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Pharmacol Ther. 2011 November ; 132(2): 146–157. doi:10.1016/j.pharmthera.2011.03.008.

Serotonin Receptors and Heart Valve Disease – it was meant 2B

Joshua D. Hutcheson1, Vincent Setola2, Bryan L. Roth2, and W. David Merryman1
1Department of Biomedical Engineering, Vanderbilt University, Nashville, TN

2Department of Pharmacology, University of North Carolina, Chapel Hill, NC

Abstract
Carcinoid heart disease was one of the first valvular pathologies studied in molecular detail, and
early research identified serotonin produced by oncogenic enterochromaffin cells as the likely
culprit in causing changes in heart valve tissue. Researchers and physicians in the mid-1960s
noted a connection between the use of several ergot-derived medications with structures similar to
serotonin and the development of heart valve pathologies similar to those observed in carcinoid
patients. The exact serotonergic target that mediated valvular pathogenesis remained a mystery for
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many years until similar cases were reported in patients using the popular diet drug Fen-Phen in
the late 1990s. The Fen-Phen episode sparked renewed interest in serotonin-mediated valve
disease, and studies led to the identification of the 5-HT2B receptor as the likely molecular target
leading to heart valve tissue fibrosis. Subsequent studies have identified numerous other activators
of the 5-HT2B receptor, and consequently, the use of many of these molecules has been linked to
heart valve disease. Herein, we: review the molecular properties of the 5-HT2B receptor including
factors that differentiate the 5-HT2B receptor from other 5-HT receptor subtypes, discuss the
studies that led to the identification of the 5-HT2B receptor as the mediator of heart valve disease,
present current efforts to identify potential valvulopathogens by screening for 5-HT2B receptor
activity, and speculate on potential therapeutic benefits of 5-HT2B receptor targeting.

1. Serotonin and its receptors

1.1. Serotonin
Serotonin or 5-hydroxytryptamine (5-HT; Fig. 1) is enzymatically transformed from the
essential amino acid tryptophan following hydroxylation and decarboxylation. Serotonin
was discovered and isolated from serum 60 years ago (Rapport, Green et al. 1948), and
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shortly after, the molecule was determined to originate from the enterochromaffin (or
Kulchitsky) cells that are found throughout the gastrointestinal and bronchopulmonary
system (Erspamer and Asero 1952). High concentrations of 5-HT are found in blood
platelets and enterochromaffin cells of the gut; lesser amounts are found around neurons
located along the raphé nuclei of the brainstem. The human brain has evolved a
sophisticated arrangement of axons stemming from the raphé nuclei to innervate nearly
every brain region.

© 2011 Elsevier Inc. All rights reserved.

For Correspondence: W. David Merryman, Ph.D. Department of Biomedical Engineering, Vanderbilt University, Room 9445D MRB
IV-Langford, 2213 Garland Avenue, Nashville TN 37232-0493, Office: 615-322-7219, Fax: 615-322-6541,
[email protected].
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 Hutcheson et al. Page 2

5-HT is involved in a diverse array of physiologic and biologic processes. In the brain, 5-HT
has been found to affect sleep, mood, appetite, anxiety, aggression, perception, pain, and
cognition (Roth, Willins et al. 1998; Edited by Roth 2006; Berger, Gray et al. 2009).
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Systemically, 5-HT contributes to vascular and non-vascular smooth muscle contraction and
platelet aggregation. Serotonin released from neurons is recaptured by an active reuptake
pump (serotonin transporter), and is then inactivated by monoamine oxidase and converted
to 5-hydroxyindoleacetic acid (Guyton and Hall 1996). In vivo, the rate of urinary 5-
hydroxyindoleacetic acid output is commonly used as an index of 5-HT metabolism.

1.2. Serotonin receptors

Signaling of 5-HT is mediated by receptors that are located on the cell membrane of neurons
and most other cells in the body (Edited by Roth 2006; Berger, Gray et al. 2009). There are
six classes of G protein-coupled 5-HT receptors (5-HT1,2,4,5,6,7) that can be subdivided into
14 unique subtypes. The 5-HT3 receptor is unique among 5-HT receptors in that it is a
ligand gated ion channel (Maricq, Peterson et al. 1991; Edited by Roth 2006).
Heterotrimeric guanine nucleotide-binding protein G protein-coupled receptors (GPCRs) are
well characterized and have been described extensively (Kroeze, Kristiansen et al. 2002;
Kroeze, Sheffler et al. 2003). Briefly, GPCRs are transmembrane proteins consisting of
seven membrane-spanning α-helical segments with an extracellular N-terminus and an
intracellular C-terminus. The binding of 5-HT to one of its receptors is thought to elicit a
conformational change that activates associated heterotrimeric G proteins and recruits other
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downstream signaling/scaffolding molecules, such as GPCR kinases and β-arrestins

(Armbruster and Roth 2005; Allen and Roth 2010). Upon activation by an agonist-occupied
GPCR, G proteins release guanosine diphosphate, which is constituitively bound to the α
subunit of the heterotrimer, and bind guanosine triphosphate (GTP). Binding of GTP to the α
subunit causes it to dissociate from the βγ subunits (which remain associated to each other);
free Gα then interacts with nearby, downstream effectors (e.g., adenylate cyclase for Gαs/
olf- and Gαi/o/z-types or phospholipase C for Gαq/11-types), generating second messengers
(e.g., cAMP produced by adenylate cyclase or inositol 1,4,5-trisphosphate and
diacylglycerol produced by phospholipase C) that modulate downstream effectors inside the
cell (e.g., protein kinases A and C activated by cAMP and diacylglycerol) [see (Raymond,
Mukhin et al. 2001) for review of 5-HT signaling pathways]

Because of the systemic presence of 5-HT and the multitude of receptor types found
throughout the body that can elicit a myriad of cellular responses, drugs targeting 5-HT
receptors are effective treatments for a variety of conditions. Each 5-HT receptor subtype
contains at least one important therapeutic target. For instance, antimigraine medications
(e.g., ergotamine, sumatriptan) activate 5-HT1B/D receptors. Clinically effective
antipsychotics block the activation of 5-HT2A, 5-HT2C (Roth et al, 1992), 5-HT6 (Roth,
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Craigo et al. 1994), and 5-HT7 (Roth, Craigo et al. 1994; Abbas, Hedlund et al. 2009)
receptors [see (Roth and Xia 2004) for review]. Antagonism of 5-HT6 receptors has been
postulated to enhance memory and learning in healthy individuals (Lindner, Hodges et al.
2003). There are many pharmaceuticals used to target the multitude of serotonergic GPCRs;
however the 5-HT2 receptors are among the most frequently targeted, highlighting their
important role in physiological and pathophysiological processes see [(Roth 2010) for recent
review].

