Invited Reviews

Toxicologic Pathology, 38: 837-848, 2010

Copyright # 2010 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623310378027

Drug-induced Valvulopathy: An Update

CHANDIKUMAR S. ELANGBAM
Safety Assessment, GlaxoSmithKline, Research Triangle Park, North Carolina, USA
ABSTRACT

Drug-induced valvulopathy is a serious liability for certain compound classes in development and for some marketed drugs intended for human use.
Reports of valvulopathy led to the withdrawal of fenfluramines (anorexigens) and pergolide (antiparkinson drug) from the United States market in 1997
and 2007, respectively. The mechanism responsible for the pathogenesis of valvulopathy by these drugs is likely a result of an ‘‘off-target’’ effect via
activation of 5-hydroxytryptamine (5-HT) 2B receptor (5-HT2BR) expressed on heart valve leaflets. Microscopically, the affected valve leaflets showed
plaques of proliferative myofibroblasts in an abundant extracellular matrix, composed primarily of glycosaminoglycans. However, the valvular effects
caused by fenfluramines and pergolide were not initially predicted from routine preclinical toxicity studies, and to date there are no specific validated
animal models or preclinical/toxicologic screens to accurately predict drug-induced valvulopathy. This review covers the structure and function of heart
valves and highlights major advances toward understanding the 5-HT2BR-mediated pathogenesis of the lesion and subsequently, development of
appropriate animal models using novel techniques/experiments, use of functional screens against 5-HT2BR, and more consistent sampling and pathologic
evaluation of valves in preclinical studies that will aid in avoidance of future drug-induced valvulopathy in humans.

Keywords: heart valve; valvulopathy; 5-hydroxytryptamine 2B receptor; anorexigens; ergot alkaloids; animal models.

HEART VALVES: STRUCTURE AND FUNCTIONAL HISTOLOGY cardiac cycle; (2) the subjacent spongiosa, composed of loose
collagen and abundant proteoglycans, provides a cushion for
Normal semilunar (i.e., aortic and pulmonary) and atrioventri-
physical forces; and (3) the fibrosa, facing the outflow surface,
cular (i.e., mitral and tricuspid) heart valves play an important
consisting predominantly of dense collagen fibers, provides
role in maintaining unidirectional blood flow through the cardiac
strength (Figure 1). Additionally, a poorly demarcated fourth
chambers of the heart, and this function requires not only struc-
layer called the aortalis (facing the aortic surface), composed of
tural integrity, but also coordinated interactions among several
scant collagen and elastin, has been described in selected publica-
critical valvular components (i.e., for aortic and pulmonary
tions (Schoen and Edwards 2001; Schoen 2005a; Schoen 2005b;
valves, cusps/leaflets, commissures, and their respective support-
Schoen 2006). The atrioventricular valve (AV) leaflets are struc-
ing structures [roots]; and for mitral and tricuspid valves, leaflets,
turally similar to those of the SV and have analogous compo-
commissures, annulus, chordae tendineae, papillary muscles, and
nents: atrialis (also known as auricularis), spongiosa, fibrosa,
atrial and ventricular myocardium). In humans, semilunar valve
and ventricularis (from inflow to outflow, respectively). Both the
(SV) leaflets are lined with endothelium and have three histologi-
AV and the SV are innervated by adrenergic and cholinergic
cally well-defined tissue layers: (1) the ventricularis, facing the
neural networks (Marron et al. 1996).
inflow surface, composed primarily of collagen and elastic fibers,
provides for elasticity when the cusp changes shape during the
VALVULAR CELLS

Address correspondence to: Chandikumar S. Elangbam, Safety Assessment,

9.3010.2D, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC
27709-3398, USA; e-mail: chandi.s.elangbam@gsk.com.
Abbreviations: 5-HT, 5-hydroxytryptamine; 5-HTT, 5-hydroxytryptamine
transporter; ALK-5, activin receptor-like kinase-5; AV, atrioventricular valve;
aVICs, activated VICs; ECM, extracellular matrix; ERK, extracellular
regulated kinase; FDA, United States Food and Drug Administration; GAGs,
glycosaminoglycans; MDA, 3,4-dimethoxyamphetamine; MDMA, 3,4-methy-
lenedioxymethamphetamine; MMPs, matrix metalloproteinases; NTP,
National Toxicology Program; obVICs, osteoblastic VICs; PGs, proteogly-
cans; pVICs, progenitor VICs; qVICs, quiescent VICs; SD, Sprague-Dawley;
SMV, spontaneous mitral valvulopathy; SV, Semilunar valve; TGFb, trans-
forming growth factor b; TIMPs, tissue inhibitors of metalloproteinases; VECs,
valvular endothelial cells; VICs, valvular interstitial cells; a-SMA, a-smooth
muscle actin.
Two types of cells are present in fully formed heart valves:
valvular endothelial cells (VECs), covering all surfaces of the
valve leaflet, and valvular interstitial cells (VICs), located deep
below the surface. It has been shown that VECs express differ-
ent transcriptional profiles and respond differently to injury at
various regions of the valve (e.g., aortic side vs. ventricular
side) (Simmons et al. 2005). For example, VECs in normal aor-
tic valves of adult pigs have been shown to differentially
express 584 genes on the aortic side versus the ventricular side
(Simmons et al. 2005). Several of these observed differences
could help explain the vulnerability of the aortic side of the
valve cusp to calcification in disease (Simmons et al. 2005).
Valvular endothelial cells also show phenotypic differences

