NSAIDs and Cardiovascular Diseases: Role of Reactive Oxygen Species.

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Title

NSAIDs and Cardiovascular Diseases: Role of Reactive Oxygen Species.

Permalink

https://escholarship.org/uc/item/0hv9b1cm

Authors

Ghosh, Rajeshwary

Alajbegovic, Azra

Gomes, Aldrin V

Publication Date

2015

DOI

10.1155/2015/536962

Peer reviewed

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Review Article

NSAIDs and Cardiovascular Diseases:

Role of Reactive Oxygen Species

Rajeshwary Ghosh,

1

Azra Alajbegovic,

1

and Aldrin V. Gomes

1,2

1Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616, USA 2Department of Physiology and Membrane Biology, University of California, Davis, CA 95616, USA

Correspondence should be addressed to Aldrin V. Gomes; avgomes@ucdavis.edu Received 22 December 2014; Revised 3 March 2015; Accepted 3 March 2015 Academic Editor: Mark J. Crabtree

Copyright Β© 2015 Rajeshwary Ghosh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs worldwide. NSAIDs are used for a variety of conditions including pain, rheumatoid arthritis, and musculoskeletal disorders. The beneficial effects of NSAIDs in reducing or relieving pain are well established, and other benefits such as reducing inflammation and anticancer effects are also documented. The undesirable side effects of NSAIDs include ulcers, internal bleeding, kidney failure, and increased risk of heart attack and stroke. Some of these side effects may be due to the oxidative stress induced by NSAIDs in different tissues. NSAIDs have been shown to induce reactive oxygen species (ROS) in different cell types including cardiac and cardiovascular related cells. Increases in ROS result in increased levels of oxidized proteins which alters key intracellular signaling pathways. One of these key pathways is apoptosis which causes cell death when significantly activated. This review discusses the relationship between NSAIDs and cardiovascular diseases (CVD) and the role of NSAID-induced ROS in CVD.

1. Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used over-the-counter drugs as well as the most prescribed class of drugs for a variety of conditions including pains, rheumatoid arthritis, osteoarthritis, musculoskeletal disorders, and other comorbid conditions [1]. Millions of people suffer from pain resulting in the prolonged use of NSAIDs being common. Besides reducing or relieving pain NSAIDs have been shown to be useful as anticancer agents in various kinds of cancers [2–4]. However, NSAIDs also have undesirable side effects including ulcers [5], bleeding [6], kid-ney failure [7,8], and increased risk of heart attack and stroke [8,9]. One of the mechanisms which has been associated with the adverse effects of NSAIDs is the generation of oxidative stress. The present review focuses on NSAIDs-induced ROS generation leading to cardiovascular diseases (CVD).

2. Types of NSAIDs

NSAIDs may be classified according to their mechanism of action. Nonselective NSAIDs like ibuprofen and naproxen,

which comprise one class, inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. A second class of NSAIDs (celecoxib and rofecoxib) targets only the COX-2 pathway and is termed as COX-2 selective inhibitors (also known as coxibs). COX selectivity is one of the determining factors that is considered when administrating NSAIDs to a patient. Administration of nonselective NSAIDs has been associated with side effects like peptic ulcer disease and gastrointestinal bleeding [10]. COX-2 selective NSAIDs have been shown to exhibit gastroprotective effects unlike the nonselective NSAIDs and are thus useful in patients with painful gastrointestinal conditions [10–12]. Another class of semiselective NSAIDs (indomethacin, meloxicam, and diclofenac) have a higher affinity for COX-2 but tend to inhibit the COX-1 pathway also [13]. However, irrespective of their mechanism of action, prolonged exposure to any class of NSAIDs has been shown to have potential adverse effects on cardiovascular events in patients with or without preexisting cardiovascular conditions, depending on the duration and dosage of these drugs [14,15] (Table1). Patients with preexisting cardiovascular conditions such as coronary

Volume 2015, Article ID 536962, 25 pages http://dx.doi.org/10.1155/2015/536962

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T a ble 1: L ist o f NSAID s w it h their re co mmended d os es an d le vels in the cir cula tio n. G ener ic n am e C hemic al na me Reco mmended d os ag es (ma y va ry dep endin g o n th e age an d con d it ion ) L ev els in th e cir cu la ti o n (πœ‡ Ma n d πœ‡g/mL) (dos e d ep enden t) Tr ad e n am e Dic lo fenac {2-[(2,6-Dic h lo ro p h en yl )a mino]p h en yl acet ic acid 25 mg – 15 0 mg /d ay * 5 πœ‡M (1.5 πœ‡g/mL) an d 7 πœ‡M( 2. 0 πœ‡g/mL) [f o r 50 m g an d 75 m g d os es, resp ec ti vel y] Z o rv ol ex ,C at afl am ,C ambi a, V o lt are n , Vo lt ar en ge l, A rt h ro te c (c o m b in edw it hm is o p ro st o l) ,F le ct o r, Z ip so r, P enn sa id In do met h acin [1 -(4-C hlo ro b enzo yl )-5-met ho x y-2-met h yl -1H-indo l-3-y l]acet ic acid 25 mg – 20 0 mg /d ay * 3 πœ‡M–6 πœ‡M( 1t o2 πœ‡g/mL) T iv o rb ex, Indo cin, In do cin SR , In do-L emmo n, In do met h ag an K et o p ro fen 2-(3-B enzo yl p h en yl ) p ro p ano ic acid 25 m g–3 0 0 m g/ da y ** 2 πœ‡Mt o2 2 πœ‡M (0.4 3 to 5.6 2 πœ‡g/mL) [5 0 m g d os e] Or u va il ,N ex cede Ib uprofe n 2-(4 -I so but ylph en yl ) prop an oi c ac id 10 0 mg – 80 0 mg /d ay * 97 πœ‡M–29 1πœ‡ M( 20 – 6 0 πœ‡g/mL) [4 0 0 m g dos e] A d vil ,M o tr in an d N ur o fen, V ico p ro fe n (c o m b in edw it hh yd ro co d o n e) ,D u ex is (co m b ined w it h fa m o tidine) Na p ro xe n (2S)-2-(6-M et ho x y-2-na p h th yl) p ro p an o ic acid 250 m g– 1000 m g/ d ay (b o tho ra la n d IV ) * 37 7 πœ‡M( 95 πœ‡g/mL) [500 m g d o se o n ce ev er y 12 h o r 1000 m g dos e fo r 5 da ys ] Na p ro sy n ,E C -Na p ro sy n ,N ap ra p ac (co p ac kag ed w it h la n so p razo le), Al ev e, A cco rd ,An ap ro x D S, An ap ro x An ta lgin, A p ra nax, T reximet (co m b ined w it h suma tr ip ta n succina te) and V imo vo (co m b ined w it h es o mep razo le mag n esi um) Su li n d ac {(1Z)-5-Fl u o ro-2-met h yl -1 -[4-(met h ylsulfin yl )b enzy lidene]-1H-inden-3-yl }acet ic acid 20 0m g– 4 0 0m g/ d ay ** 1.4 πœ‡M–6 πœ‡M (0.5–2.0 πœ‡g/mL) [co n st an t fo r all dos es ] Cl in o ril Ke to ro la c 5-B enzo yl-2,3-dih ydr o-1H-p yr ro lizine-1 -ca rb o x yl ic acid 10 mg– 4 0 m g/ d ay ** 39 πœ‡M( 10 πœ‡g/mL) T o rado l, Sp ri x Pi ro xi ca m 4-H ydr o x y-2-met h yl -N-(2-p yr idin yl )-2H-1,2-b enzo thiazine-3-ca rb o xa mide 1,1 -dio xide 0.2 m g–20 m g/ da y * 5 πœ‡Mt o6 πœ‡M (1.5 to 2 πœ‡g/mL) [sin gl e 20 m g dose ] 9 πœ‡Mt o2 4 πœ‡M( 3– 8 πœ‡g/mL) [f o r 20 m g re p ea ted dos es] Fe ld en e

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Ta b le 1: C o n ti n u ed . G ener ic n am e C hemic al na me Reco mmended d os ag es (ma y va ry dep endin g o n th e age an d con d it ion ) L ev els in th e cir cu la ti o n (πœ‡ Ma n d πœ‡g/mL) (dos e d ep enden t) Tr ad e n am e N imesulide N-(4-N it ro -2-p h eno x yp hen yl)met h an esulf o na mide 10 0 m g tw ice d ai ly ** 10 πœ‡Mt o2 0 πœ‡M (2.86 to 4.58) m g/L [1 0 0 m g] [ 17 5 ] Su lide ,N imalo x, M esulid ,X ilo x, N is e, N ex en, N ido lo n Difl unis al 2 σΈ€  ,4 σΈ€  -Difl uo ro -4 -h yd ro x y-3-b ip h en yl ca rb o x yl ic acid 250 m g– 1000 m g **16 4 πœ‡M( 4 1πœ‡ g/mL) [2 50 m g dos es] 348 πœ‡M( 87 πœ‡g/mL) [5 0 0 m g dos es] an d 49 6 πœ‡M( 12 4 πœ‡g/mL) [sin gl e 10 0 0 m g dos e] Do lo b id F lu rbiprofe n 2-(2 -F lu oro -4 -biph en yl yl )p rop an o ic ac id 50 m g– 30 0 m g/ d ay ** 60 πœ‡M (1 4 m g/L) [0.6 5 m g/kg o f flu rb iprofe n IV ] [ 17 6 ] Ans aid M ef ena mic acid 2-[(2,3- Dimet h yl p h en yl )a mino]b enzo ic acid 250 m g fo ll o w ed by 500 m g ev er y 6 h o u rs * 83 πœ‡M( 20 πœ‡g/mL) [2 50 m g sin gle dos es] Po n st el To lm et in [1 -M et h yl-5-(4-met h ylb enzo yl )-1H-p yr ro l-2-y l]acet ic acid 5m g– 18 0 0 m g/ d ay ** 15 6 πœ‡M( 4 0 πœ‡g/mL) [4 0 0 m g o ral dos e] T o lec tin, T o lec tin DS, T o lec ti n 6 0 0 Th e ta b le li st s o n ly th o se N SA ID s th at h av e b ee n m en ti o n ed in T ab le 3 al o n g w it h their re co mmended d os es an d resp ec ti ve co n cen tra tio n s in the cir cula tio n. M o st o f th e inf o rma tio n re ga rd in g the dos ag es and le ve ls in se ru m o r p lasma o f the NSAID s has b een tak en fr o m th e F D A ap p ro ved si te , h tt p ://w w w .dr u gs.co m .N imesulide is the o n ly NSAID in the list th at is no t ava ila b le in th e USA. R ef er ences fo r th e p lasma co ncen tra tio n s o f fl u rb ip ro fe n and nimesulide ar e gi ven in th e ta b le . *Se ru m co n cen tra tio n s, **Plasma co ncen tra tio n s.

