J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E VO L . - , N O . - , 2 0 1 9

ª 2 0 1 9 T H E A U T H O R S . P U B L I S H E D B Y E L S E V I E R O N B E H A L F O F T H E A M E R I C A N

C O L L E G E O F C A R D I O L O G Y F O U N D A T I O N . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R

T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o mm o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .

LEADING EDGE IN TRANSLATIONAL RESEARCH

Celecoxib Is Associated With DystrophicCalcification and Aortic Valve Stenosis

Meghan A. Bowler, PHD,a,* Michael A. Raddatz, BS,a,* Camryn L. Johnson, BS,a Brian R. Lindman, MD, MSC,b

W. David Merryman, PHDa

VISUAL ABSTRACT

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Bowler, M.A. et al. J Am Coll Cardiol Basic Trans Science. 2019;-(-):-–-.

SN 2452-302X

rom the aDepartment of Biomedical Engineering, Vanderbilt University, Nashville, Tenness

alve Center, Vanderbilt University Medical Center, Nashville, Tennessee. *Dr. Bowler and Mr.

ork, and are joint first authors. This content is solely the responsibility of the authors and

fficial views of the National Institutes of Health (NIH), the National Science Foundation (N

ork was funded by NIH grants HL135790, HL115103, and GM007347, American Heart Associat

rants 1055384 and 2013170175. The datasets used for the analyses described were obtained f

enter’s BioVU, which is supported by numerous sources: institutional funding, private ag

clude the NIH-funded Shared Instrumentation grant S10RR025141, and CTSA grants U

L1RR024975. The clinical datasets used for the analyses described were obtained from Vand

ynthetic Derivative. Dr. Lindman has received research grants from Edwards Lifesciences

cientific advisory boards for Roche Diagnostics; and has been a consultant to Medtronic and R

ave reported that they have no relationships relevant to the contents of this paper to disclos

ll authors attest they are in compliance with human studies committees and animal w

stitutions and Food and Drug Administration guidelines, including patient consent where a

isit the JACC: Basic to Translational Science author instructions page.

anuscript received November 13, 2018; revised manuscript received December 11, 2018, acce

HIGHLIGHTS

� Celecoxib use is associated with diagnosis

of aortic stenosis in analysis of electronic

medical records.

� Celecoxib treatment increases dystrophic

calcification of aortic valve interstitial

cells in vitro.

� Dimethyl celecoxib, which binds cadherin-11,

prevents TGF-b1–mediated calcification of

aortic valve interstitial cells in vitro.

https://doi.org/10.1016/j.jacbts.2018.12.003

ee; and the bStructural Heart and

Raddatz contributed equally to this

does not necessarily represent the

SF), or Vanderbilt University. This

ion grant 18PRE34070125, and NSF

rom Vanderbilt University Medical

encies, and federal grants. These

L1TR002243, UL1TR000445, and

erbilt University Medical Center’s

and Roche Diagnostics; served on

oche Diagnostics. All other authors

e.

elfare regulations of the authors’

ppropriate. For more information,

pted December 12, 2018.

ABBR EV I A T I ON S

AND ACRONYMS

ANOVA = analysis of variance

AS = aortic stenosis

AVEC = aortic valve

endothelial cell

AVIC = aortic valve interstitial

cell

CAVD = calcific aortic valve

disease

CDH11 = cadherin-11

CN = calcific nodule

COX2 = cyclooxygenase-2

EMR = electronic medical

record

FDA = Food and Drug

Administration

OR = odds ratio

SMA = smooth muscle actin

TGF = transforming growth

factor

VUMC = Vanderbilt University

Medical Center

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SUMMARY

Calcific aortic valve disease is a progressive fibrocalcific process that can only be treated with valve replace-

ment. Cadherin-11 has recently been identified as a potential therapeutic target for calcific aortic valve disease.