837
838 ELANGBAM TOXICOLOGIC PATHOLOGY

FIGURE 1.—Systematic representation of heart chambers, valves, and valvular histology. Blue arrows indicate unidirectional blood flow through
the various chambers of the heart. Histology of aortic valves, mimicking Elastic Van Giesen stain (collagen, red; elastic fibers, black). Three well-
defined layers: (1) fibrosa, facing the outflow surface, consisting predominantly of dense collagen fibers; (2) spongiosa, composed of loose col-
lagen and abundant proteoglycans (clear spaces); and (3) ventricularis, facing the inflow surface, composed of primarily collagen and elastic
fibers. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; IS, interventricular septum; and PM, papillary muscles.

in response to in vitro shear when compared with vascular
endothelial cells (Butcher et al. 2006). However, reasons for
and implications of these differences are not yet known.
Valvular interstitial cells, the most prevalent cells in all
three valvular layers, maintain the integrity and stability of nor-
mal valves and regulate repair processes during disease and fol-
lowing valve injury (Messier et al. 1994; Mulholland and
Gotlieb 1996; Mulholland and Gotlieb 1997; Rabkin et al.
2001). Valvular interstitial cells have morphologic and func-
tional features characteristic of fibroblasts, smooth muscle
cells, and myofibroblasts (Schoen 2006), and they express
muscle structural genes, including the cardiac isoforms of
troponin-T, -I, and -C; b-myosin heavy chain; and cardiac
myosin light chain 2 (Roy et al. 2000). Approximately 57%
of VICs in human valve leaflets express a-smooth muscle actin
(a-SMA, a marker for distinguishing smooth muscle cells and
myofibroblasts from fibroblasts). In vitro, however, nearly 80%
of VICs stain for a-SMA (Taylor et al. 2000). Valvular intersti-
tial cells have specific features determined at the time of
embryonic endothelial-to-mesenchymal transformation, and
therefore, the term ‘‘valve fibroblasts’’ should be abandoned
and replaced by the term ‘‘VICs’’ (Liu et al. 2007). The valves
were previously thought to be composed of a phenotypically
homogeneous population of VICs, but now there are currently
five identifiable phenotypes of VICs: (1) embryonic progenitor
endothelial/mesenchymal cells; (2) quiescent VICs (qVICs);
(3) activated VICs (aVICs); (4) progenitor VICs (pVICs); and
(5) osteoblastic VICs (obVICs) (Liu et al. 2007). These pheno-
types may exhibit plasticity and convert from one form to
another. The embryonic progenitor endothelial/mesenchymal
cells represent the cells that are derived embryonically through
endothelial-to-mesenchymal transformation, which initiates
the process of valve formation in the embryo (Norris et al.
2008). Quiescent VICs are the fibroblast-type cells in the adult
valve and maintain normal valve physiology. During valvular
injury, qVICs become aVICs, express a-SMA, and become
involved in the repair and remodeling of valves (Aikawa et al.
2006; Cushing et al. 2005; Liu and Gotlieb 2008; Walker et al.
2004). These cells are a type of myofibroblast cell similar to
those found at sites of wound repair in a variety of tissues
(Desmouliere et al. 2005; Hinz et al. 2007). The pVICs are poorly
defined and consist of a heterogeneous population of progenitor
cells that may be important in repair (Visconti et al. 2006). In
conditions promoting calcification, such as the presence of osteo-
genic and chondrogenic factors, qVICs can undergo osteoblastic
differentiation into obVICs (Chen et al. 2009).
Vol. 38, No. 6, 2010 DRUG-INDUCED VALVULOPATHY 839