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Naproxen, ibuprofen, ketoprofen

(cytosolic/secreted) (induced by thrombin, injury/inflammation, hormones like epinephrine,

angiotensin II) Cyclooxygenase pathway COX-1 (constitutive) COX-2 (inducible) Valdecoxib, celecoxib, rofecoxib(Coxibs) NSAIDS Prostaglandin G2 Prostacyclin (endothelium) NSAIDS PGD syn thas e Thr om boxa ne syn thas e PGI syn thas e PGF syn thas e PGE syn thas e Potent inhibitor of platelet aggregation, vasodilator Potent activator and stimulator of platelet aggregation, vasoconstrictor (smooth muscle) Arachidonic acid Phospholipase A2

Protein degradation Protein synthesis

Leukotriene COXIBS Atherosclerosis, acute myocardial infarction, thrombosis Platelet aggregation Lipoxygenase pathway Homeostasis disruption COX-1, COX-2 COOH CH3 Cytokines IL-1, IFN-𝛾, growth factors PGG2 Prostaglandin H2 PGH2 PGF2

Homeostasis maintained by PGF2and PGE2

(brain) neurophysiological functions PGD2 TXA2 TXB2 PGI2 (platelets) βˆ— TXA2 PGI2 βˆ— PGE2 Membrane phospholipid

Figure 1: Inhibition of cyclooxygenase pathway by NSAIDs. Coxibs as well as nonselective NSAIDs inhibit the formation of the metabolites of the cyclooxygenase pathway thereby disrupting the homeostasis maintained by these metabolites. Coxibs cause an imbalance between the levels of thromboxane and prostacyclin being more favorable towards thromboxane and decreasing prostacyclin levels leading to the aggregation of platelets and causing thrombosis.

artery disease, hypertension, and history of stroke are at the greatest risk of cardiovascular events after taking NSAIDs [14,15]. Patients who have recently had cardiovascular bypass surgery are advised not to take NSAIDs due to a high risk of heart attacks [16,17]. The increased selectivity for COX-2 has also been reported to increase the risk of various CVD [18,19]. Meta-analyses of several trials have shown that coxibs

are associated with a high risk of atherothrombotic vascular events [20].

3. Mechanism of Action of NSAIDs

NSAIDs exert their pain relieving effect mainly by inhibit-ing the cyclooxygenase pathway (Figure 1). This pathway

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is responsible for the conversion of arachidonic acid to prostaglandins and thromboxanes [21]. Although COX is officially known as prostaglandin-endoperoxide synthase (PTGS) the abbreviation β€œCOX” is commonly used for cyclooxygenase-1 and cyclooxygenase-2 in medicine. In genetics, the abbreviation PTGS is officially used for the cyclooxygenase family of genes and proteins to prevent ambiguity with the cytochrome c oxidase family of genes and proteins which are also abbreviated COX. Arachidonic acid is the main precursor to the formation of various eicosanoids in the cyclooxygenase pathway. Of all the metabolites formed in the arachidonic acid metabolism, thromboxane A2 in the platelets is the major product which along with prostacy-clin (prostaglandin I2) maintains vascular homeostasis [22] (Figure 1). Both these eicosanoids (thromboxane A2 and prostaglandin I2) have opposing effects. While thromboxane is well known for its role in vasoconstriction and aggregation of platelets, prostacyclin is important for platelet aggregation inhibition and vasodilation.

The COX enzyme is present as two isoforms, each with distinct functions: COX-1 is constitutively expressed in the stomach, kidneys, intestinal mucosa, and other tissues [23]. It protects the mucosal lining of the stomach and plays an important role in vasoconstriction and platelet aggre-gation [23]. On the other hand the inducible COX-2 is upregulated during times of inflammation where it causes vasodilation [24]. COX-1 and COX-2 are similar in molecular weights, 70 and 72 kDa, respectively, and show 65% homology with near-identical catalytic sites (based upon information from UniProtKB/Swiss-Prot database). The critical differ-ence between the isoenzymes, which permits the selective inhibition of each isoform, is the substitution of isoleucine 523 in COX-1 with valine in COX-2 [25]. The presence of valine, which is a smaller amino acid than isoleucine, allows drugs entrance to a hydrophobic side-pocket only accessible in COX-2. Expression of both isoforms, COX-1 and COX-2, may be upregulated and downregulated under various pathological conditions [26]. It is likely that the classification of the COX enzymes into two isoforms was an oversimplification [26] as COX-2 may be constitutively expressed in the brain [27], kidney [28], and testes [29]. In fact immunohistochemical studies have revealed the constitutive expression of COX-2 mRNA in the lung, thyroid gland, spleen, and adipose tissue, which was greater than COX-1 in these tissues, and in the liver both isoforms were expressed equally [29]. Therefore the idea that COX-2 can only be expressed under inducible conditions is unlikely since recent evidence suggests their occurrence in various human tissues under normal conditions.

Coxibs disrupt the balance between the levels of throm-boxane A2 and prostaglandin I2 leading to atherosclerosis, thrombosis, and other cardiovascular complications. Coxibs, through their selective inhibition of COX-2, inhibit endothe-lial cell synthesis of prostacyclin [30]. In the ApoEβˆ’/βˆ’

mice (model for atherosclerosis), deletion of the prostacyclin receptor increased atherogenesis with no such effect observed in thromboxane receptor deleted mice [30]. Apart from its role in the inhibition of cyclooxygenase pathway, NSAIDs have been shown to cause cell death by the inhibition of

the Akt signaling pathway [31], downregulation of the NF-πœ…B

pathway [32], downregulation of the Bcl pathway [33], upreg-ulation of the nonsteroidal activated gene-1 [34], and altering the p53 pathway [35], all of which have been suggested to be involved in apoptosis [36]. Apoptosis (programmed cell death) induced by NSAIDs has been suggested to be due to oxidative stress caused by increased generation of reactive oxygen species (ROS) [37].

A series of mechanisms are involved wherein NSAIDs exert their cardiotoxic effects and cause various cardiac conditions. Various non-NSAID drugs like doxorubicin, azidothymidine, and cisplatin have been shown to induce oxidative stress as a consequence of elevated ROS levels [38]. Doxorubicin, for example, induced cardiotoxicity through DNA damage and apoptosis in cardiac cells as a result of oxidative stress which were reduced by the antioxidant effect of statin [37]. It is possible that the oxidative stress induced by NSAIDs, which is known to cause apoptosis and cell death, is significantly involved in causing cardiovascular dysfunction (Figure2).

4. Incidences of CVD Induced by NSAIDs

Various clinical trials have been made during the past few years regarding the safety and effectiveness of NSAIDs in CVD (Table2). Most trials focused on the CVD outcome of NSAID use in patients with a previous history of cardiovas-cular disease. Few trials were carried out on patients with no history of CVD. An important finding is that not only nonselective NSAIDs lead to the development of hyperten-sion in both normotensive and hypertensive individuals [39], but their use interferes with the antihypertensive medications except for the calcium channel blockers [40]. The risk of atrial fibrillation, heart failure, myocardial infarction, and other cardiovascular conditions also increased in patients with a history of these pathological conditions (Table2).

