Furthermore, analyses of colonic biopsies revealed that reduced Ki-67 levels and an increased rate of apoptosis in the superficial colonic mucosa, compared to the deeper layers, correlates with reduced polyp counts in polyposis patients treated with celecoxib [252].

Statins, which reduce cholesterol synthesis, have also been studied for cancer chemoprevention potential in combination with selective COX-2 inhibition. For example, treatment of HT-29 cells, derived from a human colonic carcinoma, with a combination of a celecoxib and levostatin synergistically enhanced cell apoptosis [253]. Thus, in spite of cardiovascular problems in the APC study, the search continues for NSAIDs that may reduce cancer risk while exhibiting a reasonable, low level of adverse side effects.

	Theories and Speculations

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
The many divergent effects of eicosanoids may appear confusing. Some of the different effects of eicosanoids may be due to bimodal responses to different eicosanoid concentrations that are altered by NSAID treatment. Such bimodal responses resemble those that can be induced by, for example, TGF-ß, which stimulates and inhibits cell proliferation at low and high concentrations respectively [254–256]. Such bimodal morphogens are essential for formation of tissue structures in embryology.

In vitro, TGF-ßorganizes endothelial cells into tubular structures, verifying its ability to control radius of curvature and morphogenesis. Furthermore, it has been proposed that gradients of bimodal morphogens are involved in tissue remodeling and that aberration in their expression or the shape of their gradients may play an essential role in tissue remodeling during atherogenesis and in tumor development. Close to the source of morphogen, where concentration is high, growth is inhibited; further away, at low concentration, growth is stimulated, and in between is a neutral zone, where growth is unaffected by the morphogen. Thus a curved shape is formed, the radius of which is determined by the morphogen source concentration and the shape of the gradient [257]. Interaction of more than one gradient of bimodal morphogens could conceivably produce complex structures. Also important for basic and clinical research, determination of concentration gradients of bimodal morphogen may perhaps enhance proteomic analysis of tumor tissue samples [258].

As noted above, TGF-ß can regulate expression of COX-2. It is therefore interesting that, in vitro, some prostaglandins can mimic the effects of bimodal morphogens such as TGF-ß. As examples, PGE₁ exhibits bimodal properties; it relaxes human corpus cavernosum tissue and smooth muscle cells up to a certain threshold level, beyond which relaxation is replaced by contraction [259]. Furthermore, in vitro cyclopentenone prostaglandins J₂ and 15-deoxy-^12,14-J₂ (15d-PGJ₂) exhibit effects typical of bimodal morphogens. In cultured rat basophilic leukemia cells they induce proliferation at low, and inhibit proliferation at high, concentrations; 15d-PGJ₂ stimulates proliferation at concentrations up to ~3–4 µM, but progressively inhibits proliferation at higher concentrations, forming a neutral zone of no effect on proliferation at a concentration of about 4 µM. At a concentration of 30 µM, apoptosis develops, which is mainly caused by mitochondrial effects rather than peroxisome proliferator-activated receptor gamma (PPAR-) signaling [260].

An exciting finding is that mitochondrial NADH-ubiquinone reductase, complex I of the electron transport chain, is an important target for 15d-PGJ₂, which inhibits the enzyme and significantly increases its rate of ROS formation (Fig. 4) [261]. This may be the first experimental evidence of direct prostaglandin regulation of OXPHOS. It points to regulation of mitochondrial energy metabolism as a possible link by which prostaglandins may affect morphogenesis, tissue remodeling, benign neoplasia, and carcinogenesis, and suggests that mitochondria may play a central role in regulating the radius of curvature and architecture of curved structures in biology

Other recent findings corroborate involvement of mitochondrial energy metabolism in neoplasia. For example, mutations in SDHB, SDHC, SDHD, nuclear genes coding for subunits of succinate dehydrogenase (SDH, Complex II) of the mitochondrial electron transfer chain are associated with paraganglioma and pheochromocytoma [262]. As succinate dehydrogenase links the Krebs cycle to the mitochondrial electron transfer chain, it reinforces the notion that regulation of mitochondrial energy generation may represent a common mode of controlling radius of curvature during development, post-natal remodeling, and, when disrupted, disease and neoplasia formation. Gradients of morphogens that regulate mitochondrial energy output and thereby growth could determine the morphogenesis of curved structures.