The already approved drug celecoxib, a cyclooxygenase-2 inhibitor, binds cadherin-11, and was investigated as a

therapeutic against calcific aortic valve disease. Unexpectedly, celecoxib treatment led to hallmarks of myo-

fibroblast activation and calcific nodule formation in vitro. Retrospective electronic medical record analysis of

celecoxib, ibuprofen, and naproxen revealed a unique association of celecoxib use and aortic stenosis. (J Am

Coll Cardiol Basic Trans Science 2019;-:-–-) © 2019 The Authors. Published by Elsevier on behalf of the

American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/4.0/).

M ore than 25% of the U.S. popula-tion over 65 years of age isaffected by calcific aortic valve

disease (CAVD) (1). This degenerative diseaseis the most common cause of aortic stenosis(AS), which eventually requires surgicalreplacement of the aortic valve because thereare no effective pharmaceutical treatments.This lack of medical therapy is a result of

our inadequate understanding of the disease mecha-nism (2). CAVD is believed to be mediated by aorticvalve interstitial cells (AVICs), which become acti-vated by transforming growth factor (TGF)-b1 intomyofibroblasts (3), characterized by increasedcontractility, collagen deposition, and expression ofa-smooth muscle actin (SMA) and cadherin-11(CDH11). When these myofibroblasts are subjected tostrain, as is normal in the cardiac valve environment,this causes membrane tearing, leading to apoptosis-mediated cell death. This process has been termedthe dystrophic pathway of calcification and wasevident in 83% of excised human aortic valves(whereas only 13% of those showed osteogenicmarkers) (4), making dystrophic calcification themost prevalent mechanism of CAVD.

We recently identified and validated CDH11 as apossible therapeutic target for CAVD (5–8). CDH11 is amechanosensitive transmembrane cell adhesion pro-tein known to have increased expression in calcifiedhuman aortic valves (7), to be increased in the AVICsof the Notch1þ/� murine model of CAVD (5), and to benecessary for in vitro formation of the calcific nodules(CNs) characteristic of CAVD (7). Additionally, recentwork has shown that blocking CDH11 with a mono-clonal antibody in the Notch1þ/� model preventsCAVD progression (6). These findings motivated us toevaluate current Food and Drug Administration(FDA)-approved drugs that may block CDH11 activityfor CAVD, as the CDH11 antibody research programwas recently halted by Roche after disappointing

Phase II trials for rheumatoid arthritis. A review of thepublished reports revealed that celecoxib, brandname Celebrex (Pfizer, New York, New York), and itsinactive analog, dimethyl celecoxib, bind CDH11 withhigh affinity (9). We therefore hypothesized thateither of these drugs may prevent CAVD by blockingthe homotypic CDH11 bonds between neighboringcells.

To evaluate this hypothesis, we treated porcineAVICs and aortic valve endothelial cells (AVECs) withcelecoxib or dimethyl celecoxib. Cells were alsotreated with TGF-b1 to biochemically induce myofi-broblast differentiation. Cells were then subjected towell-established functional assays of CAVD such asCN formation (5,7,10–12) and collagen gel contraction,as well as evaluated for expression of myofibroblastmarkers a-SMA and CDH11. To assess clinical rele-vance, we performed a retrospective analysis of cel-ecoxib use and AS incidence in the electronic medicalrecord (EMR) from Vanderbilt University MedicalCenter (VUMC).

METHODS

IN VITRO EXPERIMENTS AND STATISTICAL ANALYSIS.

Porcine aortic valve cells were isolated as previouslydescribed (7,13) and used between passages 3 and 11.Cells were evaluated with a combination of molecularand functional assays in order to understand the roleof treatment with celecoxib, dimethyl celecoxib, andTGF-b1 in their propensity to calcify; details of thefollowing in vitro assays are in the SupplementalAppendix. The nodule assay allows for rapidscreening of potential drug strategies that may pre-vent dystrophic calcification in vitro (5,7,12). Briefly,cells were plated onto pronectin (AVICs) or collagenIV (AVECs) Flexcell plates (Flexcell International,Burlington, North Carolina), then treated withTGF-b1, and subsequently strained at 15% using theFlexcell Tension system, as previously described