The CVD related outcomes in patients enrolled in the REACH (REduction of Atherothrombosis for Continued Health) registry showed that in patients with established stable atherothrombosis, use of NSAIDs increased the inci-dences of myocardial infarction and cerebrovascular con-ditions [41]. Additionally, this paper reported that the use of NSAIDs with other antiplatelet drugs (except aspirin) increased the rate of cardiovascular events like cardiovascular death, myocardial infarction, and stroke [41]. Although a few studies suggest that COX selectivity does not seem to be a determining factor for myocardial infarction [13,42], several studies suggest that coxibs elevate the rate of incidences of CVD compared to nonselective NSAIDs [18–20]. Several clinical trials have been completed and are still ongoing, but some inconsistencies in the results exist regarding the effect of different types of NSAIDs on cardiovascular outcomes (Table2). One meta-analysis report found that∼75% studies investigating the cardiovascular risk in new NSAID users reported an increase in the occurrence of cardiovascular diseases within the first month of NSAID use [43]. Overall, the different trials showed that several NSAIDs increased the risk of CVD both at low and high doses [41, 43–45]. Targeted inhibition of COX-2, even for a short term, was

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T a ble 2: Summa ry o f resul ts fr o m cl inical tr ials o n th e ad ver se eff ec ts o f NSAID s o n ca rdio vas cula r dis eas es. NSAID C linical tr ial n am e/lo ca ti o n Dura ti o n o f st ud y N um b er o f p at ien ts selec te d E ff ec t Ref er ences C eleco xib ,na p ro xen so di um, an d p laceb o Alzheimer ’s D is ea se An ti -I nfl am ma to ry Pre ve n ti on T ri al (AD A PT)/USA 4y ea rs 2,5 28 p at ien ts w it h a fa mil y hist o ry o f A lzheimer ’s Dis eas e. CVD/CB V d ea th ,MI, st ro ke ,C HF ,o r TI A in the celeco xib-, n ap ro xe n-, and p la ce b o -t re at ed gr oup s we re 5. 54 % , 8.2 5%, and 5.6 8%, re sp ec ti ve ly .D ea th ra te s in the cas e o f pa tien ts ta kin g NSAID s w er e hig her b u t no t st at ist icall y significa n t [ 44 ] C eleco xib ,r o fe co xib ,va ldeco xib , d ic lo fe n ac ,n aproxe n ,i buprofe n , difl unis al ,et o d o lac, fe no p ro fen, fl u rb ip ro fe n, indo met h acin, ke to p ro fen, ke to ra lac, mec lo fena m at e, mef ena mic acid, melo xic am, nab u meto ne, o xa p rozin, p ir o xic am, su lind ac, an d tolmet in No n e/ U SA 5 ye ar s 74,8 38 us er s o f NSAID s and 23 ,5 35 us er s o f o th er dr ugs. The ad ju st ed ra te ra tio fo r Ro fe co xib w as 1.1 6 fo r M I and 1.15 fo r str o ke w hic h w as th e hig hest am o n g al lo ther NSAIDs inc ludin g o th er co xibs. N ap ro xe n mo dest ly re d u ce d the ra te ra ti o (RR fo r MI 0.0 67 an d R R fo r st ro ke 0.08 3) o f ca rdio vas cula r ev en ts [ 17 7 ] C el ec o xi b, ib uprofe n N o n e/ C h ie ti ,C h ie ti ,I ta ly 3 m on th s 24 pa ti en ts under go in g asp ir in tr ea tmen t fo r ca rd io prot ec ti on Ib u p ro fen in te rf er ed wi th th e in h ib it io n o f p la telet C O X-1 n ecess ar y fo r ca rd io prot ec ti on by as pi ri n [ 17 8 ] Dic lo fenac, ib u p ro fen, indo m et hacin, ke to p ro fen, n ap ro xe n, mef ena mic acid , p ir o xica m, te no xica m, to lf ena mic acid , acec lo fenac, ti ap ro fe nic acid ,mef ena mic acid ,et o d o lac, na b u met o ne ,nimesu lide , an d m el ox ic am ,r of ec ox ib ,c el ec ox ib , va lde co xib ,a nd eto rico xib N o ne/Fin la nd 3 ye ars 33 ,3 0 9 p at ie n ts w it h fi rs t MI, 13 8,94 9 co n tr o ls Their re sul ts sug ge st th at C O X-s elec ti vi ty do no t d et er mine the ad ve rs e eff ec t o f CVD by NSAID s, at le ast co n cer nin g MI. N o NSAID is MI-p ro te ct iv e [ 13 ] E to rico xib an d dic lo fe nac M u lt ina tio nal E to ri co xib an d D ic lo fe nac A rt hr it is L o n g-t er m (MED A L) St ud y P rogra m/M u lt ina tio nal 18 mo n ths 34,7 01 p at ien ts (2 4,9 13 wi th ost eoa rt hr it is an d 9,7 87 wi th rh eu m at o id ar th ri ti s) Thr o m b o ti c ca rdio vas cula r ev en ts o cc u rr ed in 32 0 p at ien ts in the et o rico xib gr o u p and 32 3 p at ien ts in the dic lo fe nac gr o u p w it h ev en t ra te s o f 1.2 4 an d 1.3 0 pe r 10 0p at ie n t-ye ar s an dah az ar d ra ti oo f 0 .9 5 for et or ic ox ib co m p are d to dic lo fe nac. L o n g te rm us ag e o f ei ther o f th es e NSAID s h ad simi la r eff ec ts o n the ra te s o f thr o m b o ti c ca rdio vas cula r ev en ts [ 179 ] E to rico xib an d dic lo fe nac Et o ri co xi b ve rs u s Dic lo fenac So di um Gast ro in test ina l T o lera b ili ty an d Eff ec ti ve ness (ED G E) trial /U SA 9. 3 m on th s for et o rico xib an d 8.9 mon ths for d ic lo fe n ac 7, 111 pa tien ts et o rico xib (𝑛=3 ,593 )o r dic lo fe nac so d iu m (𝑛=3 ,518 ) The ra te o f th ro m b o tic CV ev en ts was 1.3 0 an d 1.2 4 in u se rs o f et o rico xib (9 0 m g) an d dic lo fe nac (15 0 m g), re sp ec ti ve ly ,w it hin 28 da ys [ 18 0 ]

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Ta b le 2: C o n ti n u ed . NSAID C linical tr ial n am e/lo ca ti o n Dura ti o n o f st ud y N um b er o f p at ien ts selec te d E ff ec t Ref er ences Ib u p ro fen, n ap ro xe n o r lumiraco xib The Thera p eu tic Ar th ri ti s R es earch an d Gast ro in test ina lE ven t T ri al (T AR GET)/I n te rn at io nal Ex te n d ed re po rt 18,3 25 p at ien ts wi th ost eoa rt hr it is Ib u p ro fen incr ea se d risk o f thr o m b o sis an d C HF co m p ar ed to lumiraco xib (2.1 4% ve rs us 0.2 5%) amo n g asp ir in us er s. N ap ro xen co nf er s lo w er ri sk re la ti ve to lumiraco xib amo n g no nasp ir in us ers [ 181 ] D ic lofe n ac ,i b uprofe n ,a n d n aproxe n N o n e/ U SA 7 ye ar s N D P ro lo n ge d exp osur e to dic lo fe nac incr ea se s the ri sk o f AMI (r ela ti ve ra ti o = 1.9–2.0) un li ke ib u p ro fen o r na p ro xen [ 182 ] E to rico xib an d dic lo fe nac E D G E II/USA 19 .3 m o n ths for et o rico xib an d 19 .1 mon ths for d ic lo fe n ac 4,086 p at ien ts wi th rheuma to id ar th ri ti s et o rico xib 𝑛=2 ,032 ; dic lo fe nac 𝑛=2 ,054 The ra te o f o cc u rr ence o f AMI w as hig her in dic lo fe nac (0.6 8) tr ea ted pa ti en ts th an in et o rico xib us er s (0.4 3). Als o an o vera ll incr ea se in ca rd iac ev en ts w as o b se rv ed in dic lo fe nac tr ea ted gr o u p (1.1 4 ) ver sus th e et o ri co xi b gr o u p (0 .8 3) [ 18 3 ] C el ec o xi b (a t 4 0 0m g Q D ,2 0 0m g tw o ti m esad ay( B ID ),o r4 0 0 m g B ID ) No n e/ U SA 3 ye ar s 7, 95 0 p at ie n ts (w it h ar th ri ti s and o ther co ndi tio n s) The haza rd ra ti o fo r th e C VD ,MI, HF ,o r th ro m boe m b o li c ev en t w as lo w es t fo r th e 4 0 0 m g o nce a da y (1.1), in te rm edia te fo r th e 20 0 m g-BID dos e (1.8), and hig hest fo r the 4 0 0 m g-BID d os e (3.1) [ 18 4 ] Ib uprofe n ,n ap roxe n ,d ic lo fe n ac , melo xica m, indo met h acin, p ir o xica m, an d m ef ena mic acid No n e/ U K p ri m ar y ca re 20 ye ar s 72 9, 294 NSAID us er s; 44 3, 04 7 co n tr o ls In cr ea se in th er el at iv er at ef o r M I w it h cu m u la ti ve an d d ai ly d o se of ib uprofe n an d dic lo fe nac. H ig h er ri sk o f MI wi th dic lo fe nac u se (r ela ti ve ra tio = 1.21) th an ib u p ro fe n (r el at iv e rat io = 1. 0 4 ) o r n ap rox en (r el at iv e rat io = 1. 03 ) [ 185 ] E to rico xib an d dic lo fe nac The MED A L St ud y/M u lt ina tio nal 19 .4 to 20.8 mo n th s Et o ri co xi b 60 m g/ d ;𝑛=6 ,769 90 m g/ d ;𝑛=5 ,012 Dic lo fenac 𝑛=1 1, 717 The th ro m b o tic CV ri sk HR o f et o rico xib to dic lo fe nac = 0.9 6 .P ro lo n ged us e o f ei th er NSAID re su lt ed in an incr ea se d ri sk o f th ro m b o ti c C V ev en ts [ 18 6 ] N aproxe n ,i buprofe n ,d ic lo fe n ac , celeco xib ,a n d ro feco xib No n e/ U SA 6 ye ar s 4 8,5 6 6 p at ien ts re cen tl y h o spit al iz ed for m yo ca rd ia l infa rc tio n, re vas cula riza tio n, o r unst ab le an gi na p ec to ris CVD risk incr eas ed wi th sho rt ter m (< 90 da ys) u se fo r ib u p ro fen wi th incidence ra te ra ti os o f 1.67 ,dic lo fe nac 1.86, celeco xib 1.3 7, an d ro feco xib 1.4 6 ,b u t no t fo r n ap ro xe n 0.88 [ 51 ]