VALVULAR EXTRACELLULAR MATRIX but characterized as a central core of collagen fibers, reinforced
by thin layers of elastic fibers (Aupperle, Marz et al. 2009;
The primary components of valvular extracellular matrix
Aupperle, Thielebein et al. 2009; Aupperle et al. 2010; Maxie
(ECM) are proteoglycans (PGs), collagens, and elastin. The
and Robinson 2007; Plendl 2006; Van Vleet et al. 2002).
overwhelming determinant of valve durability is valvular
Among the preclinical species for drug safety screening, gross
ECM, produced and maintained by VICs. The quantity and
evaluation of valves and associated structures is routinely
quality of valvular ECM, therefore, depend on the viability and
performed in nonrodents (dogs, primates), including sampling
function of VICs. Proteoglycans are integral ECM components
and characterization of changes and location of affected areas
of valves and play an important role in mediating ECM
for further histopathological evaluation. In contrast, rodent
organization (Kinsella et al. 2004). Proteoglycans consist of
hearts are routinely fixed intact in 10% neutral buffered forma-
glycosaminoglycan (GAG) chains (linear molecules of repeat-
lin, and heart valves are not grossly examined. Inconsistencies
ing disaccharides) attached to a core protein. Depending on
in rodent heart-valve sampling are often unavoidable because
biological and biomechanical needs, the sulfation pattern of
of the inherent small heart size, thin valve leaflets, and the var-
GAG chains (i.e., whether 4-sulfated or 6-sulfated) varies.
ious trimming/sectioning methods used. For most standard
4-Sulfated GAGs (i.e., small leucine-rich dermatan sulfate
rodent studies, the heart is sectioned through its longitudinal
PGs), such as decorin and biglycan, are abundant in tensile
axis to include one or ideally both the right and left ventricles,
loading regions such as chordae tendineae; these PGs mediate
interventricular septum, portions of both atria, and major blood
collagen fibrillogenesis (Reed and Iozzo 2002) and sequester
vessels at the base of the heart. As a result, some sections may
transforming growth factor-b (Kresse and Schonherr 2001).
contain one or more valves, whereas others may not contain
Glycosaminoglycan hyaluronan and 6-sulfated GAGs (versi-
any. Review of nine long-term rat studies (four from Charles
can) are found in regions experiencing compression, such as the
River, Montreal and five from the National Toxicology Pro-
leaflet free edges; versican often co-localizes with elastic fibers
gram [NTP]) revealed a substantial variability in the presence
and numerous cell-associated molecules to regulate cell adhe-
of individual valves in heart sections, and some heart sections
sion, proliferation, and migration (Grande-Allen et al. 2004;
had no valves (Elangbam, unpublished data). Furthermore,
Wight et al. 1991; Wight 2002). Additionally, valves contain
even in the presence of prominent valvulopathy, no alteration
collagen types I, III, and IV; elastin; fibronectin (ECM protein);
in the valves was described and recorded in any of these studies
and tenascin-C (ECM glycoprotein) (Fayet et al. 2007).
(Elangbam et al. 2006). There is also a paucity of published reports
Valvular interstitial cell–regulated repair involves synth-
on age-dependent valvular changes and drug-induced valvular
esis, degradation, and reorganization of ECM, which depends
disease in rodents, and most importantly, chemically exacerbated
on matrix-degrading enzymes such as matrix metalloprotei-
spontaneous valvulopathy has not been fully explored. For exam-
nases (MMPs). Matrix metalloproteinases are calcium- and
ple, although valvular changes were not described and recorded in
zinc-dependent proteases that are produced and secreted by
the original NTP study report with dl-amphetamine in Fischer 344
VICs as inactive zymogens. Matrix metalloproteinases are
(F344) rats (NTP 1991), subsequent re-evaluation of hearts from
activated by plasmin, interleukin-1b, and tumor necrosis fac-
this study found a treatment-related exacerbation of spontaneous
tor-a, and they are inhibited by four types of specific tissue
valvulopathy (Elangbam et al. 2006). Therefore, unless specified
inhibitors of metalloproteinases (TIMPs) through the forma-
for a particular rodent study, it is not customary or mandatory prac-
tion of irreversible complexes (Li et al. 2000; Visse and
tice to examine and record heart valve findings, particularly in
Nagase 2003). TIMP-1, -2, and -4 are present in soluble form,
two-year studies. As a result, the valvular findings in rodents may
whereas TIMP-3 binds to the ECM through heparan sulphate
have been underdiagnosed and underreported, mainly because of
proteoglycans (Kassiri and Khokha 2005). Matrix metallopro-
inevitable inconsistency of histological sampling and inconsis-
teinases and TIMPs play an important role in the physiological
tency in reporting.
maintenance of ECM, as well as in the pathogenesis of a vari-
ety of diseases (Li et al. 2000; Visse and Nagase 2003). Extra-
cellular matrix changes occur in many heart valve pathologies, DRUG-INDUCED VALVULOPATHY IN HUMANS
including myxomatous mitral valve disease (Grande-Allen
Valvular abnormality from any cause can lead to hemody-
et al. 2004; Gupta et al. 2008; Mahimkar et al. 2009) and
namic overload on ventricles (left or right ventricle, or both
drug-induced valvulopathy in humans and animals (Droog-
ventricles), and this overload eventually leads to myocardial
mans, Cosyns et al. 2007; Droogmans, Franken et al. 2007;
dysfunction, congestive heart failure, and sudden death in
Elangbam et al. 2006; Elangbam et al. 2008; Gustafsson
humans. Even a subtle, nondestructive valvular thickening may
et al. 2005; McDonald et al. 2002).
result in leaflet restriction and regurgitation (Carabello and
Crawford 1997). Valvular disease, or valvulopathy, is a main
ASSESSMENT OF HEART VALVES IN TOXICOLOGIC PATHOLOGY
cause of morbidity and mortality in humans worldwide,
In veterinary medicine, valvular histology has been incon- requiring valve replacement surgery, the second most common
sistently described in recent editions of histology/pathology heart operation performed in the United States (Keane et al.
textbooks; that is, it is described as three or four well-defined 1993; Thom et al. 2006). Four drug classes that have been