1.3. 5-HT2 receptors

The 5-HT2 family consists of three GPCRs: 5-HT2A, 5-HT2B, and 5-HT2C. 5-HT induces an
increase in inositol 1,4,5-trisphosphate (which leads to release of intracellular calcium stores
and diacylglycerol production (Conn and Sanders-Bush 1984; Roth, Nakaki et al. 1984;
Kursar, Nelson et al. 1994). In addition to these known signaling mechanisms, 5-HT2

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receptors also generate second messenger signaling that leads to cell-type specific responses
depending on the organ under consideration. Some of the most notable effects of 5-HT2
receptor-preferring drugs involve the brain, and these activities are exploited therapeutically.
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Two important, common examples are atypical antipsychotics and anorexigens. These drugs
—and/or their metabolites—display activity at 5-HT2A (viz: atypical antipsychotics are
inverse agonists) (Meltzer, Matsubara et al. 1989; Weiner, Burstein et al. 2001) and 5-HT2C
receptors (anorexigens and putative atypical antipsychotic drugs are agonists) (Kozikowski,
Cho et al. 2010). 5-HT2A and 5-HT2C receptors are highly abundant in various human brain
regions with 5-HT2A being highly concentrated in cortical regions and 5-HT2C more broadly
distributed (Pazos, Cortes et al. 1985; Abramowski, Rigo et al. 1995). The 5-HT2B receptor
subtype displays a lower expression in the brain (Kursar, Nelson et al. 1994), and thus, it
plays a lesser role in the effects of psychoactive agents. Nevertheless, recent genetic and
pharmacologic studies have implicated 5-HT2B receptors in the biological activities of the
recreational psychostimulant 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)
(Doly, Valjent et al. 2008; Doly, Bertran-Gonzalez et al. 2009) and the anorexigen
fenfluramine (Banas, Doly et al. 2010).

The putative cardiovascular action of the 5-HT2A and 5-HT2B receptors may be similar.
Both of these receptors have been shown to elicit mitogenic and secretory responses in
ventricular and heart valve fibroblasts (Xu, Jian et al. 2002; Setola, Hufeisen et al. 2003;
Jaffre, Callebert et al. 2004; Yabanoglu, Akkiki et al. 2009), indicating a possible role for
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each in cardiac development and disease. An issue in isolating functional differences

between the 5-HT2 receptors has been the lack of specificity in pharmacological agents used
to target the receptors. In fact, many clinically used agents, particularly antipsychotics and
anorexigens, display some activity at all three 5-HT2 receptor subtypes. One reason for low
selectivity among 5-HT2 receptor-active compounds is the high degree of amino acid
sequence homology among the three subtypes (Roth, Willins et al. 1998; Barnes and Sharp
1999) with a structural similarity of 45–50% between the receptors (Pytliak, Vargova et al.
2010). This homology is of significant consequence as drugs intended for the 5-HT2A or 5-
HT2C receptors located in the brain may also bind to the 5-HT2B receptors expressed in the
brain or in other tissues. Therefore, special attention should be given to differential
properties of these receptor subtypes to identify functional differences and enhance
understanding of target specificity.

In 2005, we examined non-conserved residues in the transmembrane helices of the 5-HT2

receptors to identify ones that might participate in the preferential binding of (+)-
norfenfluramine to 5-HT2B receptors. We generated a series of 5-HT2B receptor point
mutants that contained one 5-HT2A- and/or 5-HT2C-like putative ligand binding residue, and
we determined whether any of the point mutations affected (+)-norfenfluramine binding
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affinity. Mutation of a valine in TM2, V2.53, to leucine (the analogous residue in the 5-
HT2A receptor, caused a 17-fold decrease in the Ki of (+)-norfenfluramine. Residue 2.53 in
the 5-HT2C receptor is also a valine, and the V2.53L point mutation caused a 12-fold
decrease in the Ki of (+)-norfenfluramine. The reciprocal point mutation (L2.53V) in the 5-
HT2A receptor had no effect on the Ki of (+)-norfenfluramine. The preceding observations
suggest that V2.53 in the 5-HT2B receptor contributes to the high-affinity binding of (+)-
norfenfluramine.

Using a rhodopsin-based 5-HT2B receptor homology model, we performed in silico ligand

docking, and molecular dynamics simulations to predict how V2.53 might contribute to (+)-
norfenfluramine binding. One result suggested that both terminal methyl groups of V2.53
formed stabilizing van der Waals (vdW) interactions with the α-methyl group of (+)-
norfenfluramine, and that the V2.53L mutation resulted in the loss of one of these
interactions. To test that prediction, we generated additional point mutants and

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norfenfluramine analogs. First, we reasoned that a V2.53A mutation would eliminate both
vdW interactions, further decreasing (+)-norfenfluramine affinity. In fact, the mutation
caused a 150-fold reduction in the Ki of (+)-norfenfluramine. Second, we synthesized a
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norfenfluramine analog lacking an α-methyl group. The affinity of α-desmethyl-

norfenfluramine for the wild type 5-HT2B receptor was reduced three-fold compared with
(+)-norfenfluramine. Further, α-desmethyl-norfenfluramine binding was less sensitive to the
V2.53L mutation than was (+)-norfenfluramine. Our molecular dynamics simulations also
predicted that a V2.53I mutation would permit two vdW interactions between the terminal γ-
and δ-methyl groups of I2.53 and the α-methyl group of (+)-norfenfluramine. Upon
experimental validation, the Ki of (+)-norfenfluramine binding to the V2.53I 5-HT2B
receptor was 35 nM, compared with 22 at the wild type 5-HT2B receptor. Together, our in
vitro and in silico studies of the 5-HT2B receptor (+)-norfenfluramine binding provide
evidence linking V2.53 to the high-affinity and subtype-selective binding of the
valvulopathogenic anorexigen.

1.4. 5-HT2B receptors

What is now called the 5-HT2B receptor (Fig. 2) was first recognized 50 years ago relating
to the putative role of a specific 5-HT receptor subtype in the contraction of the gastric
fundus from rat stomach (Vane 1959). Although there was controversy prior to the cloning
of the 5-HT2B receptor whether the stomach fundus receptor was pharmacologically distinct
from the 5-HT2A and 5-HT2C receptors (Kaumann and Levy 2006), this disappeared once all
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three were cloned and their tissue distribution illuminated. Since then, 5-HT2B receptors
have been found to be present in both rodent and human tissues, particularly in the
cardiovascular system, gastrointestinal tract, bone, and central nervous system (Ullmer,
Schmuck et al. 1995; Choi and Maroteaux 1996; Fitzgerald, Burn et al. 2000). Importantly,
the tissue distribution of 5-HT2B receptor protein in rodents and humans is similar, as are
their pharmacologies (Manivet, Schneider et al. 2002); this observation facilitates the
extrapolation of physiological and pharmacological results from rodent studies of the 5-
HT2B receptor to humans. Accordingly, Sprague-Dawley rats have been used in several
studies to gain insight into the mechanism of 5-HT-induced valvular alterations (Elangbam,
Lightfoot et al. 2005; Gustafsson, Tommeras et al. 2005; Elangbam, Wehe et al. 2006;
Hauso, Gustafsson et al. 2007; Elangbam, Job et al. 2008). Long-term serotonin
administration in these rats has been shown to induce aortic valve insufficiency and
histopathological changes similar to those observed in human carcinoid patients (discussed
in Section 3.1) (Gustafsson, Tommeras et al. 2005). The valvular changes seem to be
associated with an increase in 5-HT2B receptor expression and a decrease in the expression
of 5-HT transporter in both mitral and aortic valves (Elangbam, Job et al. 2008). These
observations may indicate an indirect interaction between these two membrane proteins;
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whereby, in normal physiology, the 5-HT transporter may control homeostatic 5-HT levels,
preventing 5-HT2B receptor over-stimulation (Elangbam 2010). In the disease state, the
cooperation between these receptors may be lost, leading to persistent 5-HT2B receptor
stimulation and the resultant valvulopathies discussed in Section 3.