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Ta b le 2: C o n ti n u ed . NSAID C linical tr ial n am e/lo ca ti o n Dura ti o n o f st ud y N um b er o f p at ien ts selec te d E ff ec t Ref er ences C eleco xib ,r o fe co xib ,va ldeco xib , ib u p ro fen, n ap ro xe n, dic lo fe nac, an d indo met h acin No n e/ U SA 7 ye ar s 61 0 ,0 01 p at ie n ts ;w it h o ut CVD 𝑛 = 525 ,249 ;w it h hist o ry o f C VD 𝑛 = 84752 Ro fe co xib (10.9 1 ev en ts/10 0 0 p er so n -y ea rs ), valdeco xib (12.41 ev en ts /1 0 0 0 p ers o n -y ea rs ), and indo met h acin (13.2 5 ev en ts/10 0 0 p ers o n -y ea rs ) inc re as ed C V D risk in p at ien ts wi th no hist o ry o f C VD . Ro fe co xib u se incr ea se d risk o f ca rdio vas cula r ev en t in p at ien ts w it h C V D( 30 .2 8e ve n ts /1 0 0 0 p er so n -y ea rs ) [ 187 ] Ib u p ro fen, dic lo fe nac, ro fe co xib , celeco xib ,a n d n ap ro xe n N o n e/ D an is h p opu la ti on 9– 34 d ay s 15 3, 4 65 h ea lt h y in d iv idu al s D o se dep enden t incr eas e in ca rd io va scul ar ev en ts d u e to u se o f dic lo fe nac (HR = 1.6 3), ro feco xib (HR = 2.13) and celeco xib (HR = 2.01) was obs er ve d [ 188 ] C el ec o xi b, ib uprofe n ,a n d n aproxe n The Pr osp ec tive R an d om iz ed E va lu at ion of C eleco xib In teg ra te d Sa fety ve rs u s Ib uprofe n O r Na p ro xe n (P RECIS IO N ) tr ial/M u lt ina tio nal 20 0 9-o ngoi ng 20 ,000 pa ti en ts w ith sym p to m ati c o st eoa rth ri ti s o r rheuma to id ar th ri ti s at h ig h ri sk for or w it h est ab lished C VD This tr ial will det er mine the ca rdio vas cula r sa fe ty o f th e NSAID s [ 18 9 ] ND N o ne/D enma rk 10 ye ar s 107 ,0 92 p at ien ts w it h fi rs t incidence o f H F The haza rd ra ti o fo r de at h d ue to MI o r HF was 1.7 0, 1.7 5, 1.3 1, 2.08, 1.22, an d 1.2 8 for rofe cox ib ,c el ec ox ib ,i buprofe n , dic lo fe nac, na p ro xen, and o ther NSAID s, re sp ec ti ve ly [ 19 0 ] Ib uprofe n ,d ic lo fe n ac ,n aproxe n , ro fe co xib ,celeco xib ,va ldeco xib ,a nd et o rico xib N o ne/The N et herl an ds 4 ye ars 4 85,05 9 sub je ct s w it h fi rs t h o spit al is at io n for ac ut e m yo ca rdial infa rc tio n, CV , an d ga str o in te sti n al ev en ts AMI risk w it h celeco xib (O R 2.5 3), ro fe co xib (O R 1.6 0 ), ib u p ro fen (O R 1.5 6), an d dic lo fe nac (O R 1.5 1) w as significa n tl y incr ea se d .S ignifica n t incr eas e in C V risk wi th cu rr en t u se o f indi vid u al C O X-2 in hib it o rs an d tNSAID s (O R fr o m 1.1 7 to 1.6 4 ). Si gnifica n t d ecr eas e in A MI wi th cu rr en t u se o f na p ro xen (O R 0.4 8) [ 191 ] Ib uprofe n ,n ap roxe n ,d ic lo fe n ac , et o d o lac, celeco xib ,r o fe co xib N o ne/N o rt h er n D enma rk 10 ye ar s 32 ,6 02 p at ie n ts w it h fi rs t at ri al fi b ri ll at io n o r fl u tt er an d 32 5, 91 8 p opu la ti on co n tr o ls In cr ea sed ri sk o f atrial fi b rilla tio n o r flutt er w as 4 0 – 70 % (l owe st for no n-s ele ct ive NSAIDs and hig hest fo r C O X -2 in h ib it or s) . [ 192 ]

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Ta b le 2: C o n ti n u ed . NSAID C linical tr ial n am e/lo ca ti o n Dura ti o n o f st ud y N um b er o f p at ien ts selec te d E ff ec t Ref er ences ND N o ne/D enma rk 10 ye ar s 83 ,67 7 p at ien ts o f w hic h 4 2.3% recei ve d NSAID s D ea th/r ec u rr en t M I d ue to NSAID tr ea tm en t (e ven sho rt ter m) was significa n tl y hig her .Dic lo fenac p o se d the hig hest ri sk o f all NSAID s [ 193 ] ND IN te rn at io n al V E rap am il T ra n do la p ril ST ud y (INVES T)/M u lt ina tio nal 7y ea rs 88 2 chr o nic NSAID us er s an d 21,6 94 n o n ch ro nic NSAID us er s. P at ien ts wi th h yp er ten sio n an d clinicall y st ab le co ro n ary ar te ry dis eas e P rima ry o u tco me li ke al l-ca us e m o rt ali ty , no nfa tal MI, o r no nfa tal st ro ke an d se co nd ar y indiv id u al o u tco mes li ke al l-ca us e m o rt ali ty ,c ar dio vas cu la r mo rt ali ty ,t o ta lMI, an d to tal str o ke o cc u rr ed at a rat e o f 4 .4 ev en ts p er 10 0 pa ti en t-y ea rs in the ch ro nic NSAID gr oup ,ve rsu s 3. 7 eve n ts p er 10 0 p at ien t-ye ar s in the no nchr o nic NSAID gr o u p [ 111 ] C eleco xib ,r o fe co xib ,ib u p ro fen, dic lo fe nac, na p ro xen, and o ther s No n e/ D en m ar k 13 ye ar s (wi th fir st MI) 12 8,418 p at ien ts (7 7% pa rt ici p at ed in th e st u d y) In cidences o f co ro na ry de at h o r n o n fa ta l re cu rr en t M I w it h N SA ID s u se re m ai n uncha n ge d w it h the ti me el ap se d even afte r 5 ye ars [ 19 4 ] ND N o ne/ d at a o b ta ine d fr o m RE d u ct io n o f A th erot h romb o si s for C o n tin ued H ea lt h (REA C H) re gist ry w hic h inc ludes p at ien ts fr o m La ti n A m er ic a, N o rt h Amer ica, E u ro p e, A sia, th e Midd le E ast, and A u st ra li a 4y ea rs 4,4 20 NSAID us er s; 39 ,67 5 NSAID no n u se rs 1.1 6 -f o ld hig her risk o f C VD ,M I in NSAID us er s [ 41 ] MI: m yo ca rdial infa rc tio n ,H F :hea rt fa il u re ,C VD: ca rdio vas cu la r d is eas es, CB V :cer eb ro vas cu la r d is eas es, CHF :co n ge sti ve h ea rt fa il ur e, HR: h aza rd ra tio ,O R :o dd ra tio ,ND: n o t defin ed ,RR: ra te ra tio ,a n d T IA : tra n sien t is chemic at tack. V ar io u s clinica lt ri al s sug ge st th e incr eas ed incidences o f CVD in N SAID us ers. The ta b le lists o n ly th e clinica lt ri al s re po rt ed b etw een 20 0 6 an d 20 14 .

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Thrombosis Nonselective (inhibits COX-1 and COX-2)

naproxen, ibuprofen, ketoprofen

COX-2 selective valdecoxib, celecoxib, rofecoxib

Apoptosis

NADPH oxidases Cell signalling pathways affected

Lipoxygenases Xanthine oxidases Nitric oxide synthases

(free radicals)

Acute myocardial infarction,

reperfusion injury, heart failure Oxidative stress

Cardiovascular diseases ROS

Cytochrome p450

Arachidonic acid metabolism

Platelet aggregation on the site of atherosclerotic plaque

rupture on the coronary artery wall of the heart NSAIDs

↑ Nonsteroidal activated gene- 1 ↑ p53 ↑ Bax ↓ Bcl-2

↑ Mitochondrial cytochrome c ↑ Apoptosis inducing factor ↓ Akt ↓ NF-πœ…B ↓ Proteasome activity O2βˆ™βˆ’, H2O2, βˆ™OH ↓Prostacyclin ↑Thromboxane Thromboxane/prostacyclin imbalance

Figure 2: Pathways involved in the development of cardiovascular diseases by NSAIDS. The figure shows the upregulation and downregulation of various pathways by NSAIDs leading to the development of CVD. Mitochondria play a major role in the generation of ROS induced by NSAIDs followed by oxidative stress and finally CVD.

found to increase the risk of atherothrombosis [46]. The COX-2 selective NSAID rofecoxib at low doses (50 mg daily) increased the occurrences of myocardial infarction by 0.5% in approximately 8000 patients with rheumatoid arthritis compared to 0.1% in those treated with naproxen (500 mg twice daily), indicating a severe thrombogenic effect of rofe-coxib compared to naproxen [47]. The main increased risk of cardiovascular events associated with COX-2 inhibitors was an increased risk of myocardial infarction [46].