tissue layers as in humans, with no mention of specific layers, linked to valvulopathy in humans are described below.
840 ELANGBAM
Anorexigens (fenfluramine, dexfenfluramine)—Appetite
Suppressants
Anti-obesity drugs, such as fenfluramine and dexfenflura-
mine (stereoisomer of fenfluramine), have been linked to val-
vulopathy in humans (Connolly et al. 1997; Dahl et al. 2008;
Greffe et al. 2007; Hershel 2000; Jick et al. 1998). Fenflura-
mine, dexfenfluramine, and their major metabolites norfenflur-
amines stimulate 5-hydroxytryptamine (5-HT, serotonin)
release from neuronal and platelet stores (Fishman 1999;
Yanovski and Yanovski 2002). Connolly et al. (1997) first
reported twenty-four cases of valvulopathy in women who had
received treatment with the fenfluramine and phentermine
combination (‘‘Fen-Phen’’) for a mean duration of eleven
months. Five of these patients subsequently underwent cardiac
surgery and valve replacement. Microscopic examination of
the valve leaflets showed ‘‘stuck-on’’ plaques of proliferative
myofibroblasts in an abundant extracellular matrix. A subse-
quent larger study with valve samples from sixty-four patients
described an ‘‘atypical plaque’’ containing myxoid stroma, pro-
liferative myofibroblasts, and often small vessels and lympho-
cytic infiltrations (Steffee et al. 1999; Volmar and Hutchins
2001). These valvular changes resembled lesions associated with
carcinoid heart disease (valvular disease in patients with carci-
noid tumors of the gastrointestinal tract that have metastasized
to the liver) or ergotamine-induced valvulopathy. Prevalence var-
ies considerably between studies, ranging from approximately
6% to just over 30% (Bowen et al. 1997). One case-control study
reported a prevalence of 23% in patients treated with fenflura-
mine. A meta-analysis of the published studies of patients treated
with fenfluramine estimated that one in eight patients receiving
these agents for more than ninety days had valvulopathy (Sachdev
et al. 2002). It has also been shown that the duration of treatment
played a role in the development of clinical valvulopathy, namely,
that low prevalence rates are associated with shorter durations of
therapy, and higher prevalence rates are associated with longer
exposures (Jollis et al. 2000). In September 1997, both fenflura-
mine and dexfenfluramine were withdrawn from the United
States (U.S.) market, including a multibillion-dollar settlement
by the manufacturer of these drugs (SoRelle 1999). Recently,
several cases of fenfluramine-induced valvulopathy requiring
surgery have been identified, seven years after discontinuing the
medication, suggesting that anorexigen-induced valvulopathy
may continue to manifest well into the future (Dahl et al. 2008;
Greffee et al. 2007).
Dopamine Agonists (pergolide, cabergoline)—Parkinson’s
Disease and Hyperprolactinemic Disorders
Over the past three decades, there has been widespread
use of ergot-derived dopamine agonists (pergolide and caber-
goline) to treat Parkinson’s disease, restless leg syndrome, and
hyperprolactinemic disorders. An association between valvulo-
pathy and pergolide was first reported in 2002 (Pritchett et al.
2002), and this association is now widely accepted. Typically,
the morphological and histological features of pergolide-
related valvulopathy resemble fenfluramine- and ergotamine-
TOXICOLOGIC PATHOLOGY
induced valvulopathy and carcinoid heart disease (Baseman
et al. 2004). Recently, two large European studies indepen-
dently verified the association of valvulopathy with pergolide
and cabergoline. In the first study, Zanettini et al. (2007)
reported moderate or severe valvular regurgitation with a
significantly greater frequency in patients given pergolide
(23.4%) or cabergoline (28.65%) than in those given non-
ergot dopamine agonists such as pramipexole and ropinirole
(0%) or controls (5.6%). In the second study, Schade et al.
(2007) concluded that the incidence of heart valve regurgita-
tion was increased in pergolide (7.1-fold) and cabergoline
(4.9-fold) groups, but not with use of other dopamine agonists
such as ropinirole, pramipexole, bromocriptine, or lisuride.
This study also noted that the severity of valvular disease was
associated with higher cumulative doses of either cabergoline
or pergolide. Similarly, a recent systematic literature review
on valvulopathy in Parkinson’s disease patients showed that
in eleven of thirteen studies, the use of cabergoline and pergo-
lide was associated with significantly increased frequency of
heart valve disease compared with non-ergot group and con-
trols (Steiger et al. 2009). Pergolide was withdrawn from the
U.S. market in August 2007 because of increased risk for
valvulopathy. Although the European Medicines Agency has
added new warnings and contraindications to the product infor-
mation, cabergoline and pergolide remain in use in the rest of
the world (Antonini and Poewe 2007; EMEA 2008).
Ergot Alkaloids (methysergide, ergotamine)—Migraine
Prophylaxis
Migraine is a complex, common, and disabling disorder of
the brain that occurs in 34.5% of adult women and 20.1% of
adult men in the general population (Bigal et al. 2006). Ergot
alkaloid drugs include methysergide and ergotamine, which are
very effective drugs for migraine headache prophylaxis. In
1967, Graham reported the development of valvular insuffi-
ciency and retroperitoneal fibrosis in twenty-seven patients
taking methysergide. Although initially reported as left-sided
valve effect, autopsy studies and case reports showed tricuspid
valve involvement (Bana et al. 1974; Hendrikx et al. 1996;
Misch 1974). Furthermore, several patients developed dyspnea
and eventually required heart valve replacement. Ergotamine,
an ergot alkaloid structurally similar to methysergide, has been
shown to cause left-sided valvulopathy primarily affecting the
mitral valve. Ergotamine- or methysergide-associated valvulo-
pathy is morphologically similar to that seen in carcinoid heart
disease (Hauck et al. 1990; Redfield et al. 1992; Van Camp
et al. 2004). Although rarely used now because of their adverse
effects, methysergide and ergotamine remain licensed for
migraine prophylaxis.
Recreational Drug:
3,4-Methylenedioxymethamphetamine (Ecstasy)
3,4-Methylenedioxymethamphetamine (MDMA), an
amphetamine-based drug, is a psychoactive stimulant used for
recreational purposes. Use of MDMA has increased over the
Vol. 38, No. 6, 2010 DRUG-INDUCED VALVULOPATHY 841