J A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L . - , N O . - , 2 0 1 9 Bowler et al.- 2 0 1 9 :- –- Celecoxib Is Associated With Aortic Stenosis

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(10,11). In a separate cohort, AVICs were treated withconditioned medium harvested from AVEC culturesafter strain. AVICs were also evaluated for contrac-tility using a free-floating collagen gel system inwhich cells were plated onto gels and imaged overtime to quantify the gel area. Western blots andimmunofluorescence were used to evaluate expres-sion of myofibroblast markers CDH11 and a-SMA aftervarious treatments. In all cases, cells were platedsimultaneously with celecoxib (Tocris 3786, TocrisBioscience, Bristol, United Kingdom), dimethyl cele-coxib (Sigma-Aldrich D7196, Sigma-Aldrich, St. Louis,Missouri), or no drug to allow for interactions withCDH11 before homotypic bonds were formed. 10mmol/l celecoxib and dimethyl celecoxib was chosento match the plasma concentration found after typicaldoses of celecoxib in humans (9). For all experiments,n $ 3; more detailed methodology can be found in theOnline Appendix. All groups were compared withanalysis of variance (ANOVA) in SigmaPlot softwareversion 11.0 (Systat Software, San Jose, California), anda p value <0.05 was considered significant. Normality(Shapiro-Wilk) and equal variancewere tested. Normaldatasets with equal variance were analyzed via 1-wayANOVA with pairwise multiple comparisons madeusing the Holm-Sidak post hoc testing method.Non-normal datasets were analyzed via Kruskal-Wallis1-way ANOVA on ranks with pairwise multiplecomparisons made using Dunn’s post hoc testingmethod. In vitro data are presented as mean � SEM.

CLINICAL DATA AND STATISTICAL ANALYSIS. ASpatients 60 to 89 years of age on January 27, 2018,were identified using the Synthetic Derivative, ade-identified version of VUMC’s EMR containing>2.5 million unique records. Date gating and clinicalcovariates were identified a priori on the basis ofcelecoxib’s approval history and known risk factorsfor AS, respectively. Ibuprofen and naproxen werechosen for comparison due to their similar indicationsand pattern of use (14,15), and their previous useas comparators for celecoxib in the PRECISION(Prospective Randomized Evaluation of CelecoxibIntegrated Safety vs Ibuprofen or Naproxen) trial (16).Detailed cohort definition criteria can be found in theSupplemental Appendix. Mean available follow-upwas 10.16 � 3.14 years. Unadjusted odds ratios (ORs)and differences between cases and controls werecalculated using the Fisher exact and Mann-WhitneyU tests, respectively. Given the significant associa-tion of several clinical variables with incident AS inour preliminary models, a multivariable logisticregression based on age, sex, body mass index,hypertension, diabetes, and drug use was used to

calculate adjusted ORs and p values (17). All analyseswere performed using the statistical programminglanguage R, version 3.4.4 (18). Clinical data are pre-sented as mean � SD. Use of the Synthetic Derivativeis classified as nonhuman research by VanderbiltUniversity’s institutional review board, and approvalwas given for this study.

RESULTS

IN VITRO DYSTROPHIC CALCIFICATION ANALYSIS.

Alizarin Red staining of calcium shows the charac-teristic rounded morphology of CNs formed by AVICs(Figure 1A). As expected, treatment with TGF-b1increases the number of CNs under all pre-treatmentconditions (Figures 1A and 1B). Unexpectedly, cele-coxib pre-treatment causes a greater increase in CNnumber, whereas dimethyl celecoxib pre-treatment,as hypothesized, prevents TGF-b1–induced CN for-mation (Figures 1A and 1B). A gel contraction assayreveals that celecoxib-treated AVICs appear morecontractile than their untreated or dimethylcelecoxib–treated counterparts, though not signifi-cantly (Figure 1C). TGF-b1 treatment increasescontractility as well and compounds with celecoxibtreatment to cause significantly more contractionthan the no drug pre-treated with TGF-b1 (Figure 1C).Expression of myofibroblast markers a-SMA andCDH11 were evaluated by Western blot (Figures 1D and1E, Supplemental Figure S1) and immunofluorescence(Figure 1F, Supplemental Figure S2). Densitometrydemonstrates a significant increase in both markersonly in the celecoxib pre-treated AVICs (Figures 1Dand 1F). We observe no calcification of AVECsalone, as was expected, and very little calcification inAVICs treated with AVEC conditioned medium(Supplemental Figure S3).