The report of Adenomatous Polyp Prevention on Vioxx (APPROVe) trial on the adverse effect of rofecoxib on CVD ultimately led to the discontinuation of this drug in several countries [48]. Increased thromboembolic events have been associated with rofecoxib compared to naproxen [49]. In another trial rofecoxib was found to be associated with cardiac arrhythmias and renal conditions [50]. However similar adverse effects were not encountered in patients treated with other COX-2 inhibitors [50]. More importantly, several semiselective NSAIDs like diclofenac and meloxicam and nonselective NSAIDs including naproxen and ibuprofen have also been shown to increase the incidences of CVD [46,51]. Of these drugs, diclofenac has been shown to increase

the occurrences of myocardial infarction and stroke even at lower doses of <150 mg/day compared to naproxen at doses of >1000 mg/day [51]. This effect of diclofenac has been attributed to its greater selectivity for COX-2 inhibition. It has also been reported that ibuprofen is associated with CVD comparable to the effect of COX-2 selective inhibitor, celecoxib [20]. It has been suggested that the difference between the effect exhibited by rofecoxib and other NSAIDs of the same class on cardiovascular incidences is due to the distinct chemical properties and prooxidant activity of rofecoxib [52]. The toxic effect of rofecoxib was reportedly due to its ability to reduce the low density lipoprotein (LDL) antioxidant capacity as a result of increased lipid peroxidation [52]. Merck voluntarily withdrew rofecoxib (Vioxx) in 2004. Pfizer was asked by the US Food and Drug Administration (FDA) to withdraw valdecoxib (Bextra) from the market in 2005 because of a higher than expected number of reports of serious and potentially life-threatening skin reactions and deaths.

Naproxen seems to show less cardiovascular events than other commonly used NSAIDs, possibly because it mimics the activity of acetylsalicylic acid (aspirin) by suppressing

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cyclooxygenase platelet thromboxane B2 [53]. However, the role of naproxen in the development of CVD has been con-troversial. Several studies have reported the cardioprotective effect of the compound [54–56], and on the other hand increased cardiovascular risk has been associated with the use of this NSAID [57–59]. The only NSAID which has not been associated with increased cardiovascular events is aspirin. Aspirin is the only known NSAID which has antithrombotic activity through the inhibition of platelet aggregation in the artery of the heart thereby exhibiting cardioprotective effects [60]. It does so by acetylating the platelet COX-1 and irreversibly suppressing thromboxane A2 production, which is required for platelet aggregation and thrombus formation [60]. Naproxen on the other hand causes reversible inhibition of cyclooxygenase by binding to the enzyme [61]. It was found that inhibition in thromboxane A2 synthesis was more pronounced (75%) compared to prostacyclin inhibition (50%) after the oral administration of 500 mg naproxen in healthy volunteers [61].

5. NSAIDs and Reactive Oxygen

Species Generation

NSAIDs have been shown to be associated with increased ROS production (Table3). In the heart the main producer of ROS is the mitochondria [62]. Under normal physiological conditions, mitochondria generate ROS as a consequence of aerobic respiration [63]. During aerobic respiration about 5% of O2consumed via aerobic reaction is converted into ROS [63]. The mitochondria-dependent overproduction of ROS has been reported under numerous pathological conditions including myocardial heart failure, inflammatory diseases, cancer, hypertension, and diabetes [64–67]. In myocardial heart failure, cardiomyocytes have been shown to be targeted by excessive ROS generation [64].

ROS levels and the redox state of a cell are considered to be important in the dysfunction of various biological signaling pathways. The formation of ROS via the reduction of molecular oxygen or by the oxidation of water leads to the formation of free radicals such as superoxide anion (O2βˆ™βˆ’), hydroxyl radical (βˆ™OH), and hydrogen peroxide (H2O2). Oxidative stress arises when the oxidant production (sum of all the ROS) surpasses the antioxidant capacity in the cells. Under normal physiological conditions low amounts of O2βˆ™βˆ’ in the cardiomyocytes are converted to the less toxic H2O2 by the enzyme superoxide dismutase (SOD). The reaction further proceeds by the formation of water by the action of the enzyme catalase or glutathione peroxidase (GPx) system [68]. However, when a homeostatic imbalance between the cellular antioxidant capacity and ROS levels occurs, elevated ROS levels can damage cellular macromolecules including lipids, proteins, and nucleic acids. High levels of ROS also accelerate cell death due to apoptosis as well as necrosis by the activation of poly(adenosine diphosphate ribose) polymerase [69] and thus significantly contribute to the development of various pathological conditions [70].

The generation of ATP in the mitochondria utilizes the electrons from reduced substrates that are transferred to an acceptor molecule of the electron transport chain (ETC).

Leakage of electrons from the mitochondria ETC results in O2βˆ™βˆ’formation [71]. Electron leakage is potentially possible at 9 sites in the ETC; however most of the ROS seem to be associated with complexes I and III which have been well studied [72]. The generation of ROS increases in intact mitochondria as well as in submitochondrial particles due to the oxidation of complex I substrates as a result of inhibiting complex III by antimycin A [72]. On the other hand, rotenone, an inhibitor of complex I, prevented the antimycin A induced ROS generation in mitochondria but not in the submitochondrial particles [72].

The ROS status in the cellular system regulates many biological processes. While increased levels of ROS have been shown to be involved in various pathological conditions, under basal conditions, the generation of free radicals in the heart is needed for cellular responses including regulating myocyte growth and maintaining vascular smooth muscle tone [73]. Basal levels of ROS play an important role in the increase of cell cycle progression and intracellular signaling associated with phosphorylation of several signaling proteins like mitogen-activated protein kinases (MAPKs) and protein kinase B [74]. Under normal physiological conditions, ROS upregulates the Akt signaling pathway and promotes cell survival [75]. ROS also behaves as second messengers in sig-naling pathways wherein it has been demonstrated that, in the presence of basal ROS levels, tyrosine phosphatase activity is higher relative to its kinases [76]. Ligand stimulation (as in the case of neutrophils, through the binding of cytosolic proteins to a membrane bound oxidase) leads to increased ROS levels and deactivation of tyrosine phosphatases with a consequent increase in the kinase activity [76]. This condition is transient and is reversed by reductions in ROS levels. ROS has also been demonstrated to be involved in the modulation of transcription factors like NF-πœ…B, Hif-1 [77], and several cardiac transcription factors like SRF, Sp1, AP-1, GATA-4, and MEF2C [78].

Current experimental data suggest that the main mecha-nism through which NSAIDs exert their anticancer activity is through the generation of ROS leading to oxidative stress and finally apoptosis in cancer cells [79–82]. ROS is accompanied by the activation and inhibition of several signaling pathways associated with cell death and cell survival although contro-versies exist regarding the role of ROS in the downregulation and upregulation of these pathways [83,84]. The Akt pathway is one of the most important pathways for promoting cell survival and growth, and it has been shown that high ROS levels are lethal and can inactivate the Akt signaling pathway [85]. NF-πœ…B is an important transcription factor involved in

cell survival, inflammation, and stress responses. Its down-regulation was demonstrated by exposure of HLEC (human lymphatic endothelial cells) cells to sustained oxidative stress [86]. Increased levels of H2O2caused by addition of glucose oxidase to HLECs prevented NF-πœ…B(p65) regulated gene expression by blocking translocation of NF-πœ…B to the nucleus [86]. NF-πœ…B regulated gene expression is important for a

broad range of physiological processes [86].

Sulindac, a nonselective NSAID, as well as its metabolites, generates ROS in different cancer cell lines [80–82]. Similarly, diclofenac-induced apoptosis of various kinds of cancer cells

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T a ble 3: NSAID s ind uced R O S genera tio n in diff er en t cel lt yp es. (a) NSAID s N o ns ele ct ive/s emis ele ct ive So ur ces o f R O S genera tio n C ells/mo d els/a nimals st udied O u tco mes Ref er ences Dic lo fenac, indo met h acin, ke to profe n M it o ch on d ri al re spi ra ti on Sacc ha ro m yces cer ev isiae Y ea st cells B Y4 74 1 and mi to ch o n dr ial D N A d elet io n (rho 0 )s tr ai n s Thes e NSAIDs ta rget m it o cho ndr ia to ind uce cel lt o xici ty [ 95 ] Ib uprofe n an d n ap roxe n Thes e NSAIDS ind uce to xici ty indep enden t o f m it o ch on d ri al re spi ra ti on [ 95 ] In do met h acin, so di um dic lo fe nac, fl u rb ip ro fe n, za lt oprofe n ,an d m ofe zol ac ND H u ma n gast ric ep it helial cel lline AG S Al lNSAID s ex cep t m o fezo lac incr ea se d ap opt ot ic DN A fr ag m en ta ti on an d ex p re ss io n o f C O X-2 m RN A. D N A frag m en ta ti o n ind u ced by indo met h acin o r fl urb ip ro fen was red uced by an ti o xida n ts. Indo m et hacin at 1 mM was a p o te n t ind u cer o f R O S genera tio n in the cel ls [ 79 ] Dic lo fenac an d n ap ro xe n N AD PH o xidas es Sp ont an eou s h yp er te n si ve ra ts N A D P H o xidas e exp ressio n incr ea se d in the he ar t and ao rt a by diclo fenac and na p ro xen. ROS le vels incr eas ed d u e to N AD PH o xid as e [ 11 4 ] Hu m an E A .h y 92 6 Endo th eli al cel ls N o x2 is ofor m o f N A D P H ox id as e is in cre as ed by dic lo fe nac [ 11 4 ] As pi ri n ,n aproxe n ,a n d pi rox ic am N A D P H o xidas es R at adi p o cyt es A ct iv at ion of NO X 4 is ofor m o f N A D P H o xidas e resu lts in th e genera tio n o f H2 O2 [ 115 ] In do met h acin X an thine o xidas es H u ma n co lo nic adeno ca rcino ma cel ls (C aco-2 cel ls) Xa n thine o xidas e ac ti vi ty incr ea se s by m o re th an 10 0 % 1h o u ra ft ert re at m en t [ 96 ] In do met h acin M it o ch o n dr ia su p er o xide le aka ge R at gast ric ep it helia lcel lline RG M 1 In do met h acin ind uce d th e le akage o f su p er o xide anio n in the is o la ted mi to ch o n dr ia fr o m th e gast ric R G M1 cells [ 93 ] In do met h acin, dic lo fe nac so di um, an d asp ir in ND R at gast ric ep it helia lcel lline RG M 1 and ra t sm al li n te st ina l ep it helial cel lline IEC6 In do met h acin, dic lo fe nac, an d asp ir in in gast ri c R GM1 cel ls an d smal lin te st inal IEC6 cells incr eas ed li p id p er o xida tio n [ 93 ] Su lindac, su lindac su lfide , an d sul in da c sul fo n e ND P an cre as ca rc in om a B X P C 3, gl io bl astoma A 17 2, col on ca rc ino m a D LD-1, o ral sq ua mo us SAS, an d ac u te m ye loc yt ic le uk em ia H L 60 ce ll s The NSAID su lind ac an d its met ab o li tes sulindac sulfide an d sulindac sulf o ne all incr ea se d R OS ge nera ti o n .S ulindac sulfide sho w ed th e gr ea test R OS genera ti o n [ 80 ] In do met h acin, p ir o xica m, an d asp ir in Li po x yg en ase s Gast ri c and in te st inal m u cos a in mice Resul ts in an o ve rp ro d u ct io n o f leuk o tr ienes an d p ro d u ct s o f 5-li p o x yg en as e acti vi ty . In cr ea se s in leu ko tr iene an d 5-li p o x yg enas e ac ti vi ty ha ve b een ass o cia ted wi th R O S ge ne ra ti on [ 123 , 12 4 , 127 ] In do met h acin L ip o x yg enas es Eff er en t gast ric cir cu la tio n o f p igs T ime d ep enden t fo rm at io n o f leu ko tr iene-C4 [ 128 ]