The complexity and variety of 5-HT2B receptor expression is paralleled by its signal
transduction. Activation of 5-HT2B has been found to stimulate phospholipase C
(Kellermann, Loric et al. 1996) and phospholipase A2 (Tournois, Mutel et al. 1998), both of
which increase intracellular calcium levels. Activation of 5-HT2B receptors in some cell
types has also been shown to stimulate nitric oxide synthase (Manivet, Mouillet-Richard et
al. 2000). In fibroblasts and smooth muscle cells, the biological result of activating 5-HT2B
receptors is mitosis (Fitzgerald, Burn et al. 2000; Nebigil, Choi et al. 2000; Nebigil, Launay
et al. 2000; Setola, Hufeisen et al. 2003) and secretion of inflammatory cytokines and
extracellular matrix (ECM) components (Jaffre, Bonnin et al. 2009). As such, 5-HT2B

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receptors appear to play a crucial role in allowing these cells to maintain the structural
homeostasis of the tissues comprising them (e.g., myocardium, heart valves, blood vessels).
For example, over-expression of 5-HT2B receptors in hearts of transgenic mice results in
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cardiac hypertrophy and decreased ventricular function due to enhanced ECM and
remodeling (Nebigil, Jaffre et al. 2003). Likewise, genetic deletion of 5-HT2B receptors was
shown to lead to ventricular dilation and incomplete cardiac development (Nebigil, Choi et
al. 2000).

The molecular signaling pathways associated with these matrix remodeling responses
appears to involve typical mitogenic signal transduction as well as coordination between the
signaling of 5-HT2B receptors and various growth factor and cytokine receptors (Nebigil,
Choi et al. 2000; Nebigil, Launay et al. 2000; Jaffre, Callebert et al. 2004). Specifically, 5-
HT2B activation in mouse fibroblasts was initially shown to lead to the p21Ras- and
heterotrimeric G protein-dependent activation of mitogen activated protein kinase (MAPK)
(Launay, Birraux et al. 1996). Further studies in fibroblasts revealed that 5-HT2B activation
also leads to phosphorylation of the cytoplasmic tyrosine kinase Src (Nebigil, Launay et al.
2000). Src phosphorylation appears to enhance signaling of the platelet derived growth
factor and epidermal growth factor (Nebigil, Launay et al. 2000; Li, Zhang et al. 2008); Src
inhibitors have been found to negatively affect signaling at both these receptors (Hsu,
Persons et al. 1991; Nebigil, Launay et al. 2000). A more recent study connected these 5-
HT2B signaling pathways using pharmacological agents to selectively inhibit key signal
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transduction proteins in cardiac fibroblasts (Jaffre, Bonnin et al. 2009). Accordingly, 5-

HT2B receptor-mediated Src activation was found to lead to an increase in matrix
metalloproteinase activity, which leads to a release of heparin-bound epidermal growth
factor. In turn, epidermal growth factor signaling leads to MAPK activation and subsequent
upregulation of various cytokines. In addition, Jaffré et al. (Jaffre, Bonnin et al. 2009)
demonstrated that 5-HT2B receptors work in concert with the angiotensin II type 1 receptor
(AT1R) to mediate hypertrophic signaling in cardiac fibroblasts. These results indicate that,
in cardiac fibroblasts at least, the function of both GPCRs is required for the activity of
either one. Consequently, by inhibiting one receptor the action of the other receptor is
likewise inhibited.

Many of the cardiac-related studies into the molecular signaling pathways of the 5-HT2B
receptors have focused on the role of this receptor in ventricular fibroblasts. In this review,
we will highlight the role 5-HT2B receptors may play in mediating changes in heart valves.
Therefore, to make the connection between the action of 5-HT2B receptors in ventricles and
heart valves, it is interesting to note that the signaling pathways discussed previously seem
to play a major role in both regulating and responding to the biomechanical properties of
ventricular tissue, and as noted below, biomechanical integrity is crucial to the appropriate
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function of heart valves. Thus, both AT1 and 5-HT2B receptors demonstrate a mechano-
dependent upregulation and signaling activation during ventricular pressure overload that
results in an increase in tissue stress levels (Rosenkranz 2004; Liang, Lai et al. 2006).
Moreover, agonist signaling at these GPCRs has been shown to lead to an increase in
expression of the cytokine transforming growth factor-β1 (TGF-β1) (Jian, Xu et al. 2002;
Rosenkranz 2004), which has also been shown to exhibit mechano-responsive signaling
(Wipff, Rifkin et al. 2007) and seems to be a key mediator in the tissue changes that lead to
heart valve disease (Walker, Masters et al. 2004; Merryman, Lukoff et al. 2007).

2. Degenerative heart valve disease

2.1. Heart valve overview
Heart valves (HVs) control the direction of blood flow through the heart during the cardiac
cycle by directing oxygen-poor blood to the lungs and oxygen-rich blood to the vital organs

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via the systemic circulation. There are four valves in the heart: two on pulmonic side and
two on the systemic side (Fig. 3). Progressing through the heart from the vena cava,
deoxygenated blood enters the right atrium and passes through the tricuspid valve to the
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right ventricle, and then from the right ventricle through the pulmonary valve into the
pulmonary artery, which directs blood flow to the lungs. Upon reoxygenation by perfusion
through the lungs, the blood returns to the left atrium of the heart via the pulmonary vein,
passes through the mitral valve into the left ventricle, and then enters the aorta through the
aortic valve. The anatomies of the pulmonary and aortic valves are very similar, as are the
anatomies of the tricuspid and mitral valves. The pulmonary and aortic valves are referred to
as semilunar valves because the three leaflets of the valves look like a half-moon when
excised (Fig. 4a). Semilunar valves open and close solely due to inertial forces from the
blood during the cardiac cycle; whereas, the structure and function of the tricuspid and
mitral valves, referred to as atrioventricular valves, are more complex than the semilunar
valves (Fig. 4b). Briefly, their leaflets are tethered to the papillary muscles of ventricular
wall via chordae tendineae, which act as anchors to prevent the leaflets from prolapsing into
the atria during ventricular contraction. The primary difference between the tricuspid and
mitral valve is the number of leaflets: the tricuspid has three and the mitral only two. The
atrioventricular valves ensure that blood does not back-flow during myocardial contraction
(systole), and the semilunar valves keep blood from re-entering the heart during relaxation
(diastole).
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All of the valves rely on hemodynamic forces to regulate the opening and closing of the HV
leaflets. As such, the biomechanical integrity of the leaflet tissues is crucial to appropriate
function of the HVs. Two distinct cell types have been shown to play a role in regulating
tissue homeostasis within the HV: valve endothelial cells and valve interstitial cells (VICs)
(Chester and Taylor 2007). Valve endothelial cells exhibit regional heterogeneity along the
surface of the HV leaflets and may function to direct spatial-specific extracellular matrix
(ECM) production through paracrine signaling to the VICs (Simmons, Grant et al. 2005).
During HV development, activated VICs synthesize ECM components to form the leaflet
structure that is crucial to correct HV function; however, during disease, over-activation of
VICs can lead to inappropriate ECM accumulation and/or valvular architecture and
subsequent loss of HV function that leads to heart valve disease (HVD). Under healthy
physiologic conditions, the left side VICs are much more active and dynamic with greater
levels of cytoskeletal proteins and ECM biosynthesis, likely due to the increased pressures
imposed on the leaflets (Merryman, Huang et al. 2006; Merryman, Youn et al. 2006;
Merryman, Liao et al. 2007).