RETROSPECTIVE CLINICAL ANALYSIS. The resultsobtained from these in vitro experiments led us toinvestigate possible clinical significance of celecoxibuse. Approximately 8,300 deidentified patient recordsfrom VUMC met inclusion criteria and were queriedfor possible association of AS with celecoxib, nap-roxen, or ibuprofen use (Figure 2). In unadjustedanalyses, celecoxib use is associated with an increasedodds of developing AS (OR: 1.36; 95% confidence in-terval: 1.11 to 1.67; p ¼ 0.003) (Table 1). After adjust-ment, this association persists (adjusted OR: 1.24; 95%confidence interval: 1.00 to 1.53; p ¼ 0.046). Identicalanalyses were performed with ibuprofen and nap-roxen, and no associations were found. To assess theconsistency of this cohort with those in other cele-coxib studies, we cursorily examined unadjusted ORsof celecoxib with myocardial infarction and ischemic

FIGURE 1 Dimethyl Celecoxib Prevents CN Formation in AVICs but Celecoxib Promotes CN Formation Through Myofibroblast Induction

(A) Cyclic biaxial strain and TGF-b1 induce CN formation, identified by Alizarin Red staining. (B) Treatment with celecoxib increases the number of CNs formed in the

untreated and TGF-b1–treated cases. Dimethyl celecoxib treatment reduces the number of TGF-b1–induced CNs. (C) TGF-b1 treatment increases contractility. Celecoxib

pre-treatment also increases contractility to the level of ND þ TGF-b1. Treatment with celecoxib increases expression of a-SMA (D and F) and CDH11 (E and F). n $ 3.

*p < 0.05 versus ND, #p < 0.05 versus ND þ TGF-b1, **p < 0.001 versus same pre-treatment, ##p < 0.001 versus ND þ TGF-b1. Scale bars indicate 1 mm (A) and 100

mm (F). AVIC ¼ aortic valve interstitial cell; CCB ¼ celecoxib; CN ¼ calcific nodule; DMC ¼ dimethyl celecoxib; ND ¼ no drug; TGF ¼ transforming growth factor.

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stroke, and found no association, as has been reportedpreviously (Supplemental Table S1) (16).

DISCUSSION

Our investigation was motivated by the need forpharmaceutical alternatives to aortic valve replace-ment and the unique ability of celecoxib anddimethyl celecoxib to bind CDH11, a recently identi-fied target for CAVD and AS. We have previouslydemonstrated that targeting CDH11 in vivo preventsthe pathological increase in aortic jet maximum ve-locity (6), a clinical metric used to define the severityof AS. Others have found that celecoxib and its inac-tive analog, dimethyl celecoxib, were able to bindCDH11 (9), presenting an opportunity to exploit theoff-target effects of celecoxib to treat CAVD with an

already FDA-approved drug. The main finding of thiswork was unexpected. Primarily, celecoxib, the FDA-approved drug we anticipated being a potentialtherapeutic for CAVD, causes calcification in vitro andis associated with AS in patients. Conversely, theinactive analog dimethyl celecoxib showed the ex-pected benefit of CDH11 blockade. Although furtherstudies of dimethyl celecoxib are warranted, the newrisk of celecoxib, a commonly prescribed drug, is thefocus of our studies and discussion.

CELECOXIB PROMOTES MYOFIBROBLAST

DIFFERENTIATION AND CALCIFICATION IN VITRO.