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(a) C o n tin u ed. NSAID s N o ns ele ct ive/s emis ele ct ive So ur ces o f R O S genera tio n C ells/mo d els/a nimals st udied O u tco mes Ref er ences Dic lo fenac C yt o ch ro me P45 0 enzymes Sacc ha ro m yces cer ev isiae ye ast ce ll s ex p re ss in g th e mu ta nt P 4 50 BM3 M11 (ca p ab le o f met ab o lizin g dic lo fe nac simila r to h u mans ) In cr ea se d R O S pro d u ct ion by ∼ 1.5 and 1.8 ti m es at co n ce n tra ti o n s o f 30 πœ‡Ma n d 50 πœ‡M, re sp ec ti ve ly ,c om p are d to w il d ty p e [ 14 0 ] Dic lo fenac, indo met h acin, ke to profe n ,a n d n aproxe n C yt o ch ro me P45 0 enzymes Sacc ha ro m yces cer ev isiae Y ea st cells B Y4 74 1 and mi to ch o n dr ial D N A d elet io n (rho 0 )s tr ai n s N SA ID sm et ab o li sma ss o ci at edw it hi n cr ea se d P45 0 -r ela ted to xici ty [ 95 ] (b) NSAID s co xibs So ur ces o f R O S genera tio n C ells/mo d els/a nimals st udied O u tco mes Ref er ences Ib u p ro fen, ket o p ro fe n, an d indo m et hacin C yt o chr o m e P 45 0 enzymes Ba ci llus m ega te ri u m A llt h e N SA ID sc au se dam ar ke d in cr ea se in th e to tal cy to ch ro me P45 0 le ve l [ 14 2 ] A sp ir in, dic lo fe nac, difl unis al , flu rb iprofe n ,i b uprofe n ,i n d om et h ac in , ke to p ro fen, m ef ena mic acid ,na p ro xen, p ir o xic am, su lind ac, an d tolmet in C yto chr o me P45 0 enzy mes R at hep ato cy tes C yt o to xici ty o f dic lo fe nac, ke to p ro fen, an d p ir o xica m was incr eas ed by cy to ch ro me P45 0 ca usin g h epa to to xici ty [ 13 8 ] Dic lo fenac an d n ap ro xe n Endo th elial n it ri c o xide syn thas e Sp ont an eou s h yp er te n si ve ra ts In cr ea se d exp re ssio n o f eN O S m RN A d ue to th e genera tio n o f H2 O2 w hic h is re sp ons ibl e for upre gu la ti o n o f eNO S at th e tr ans cr ipt ion al an d p o st -t ra ns cr ipt ion al le ve ls [ 11 4 ] Ro fe co xib and celeco xib N AD PH o xidas es Sp ont an eou s h yp er te n si ve ra ts N A DP H ex p re ss io n in cre as ed in th e h ea rt an d ao rt a by ro fe co xib and celeco xib [ 11 4 ] Hu m an E A .h y 92 6 Endo th eli al cel ls N o x2 E xpre ss ion in cr ea se d by rofe cox ib [ 11 4 ] Ro fe co xib and celeco xib Endo th elial n it ri c o xide syn thas e Sp ont an eou s h yp er te n si ve ra ts In th e ao rt a, the co xibs did no t sho w an y eN OS mRN A exp ressio n .I n the he ar t o n ly ro fe co xib sho w ed a significa n t incr ea se in th e exp re ssio n o f eN O S [ 11 4 ] Et o d o la c N D H u m an ga st ri c epit h el ia lc el l line A G S In cr ea se d ap opt ot ic DN A fr ag m en ta ti on an d exp re ssio n o f C O X -2 mRN A [ 79 ] N imesu lide N A D P H o xidas es R at adi p o cyt es A ct iv at ion of NO X 4 is ofor m o f N A D P H o xidas e resu lts in th e genera tio n o f H2 O2 [ 115 ] * Al th o u gh o ther rep o rts ar e ava ila b le sug gestin g th e ro le o f N SAID s in R OS fo rm at io n, th e ta b le lists o nl y thos e NSAID s fo r w hic h re sul ts sho w tha t thes e N SAID s ar e ass o cia ted wi th CVD o r N SAID s m en tio n ed in th e text.

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has been reported [87]. This apoptosis is mainly mediated by an increase in the intracellular levels of the ROS [87]. In the cardiovascular system the major sources of ROS gener-ation include the mitochondria, NADPH oxidases, xanthine oxidoreductases, lipoxygenase, cyclooxygenases, nitric oxide synthases, and cytochrome P450 based enzymes (Figure2). Continuous exposure of cardiovascular cells to oxidative stress associated with elevated ROS levels would result in altered cellular homeostasis which could be an important contributing factor for various cardiovascular conditions. Endothelial cells play a critical role in maintaining vas-cular homeostasis which is important for the control of many cardiovascular diseases including atherosclerosis and thrombosis [88]. In human umbilical vein endothelial cells (HUVEC), 160πœ‡M sulindac induced endothelial apoptosis as determined by a time dependent increase in annexin V positive cells. Both sulindac and indomethacin significantly increased cleaved poly(ADP-ribose) polymerase (PARP) lev-els as well as increasing the level of the apoptotic activating factor caspase-3 [89]. The apoptosis in HUVEC cells induced by the NSAIDs was associated with reduced PPAR𝛿 and 14-3-3-πœ€ expression. Under normal conditions 14-14-3-3-πœ€ binds to phosphorylated Bad inhibiting translocation of Bad to the mitochondria and preventing apoptosis through the mitochondrial pathway. However, sulindac through the sup-pression of 14-3-3-πœ€ exsup-pression increased Bad translocation to mitochondria thereby inducing apoptosis [89].

6. Mitochondria Are the Main Target

Organelles of the NSAIDs

It has been shown that the heart is more susceptible to ROS generation induced by drugs like doxorubicin compared to other tissues of the body although the drugs are evenly distributed throughout the body [90]. This is possibly due to high levels of mitochondria in the heart which are the major producers of ROS in the cardiovascular system. Over 90% of ATP required for the normal functioning of the heart is provided by the mitochondria, which utilizes an efficient oxidative phosphorylation system. ATP production may increase depending upon the requirements of the body, especially at times of excessive physical exertion or other hor-monal stimulations [91]. Mitochondria are not only the major producers of the free radicals [72], but excessive generation of ROS in turn targets the mitochondria itself [92]. NSAIDs have been shown to have adverse effects on the mitochondria resulting in the increased production of ROS [93,94]. In yeast cells different NSAIDs generated ROS which was associated with delayed growth in wild-type cells [95]. Yeast cells lacking mitochondrial DNA were resistant to a delay in cell growth [95]. More specifically the yeast deletion strains lacking the genes encoding subunits of the mitochondrial complexes III and IV were significantly resistant to diclofenac as well as indomethacin and ketoprofen [95]. This data suggests that mitochondria are the main target organelles through which these compounds exert their toxic effect on these cells. The data also suggest that mitochondrial complex III and/or

complex IV are affected by NSAIDs resulting in increased ROS production.

The nonselective NSAID indomethacin has been shown to target mitochondria by directly inhibiting mitochondrial complex 1 of human colonic adenocarcinoma cells. The inhibition of complex I was accompanied by a decrease in the intracellular ATP levels within 10–30 minutes [96]. Studies directly related to mitochondrial dysfunction or damage in the presence of NSAIDs in CVD have not been carried out but are needed (Table 2). Although the mechanism by which NSAIDs can cause mitochondrial dysfunction in cardiomyocytes is not fully understood, preventing increased mitochondrial ROS levels will reduce the adverse effects of elevated ROS levels and may reduce the incidences of CVD caused by NSAIDs.