2.2. Disease etiology and pathology

There exist two types of HVD: congenital and acquired (Otto 2004). Congenital valve
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disease is an abnormality that develops before birth and may be related to improper valve
size, malformed leaflets, or an irregularity in the way the leaflets are attached. This most
often affects the pulmonary and aortic valves. A very common congenital defect is bicuspid
aortic valve disease where instead of the normal three leaflets or cusps, the bicuspid aortic
valve has only two. Without the third leaflet, the valve may be stenotic (narrowing,
obstructing flow) or regurgitant (‘floppy,’ allowing backward flow). It is estimated that
some form of bicuspid aortic valve disease affects about 1.4% of the population (Lloyd-
Jones, Adams et al. 2010), but this is difficult to verify since in some cases it goes
undetected and does not result in significant changes in cardiovascular function. Acquired
HVD pertains to problems that develop within valves that were at one time normal. This is
typically referred to as age-related degenerative valve disease. In the early part of the 20th
century, the primary cause of acquired valve disease stemmed from rheumatic valve disease;

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however, this trend has changed dramatically with a decrease in rheumatic valve disease and
a concomitant increase in age-related degenerative valve disease (Otto 2004).
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The risk factors for HVD are similar to traditional clinical risk factors for atherosclerosis
and coronary artery disease and include age, male gender, hypertension, diabetes,
triglycerides, and smoking (Mohler, Sheridan et al. 1991; Stewart, Siscovick et al. 1997;
Agmon, Khandheria et al. 2001). In fact, the similarities between the two have led to the
hypothesis that acquired HVD is primarily a manifestation of atherosclerosis (O’Brien
2006). The hallmark of HVD pathogenesis is the formation of lesions containing cell types
that are characteristic of chronic inflammation (Olsson, Dalsgaard et al. 1994; Olsson,
Rosenqvist et al. 1994; Otto, Kuusisto et al. 1994). These include macrophages, T
lymphocytes, and mast cells. Additionally, there are lipoproteins [LDL and Lp(a)] found in
human diseased valve lesions, and accumulation of these lipoproteins is mediated, in part,
by ECM proteoglycans (Walton, Williamson et al. 1970; O’Brien, Reichenbach et al. 1996).
Besides lipoproteins, recent evidence has shown that the renin-angiotensin system,
particularly angiotensin converting enzyme and angiotensin II, may play a role in HVD
pathogenesis (O’Brien, Shavelle et al. 2002). Angiotensin II has a number of potential
lesion-forming effects, including inflammation and macrophage and cholesterol
accumulation. Therefore, there are multiple mechanisms that initiate HVD and further
perpetuate the disease in otherwise normally functioning valve.
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2.3. Current theories

As noted previously, degenerative HVD is believed to be caused by an over-activation of
VICs through a process that depends on signaling associated with TGF-β1. This cytokine is
secreted from cells in a latent form and stored in the ECM. Various ECM cues can result in
the activation of TGF-β1, which then binds to and activates the TGF-β1 serine/threonine
kinase receptors (TβRs). Following cytokine binding, TβRs modulate a wide variety of
cellular processes by signaling through a family of transcription factors known as Smads
(Howe 2003). In VICs, active TGF-β1 leads to myofibroblast activation in vitro (Walker,
Masters et al. 2004), and the combination of active TGF-β1 and mechanical strain leads to a
synergistic increase in hallmarks of HVD within porcine HV leaflets (Merryman, Lukoff et
al. 2007). Confounding the problems associated with its mediation of cellular changes that
lead to HVD, TGF-β1 signaling also leads to increased synthesis of the latent form of TGF-
β1, which in turn may be activated by VICs (Merryman 2008). This feed-forward signaling
mechanism may drive the progression of HVD.

2.4. Treatments
Currently, the only viable long-term treatment option for HVD is valve replacement surgery.
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Typically, the native diseased HV is either replaced by a decellularized HV of bovine or

porcine origin or supplanted by a mechanical HV. While the techniques and resultant risks
associated with HV replacement procedures have dramatically improved, surgery still
remains a poor option for the two patient populations most affected by HVD: children and
elderly.

In pediatric patients, the main concern is the integration of replacement HVs within tissues
that are rapidly growing and changing. Compounding the problem in children, replacement
HVs tend to wear quickly and typically need to be replaced within 20 years (Merryman,
Engelmayr Jr et al. 2006). To circumvent these problems, many surgeons elect to perform
the Ross procedure whereby the pulmonic HV is resected and placed in the aortic position.
The replacement HV is then placed in the position formerly occupied by the pulmonic HV.
The advantage of this procedure is that the pulmonic HV is able to incorporate within native
tissue in the aortic position, and the replacement HV wears more slowly due to the lower

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pressures associated with the pulmonic circulation. The major disadvantage of the Ross
procedure is that it requires two very invasive surgical procedures (i.e., resection of both the
aortic and pulmonic HVs), and often, the pulmonic HV is not able to adapt to the increased
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pressure associated with systemic circulation. For these reasons, the Ross procedure is rarely
performed in elderly patients. Faced with the problems associated with HV replacement,
physicians often delay surgical options until they are absolutely necessary; therefore, many
patients would greatly benefit from an early treatment to delay or prevent the onset of HVD.
Elderly patients face a difficult recovery following open-chest surgery. While these
procedures may improve the cardiac function of these patients, their quality of life declines
for a prolonged period of time (up to a year). Thus, elderly patients would benefit greatly
from a pharmacological strategy against HVD.

2.5. Statins
The hopeful story in recent years has been that lipid-lowering drugs (i.e., statins) might
prevent HVD (Rajamannan, Subramaniam et al. 2002; Rajamannan, Edwards et al. 2003;
Benton, Kern et al. 2009), similarly to their promise against atherosclerosis. In 2001, two
retrospective studies indicated that statins may inhibit the progression of aortic valve
stenosis (Aronow, Ahn et al. 2001; Novaro, Tiong et al. 2001), and it was suggested that
large prospective studies be conducted. When these prospective studies were finally
completed, the efficacy of statins proved disappointing. In 2005, a small double-blind,
placebo controlled study showed no benefit of statins to reduce HVD (Cowell, Newby et al.
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2005). Moreover, a recent report of a large clinical study demonstrated, rather conclusively,
that statins do not reduce major cardiovascular outcomes, including aortic valve
replacement, in patients with aortic valve stenosis (Rossebo, Pedersen et al. 2008). In light
of these findings, the enthusiasm for statin therapy as a potential preventive treatment for
HVD has been severely dampened. Thus, there is no pharmacological strategy currently
available or being developed (to our knowledge) that has the potential to prevent or delay
HVD progression.