TABLE 1.—Drugs with 5-HT2BR functional profiles and their association with valvulopathy in humans.

Drug/compound name Chemical class and clinical uses Interactions with 5-HT2BR Valvulopathy
Fenfluramines (fenfluramine and dexfenfluramine) and Amphetamine derivative for obesity, withdrawn High-affinity full 5-HT2BR Yes
their active metabolite norfenfluramine from the market in 1997 agonist (norfenfluramine)
3,4-methylenedioxymethamphetamine Amphetamine derivative, a commonly abused illicit High-affinity potent 5-HT2BR Yes
drug agonist
Ergotamine and dihydroergotamine Ergot alkaloid for migraine prophylaxis High-affinity potent partial 5- Yes
HT2BR agonist
Methysergide and its active metabolite Synthetic ergot alkaloid for migraine prophylaxis High-affinity partial 5-HT2BR Yes
methyergonovine agonist (methylergonovine)
Pergolide Ergot derivative for Parkinson’s disease, withdrawn High-affinity potent 5-HT2BR Yes
from the US market in August 2007 agonist
Cabergoline Ergot derivative for Parkinson’s disease and High-affinity potent 5-HT2BR Yes
hyperprolactinemia agonist
Bromocriptine Ergot derivative for hyperprolactinemia and Par- Low-affinity partial 5-HT2BR Case reports
kinson’s disease agonist
Terguride Ergot derivative for hyperprolactinemia 5-HT2BR antagonist No
Lisuride Ergot derivative for Parkinson’s disease 5-HT2BR antagonist No
Quinagolide Non-ergot derivative for hyperprolactinemia Low affinity 5-HT2BR agonist No
Pramipexole Non-ergot derivative for Parkinson’s disease Low affinity 5-HT2BR agonist No
Ropinirole Non-ergot derivative for Parkinson’s disease and Low affinity, less potent 5-HT2BR No
Restless leg syndrome agonist