Apart from the role of NSAIDs in causing cardiovascular incidences, NSAIDs affect other cardiovascular (CV) param-eters including left ventricular function, infarct size, and blood pressure.In elderly patients (71.8 Β± 7.6 years) NSAID exposure for<14 days showed a significantly higher left ven-tricular end-systolic dimension (+1.74 mm) and left ventric-ular end-diastolic dimension (+3.69 mm) with a significantly lower fractional shortening (βˆ’6.03%) when compared to non-NSAID users. Patients with non-NSAID use for>14 days were found to have higher left end-diastolic dimension (+1.96 mm) but no significant changes in other echocardiographic param-eters compared to non-NSAID users [97].

The impact of NSAIDs on infarct size during myocardial infarction has only been investigated by a few laboratories. Indomethacin (10 mg/kg) was found to increase myocardial infarct size in animals, while ibuprofen (6.25 mg/kg/h) had the opposite effect [98–100]. The authors suggested that the opposite effect on the infarct size could be attributed to variable doses of the NSAIDs, different degrees of inhibition of prostaglandin and its by-products, and others factors like myocardial oxygen consumption [99]. The same group reported that the frequency of acute infarct expansion syn-drome in patients with symptomatic pericarditis treated with indomethacin was 22% compared to ibuprofen which was only 8% [101]. The degree of infarct expansion was also greater in patients treated with indomethacin compared to ibuprofen [101].

Another important CV parameter is hypertension, which is one of the major contributors to the development of CVD. A series of events occur in patients with prolonged high blood pressure including left ventricular hypertrophy, systolic and diastolic dysfunction leading to arrhythmias, and heart failure [102]. Except for aspirin, all NSAIDs could potentially increase blood pressure when taken at doses necessary to alleviate pain and inflammation in both hypertensive and normotensive individuals [39].

NSAIDs through their ability to block COX enzymes lead to the inhibition of renal prostaglandin [103]. Renal prostaglandins modulate several renal functions which include maintaining renal homeostasis and exerting diuretic and natriuretic effects [104,105]. Inhibition of the natriuretic effect of COX-2 leads to an increase in sodium retention thereby leading to excess water retention in humans [106]. The inhibition of renal vasodilating prostaglandins induces

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the production of vasoconstricting factors like vasopressin and endothelin-1. This also results in water retention, thereby leading to an increase in the total blood volume and caus-ing altered systolic and diastolic blood pressure [103, 106,

107]. NSAIDs like ibuprofen, indomethacin, and naproxen increase the mean arterial pressure by 5 to 6 mmHg in patients with established hypertension [108,109], which may be enough to raise medical concerns under certain conditions [110]. Additionally, the efficiency of all antihypertensive medications except for calcium channel blockers may be sub-stantially reduced by NSAIDs [40]. Since CV outcomes would have different pathogenesis, increased ROS levels may not be the underlying mechanism for some occurrences of CVD. Other CV related parameters (such as hypertension), which are important for the normal functioning of the heart, are also likely to affect the CV outcome. Although direct comparisons between the different clinical studies shown in Table2are not possible, these studies suggest that CVD in NSAID users are influenced by the study populations used (such as with versus without preexisting acute myocardial infarction). Chronic use of NSAIDs by hypertensive individuals has been shown to increase the incidences of myocardial infarction, stroke, and cardiovascular mortality compared to nonchronic NSAID users [111] (Table2).

7. NSAIDs and Nicotinamide Adenine

Dinucleotide Phosphate-Oxidase

Apart from mitochondria being the major source of ROS gen-eration, the plasma membrane bound nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) has also been indicated in the production of ROS in phagocytes like neutrophils and macrophages where it produces an β€œoxidative burst” of O2βˆ™βˆ’[112]. NADPH oxidases are considered to be the major source of nonmitochondrial ROS generation in many cells. The formation of free radicals is due to the electron transferred from the NADPH to molecular oxygen during the process of phagocytosis. The phagocytic NADPH oxidases play an important role in the host defense mechanism and are involved in killing pathogens ingested during phagocytosis. Apart from the phagocytic NADPH oxidase, in the last decade NADPH oxidase-dependent ROS generation was also identified in nonphagocytic cells including the endothelial cells, vascular smooth muscle cells, and cardiomyocytes of the cardiovascular system [113]. Compared to the phago-cytic NADPH oxidases, the nonphagophago-cytic enzymes produce lower amounts of ROS continuously and these levels may increase in the presence of specific extrinsic stimuli [113]. More interestingly, the ROS generated by NADPH oxidases lead to an enhancement in levels of ROS produced from other sources [112]. In one such study, mitochondria and NADPH oxidase 1 isoenzyme (Nox 1) were found to be closely coordinated for sustained generation of ROS leading to oxidative stress and cell death [112]. When human embryonic kidney 293T cells were exposed to serum-free media elevated ROS levels occur within 5 minutes of exposure and persisted for 8 hrs [112]. Utilizing RNA interference Nox isoenzymes were demonstrated to play a role in the induction of serum-withdrawal ROS generation. Although low levels of Nox 2

and Nox 4 did not affect the generation of serum-withdrawal induced ROS generation, low levels of Nox 1 led to a decrease in the ROS formation in these cells at 8 h (late phase). Using different mitochondrial complex inhibitors such as rotenone and potassium cyanide (KCN) mitochondria were found to play a role in the early phase (first 30 minutes) of ROS generation in the cells exposed to serum-free media [112]. The authors of the study suggested that mitochondrial generation of ROS occurs first, followed by the action of Nox 1 in the serum deprived cell system [112].

The adverse effect of NSAIDs on the cardiovascular system was evident by a study which reported that when male spontaneously hypertensive rats (SHR) were treated either with coxibs or with nonselective NSAIDs like diclofenac and naproxen, the mRNA expression of NOX enzymes 1, 2, and 4 was markedly increased in the heart and aorta [114]. Furthermore the oxidative stress in the heart and aorta of the NSAID treated animals was also elevated along with an increase in the O2βˆ™βˆ’ production. Of all the NSAIDs used, diclofenac was the most potent inducer of NADPH oxidases [114]. The role of NADPH oxidases in the generation of ROS in the animals was ascertained by studying the effect of apocynin (an NADPH oxidase inhibitor) in the reversal of the oxidative stress induced by diclofenac [114]. Activation of Nox 4 by NSAIDs like aspirin, naproxen, nimesulide, and piroxicam has also been reported in rat adipocytes resulting in higher production of H2O2[115].

8. NSAIDs and Xanthine Oxidase

Xanthine oxidoreductase (XOR) is a conserved molybd-oflavoenzyme occurring in milk and some tissues includ-ing the liver, heart, and small intestine [116]. It has two interconvertible forms: xanthine dehydrogenase (XDH) and xanthine oxidase (XO). Both enzymes catalyze the conversion of hypoxanthine to xanthine and xanthine to uric acid [116]. Although both enzymes are involved in purine degradation, XO is involved only in the reduction of oxygen while XDH is involved in the reduction of both oxygen and to a larger extent NAD+. The enzyme XDH has the ability to bind to NAD+and, following the oxidation of hypoxanthine to xanthine, reduces NAD+to NADH. On the other hand, XO, not being able to bind to NAD+, produces the free radical, O2βˆ™βˆ’by the transfer of electrons to molecular oxygen. Apart from O2βˆ™βˆ’, H2O2 was also shown to be a major free radical generated by XO [117]. XDH can utilize molecular oxygen and generate ROS but to a lesser extent than XO. Compared to the other sources of ROS generation like mitochondria and NADPH oxidases, the role of XOR as a producer of ROS has been previously overlooked due to its relatively low activity in the heart of animals and human. Recently the role of XOR in mediating various pathological conditions has been demonstrated in the cardiovascular system due to significant increases in cellular XOR levels [118]. This was evident by the inhibition of XOR by compounds like allopurinol and oxypurinol which lead to a decrease in the oxidative tissue damage. Apart from their property of inhibiting XOR, allopurinol and oxypurinol have also been shown to act as free radical scavengers and inhibit lipid peroxidation as well as inducing heat shock proteins

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during times of oxidative stress [118]. Interestingly, a high level of serum uric acid, the end product of XOR, has been shown to be a marker for impaired oxidative metabolism [119].

Although the prevalence of this enzyme has been reported in human hearts [116,120] and although increased levels of uric acid in myocardial heart failure have been reported in the literature [119], the role of NSAIDs on XO in the development of various cardiovascular conditions has not been investigated. In a study to investigate the adverse effect of aspirin on gastric mucosal lining leading to peptic ulcers and intestinal bleeding, rat gastric mucosal cells were incubated with aspirin and cytotoxicity already induced by hypoxanthine/XO was determined [121]. Aspirin was shown to increase the cytotoxicity induced by XO. This increase in cytotoxicity in the presence of hypoxanthine/XO was incor-porated with an increase in uric acid levels which suggests ROS generation in these cells. Aspirin had an additive effect to these changes induced by hypoxanthine/XO [121]. In another study, it was seen that indomethacin augmented the XO activity in human colonic adenocarcinoma cells by more than 100% upon 60 minutes of exposure which resulted in a time dependent increase in the rate of lipid peroxidation [96].

9. NSAIDs and Lipoxygenase

Arachidonic acid that is formed by the action of diacylglyc-erol and cytosolic phospholipase A2 (cPLA2) from the mem-brane phospholipids is a substrate involved in the formation of either prostaglandins and thromboxanes or leukotrienes by the cyclooxygenase and lipoxygenase pathway respectively. The oxidation of arachidonic acid by these enzymes leads to the formation of ROS as the by-product [122]. It was shown that cPLA2-arachidonic acid linked cascade could lead to an increase in ROS generation via the lipoxygenase pathway in the rat fibroblasts [123]. The same group reported that the generation of ROS triggered by tumor necrosis factor- (TNF-)𝛼 was mediated by the activation of cPLA2 and the metabolism of arachidonic acid by 5-lipoxygenase [124]. Lipoxygenase has been reported to be involved in the upregulation of NADPH oxidases and increased ROS generation [125].