3. Serotonergic drugs and heart valve disease

3.1. Carcinoid syndrome
Carcinoid syndrome occurs as a result of the formation of neuroendocrine tumors arising
from oncogenic enterochromaffin cells. These cells synthesize and deliver 5-HT to the
bloodstream for controlled systemic distribution; however, when tumor-forming
enterochromaffin cells metastasize to the liver, 5-HT more readily enters systemic
circulation resulting in an increase in plasma 5-HT levels and leading to the cardiac changes
associated with carcinoid heart disease (CHD) (Druce, Rockall et al. 2009). CHD was first
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noted 80 years ago by the Dutch pathologist A.J. Scholte, who observed that a patient with a
carcinoid tumor also had thickened tricuspid valve leaflets (Gustafsson, Hauso et al. 2008).
Interestingly, CHD differs from most forms of HVD in that it mostly affects right-side HVs
(i.e., the pre-lung tricuspid and pulmonary valves). Elevated blood levels of 5-HT are carried
to the right side of the heart through the inferior vena cava, where it is believed that
interaction with 5-HT2B receptors on cells in the tricuspid and pulmonic HVs leads to the
extracellular matrix secretion and the thickening of the HV leaflets that characterize HVD.
As the blood continues into pulmonary circulation, the 5-HT is inactivated by
monoaminoxigenase in the lungs and, therefore, does not induce HV changes when the
blood returns to the left side of the heart. For many years, the serotonergic receptor subtype
responsible for CHD was not known due to the fact that serotonin has a similar affinity for
many of its receptors, and several of the subtypes are expressed throughout the
cardiovascular system. The current hypotheses identifying 5-HT2B receptor as the major HV

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 Hutcheson et al. Page 9

target came about only as a result of inadvertent targeting of the 5-HT2B receptors with Fen-
Phen.
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3.2. Fen-Phen
Among all the serotonergic drugs on the market, there may be none more recognizable to the
general public than fenfluramine, one of the components of the ‘Fen-Phen’ anorexigen
combination. This popular diet drug regimen consisting of fenfluramine and phenteramine
was shown to be better tolerated than either alone in 1984 and the drug combination was
subsequently and widely prescribed (Weintraub, Hasday et al. 1984). However, a study
published in late 1997 identified both right-sided and left-sided HV defects in a number of
patients who had been taking Fen-Phen for an average of 12 months (Connolly, Crary et al.
1997). Soon thereafter, the drugs (including other fenfluramine formulations) were
voluntarily withdrawn from the market based upon recommendations by the FDA. In a
retrospective study, the highest incidence of HVD development from Fen-Phen was found to
be 25.2% among patients treated for an average of 20 months (Khan, Herzog et al. 1998).

Early histological analyses of diseased tissue from Fen-Phen patients indicated a

pathophysiology similar to that observed in carcinoid patients and from tissues of subjects
who had taken specific ergot-derived formulations. In this regard, in the mid-1960s,
researchers and physicians noticed a strong connection between the ergot agents
methysergide and ergotamine and the development of fibrotic pathologies including HVD
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(Graham, Suby et al. 1966; Graham 1967; Mason, Billingham et al. 1977). Some even went
as far as to note that the “similarities in chemical structure of serotonin, methysergide, and
ergotamine” may “suggest a common pathophysiologic mechanism for ergot alkaloid-
associated valve disease and carcinoid valve disease” (Redfield, Nicholson et al. 1992).
Much like with CHD, however, the exact molecular cause of the tissue changes that lead to
HVD remained undetermined.

The Fen-Phen episodes sparked new interest in identifying the specific 5-HT receptor
subtype involved in drug-induced HVD. Due to the known mitogenic roles of the 5-HT2
subfamily, Fitzgerald et al. (Fitzgerald, Burn et al. 2000) examined the interaction of
fenfluramine, norfenfluramine (the main metabolite of fenfluramine), ergotamine, and
methysergide on human 5-HT2A, 5-HT2B, and 5-HT2C receptors. The ergot-derived
compounds were found to possess high affinity for all three receptor subtypes. In contrast,
fenfluramine rotamers demonstrated weak affinities for the 5-HT2 receptors; however, the
norfenfluramine rotamers exhibited relatively high affinity for both 5-HT2B and 5-HT2C
receptors (Table 1) and slightly lower affinity for 5-HT2A receptors. These results suggested
that norfenfluramine, the main metabolite of fenfluramine, was chiefly responsible for 5-
HT2 receptor activation, and thus, the most likely candidate for causing Fen-Phen-mediated
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HVD. Furthermore, norfenfluramine was found to be two orders of magnitude more potent
at 5-HT2B and 5-HT2C receptors compared with 5-HT2A receptors (Table 2). Additionally,
5-HT2C receptors, which are restricted to the central nervous system, were found to be
expressed at exceedingly low levels within HV tissues (Fig. 5); whereas 5-HT2B receptors
are expressed in relatively high quantities within the HV leaflets. Simultaneously, Rothman
et al. (2000) compared the in vitro pharmacology of norfenfluramine with that of known
inducers of HVD (5-HT, methysergide), as well as with that of serotonergic drugs not
associated with HVD (negative controls), and found that the HVD-associated compounds
were, unlike the negative controls, potent 5-HT2B receptor agonists (see below). Taken
together with the expression of 5-HT2B receptors in heart valve tissue (Fitzgerald, Burn et al.
2000) and the mitogenic effect of 5-HT2B receptors in cardiac and other cell types (Nebigil,
Choi et al. 2000; Nebigil, Launay et al. 2000), these results pointed to the 5-HT2B receptor
as the serotonergic target leading to HVD.

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As noted above, the results of the Fitzgerald et al. study were supported in a seminal report
published simultaneously by Rothman et al. (Rothman, Baumann et al. 2000) in which the
affinities and potencies of 15 different molecules were tested at 11 different 5-HT receptor
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subtypes: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5A, 5-HT6,
and 5-HT7. In this study, all but the 5-HT2B receptor subtypes were systematically ruled out
based on pharmacological similarity with HVD-associated molecules and pharmacological
differences from negative control molecules that were not suspected mediators of HVD
(e.g., fluoxetine and its metabolite norfluoxetine). The 5-HT2 receptor subtypes were the
only receptors to display relatively high affinity (Table 1) and high potency (Table 2) for
both the norfenfluramine rotamers and for the HVD-associated ergot-derived compounds
(Tables 1 and 2, italicized). Similar to the Fitzgerald et al. study, the data indicate both high
affinity (Ki) and potency (shown by phosphatidylinositol hydrolysis, Kact) at 5-HT2B over 5-
HT2A for norfenfluramines and the known inducers of HVD. Taken together, these studies
indicate that norfenfluramine, the main fenfluramine metabolite, is the likely culprit in
fenfluramine-mediated HVD. It is important to note that in both the Fitzgerald et al. (2000)
and Rothman et al. (2000) studies, phenteramine did not exhibit high affinity agonism of 5-
HT2B receptors (Table 1); correspondingly, formulations consisting of phenteramine alone
(i.e., in the absence of fenfluramine)—in use for decades prior to the Fen-Phen HVD
outbreak—have not been associated with the disease (Rothman, Baumann et al. 2000).