past two decades and has become one of the most popular party
drugs in Western Europe and the United States (EMCDDA
2005; Martins et al. 2005). Setola et al. (2003) first reported
that MDMA and its metabolite 3,4-dimethoxyamphetamine
(MDA) are potent 5-HT2BR agonists, and these drugs elicited
prolonged mitogenic responses in human VICs in vitro. As pre-
dicted by Setola et al. (2003), Droogmans, Cosyns et al. (2007)
reported the development of significant valvular regurgitation
in eight (28%) people (mean age of 24.3 years) who took
MDMA (average of 3.6 tablets of MDMA per week for six
years) compared with none in the control group. Analysis of
valve morphology identified restrictive valvulopathy similar to
that seen in patients taking pergolide or cabergoline. The investi-
gators also noted a correlation between dose and severity of val-
vulopathy. Most MDMA use is intermittent and recreational, but
in heavy and frequent MDMA users, an increased risk of valvulo-
pathy seems probable. 3,4-Methylenedioxymethamphetamine is
currently being evaluated as a treatment for post-traumatic stress
disorder.
MOLECULAR MECHANISM OF DRUG-INDUCED VALVULOPATHY
Pharmacologically, anorexigens (fenfluramine, dexfenflura-
mine, and their active metabolite norfenfluramine), dopamine
agonists (pergolide and cabergoline), amphetamines (MDMA
and its metabolite MDA), and ergot alkaloids (ergotamine, dihy-
droergotamine, and methysergide and its active metabolite
methylergonovine) are potent 5-HT2B receptor (5-HT2BR)
agonists (Table 1). Therefore, the mechanism responsible for the
pathogenesis of valvulopathy by these drugs is likely a result of
an ‘‘off-target’’ effect via activation of 5-HT2BR expressed on
heart valve leaflets (Fitzgerald et al. 2000; Roth 2007; Rothman
and Baumann 2009; Rothman et al. 2000; Setola et al. 2003).
The critical role of 5-HT2BR activation in the pathogenesis of
valvulopathy is further highlighted by the observation that che-
mically similar drugs, such as lisuride and terguride, which are
selective agonists for 5-HT2C and 5-HT2A receptors and
antagonists for 5-HT2BR, were not associated with valvulopa-
thy in humans (Jahnichen et al. 2005; Roth 2007). Bromocrip-
tine and quinagolide have a weaker affinity for the 5-HT2BR
and, therefore, are thought to be less valvulopathic. However,
evidence from a relatively small study suggests that high doses
of bromocriptine are associated with an increased risk of valvu-
lopathy in patients with Parkinson’s disease (Antonini and
Poewe 2007; Serratrice et al. 2002). Similarly, no increase in the
risk of valvulopathy was observed in patients treated with the
dopamine agonist pramipexole, which has low affinity to the
human 5-HT2BR (Mulholland and Gotlieb 1997; Millan et al.
2002; Schade et al. 2007; Zanettini et al. 2007).
There are seven distinct families of 5-HT receptors (5-HT1-7)
and several subtypes (Nichols and Nichols 2008). However,
only 5-HT2BR has been implicated in valvulopathy because of its
high concentration in human heart valves and pulmonary arteries
(Fitzgerald et al. 2000; Rothman et al. 2000; Roth 2007). Recent
studies have implicated preferential activation of 5-HT2BR as a
key initiating step in the pathogenesis of anorexigen-induced
valvulopathy in humans. 5-Hydroxytryptamine has a direct
mitogenic effect on VICs, and this mitogenic effect is mediated
by 5-HT receptors (Fanburg and Lee 1997; Lopez-Ilasaca 1998;
Rajamannan et al. 2001). 5-Hydroxytryptamine 2B receptors are
G protein coupled and known to activate mitogenic pathways
through the phosphorylation of Src kinase and extracellular-
regulated kinases (ERK) (Roth 2007). Activation of 5-HT2B
receptors also leads to activation of phospholipase C-b and subse-
quent activation of protein kinase C. 5-Hydroxytryptamine and
Src-P may mediate and enhance the activity of the transforming
842 ELANGBAM
growth factor b1 receptor (also known as activin receptor–like
kinase, ALK-5) (Ignotz and Massague 1986; Jian et al. 2002; Roth
2007; Waltenberger et al. 1993). Once activated, ALK-5 subse-
quently phosphorylates the major downstream signaling mole-
cules SMAD2 and 3 proteins. Phosphorylated SMAD2 and 3
form a complex with SMAD4, and this complex translocates into
the nucleus and regulates the transcription of specific genes
involved in cell growth, differentiation, development, and
immune response (Gordon and Blobe 2008; Heldin et al. 1997;
Liu and Gotlieb 2008; Massague et al. 2005) (Figure 2). It has also
been suggested that the final common pathway by which activa-
tion of 5-HT2B receptors leads to mitogenesis probably involves
phosphorylation of retinoblastoma protein (Roth 2007).
In addition to transcriptionally controlled proliferative
effects on VICs, there is also evidence of oxidative stress
(i.e., generation of reactive superoxide, O2–) in heart valves
by serotonin and dopamine in vitro (Pena-Silva et al. 2009).
Increased O2– levels in homogenates of heart valves and
blood vessels after incubation with serotonin and dopamine
were prevented by inhibitors of flavin-oxidases (diphenyliodo-
nium) or monoamine oxidase (MAO) (ranylcypromine and/or
clorgyline), but not by inhibitors of nicotinamide adenine dinu-