Several observations verify that eicosanoid metabolites are involved in development and remodeling. As examples, COX-1 regulates endothelial cells in vitro tubulogenesis [263]. Prostacyclin plays a vital role in embryogenesis; it is synthesized in the inner part of the blastocyst and is essential for normal embryogenesis [264]. Prostaglandins play a role in the perinatal closure of the ductus arteriosus (DA), and indomethacin and ibuprofen are efficacious in treating preterm infants with patent DA [265].

Proper arachidonic acid metabolism is vital to embryonic events that involve cellular movements and fusion and interference with eicosanoid metabolism can cause malformations. A study of the teratogenic potential of 5 common NSAIDs showed that sulindac was the most and indomethacin the least teratogenic drug [266]. As noted above, sulindac inhibits cPLA₂ and hence inhibits eicosanoid synthesis by both COX-1 and COX-2. This may explain why sulindac was the most teratogenic NSAID.

Common NSAIDs can prevent mouse palatal fusion. Both indomethacin and glucocorticoids inhibit palate fusion in mice palate explants. Adding PGE₂ to the media restores normal fusion. In rats, arachidonic acid prevents cleft palate formation and defects in neural tube development [267]. Death of apposing medial palate shelf edge epithelium and fusion is inhibited in mice by injection of glucocorticoids, which restrain COX-2 transcription. Administration of arachidonic acid, the substrate for COX-2 that stimulates COX-2 prostaglandin synthesis, rescues fusion. During normal palate fusion of apposing shelves, the epithelium dies at the point of fusion. Remarkably, the epithelium dies even if fusion fails [268]. As noted above, at highest concentrations 15d-PGJ₂ induces apoptosis. By analogy, loss of the epithelium in spite of lack of fusion is perhaps due to summation of morphogen concentration by morphogen release by opposing shelves. Normally, such high concentrations of bimodal morphogens would induce apoptosis of epithelium of the apposing shelves, which, when apposing shelves normally touch, results in shelf fusion.

Morphostats are morphogens that may serve to protect tissue architecture once established, and alteration in morphostat expression may alter tissue architecture [269]. Atherogenesis involves arterial remodeling. A speculative interpretation about NSAID effects on morphogen expression may explain why rabbits fed an atherogenic diet supplemented with cholic acid, a pro-inflammatory agent, with long-term aspirin supplementation, exhibited markedly intensified atherosclerosis compared to rabbits fed the same diet but not treated with aspirin [270]. As theories and evidence point to eicosanoid involvement in regulation of curvature of tubular structures, it follows that long-term alteration in eicosanoid metabolism caused by NSAID treatment could possibly have caused remodeling of the vessel wall, at least in part independently of its effect on lipid metabolism.

It is possible that during long-term inhibition of cyclooxygenase there may be a potential for complications caused by alterations in the radius of curvature of cystic, tubular, and glandular structures. Long-term inhibition may alter the diameter of blood vessels and alter cystic and glandular sizes as observed in many type of neoplasia. On the other hand, NSAIDs treatment may conceivably reverse neoplastic morphogenesis by modifying the synthesis of eicosanoids that affect mitochondrial OXPHOS. This may be one of the avenues by which aspirin, which inhibits the Krebs cycle, can exhibit anticancer properties. Other NSAIDs may possibly restore the architecture of neoplastic tissues by inhibiting overexpression of bimodal morphogen signalling that is associated with alterations in eicosanoid synthesis.

Combined, these findings, theories, and speculations support the notion that cyclooxygenase and eicosanoids can play important roles in morphogenesis of curved structures, the formation of cystic structures and tubular structures, such as arteries, folds and invaginations, and postnatal tissue remodeling such as observed in atherosclerosis and carcinogenesis. Long-term, continuous inhibition of COX-2, except perhaps for treatment of highly malignant tumors, should be used with caution, and possible benefits of intermittent treatment investigated, as it may permit a reset of natural biofeedback circuits during the off-periods between treatments, possibly permitting short-term beneficial effects of the therapy to dominate.