3.3. Pergolide and Cabergoline

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Following the Fen-Phen episode, a number of additional HVD-inducing drugs have been
identified. First among these were the non-specific dopamine agonists pergolide and
cabergoline, prescribed for Parkinson’s disease (Antonini and Poewe 2007; Schade,
Andersohn et al. 2007; Zanettini, Antonini et al. 2007). The stories of pergolide and
cabergoline are similar to that of fenfluramine. The first preliminary account of potential
pergolide-mediated HVD was reported in 2002 (Pritchett, Morrison et al. 2002). Dopamine
agonists had become popular anti-Parkinsonian therapeutics, and many feared that these
drugs would suffer the same fate as fenfluramine. Around the same period of time, many
drugs were beginning to be profiled at 5-HT2B receptors including anti-Parkinsonian drugs,
amphetamine derivatives and other medications (Newman-Tancredi, Cussac et al. 2002;
Setola, Hufeisen et al. 2003). Of the large number of drugs tested, pergolide, cabergoline,
MDMA and its active N-demethylated metabolite 3,4-methylenedioxyamphetamine (MDA)
were found to exhibit potent agonist activity at 5-HT2B (Newman-Tancredi, Cussac et al.
2002; Setola, Hufeisen et al. 2003). It is interesting to note in this regard that Newman-
Tancredi and colleagues were solely interested in differentiating activity of several
Parkinsonian drugs at various serotonergic receptor subtypes and suggested that 5-HT2B
activity might be associated with some sort of therapeutic action of pergolide and
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cabergoline rather than mediating side-effects. On the other hand, Setola et al. correctly
predicted that pergolide and other drugs which were 5-HT2B agonists (including MDMA
and MDA) would be associated with a high incidence of HVD. Accordingly, the incidence
of HVD from pergolide and cabergoline has been found to be 23.4% and 28.6%,
respectively (Zanettini, Antonini et al. 2007); no significant increase in HVD incidence has
been associated with any of the other dopamine agonists. Further, three of the other drugs
(lisuride, bromocripine, and terguride) were found to be potent agonists at 5-HT2A receptors
(Newman-Tancredi, Cussac et al. 2002), but have little to no activity at 5-HT2B receptors
(Huang, Setola et al. 2009). None of these drugs has been subsequently reported to be
associated with HVD (Elangbam 2010). These findings lend further credence to the theory
that the 5-HT2B receptor is the molecular culprit for drug-mediated HVD. Tables 1 and 2
contain partial compilation of the results from these early studies and offer a representative
view of the data that first indicated agonist activity at 5-HT2B receptors leads to the
development of HVD.

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3.4. A search for others

Following the Fen-Phen and pergoline/cabergolide findings, many other drugs have been
tested for agonist activity at 5-HT2B. Specifically, the NIMH Psychoactive Drug Screening
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Program has sought to screen large numbers of FDA-approved drugs and drug-like
compounds for agonist activity at 5-HT2B receptors in order to identify drugs/chemotypes
with HVD liability (Setola, Dukat et al. 2005; Roth 2007; Huang, Setola et al. 2009). The
most recent findings in this effort identified 27 5-HT2B receptor agonists among 2200 drugs
and drug-like compounds (Table 3; (Huang, Setola et al. 2009)). Of these, 14 had previously
been identified as 5-HT2B receptor agonists, including 7 known HVD-associated drugs
(Table 3). Six of the compounds that had not previously been identified as 5-HT2B receptor
agonists are currently approved medications (Table 3, italics), with five of these acting as
potent agonists: guanfacine, oxymetazoline, quinidine, xylometazoline, and fenoldopam.
Guanfacine and quinidine were noted to be of particular concern. Guanfacine is often
prescribed as an antihypertensive agent, and quinidine is used to treat arrhythmia. As such,
both tend to be prescribed for sustained periods and could potentially lead to valvular
problems. The recent indication of guanfacine to treat attention deficit hyperactivity disorder
in pediatric patients is of particular concern (Huang, Setola et al. 2009). Of less concern
(perhaps) are xylometazoline, oxymetazoline, and fenoldopam, which are typically
prescribed for relatively short time periods. However, all of these compounds should be
studied closely to ensure that there is no link to HVD (Bhattacharyya, Schapira et al. 2009).
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One of the identified agonists, ropinirole, was found to be much less potent than the other
compounds (active only at 1 μM) and therefore may not be associated with inducing HVD.
Indeed, ropinirole, which has been used for several years to treat restless legs syndrome, has
been considered ‘safe’ vis-à-vis HVD. However, a very recent study documented a case of
retroperitoneal fibrosis in a patient using ropinirole (Parissis, Papachristodoulou et al. 2010).
Additionally, at least four other cases of HVD have been reported in patients using
ropinirole (Parissis, Papachristodoulou et al. 2010). While these relatively few reports may
not indicate a statistically increased risk of HVD with ropinirole use, these types of cases
should be continuously monitored to ensure that no causal link exists.

3.5. Learning from previous mistakes

Though unfortunate in terms of human and economic loss, the fenfluramine case has led to
some important insights. The first and most impactful of these is that screening drugs for 5-
HT2B receptor agonist activity is the best in vitro method for identifying potential drug-
inducing HVD candidates (Rothman, Baumann et al. 2000; Setola, Hufeisen et al. 2003;
Roth 2007; Elangbam 2010). The case of MDMA supports this recommendation. In 2003,
we (VS and BLR), in collaboration with Richard Rothman’s group at NIDA, profiled the
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serotonergic amphetamine derivative MDMA to identify targets other than the monoamine
plasmalemmal transporters. The screening campaign revealed that MDMA and MDA were
both moderately potent 5-HT2B receptor agonists, and that they stimulated the proliferation
of primary human VIC cultures in a 5-HT2B receptor-dependent manner (Setola, Hufeisen et
al. 2003). These findings led us to predict that MDMA use may be a risk factor for HVD. In
2007, a clinical study reported an increased prevalence of HVD in MDMA users
(Droogmans, Cosyns et al. 2007). Similarly, recent clinical findings have indicated that
long-term usage of the fenfluramine-derivative benfluorex can lead to HVD (Noize, Sauer et
al. 2006; Boutet, Frachon et al. 2009; Le Ven, Tribouilloy et al. 2010; Weill, Paita et al.
2010). Benfluorex is an anorexigen and hypolipidemic agent commonly prescribed to
overweight diabetic patients in Asia, Europe and South America; however, the severity of
the clinical case reports led the European Medicines Agency to withdraw benfluorex from
the European market in December 2009 (Droogmans, Cosyns et al. 2010). Given that
norfenfluramine is a known metabolite of benfluorex, the association of this drug with HVD