cleotide phosphate (NADPH) oxidase, suggesting a novel
mechanism for MAO’s role in oxidative stress in human heart
valves by serotonin and dopamine (Pena-Silva et al. 2009).
Another study by Connolly et al. (2009) showed a significant
imbalance of ECM production versus proliferation in mitral
VIC cultures with fenfluramine plus 5-HT exposure, suggest-
ing that this imbalance may in part explain the pathophysiology
of fenfluramine-induced valvulopathy. Because of the clear
association between 5-HT2BR agonism and drug-induced
valvulopathy, it has been strongly recommended to screen
future drugs with 5-HT activity and their metabolites at the
5-HT2BR comprehensively before launching clinical trials
(Huang et al. 2009; Roth 2007; Rothman et al. 2000; Setola and
Roth 2005).
RENEWED INTEREST IN CURRENTLY PRESCRIBED DRUGS WITH
5-HT2BR AGONISM
Huang et al. (2009) identified a number of United States
Food and Drug Administration (FDA)–approved drugs that are
currently on the market as 5-HT2BR agonists and their poten-
tial for development of valvulopathy. The study included the
screening of approximately 2200 FDA-approved or investiga-
tional drugs for 5-HT2BR agonism using calcium-based,
high-throughput screening and five additional readouts of 5-
HT2BR activation such as nuclear factor of activated T cells-
mediated transcription of a b-lactamase reporter gene, ERK2
phosphorylation, b-arrestin recruitment to agonist-occupied
5-HT2B receptors, accumulation of inositol phosphates, and
cell proliferation. Of the 2200 drugs screened, twenty-seven
were 5-HT2BR agonists, including seven drugs known to be
associated with valvulopathy and six currently prescribed drugs
(guanfacine, quinidine, fenoldopam, oxymetazoline, xylometa-
zoline, and ropinirole). Furthermore, the study revealed that the
TOXICOLOGIC PATHOLOGY
patterns of 5-HT2BR functional selectivity might help to dif-
ferentiate drugs that are likely to cause valvulopathy in humans
from those that are not. For example, ropinirole, a 5-HT2BR
agonist approved for treating Parkinson’s disease and restless
leg syndrome, is not associated with valvulopathy (Schade
et al. 2007; Zanettini et al. 2007) and is distinct from the known
valvulopathic 5-HT2BR agonists in 5-HT2BR functional
assays (Huang et al. 2009). Additionally, ropinirole has low
affinity to the human 5-HT2BR. The authors also recom-
mended five currently prescribed drugs, such as guanfacine,
oxymetazoline, quinidine, xylometazoline, and fenoldopam to
be studied further in terms of their pharmacodynamics and
potential for valvulopathy. Xylometazoline and oxymetazoline
are nasal decongestants for short-term use, whereas fenoldo-
pam is an antihypertensive agent used postoperatively and
in-hospital during a hypertensive crisis for one-time use. In
contrast, guanfacine (an antihypertensive agent) and quinidine
(an antiarrhythmic agent) are indicated for prolonged use and
therefore carry a greater risk for development of valvulopathy
than drugs for short-term use. Additionally, recent FDA
approval of guanfacine for the treatment of attention-deficit–
hyperactivity disorder might further expose an increasing
number of patients, including children, to a potentially valvulo-
pathic agent (Huang et al. 2009).
DRUG-INDUCED VALVULOPATHY IN ANIMAL MODELS: NEED FOR
UNDERSTANDING OF VALVULAR PATHOPHYSIOLOGY AND ITS CAUSES
Given the serious consequences of drug-induced valvular
effects, there is a growing demand for sensitive preclinical
screening strategies, including detailed and systematic examina-
tion of heart valves and comprehensive diagnosis and recording
of valvular lesions in rats and mice, two commonly used rodent
species. To date, there are no validated animal models or precli-
nical/toxicologic screens to accurately predict drug-induced
valvulopathy in humans (Greaves 2007), and therefore, establish-
ing animal models to understand mechanisms and pathogenesis
of valvulopathy is warranted (Donnelly 2008). The first evidence
of valvular changes caused by anorexigens was reported in rats by
Bratter et al. in 1999, two years after the withdrawal of fenflura-
mine and dexfenfluramine from the U.S. market in 1997. In that
study, there was obvious thickening of mitral valves in prenatal
rats co-administered with dexfenfluramine and phentermine for
fourteen days. However, microscopic evaluations of these thick-
ened valves were not performed. It is therefore unknown whether
mitral valve thickening in rats (Bratter et al. 1999) has micro-
scopic features similar to those of human valvulopathy caused
by dexfenfluramine and phentermine combinations. Pharmacolo-
gically, fenfluramines are potent 5-HT releasers as well as
re-uptake inhibitors, whereas phentermine inhibits monoamine
oxidase (an enzyme responsible for degradation of 5-HT), and
as a result, the major emphasis has been placed on 5-HT and
5-HT receptors and their role in the pathogenesis of drug-
induced valvulopathy. In 2005, Gustafsson et al. showed that
daily subcutaneous injection of serotonin (20 mg/kg) in Sprague
Dawley (SD) rats for three months resulted in valvular changes
Vol. 38, No. 6, 2010 DRUG-INDUCED VALVULOPATHY 843