	Risk Reduction Strategies

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
The purpose of clinical risk management is to reduce severity of complications during NSAID treatment as well as to lower risks after cessation of NSAID use [271]. This discussion is focused on strategies that primarily target prevention of cardiovascular events during NSAID therapy.

Estimating cardiovascular risk is controversial. Strong opinions have been expressed that short-term studies cannot answer relevant coxib safety issues, and that coxibs treatment, for example for patients with osteoarthritis of the hip, do more harm than good [272]. The findings noted above that in apoE-null-mouse models longer duration of celecoxib treatment reverses its initial beneficial effects [19] may lend credence to such concerns.

Risk analyses and evaluations of therapeutic achievements are sometimes based on inaccurate clinical data and may suffer from use of imprecise terminology. Also, prescription practices may affect clinical findings: a recent Canadian study of 9,000 patients found that, paradoxically, patients at highest risk for cardiovascular complications were more likely to be treated with COX-2-selective NSAIDs than with traditional NSAIDs [273]. Furthermore, differences in various population groups in the frequency of certain polymorphism such as the -765GG alleles in the COX-2 promoter may lead to bias in large-scale comparison studies. It has been suggested that such factors may in part explain the different rates of cardiovascular disease in the north versus the south of Europe [194].

Proper information about clinical studies is essential to determine and communicate true risks to clinicians and patients. Employment of ghost writers for clinical studies should be strongly discouraged (or at least made clear when a manuscript is published [102]), and use of the rules initiated in 1978 by the Vancouver Group should be encouraged (International Committee of Medical Journal Editors, latest revision 2004; www.icmje.org/).

Risk factors such as CRP, oxLDL, fibrinogen, LP(a), cholesterol, isoprostanes, and asymmetrical dimethylarginine (ADMA) should be considered [274,275]. A meta-analysis that focused on four risk factors, blood pressure, platelet function, LDL and cholesterol, and homocysteine levels suggested that significant risk reduction could be achieved, with minimal side effects, using a combination of aspirin, anti-hypertension drugs, statin, and folic acid [276]. Homocysteine induces endothelial cell MCP-1, which can enhance monocyte chemotaxis [277]. Curcumin can prevent endothelial damage by homocysteine to porcine coronary arteries [278]. Homocysteine levels can be reduced by administration of folic acid and B vitamins. However, clinical studies have failed to show risk reduction through therapeutic lowering of homocysteine levels in patients with coronary artery disease [279].

In an attempt to retard the progression of coronary atherosclerosis, a clinical trial was undertaken using rofecoxib plus aspirin treatment in patients with acute coronary syndrome (ACS). The combined regimen significantly lowered blood levels for CRP; but IL-6 levels were only temporarily reduced. It was hoped that the reduction of CRP levels would lead to retardation of disease progression [280]. Similarly, treatment with a combination of rofecoxib and atorvastatin rapidly lowered post-operative CRP levels in patients with unstable angina [281]. A double-blind study involving 35 patients (with a history of at least one myocardial infarction) treated daily for 6 mo with a combination of enteric-coated aspirin (160 mg) plus rofecoxib (25 mg) resulted in long-lasting reduction in CRP and IL-6. However, endothelial expression of P-selectin (CD62), an adhesion molecule, and MMP-9 were unaffected, indicating unaltered endothelial function [282].

Low-dose (80mg daily) aspirin treatment alone lowers platelet activation and reduces thromboembolic events and atherosclerosis in humans [283]. In a rat model of arterial thrombosis, aspirin inhibited thrombus formation in a dose-dependent manner [284]. A short clinical trial of 9 healthy subjects showed that the antiplatelet effect of naproxen (500 mg, twice daily) is similar to aspirin (100 mg, daily). Both aspirin and naproxen reduced urinary 11-dehydro-TXB₂, an indicator of in vivo systemic biosynthesis of TXA₂. Naproxen, but not aspirin, also significantly reduced systemic prostacyclin biosynthesis [40]. On the other hand, as noted above, another study showed that naproxen significantly increases cardiovascular risk in patients administered naproxen, compared to patients treated with celecoxib [81,82]. Adding low-dose aspirin to anti-inflammatory treatments that employ selective COX-2 inhibitors should be seriously considered [285], as some even propose that treatment using selective COX-2 inhibitors without aspirin supplementation should be avoided [43].