Although AVICs are widely believed to be the cellsdriving CAVD, we evaluated the effects of celecoxiband dimethyl celecoxib on both AVICs and AVECs, aswell as effects on AVICs from drug-treated AVEC

FIGURE 2 Retrospective Cohort Study Design

The retrospective clinical analysis described here was designed based on the approval and clinical trial history of celecoxib. Celecoxib was

approved on December 31, 1998, and clinical trial results revealed a potential cardiovascular risk in early 2005, defining our drug surveillance

period. Body mass index (BMI) data were collected within 1 year of January 1, 2005. The aortic stenosis surveillance period extended from

January 1, 2005, to January 27, 2018. APC ¼ Adenoma Prevention With Celecoxib; FDA ¼ Food and Drug Administration.

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conditioned medium. Because AVECs showedno response to celecoxib or dimethyl celecoxib(Supplemental Figure S3), we focused on directeffects of the drugs on AVICs. We show here that

TABLE 1 Celecoxib Use Is Associated With AS

Cases Controls

Celecoxib (n ¼ 574) (n ¼ 6,397)

Male 57.49 (330) 43.71 (2,796)

Age, yrs 76.70 � 4.80 72.89 � 7.91

BMI, kg/m2 30.67 � 6.94 28.04 � 6.05

Hypertension 54.88 (315) 40.24 (2,574)

Type 2 diabetes 24.74 (142) 15.85 (1,014)

Celecoxib use 23.34 (134) 18.21 (1,165)

Ibuprofen (n ¼ 427) (n ¼ 4,724)

Male 57.14 (244) 44.86 (2,119)

Age, yrs 76.86 � 7.82 73.15 � 7.87

BMI, kg/m2 30.83 � 6.89 29.93 � 6.94

Hypertension 54.33 (232) 40.60 (1,918)

Type 2 diabetes 25.76 (110) 16.49 (779)

Ibuprofen use 26.46 (113) 30.25 (1,429)

Naproxen (n ¼ 509) (n ¼ 5,342)

Male 57.37 (292) 45.17 (2,413)

Age, yrs 76.50 � 7.86 73.07 � 7.88

BMI, kg/m2 30.58 � 6.81 29.83 � 6.96

Hypertension 55.80 (284) 40.21 (2,148)

Type 2 diabetes 24.75 (126) 16.17 (864)

Naproxen use 16.50 (84) 18.12 (968)

Values are % (n) or mean � SD. Odds ratios (ORs) for age and BMI are reported per un

AS ¼ aortic stenosis; BMI ¼ body mass index; CI ¼ confidence interval.

celecoxib causes an increase in both a-SMA andCDH11 expression, pointing to an induction of themyofibroblast phenotype, which then leads to CNformation. Conversely, although dimethyl celecoxib

Unadjusted OR(95% CI)

Unadjustedp Value

Adjusted OR(95% CI)

Adjustedp Value

1.73 (1.46–2.06) <0.001 1.70 (1.43–2.03) <0.001

1.06 (1.05–1.07) <0.001 1.06 (1.05–1.07) <0.001

1.02 (1.01–1.03) 0.002 1.02 (1.01–1.03) 0.003

1.81 (1.52–2.14) <0.001 1.42 (1.19–1.70) <0.001

1.75 (1.43–2.13) <0.001 1.35 (1.09–1.67) 0.006

1.36 (1.11–1.67) 0.003 1.24 (1.00–1.53) 0.046

1.64 (1.34–2.00) <0.001 1.59 (1.30–1.95) <0.001

1.06 (1.05–1.07) <0.001 1.06 (1.05–1.07) <0.001

1.02 (1.00–1.03) 0.011 1.02 (1.01–1.04) 0.006

1.74 (1.43–2.12) <0.001 1.40 (1.13–1.72) 0.002

1.76 (1.40–2.21) <0.001 1.38 (1.08–1.77) 0.010

0.83 (0.66–1.04) 0.102 0.98 (0.78–1.23) 0.852

1.63 (1.36–1.96) <0.001 1.55 (1.29–1.87) <0.001

1.06 (1.04–1.07) <0.001 1.05 (1.04–1.07) <0.001

1.01 (1.00–1.03) 0.021 1.02 (1.00–1.03) 0.025

1.88 (1.56–2.25) <0.001 1.55 (1.28–1.88) <0.001

1.71 (1.38–2.11) <0.001 1.35 (1.07–1.88) 0.010

0.89 (0.70–1.14) 0.364 0.92 (0.71–1.18) 0.498

it increase.