FIGURE 2.—Schematic representation of 5HT signaling pathways involved in the pathogenesis of drug-induced valvulopathy by 5-HT2BR
agonists (fenfluramine, dexfenfluramine, pergolide, cabergoline, 3,4-methylenedioxymethamphetamine, ergotamine, and methylsergide). Activa-
tion of 5-HT2BR by these drugs causes dissociation of guanine nucleotide regulatory proteins (G-proteins), which leads to activation of protein
kinase C (PKC) via activated phospholipase C-b (PLC-b) resulting from intracellular calcium mobilization and diacylglycerol (DAG) release.
G-proteins may also cause phosphorylation and activation of Src with the formation of phosphorylated Src (Src-P) and activation of extracellular
regulated kinases 1 and 2 (ERK1 and ERK2), facilitated by b-arrestin (bArr) binding. Extracellular regulated kinases 1 and 2 are also activated by
PKC. Additionally, 5-HT and possibly Src-protein enhance the activity of TGFb receptors. Activation of TGFb receptor 1 (also known as activin-
like kinase-5, or ALK-5) leads to phosphorylation of SMAD 2 and 3 proteins and formation of a complex with SMAD4; this complex translocates
to the nucleus and regulates the transcription of specific target genes. All of these pathways lead to proliferation of aVICs, matrix secretion/
accumulation, and subsequent valvulopathy. PM, plasma membrane; NEN, nuclear envelope.
844 ELANGBAM
that are morphologically and echocardiographically similar to
those seen in human carcinoid heart disease. In a separate study,
Droogmans, Franken et al. (2007) showed that rats given 5-HT
(20 mg/kg/day, subcutaneously) and pergolide (0.5 mg/kg intra-
peritoneally) for five months induced valvular regurgitation and
microscopic changes similar to those seen in anorexigen-
induced valvulopathy in humans. A subsequent study by Droog-
mans et al. (2009) showed that cyproheptadine (5-HT2BR
antagonist) co-treatment prevented the development of
pergolide-induced valvulopathy in rats with a reduced number
of 5-HT2BR–positive VICs, suggesting that 5-HT2BR plays an
important role in the pathogenesis of valvulopathy.
We previously reported the immunostaining and quantitative
transcript levels of 5-HT receptors (5-HT2BR and 5-HT1BR)
in the normal mitral, aortic, tricuspid, and pulmonary valves
of SD rats and cynomolgus monkeys (Elangbam et al. 2005).
Our results showed that the 5-HT receptor expression in four
heart valves was comparable among cynomolgus monkeys, rats,
and humans, and therefore, the potential exists to gain mechan-
istic insight to anorexigen-induced valvulopathy from animal
studies. A possible association between spontaneous mitral val-
vulopathy (SMV) and increased number of 5-HT2BR–positive
cells in valve leaflets has been described in SD rats, suggesting
that 5-HT2BR may play a role in its pathogenesis. Additionally,
5HT2BR may play a role in the exacerbation of SMV (i.e., sig-
nificant increase in incidence and severity of SMV) in Fischer
344 rats with dl-amphetamine treatment (Elangbam et al.
2006). Amphetamine analogs such as MDMA and MDA are
potent 5-HT2BR agonists, which have been shown to cause
valvular cell proliferation in vitro (Setola et al. 2003) and valvu-
lopathy in people who took MDMA for six years (Droogmans,
Cosyns et al. 2007). Interference with 5-HT transmembrane
processing by knocking out the 5-HT transporter (5-HTT) gene
has been shown to cause cardiac fibrosis and valvulopathy in
mice, establishing a link between 5-HTT deficiency and the
development of valvulopathy in vivo. Interference with 5HTT
processing may also result in increased and persistent 5-HT
receptor interactions, and increased valvular mitogenic activity
and extracellular matrix production in the 5-HTT–knockout
mice (Mekontso-Dessap et al. 2006).
Valvular thickening and subendocardial fibromyxoid
proliferation noted in SMV in rats were similar to those seen
in the heart valves of patients with carcinoid heart disease and
in people medicated with ergot alkaloids, anorectics, or
MDMA (Droogmans, Franken et al. 2007; Elangbam et al.
2006). McDonald et al. (2002) reported distinctive morphologi-
cal and compositional changes to aid in the differentiation of
anorexigen-induced valvulopathy from floppy (also known as
myxomatous valvular degeneration), rheumatic, and carcinoid
heart diseases. The size and number of onlay (refers to neo-
tissue or thickening) lesions, the amount of GAGs, the location
of inflammation and neovascularization are important in this
distinction. Normal valves are composed of roughly equal
amounts of GAGs and collagens in humans, and they rarely
harbor leukocytes and blood vessels, even as they age.
McDonald et al. (2002) demonstrated that anorexigen-
TOXICOLOGIC PATHOLOGY
exposed valves contain more GAGs than normal or floppy
valves. The valvular lesions associated with carcinoid heart
disease have GAG-rich fibromyxoid tissue and contain a large
number of leukocytes and vessels per square millimeter of tis-
sue area. Floppy valves have no significant neovascularization
but have a number of onlays. In contrast, rheumatic valves have
few large, fibrous onlays. Although the neovascularization is
more prominent as compared with floppy valves, anorexigen-
exposed valves are still less vascular but more GAG rich than
valves in carcinoid heart disease. These features are indicative
of a distinctive pathologic process in anorexigen-exposed
valves (McDonald et al. 2002). Spontaneous mitral valvulopa-
thy in SD rats has a morphology and matrix composition that
are strikingly similar to anorexigen-induced valvulopathy in
humans. Compared with normal valve leaflets, SMV exhibited
a greater valve thickness, a higher amount of GAGs, and a
lower amount of collagen (Figure 3) (Elangbam et al. 2006).
Recently, we investigated the role of 5-HT in the develop-
ment of valvulopathy in SD rats using morphological and com-
positional valvular analyses, as well as transcriptomic
modulation of 5-HT2BR and 5-HTT genes in the aortic and
mitral valves. Subcutaneous injections of 5-HT for seven days
resulted in valvular thickening and compositional alterations
(higher GAGs and lower collagen contents) in aortic and mitral
valves of SD rats. These compositional alterations were associ-
ated with up-regulation of 5-HT2BR and down-regulation of
5-HTT genes (Elangbam et al. 2008), and therefore, 5-HT2BR
activation and 5-HTT inhibition may play a significant role in
the pathogenesis of 5-HT–induced valvulopathy in SD rats. In
this study, we were able to recognize a distinctive 5-HT–related
valvular matrix alteration after seven days in the aortic and
mitral valve leaflets, thus providing a possible short-term inves-
tigative tool to study in vivo drug-induced valvulopathy by
serotonergic compounds, as a way to determine or support in
vitro selectivity data. Our findings further highlight the necessity
and use of animal models to screen potential valvular effects of
serotonergic compounds in humans.
In conclusion, valvulopathy is a serious liability for certain
compound classes in development and for some marketed
drugs intended for human use. Reports of valvulopathy in
1997 led to the withdrawal of anorexigens such as fenfluramine
and dexfenfluramine from the U.S. market, as well as a
multibillion-dollar settlement by the manufacturer of these
drugs. Similarly, pergolide (for Parkinson’s disease) was with-
drawn from the U.S. market in August 2007 because of
increased risk of valvulopathy. The valvular effects of both fen-
fluramines and pergolide were not initially predicted from rou-
tine preclinical toxicity studies, although subsequent studies
have shown standard laboratory models are capable of develop-
ing valvular lesions. It is probable that inconsistent sampling
and interpretation may have contributed to this detection fail-
ure. To date, there are no specific validated animal models or
preclinical/toxicologic screens to accurately predict drug-
induced valvulopathy. Functional screens against 5-HT2BR
and more consistent sampling and pathologic evaluation of
valves in preclinical studies appear to hold the greatest promise
Vol. 38, No. 6, 2010 DRUG-INDUCED VALVULOPATHY
FIGURE 3.—Mitral valve leaflets, Movat’s pentachrome stain. (A) Normal mitral valve leaflet from a Sprague Dawley rat. Movat’s pentachrome
highlighted the glycosaminoglycans (GAGs) and collagen as cyan blue and dark yellow, respectively. Bar ¼ 100 mm. (B) Mitral valve leaflet with
spontaneous mitral valvulopathy from a Sprague Dawley rat. Note marked thickening of the valve leaflet resulting from GAGs-rich fibromyxoid
tissue, and apparent segmental loss or disorganization of collagen. Bar ¼ 100 mm.
for detecting drugs with the potential to cause valvulopathy.
A recent report suggests that some drugs with 5-HT2BR
activity with the potential for developing drug-induced valvu-
lopathy are still in the U.S. market for long-term use, which
may require careful risk–benefit analysis.
ACKNOWLEDGMENTS
The author thanks Denise M. Frailey for illustrations and
Drs. Roger H. Brown, Richard T. Miller, Alan H. Stokes, and
Christine L. Merrill for their critical review of the manuscript.
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