Older patients are at higher risk when using NSAIDs [286]. For example, cardiovascular risk is higher in older compared with younger patients treated with rofecoxib [287]. Gastrointestinal risk must be evaluated before administration of aspirin, even low-dose aspirin. Primary or secondary cardioprotection and gastric protection, by administering proton pump inhibitors, or misoprostol, should be given serious consideration [288]. Also, aspirin inhibits in vitro and in vivo wound healing in experimental models [289,290]. Consideration should also be given to possible drug interactions between aspirin and other NSAIDs. For example, in a rat model of arterial thrombosis, aspirin treatment resulted in a dose-dependent reduction in thrombus weight. However, rofecoxib, celecoxib, and ibuprofen inhibited the antithrombotic effect of aspirin, but not the antithrombotic effects of diclofenac or flurbiprofen [284].

Intermittent hypoxia, such as may occur during sleep apnea, increases COX-2 expression [291] and may alter a patient’s response to COX-2 inhibition. However, the effect of use of coxibs in patients afflicted by sleep apnea is unknown at present.

Newer NSAIDs.  Nitric-oxide-aspirin (NO-aspirin, NCX-4016) has shown cardioprotective effects in animal models [292]. It consists of an acetylsalicylic acid group, and joined by an ester linkage a substituted benzene spacer, and a NO releasing group. In vivo, esterases separate the latter from the former and nitric oxide is slowly released from the liberated NO releasing group [293]. Nitric oxide induces vasodilation, and inhibits platelet aggregation and inflammation [294]. In a crossover study involving 48 healthy subjects, NCX-4016 prevented monocyte activation and matched the inhibition of cyclooxygenase by aspirin without the gastric side effects of aspirin [295]. On the other hand, it has been suggested that gastric formation of N-nitroso compounds may occur, with urinary excretion of N-nitrosodimethylamine (NDMA), a carcinogen. The benefit/risk ratio of long-term use of NO-aspirin is unclear [296].

Pharmacogenetic screening.  As has been previously discussed, the -765GC and, even more, the -765CC polymorphism of the COX-2 promoter or the presence of a 2G allele in the MMP1 promoter are associated with cardioprotection. Presence of a 6A allele is an independent risk factor for carotid artery stenosis. Also, presence of a second allele, a G allele, tripled the risk [297]. Polymorphism of the CCR2 gene may also influence the rate of atherogenesis [298]. These findings suggest that analyses of such polymorphism may become useful to identify patients who may be genetically at higher risk for myocardial infarction and stroke during treatment with selective COX-2 inhibitors.

Metabolism of NSAIDs may interact with the metabolism of other drugs such as warfarin, resulting in complications, caused, for example, by variant alleles of the common drug metabolizing enzyme cytochrome P450. Use of celecoxib by a patient on warfarin therapy led to bleeding due to reduced cytochrome P450 metabolizing capacity. The patient carried heterozygous CYP2C9*2 and *3 variant alleles [299].

Use of some coxibs has also been associated with reversible blurred or decreased eyesight, visual field defects, temporary blindness [300], and psychiatric problems induced by rofecoxib or celecoxib. It was speculated that patients with genetic variants of cytochrome P450 (CYP) 2C9, involved in drug metabolism, or of P-glycoprotein (P-gp) may be more susceptible to such adverse drug reactions. However, a small pharmacogenetic screening pilot study that examined buccal swab samples from patients for variants of CYP2C9 and P-gp failed to identify patients who are susceptible to vision disturbances [301].

Diet, life-style, and supplements.  Experiments with rat hearts suggest that the aging myocardium may be more sensitive to stress than young hearts, and that old hearts may benefit from oral CoQ₁₀ supplementation. Senescent isolated rat hearts recovered more slowly than hearts from young rats, but pretreatment with CoQ₁₀ restored old hearts to young levels [302].