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treatment does not appear to change the AVICphenotype—contractility, a-SMA expression, andCDH11 expression remain unchanged—it does signifi-cantly reduce CN formation. This supports ourhypothesis that this beneficial effect is likely theresult of dimethyl celecoxib preventing homotypicinteractions of CDH11 between neighboring cells.With CDH11’s cell–cell adhesions blocked, the tensionbetween AVICs is reduced, which prevents themembrane tearing and subsequent apoptosis-mediated cell death that leads to CN formation.Given that activity in the cyclooxygenase-2 (COX2)axis is the key difference between celecoxiband dimethyl celecoxib, we attribute celecoxib’spromyofibroblast effect to COX2 inhibition, whichsupports the notion of a protective role for COX2 indystrophic CN formation.

CELECOXIB IS ASSOCIATED WITH AS. This is not thefirst investigation into the impact of COX2 inhibitorson heart disease. Most COX2 inhibitors were pulledfrom the market because of adverse cardiovasculareffects by 2005 (19,20). Celecoxib had not displayedthe same adverse effects and has retained FDAapproval; however, the FDA mandated a cardiovas-cular safety trial. This study showed no increased riskof cardiovascular death, nonfatal myocardial infarc-tion, or nonfatal stroke with celecoxib use whencompared with ibuprofen or naproxen (16). However,these outcomes focus on acute, relatively short-term,and thrombotic events, and do not include valvularpathologies. On the basis of our in vitro data, ratherthan an acute thrombotic event, we suspected anincrease in long-term risk of AS in these patients.Therefore, we tested our hypothesis using longitudi-nal clinical data. In retrospective analysis, weobserved a unique association of celecoxib use withthe presence of AS. The association of celecoxib andAS remained significant when adjusted for age, sex,body mass index, and known AS risk factors (17,21).The same is not observed in patients taking ibuprofenor naproxen, which have comparable major in-dications and clinical uses to celecoxib (acute pain,inflammatory or rheumatoid disorders, osteoarthritis,and primary dysmenorrhea), but inhibit COX1 inaddition to COX2. This suggests there is somethingunique about celecoxib or selective COX2 inhibitionthat is associated with AS.

THE UNKNOWN ROLE OF COX2 IN AS. COX2expression is increased in calcified human aorticvalves (22), yet there are conflicting data as towhether it is a disease initiator or part of a protectiveresponse. COX2 inhibition has been shown in theKlotho-deficient mouse to lead to decreased aortic

valve calcification via an osteogenic mechanismassessed by cell- and tissue-level pathology (22).Although calcification is the most common pathologyfinding in AS, clinical decision making is driven byfunctional measures (such as aortic jet maximumvelocity). However, clinical studies of celecoxib havenot yet focused on valvular function or pathology, orlong-term effects (>4 years) of the drug (16,19,23). Wehave shown that COX2 inhibition can promoteCN formation in porcine AVICs through the moreprevalent dystrophic pathway of calcification, and asignificant association between celecoxib use and ASin humans.

Collectively, these findings support further inves-tigation of celecoxib or COX2’s role in other models ofCAVD and AS, such as Notch1þ/� or Apoe�/� mice, toclarify whether COX2 is protective or disease-driving.COX2 also plays a key role in modulating variousimmune processes, and investigating the impact ofcelecoxib in immunocompetent CAVD models mayprovide new insights into the in vivo mechanismsimplicated. Clinically, a multifaceted retrospectivestudy of functional and imaging-defined AS progres-sion with celecoxib use may further clarify this risk. Itis still unclear whether celecoxib introduces novelrisk or is a modifying risk factor in those already atrisk. Additionally, although the efficacy of targetingCDH11 in vivo has been shown (6), further studies ofdimethyl celecoxib in relevant murine models andeventually humans could reveal a novel therapeuticfor CAVD.STUDY LIMITATIONS. Our in vitro experiments relyon porcine cells, which are a standard model forCAVD research and are potentially better examples ofhealthy valve cells than samples from humans,because most heart valve donors are not free of othercardiovascular pathologies. Future work in humanAVICs or a variety of in vivo mouse models couldconfirm our proposed mechanism.