While NSAIDs are well known to inhibit the cyclooxyge-nase pathway, they do not inhibit the formation of leukotri-enes by the lipoxygenase pathway. NSAIDs increases arachi-donic acid levels [82, 126] and arachidonic acid itself can increase ROS generation [82]. Increased levels of leukotrienes and other metabolites of the lipoxygenase pathway induced by NSAIDs lead to gastric mucosal damage [127]. The effect of NSAIDs like indomethacin, piroxicam, and aspirin on the development of gastric lesions could be reversed by 5-lipoxygenase inhibitors and leukotriene antagonists [127]. This is consistent with NSAIDs increasing the lev-els of leukotrienes and products of 5-lipoxygenase activ-ity. Increased occurrences of leukotriene C4 production were also observed in the gastric circulation induced by indomethacin [128]. The gastric and intestinal lesions in the presence of indomethacin could be reverted by 5-lipoxygenase inhibitor. Increased gastric mucosal leukotriene

B4 production was observed in patients suffering from arthritis due to prolonged NSAID treatment [129]. NSAIDs have also been reported to increase the expression of 15-lipoxygenase-1 protein in colorectal cancer cells resulting in the inhibition of cell growth and apoptosis [130]. Although a growing body of evidence supports the idea that NSAIDs upregulate the lipoxygenase pathway, no direct evidence is available on the precise biochemical mechanism by which NSAIDs induced lipoxygenase pathway activity increase the rate of ROS generation. It is possible that the upregulation of the lipoxygenase pathway by NSAIDs may contribute to the net increase in free radicals leading to apoptosis and oxidative cell damage and ultimately cell death.

10. NSAIDs and Cytochrome P450

Cytochrome P450 (P450) is a family of biotransformation enzymes involved in critical metabolic processes [131]. They are a class of more than 50 enzymes that catalyze a diverse set of reactions with a wide range of chemically dissimilar substrates [131]. In humans they are primarily membrane-associated proteins that can be found in the inner mem-brane of the mitochondria or endoplasmic reticulum of cell [132]. They are predominantly expressed in the liver but are present in other tissues of the body as well.This group of proteins belongs to the heme-thiolate enzyme family, and they are involved in the oxidative metabolism of numerous compounds of endogenous and exogenous origin [133]. The role of P450 in drug metabolism has been considered to be a key factor in overcoming the adverse and toxicological effects of drugs [134]. The P450 system has been shown to play an important role in activation of oxygen and ROS generation [135,136]. Normally, the active oxygen species are formed in situ during the P450 cycle when it reacts with a substrate. However, uncoupling of the P450 system results in excessive ROS generation and the P450 system being unable to metabolize a substrate, which leads to oxidative stress and subsequently cellular damage [133,137].

Several studies have indicated the role of P450 in toxicity induced by NSAIDs [138–140]. One such NSAID, diclofenac, has been associated with hepatotoxicity due to poor metabolism by P450 [140]. Expression of a drug metab-olizing mutant of P450, BM3 M11, in yeast mimicked the oxidative metabolite profiles of diclofenac metabolized by human P450s [141]. The treatment of the yeast cells expressing the mutant BM3 M11 with 30πœ‡M and 50 πœ‡M diclofenac increased the ROS production in the cells significantly by 1.5 and 4 times, respectively, compared to cells not treated with the compound [140]. In another study by the same group, it was shown that diclofenac as well as indomethacin and ketoprofen showed an increase in ROS generation by 1.5–2 times compared to control [95]. On the other hand, P450 has been shown to be upregulated in bacterial cells by NSAIDs like ibuprofen, ketoprofen, and indomethacin by 11.8-fold, 3.9-fold, and 3.0-fold, respectively, relative to control cells [142]. Although no direct evidence is yet available on the involvement of P450 in the generation of ROS by NSAIDs in CVD, experimental data available in other cells suggest

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that NSAIDs may lead to increased ROS levels via the upregulation of P450 in cardiovascular related cells.

11. NSAIDs and Nitric Oxide Synthases

Nitric oxide (NO) is among the few gaseous biological mes-sengers and can be synthesized from l-arginine by endothelial nitric oxide synthase (eNOS). NO has emerged as a key signaling molecule for maintaining vasodilation and the dysfunction of enzymes associated with NO production has been implicated in various pathological conditions including CVD, diabetes, and hypertension. When eNOS, which is responsible for regulating vascular homeostasis, is impaired due to a lack of the cofactor tetrahydrobiopterin (BH4), it results in the generation of O2βˆ™βˆ’rather than NO [143]. This state is referred to as the uncoupled state of eNOS and is mainly attributed to the deficiency or lack of BH4 leading to the generation of free radicals [144]. The depletion of the BH4 can be the result of severe oxidative stress.

A 1.5-fold decrease in plasma nitrite levels was demon-strated in SHR treated with nonselective COX inhibitors and coxibs [114]. Although earlier reports demonstrated a cardioprotective effect of naproxen, a twofold decrease in plasma nitrite levels was observed after treatment of SHR with naproxen suggesting diminished levels of bioactive NO [114]. Additionally, the authors reported an upregulation in the eNOS mRNA expression levels rather than a decrease by diclofenac and naproxen by>2-fold. This increase has been attributed to the generation of H2O2which increases eNOS expression at the transcriptional and posttranscriptional levels [145]. Diclofenac treated animals showed an increase in O2βˆ™βˆ’ production which could be inhibited by the NOS inhibitor l-NAME. NSAIDs have also been shown to impair the NO induced vasodilation in healthy individuals possibly due to increased oxidative stress induced by NSAIDs due to the uncoupling of eNOS [114].

12. NSAIDs, ROS, and Cardiovascular Diseases

Li et al. investigated the effect of several NSAIDs on free radical generation, NADPH oxidase expression, eNOS expression, and nitrite levels in the rat aorta and heart using a mouse model of hypertension as well as vasodilation in human subjects [114]. As previously described in the sections NSAIDs and nicotinamide adenine dinucleotide phosphate-oxidase and NSAIDs and nitric oxide synthases, this study demonstrated that the heart and aorta of the NSAID treated animals showed increased O2βˆ™βˆ’ production and oxidative stress. The oxidative stress was due to ROS formation by many enzymes including NADPH oxidases, with diclofenac being the most potent inducer of NADPH oxidases [114].

Rat embryonic H9c2 cardiac cells exposed to celecoxib, at concentrations of 10πœ‡M and 100 πœ‡M, demonstrated a decrease in cell viability by 45% and 92%, respectively, and this was associated with a decrease in the expression levels of the cell survival protein Bcl2 [146]. However, 1πœ‡M of celecoxib had no significant effect on cell viability and was associated with an upregulation of the Bcl2 transcript level

by ∼30%. This effect of such a low amount of NSAID in the upregulation of cell survival gene expression is rather comparable to the effect of proteasome inhibitors like MG-132 or epoxomicin which at nanomolar ranges has been reported to actually increase the proteasome activity in the pretreated neocortical neurons [147]. Also, this was the first study that measured COX-2 levels directly in the cardiac cells [146]. Although no significant changes in the COX-2 levels were observed in cells treated with 1πœ‡M celecoxib, at 10 πœ‡M the COX-2 levels decreased [146].

Apart from studying the effect of NSAIDs on cardiomy-ocytes, the role of NSAIDs on the COX enzyme system in platelets is also of prime importance. Platelets are impor-tant for the life-saving blood coagulation process [148] and are closely associated with CVD [149]. The aggregation of platelets by agonists like ADP, collagen, thrombin, or epinephrine leads to the formation of thrombus (microaggre-gate of platelets in fibrin mass), at the site of atherosclerotic plaque fissure on the coronary artery wall of the heart which results in thrombosis or CVD [150]. As expected because of the importance of platelets in CVD, the generation of ROS in platelets has been well-studied [149]. Measurement of O2βˆ™βˆ’ in platelets activated by thrombin as well as in inactivated platelets demonstrated that thrombin significantly increased the free radical (O2βˆ™βˆ’) generation by∼40% in the activated platelets compared to the inactivated platelets [149]. Although this result suggests the possibility of increased ROS formation in activated platelets, no direct role of NSAIDs in the production of ROS in the platelets has been reported [151]. However, it has been demonstrated that selective COX-2 inhibition led to an increase in platelet activation [152]. The increased platelet activation by COX-2 inhibition facilitated thrombotic vessel occlusion after the disruption of the vessel wall supporting a critical role for COX-2 inhibition in platelet activation and aggregation leading to thrombosis [152]. All these effects were accompanied by a decline in the production of endothelial prostacyclin. Diclofenac (1 mg/kg), a nonselective NSAID, caused significantly increased platelet vessel wall interaction with an increase in the number of adherent platelets on the endothelium of diclofenac treated animals compared to the control group [153]. Thrombotic vessel wall occlusion was also increased in animals treated with diclofenac once the vessel wall had been injured [153]. The direct contribution of ROS induced by NSAIDs to platelet activation and aggregation is yet to be determined. The role of aspirin in cardioprotection by the inhibition of platelet aggregation due to the inhibition of platelet thromboxane A2 is well established [154,155]. Similarly, naproxen has also been suggested to have significant antiplatelet effect comparable to that of aspirin [156].

13. Possible Alternatives to NSAIDs for

Reduced Cardiovascular Events

With the availability of a larger selection of synthetic drugs, people are more aware of the side effects of taking drugs on a regular basis. Nutraceuticals or phytoceuticals are a class of compounds that are derived from plants with fewer or no side effects compared to synthetic drugs [157] but

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