Moderate alcohol consumption increases blood HDL levels and lowers the risk of cardiovascular mortality [303]. Red wine given to rats dilates arteries and lowers blood pressure. Likewise, moderate beer consumption reduces cardiovascular risk [304]. These findings may help to explain the cardioprotective effects of red wine consumption observed in epidemiological studies (ie, the French paradox) [305]. Flavenoids in red wine are strong antioxidants. Moreover, certain compounds from cabernet sauvignon grape skin selectively inhibit COX-2 [306].

Red wine consumption may reduce cardiovascular risk because it contains resveratrol, a stilbene found in grapes that has anti-inflammatory, cardioprotective, and anticancer properties. It is an inhibitor of the peroxidase and cyclooxygenase sites of COX-1 and a weak inhibitor of the peroxidase site of COX-2 [307]. Nevertheless, temperance is advised in ethanol consumption, because ethanol can induce iNOS and COX-2 via NF-B [308]. Aspirin can increase blood ethanol levels after alcohol consumption [309], and the rate of major upper gastrointestinal bleeding is increased in heavy drinkers who use aspirin or ibuprofen [310].

Carrageenan is a sea weed product used as an emulsifier in many foods, and, as noted above, it induces inflammation. Importantly, it was recently found that even if injected into the hind paw of rats, it induces COX-2 and PGE₂ expression in endothelial cells throughout the murine central nervous vascular system and CSF [311]. If the same occurs in coronary arteries, which seems likely, it might be advisable to limit intake of carrageenan-containing foods to lower the risk of coronary atherosclerosis. A diet rich in omega-3 PUFA competes with arachidonic acid entry into cellular membranes and may contribute to the synthesis of less inflammatory prostaglandins [30].

Healthy sleep and regular exercise are advisable. As noted, the pineal sleep hormone, melatonin, is a potent antioxidant. Sleep-disordered breathing elevates [312], but exercise-induced weight loss lowers, serum CRP levels [313]. In apparently healthy subjects, serum CRP levels between 1 and 3 mg/L predict an average risk for subsequent cardiovascular disease; lower levels predicts lower, and higher levels higher, risk [28].

Natural anti-inflammatory substances.  The supply of arachidonic acid determines the effectiveness of COX-2 inhibition [174]. Agents that inhibit arachi-donic acid synthesis may therefore become important. As an example, a hydroalcoholic extract of Trichilia catgua (catuaba, Mileacea), a natural anti-inflammatory substance, has been employed for various healing purposes. Recently, it was found that catuaba dose-dependently decreases platelet PLA₂ activity. It completely inhibites PLA₂ at a concentration of 120 mg/L [32]. It may therefore find use in controlling the build-up of arachidonic acid caused by cyclooxygenase inhibition by NSAIDs. Also, -mangostin, derived from Garcinia mangostana, a medicinal plant, inhibits in vitro LPS-induced COX-2 expression in rat glioma cells, without affecting their COX-1 expression or viability [314].

Cardiovascular risk may also be reduced by gingko biloba, but possible complications should be considered when herbal supplements are ingested along with NSAIDs. For example, ibuprofen use in a patient taking gingko biloba extract led to massive fatal intracerebral bleeding [315]. Furthermore, aloe vera, which has antioxidant and anti-inflammatory properties, harbors compounds that affect eicosanoid synthesis, hamper platelet aggregation, and extend bleeding time [316]. Importantly, because gingko biloba and aloe vera extracts can extend bleeding time, they should be used with caution before surgery [317].

	Conclusions

Top Abstract Introduction Cyclooxygenase NSAIDs Pathophysiology Molecular Pathology Neoplasia Theories and Speculations Risk Reduction Strategies Conclusions References