Although we have imposed strict time gates andcohort selection criteria, retrospective EMR studydoes not allow for controlled assessment or follow-upof study participants. The retrospective nature of theclinical analysis does not allow us to quantify dosageof patients included, but contemporary publishedreports conclude that >80% of users at the time hadstandard 200 mg prescriptions (14,15). It is difficult toconfidently rule in or rule out AS for patients inthis large deidentified cohort of clinical records, butwe tried to use definitions that would increasethe accuracy of these designations. We cannot ruleout that CAVD may have been present in some in-dividuals during the drug exposure period. In addi-tion, the retrospective nature of the study precludes

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: A recent

cardiovascular safety trial showed that celecoxib is noninferior to

ibuprofen and naproxen with regard to cardiovascular death,

nonfatal myocardial infarction, and nonfatal stroke, and is

associated with a lower risk of gastrointestinal bleeding

than those medications. Those findings may encourage more

widespread use of celecoxib to treat inflammatory pain. Our

findings, including both in vitro studies and a retrospective

review of the electronic medical record, indicate that celecoxib is

associated with calcific nodule formation and the development

of aortic stenosis.

TRANSLATIONAL OUTLOOK: Additional clinical studies are

needed to confirm the link between celecoxib use and the

development or progression of aortic stenosis, as this could

influence prescribing patterns for medications that relieve

inflammatory pain. Our studies show that dimethyl celecoxib

impedes calcific nodule formation in vitro. Thorough in vivo

animal studies should be performed to assess dimethyl

celecoxib’s translational potential for the prevention or

treatment of calcific aortic valve disease.

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conclusions about causality of CAVD. The variousdifferences between the celecoxib and control co-horts are adjusted for when possible, but may implyadditional underlying differences that are bettercontrolled in a randomized controlled trial. Forexample, in our preliminary models, we assessed theimpact of hyperlipidemia on AS incidence, but it wasnot significantly associated with AS and had no effecton the model. This may be due to incomplete retro-spective data or the lack in the era queried ofconsistent laboratory values such as lipoprotein(a),which has since proven a reliable biomarker for theassociation of dyslipidemia with AS (21). Addition-ally, valve morphology is a highly prevalent riskfactor for CAVD, but it could not be accuratelyassessed in this retrospective study without consis-tent imaging for all subjects. An echo-driven studymay provide more clarity on the impact of celecoxibin patients with a bicuspid aortic valve. Reanalysis ofthe PRECISION trial data may be an effective optionfor assessing the potential risk outlined in thisanalysis.

CONCLUSIONS

Overall, these data suggest that celecoxib use isassociated with the development of CAVD. Althoughfurther studies are necessary, it is likely that dimethylcelecoxib or a monoclonal antibody against CDH11would be safer therapeutic options than celecoxib topursue for patients with CAVD or other CDH11-mediated diseases. Considering the indications forcelecoxib, these results suggest that physicians mustcarefully balance the risks of COX1 inhibition in thegut with those of COX2-specific inhibition in the

aortic valve when choosing a pain control regimen,and use celecoxib with caution in elderly patientswith risk factors for AS.

ADDRESS FOR CORRESPONDENCE: Dr. W. DavidMerryman, Vanderbilt University, 2213 GarlandAvenue, 9445D MRB4, Nashville, Tennessee 37232.E-mail: david.merryman@vanderbilt.edu.

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KEY WORDS aortic stenosis, aortic valve,calcification, celecoxib

APPENDIX For an expanded Methods sectionas well as supplemental figures and tables,please see the online version of this paper.