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 Hutcheson et al. Page 12

should not have come as a surprise (Droogmans, Cosyns et al. 2010). Thus, the
aforementioned studies support the claim that in vitro screening of drugs at 5-HT2B
receptors can identify previously unknown drug-inducing HVD candidates.
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Another lesson that can be learned from the role of 5-HT2B receptors in HVD is that 5-HT2B
receptor antagonists may be potential prophylactics and/or treatments. Indeed, if the
activation of 5-HT2B receptors (on heart valve interstitial cells and/or other cells) contributes
to HVD, it is likely that blockade of 5-HT2B receptors may antagonize the onset and/or
progression of the disease (Roth 2007). Furthermore, there are a number of 5-HT2B receptor
antagonists that are FDA-approved medications (e.g., antidepressants, antipsychotics,
antihistamines) with established records of safety and tolerability (Rothman, Baumann et al.
2000; Setola, Hufeisen et al. 2003; Roth 2007; Huang, Setola et al. 2009). Along these lines,
Droogmans and colleagues recently reported that the antihistamine cyproheptadine—which
is also a potent 5-HT2B receptor antagonist (Young, Khorana et al. 2005)—mitigated
pergolide-induced HVD in rats (Droogmans, Roosens et al. 2009), as predicted by Roth
(2007). Similar experiments using other 5-HT2B receptor antagonists, and analyses of HVD
prevalence among large numbers of patients taking medications with 5-HT2B receptor
antagonist activity (e.g., antihistamines, antidepressants, antipsychotics), will be informative
in terms of HVD prevention/treatment strategies.

4. 5-HT2B receptor as a novel treatment strategy

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The development of a suitable therapeutic to prevent/retard HVD depends on the ability to

target the root cause of the disease, which ultimately manifests itself as thickening and
stiffening of HV leaflets, which diminishes the ability of the HV to maintain directionality in
blood flow. At a cellular level, HV stiffening is believed to be caused by activation of VICs
to a myofibroblast phenotype (Rabkin, Aikawa et al. 2001; Rabkin, Hoerstrup et al. 2002;
Rabkin-Aikawa, Farber et al. 2004; Aikawa, Whittaker et al. 2006). Once activated, these
cells increase ECM deposition, which directly leads to the decreased compliance of the
leaflets observed in HVD. Additionally, increased mechanical strain has been shown to
exacerbate VIC activation (Balachandran, Konduri et al. 2006; Merryman, Lukoff et al.
2007; Balachandran, Sucosky et al. 2009; Balachandran, Sucosky et al. 2010). In order to
develop a strategy to prevent or treat HVD, a more thorough understanding of the cellular
signaling and subsequent tissue-level changes involved in the progression of HVD is needed
to elucidate the relevant molecular targets. Given the large number of currently-approved
medications that exhibit antagonist/inverse agonist activity at 5-HT2B receptors (Huang,
Setola et al. 2009), we believe that the 5-HT2B receptor is a tractable target to achieve these
therapeutic goals.
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Given its negative history, the idea of targeting 5-HT2B for therapeutic gain may initially
seem counter-intuitive; however, studies have begun to explore the potential benefits of
controlling 5-HT2B receptor signaling (Fabre, Marchal-Somme et al. 2008; Monassier,
Laplante et al. 2008; Porvasnik, Germain et al. 2010). Just as agonists of the 5-HT2B
receptor have been observed to lead to HVD, many of these agonists have also been
implicated in fibrotic responses and ECM alterations that lead to other pathologies such as
ventricular hypertrophy (Jaffre, Callebert et al. 2004; Jaffre, Bonnin et al. 2009) and
pulmonary arterial fibrosis and hypertension (Esteve, Launay et al. 2007). Correspondingly,
genetic deletion of 5-HT2B receptor expression in mice has been shown to lead to
incomplete cardiac development characterized by ventricular dilation and a lack of tissue
integrity (Nebigil, Choi et al. 2000; Nebigil, Hickel et al. 2001). Taken together, these
results indicate that 5-HT2B receptors play a crucial role in the maintenance of ECM
homeostasis in cardiac tissues, and with a better understanding of the downstream effectors

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 Hutcheson et al. Page 13

of these receptors, the pathways may be able to be manipulated to therapeutically target

cardiac fibrotic diseases (Hauso, Gustafsson et al. 2007).
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As an illustration of this, studies in pulmonary fibrosis have shown that 5-HT2B receptor
antagonists can effectively reduce fibrotic lesions in a mouse model (Fabre, Marchal-
Somme et al. 2008), and the selective 5-HT2B receptor antagonist PRX-08066 has been
shown to increase ventricular ejection fraction and reduce hypertrophy and vascular
remodeling in a rat model of pulmonary arterial hypertension (Porvasnik, Germain et al.
2010). A 5-HT2B receptor antagonist may be able to function similarly in preventing HVD
by blocking the fibrotic response of VICs to other, non-serotonergic stimuli.

As mentioned previously, TGF-β1 is believed to be a key mediator of the cellular changes

that lead to HVD. Unfortunately, the ubiquity of TGF-β1 signaling makes this molecule a
poor therapeutic target. A more appropriate therapeutic goal would be to interrupt TGF-β1
signaling through a separate pathway that is more localized to HV tissues, and the 5-HT2B
receptor signaling pathway may be a promising candidate to achieve this goal. Evidence
suggests that the signaling pathway from 5-HT2B receptors may crosstalk with TGF-β1
signaling pathways (Jian, Xu et al. 2002), which may be mediated in part by the tyrosine
kinase Src (Fig. 6). Src is known to play an important role in both 5-HT2B and TGF-β1
receptor signaling pathways and has been shown to be involved in the 5-HT2B receptor-
dependent regulation of the platelet derived growth factor receptor (Nebigil, Launay et al.
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2000; Xu, Jian et al. 2002; Mishra, Zhu et al. 2007; Pechkovsky, Scaffidi et al. 2008;
Samarakoon, Higgins et al. 2008; Skhirtladze, Distler et al. 2008). Therefore, 5-HT2B
receptor blockade has the potential to function in two ways: 1) preventing proliferation and
fibrotic ECM accumulation by VICs directly, and 2) interacting with TGF- β1 signaling
pathways to prevent VIC myofibroblastic differentiation (Fig. 6).

Additionally, AT1R antagonists have been observed to inhibit TGF-β1 signaling in Marfan’s
syndrome (Habashi, Judge et al. 2006; Cohn, van Erp et al. 2007; Brooke, Habashi et al.
2008), and as such, the AT1R antagonist losartan has shown tremendous clinical promise in
treating afflicted patients. Given that AT1R and 5-HT2B receptors may be functionally
linked in other cell types (Jaffre, Bonnin et al. 2009), 5-HT2B receptor antagonists may work
in a similar manner to mitigate TGF-β1 signaling. Therefore, 5-HT2B receptors may provide
a localized pharmacological target to prevent the VIC-mediated fibrotic changes that
characterize HVD. As noted above, many 5-HT2B receptor antagonists are currently FDA-
approved and used clinically to treat other diseases, with acceptable tolerance (including but
not limited to: Amesergide, Amisulpride, Bromocryptine, Lisuride, Chlorpromazine,
Clozapine, Cyproheptidine, Metergoline, Mianserin, Olanzapine, Aripiprazole, Risperidone,
9-OH-risperidone, Terguride, Yohimbine, Ziprasidone, Roxindole). Furthermore, it is
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interesting to note that lisuride has been shown to be an antagonist at 5-HT2B receptors
(Newman-Tancredi, Cussac et al. 2002; Elangbam 2010) and was prescribed for more than
30 years without a single known report of HVD (Hofmann, Penner et al. 2006). While the
absence of documented cases does not necessarily lead to the conclusion that lisuride
prevented HVD in these patients, it does seem reasonable to believe that given the large
population of patients that received lisuride and its known serotonergic activity, even
background levels (i.e., not significantly higher than the total population) would have been
reported. In conclusion, we believe that these types of molecules should be tested for
efficacy in preventing or treating HVD, as has been suggested (Roth 2007).