 
Cyclooxygenase activity impacts thrombogenesis, atherogenesis, and the risks of myocardial infarction and stroke in several ways. Elevated COX-1-dependent thromboxane synthesis enhances platelet aggregation and thrombosis, contracts blood vessels, and induces hypertension. By comparison, the effect of the inducible pro-inflammatory isoform, COX-2, appears to be far more complicated. Its expression can increase leukocyte-endothelial cell interaction and chemotaxis, which accelerate atherogenesis. On the other hand, increased COX-2-dependent synthesis of prostacyclin reduces monocyte chemotaxis, dilates blood vessels, reduces the risk of hypertension, and retards the development of atherosclerosis (Fig. 6).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Summary diagram of evidence based and hypothetical effects of NSAIDs on interaction of eicosanoid and mitochondrial metabolism associated with cardiovascular (CV) risk. Low-dose aspirin (ASA) inhibits platelet (PLT) thromboxane (TXA2) synthesis and reduces risk. NSAIDs such as coxibs that selectively inhibit COX-2 may have beneficial as well as detrimental effects. Whereas COX-2 is expressed at sites of inflammation, it may serve other functions in different cells and at different times. Inhibition of COX-2 reduces inflammation but also cardioprotection induced by HDL and apoE, which induce COX-2 that enhances prostacyclin (PGI2) that dilates arteries and opposes prothrombotic effects of TXA2. Statins (STAT) and C-reactive protein (CRP) inhibit PGI2 synthesis. Inhibition of cyclooxygenase increases generation of reactive oxygen species (ROS, circles with dots), which uncouples mitochondria by opening membrane pores (reducing membrane potential m).Arachidonic acid (AA) accumulates. It inhibits electron (e) transport from complex I to coenzyme Q10 (CoQ10, Q10) and from Q10 to complex III; both sites of inhibition increase production of ROS, which generates prostaglandin J2 (PGJ2) from AA and inhibits complex I and generates further ROS. STAT inhibits Q10 synthesis; dietary Q10 supplementation is recommended during STAT treatment. Oxidative phosphorylation (OXPHOS, partly illustrated) is powered by electrons from the Krebs cycle, which produces ROS when inhibited by aspirin. Importantly, aging reduces aconitase activity (Krebs cycle), reducing m and adenosine triphosphate (ATP) generation, which may in part explain why older patients are more at risk for CV complications during selective inhibition of COX-2. For details see text.

However, in several, but not all, clinical studies, use of coxib has been associated with increased rates of cardiovascular complications, which also have been observed in a few, but not in most, clinical studies of traditional NSAIDs, such as aspirin and naproxen. The very high selectivity of coxibs may contribute to their increased cardiovascular risk. First, lacking affinity for COX-1, most coxibs leave platelet thromboxane synthesis unaltered. Second, coxibs significantly lower COX-2-dependent endothelial synthesis of prostacyclin, a cardioprotective and antithrombotic eicosanoid that relaxes vascular smooth muscle cells, dilates blood vessels, lowers blood pressure, and defends against the formation of atherosclerotic lesions. Rofecoxib and valdecoxib have now been removed from the market. Celecoxib, which is less COX-2-selective, is still being marketed, but only with a black-box FDA warning label. A clue to explain the varying cardiovascular risks found in different clinical studies of coxib use may perhaps be emerging from animal models, in which differing effects of selective inhibition of COX-2 were related to the durations of drug administation.

Current evidence does not completely support the suggestion that cardiovascular complication of coxibs represents a class effect associated with all coxibs. Rather, each COX-2-selective inhibitor may have its own benefit/risk ratio. Some also exhibit properties that are independent of inhibition of COX-2. For example, celecoxib can regulate expression of MCP-1, reducing it during short-term, but increasing it during long-term, treatment. Whether the inconsistent findings emerging from clinical coxib studies are related to similar mechanisms associated with the duration of NSAID treatment is unknown. Until further studies on new and improved COX-2-selective inhibitors are completed, an interim solution may be to use coxibs that are less COX-2-selective and to compensate for the lack of thromboxane synthesis inhibition by adding low-dose aspirin to treatments, and to consider combining COX-2 inhibition with statin therapy., including Q₁₀ supplementation An alternative to coxibs is to use less-selective COX-2 inhibitors such as meloxicam.

It is recommended to evaluate clinical findings and laboratory tests of risk factors such as CRP before prescribing NSAIDs, especially highly selective coxibs. Special care is prudent in older patients who tend to show higher rates of cardiovascular complications associated with NSAID treatment. Importantly, recent studies on polymorphism of genes for COX-2, PGIS, CCR2, MMP1, and other genes, suggest that laboratory analyses may become useful to screen for patients who are genetically at higher risk for NSAID-associated myocardial infarction and stroke.

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