Acknowledgments
JDH was supported by an AHA Pre-doctoral Fellowship (10PRE4290020). BLR and VS were supported by the
NIMH Psychoactive Drug Screening Program, U19MH82441, RO1MH61887. BLR was also supported by the

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 Hutcheson et al. Page 14

Michael Hooker Distinguished Professorship in Pharmacology. WDM was supported by the AHA (0835496N and
09GRNT2010125), Wallace H. Coulter Foundation (Early Career Award), and NIH (HL094707).
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Abbreviations
5-HT 5-hydroxytryptomine
AT1R Angiotensin II type 1 receptor
CHD Carcinoid heart disease
ECM Extracellular matrix
GPCR G protein-coupled receptor
GTP Guanosine triphosphate
HV Heart valve
HVD Heart valve disease
MAPK Mitogen activated protein kinase
MDA 3,4-methylenedioxyamphetamine
MDMA 3,4-methylenedioxymethamphetamine
TGF-β1 Transforming growth factor – β1
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VIC Valve interstitial cell

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Fig. 1.
Molecular structure of serotonin.
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Fig. 2.
A three-dimensional homology model of the 5-HT2B receptor. The model is based on the
crystal structure of squid rhodopsin, which has a high degree of homology with the 5-HT2B
receptor. Shown are two TM3 residues and one TM6 residue known to interact with
serotonin.

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Fig. 3.
Schematic of blood flow through the four heart chambers. Blood from the body returns to
the right atrium of the heart through the vena cava and proceeds to the right ventricle
through the tricuspid valve. The ventricle pumps this deoxygenated blood through the
pulmonary valve to the lungs. Oxygenated blood from the lungs returns to the left atrium
through the pulmonary veins. The blood then proceeds through the mitral valve to the left
ventricle; whereupon, it is pumped through the aortic valve for systemic distribution.
Reprinted with permissions from the National Heart, Lung, and Blood Institute, a part of the
National Institutes of Health and the U.S. Department of Health and Human Services.
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Fig. 4.
Structures of heart valves. (a) The three-leaflet aortic valve is shown in both its open
(systolic) and closed (diastolic) states. The pulmonary valve is very similar in structure to
the aortic valve. Reprinted with permissions from (Schoen and Edwards 2001). (b) The two
leaflet mitral valve is anchored to the ventricular wall by chordae tendineae and papillary
muscles to prevent prolapsed into the atrium during systole. The tricuspid valve is anchored
in a similar manner as the mitral valve but contains three leaflets. Reprinted with
permissions from (Grashow, Yoganathan et al. 2006).
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Fig. 5.
5-HT2 receptor subtype expression in the aortic valve. 5-HT2A and 5-HT2B receptors are
expressed in much greater numbers than 5-HT2C as shown by random primed cDNA (solid
bars) or oligo(dT) data (hatched bars). Reprinted with permissions from (Fitzgerald, Burn et
al. 2000).
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 Hutcheson et al. Page 26
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Fig. 6.
Potential pathways of 5-HT2B. 5-HT2B activation may lead to proliferation through
pathways that depend on ERK1/2, Src, and PKC. Additionally, 5-HT2B may crosstalk
directly with TGF-β1 signaling via interaction with Src. A 5-HT2B antagonist may function
as an inverse agonist to inhibit these pathways and prevent the fibrotic response of VICs that
leads to HVD. Reprinted with permissions from (Roth 2007).

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Table 1
Comparison of Receptor Subtype Affinity (Ki in nM)
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Receptor Subtype

Drug 5-HT2A 5-HT2B 5-HT2C

5-HT1 614* 4.0 12.2

(+)-Fenfluramine2 2470 2080 3920

(−)-Fenfluramine2 1430 1620 680

(+)-Norfenfluramine2 187 56 27

(−)-Norfenfluramine2 267 99 65

Cabergoline4 7.8 2.6 190

Pergolide3,4 1.6 14 45

Ergotamine1 9.0* 3.0 12.0

Methysergide1 15.0* 9.1 1.8

Methylergonovine1 12.6* 0.49 12.4

Phenteramine1 >10,000* >10,000 >10,000

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*
from rat 5-HT2A

1
Rothman, R.B., et al., Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and
other serotonergic medications. Circulation, 2000. 102(23): p. 2836–41.
2
Fitzgerald, L.W., et al., Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine. Mol Pharmacol,
2000. 57(1): p. 75–81.
3
Setola, V., et al., 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) induces fenfluramine-like proliferative actions on human cardiac
valvular interstitial cells in vitro. Mol Pharmacol, 2003. 63(6): p. 1223–9.
4
Newman-Tancredi, A., et al., Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and
antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes. J Pharmacol Exp Ther, 2002. 303(2): 815–22.
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Table 2
Comparison of Receptor Subtype Potency (Kact or EC50 for phosphoinositide hydrolysis in nM)
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Receptor Subtype

Drug 5-HT2A 5-HT2B 5-HT2C

5-HT1 66 2.4 0.6

(+)-Fenfluramine1 >10,000 379 362

(−)-Fenfluramine1 5,279 1,248 360

(+)-Norfenfluramine2 3,100 24 190

(−)-Norfenfluramine2 26,600 292 727

Cabergoline3 7.76 2.57 2.40

Pergolide3 1.62 6.03 4.47

Ergotamine1 16 9.8 5

Methysergide1 3.5 150 2.9

Methylergonovine1 1.3 0.8 2.5

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Lisuride3 8.13 0* 7.76

*
Lisuride exhibits antagonist signaling at the 5-HT2B receptor.3

1
Rothman, R.B., et al., Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and
other serotonergic medications. Circulation, 2000. 102(23): p. 2836–41.
2
Fitzgerald, L.W., et al., Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine. Mol Pharmacol,
2000. 57(1): p. 75–81.
3
Adapted from: Newman-Tancredi, A., et al., Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III.
Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes. J Pharmacol Exp Ther, 2002. 303(2): p. 815–22.
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Table 3

Agonist efficacies and potencies at 5-HT2B (Emax given as % of 5-HT, EC50 for calcium flux in nM)1
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Drug Emax EC50

5-HT 100.0 1.78

BW 723C86 92.9 1.41
DOI 88.2 1.45
Norfenfluramine 107.4 2.45
Cabergoline 98.5 398
Pergolide 88.5 74.1
Methylergonovine 49.5 21.4
Roprinirole 73 2570
Guanfacine 93 123
Oxymetazoline 70.9 45.7
Quinidine 55.9 186
Xylometazoline 55.7 240
Fenoldopam 92.5 77.6
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1
Adapted from: Huang, X.P., et al., Parallel functional activity profiling reveals valvulopathogens are potent 5-hydroxytryptamine(2B) receptor
agonists: implications for drug safety assessment. Mol Pharmacol, 2009. 76(4): p. 710–22.
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