Expert Opinion on Drug Discovery

ISSN: 1746-0441 (Print) 1746-045X (Online) Journal homepage: http://www.tandfonline.com/loi/iedc20

Models for erectile dysfunction and their

importance to novel drug discovery

Christopher Wu & Jason R. Kovac

To cite this article: Christopher Wu & Jason R. Kovac (2015): Models for erectile dysfunction
and their importance to novel drug discovery, Expert Opinion on Drug Discovery, DOI:
10.1517/17460441.2016.1126243

To link to this article: http://dx.doi.org/10.1517/17460441.2016.1126243

Accepted author version posted online: 21

Dec 2015.
Published online: 22 Dec 2015.

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 EXPERT OPINION ON DRUG DISCOVERY, 2015
http://dx.doi.org/10.1517/17460441.2016.1126243

REVIEW

Models for erectile dysfunction and their importance to novel drug discovery
Christopher Wua and Jason R. Kovacb
a
McMaster Institute of Urology, Hamilton, ON, Canada; bMen’s Health Center, Indianapolis, IN, USA

ABSTRACT ARTICLE HISTORY

Introduction: Erectile dysfunction (ED) affects quality of life and is a barometer for vascular Received 22 September 2015
health. The pathophysiology is complex and multifactorial. Animal models have been critical in Accepted 26 November 2015
elucidating an improved comprehension of erectile function. They provide experimental plat- Published Online 23
forms where desired physiologic, and non-physiologic perturbations can be performed. Results December 2015
have led to the development of novel therapeutic targets. KEYWORDS
Downloaded by [University of California, San Diego] at 01:51 30 December 2015

Areas covered: The current article provides an overview of history of animal models in ED Animal models; erectile
research as well as a review of the current roles in the study of ED. The authors highlight the dysfunction;
advantages and disadvantages of each model while illustrating the similarities to the human phosphodiesterase type 5
condition and summarizing the major preclinical studies investigating novel therapeutic targets inhibitors; stem cells
in the treatment of ED.
Expert opinion: Animal models have been instrumental in the discovery of the current thera-
peutic agents. Advances in molecular biology and proteomics have uncovered many novel
potential targets including tissue regeneration and stem cell applications. Rodent models are
the current animal model of choice for ED research due to lower cost, well-established modeling
protocol, and the ability to manipulate genetically. Future clinical trials should directly assess the
translatability of these animal models to humans as well as the safety risks and long-term efficacy
that the results generate.

1. Introduction been presented as cures to ED. Da Vinci also challenged

contemporary beliefs that moving air led to stiffness in
Erectile dysfunction (ED) is defined as the consistent or
the penis after noting the abundance of blood in the
recurrent inability to achieve penile erection sufficient for
erect penis.[5]
sexual activity.[1] The condition is widespread, with a
These early observations led to further investigations
combined prevalence of 52% in men aged 40–70 years.
on erectile physiology. In this article, we highlight some
[2] ED has profound effects on quality of life and can
of these initial studies while describing the subsequent
cause significant distress to patients and their partners.
evolution to newer techniques and more novel experi-
There are well-established risk factor categories asso-
mentation using animal models. Regnier De Graaf, the
ciated with ED including diabetes mellitus (DM), athero-
inventor of the syringe, described the induction of an
sclerotic heart/cardiovascular disease, hypertension,
erection after injecting saline into the penile blood
psychiatric/psychological disorders, smoking, medica-
vessels of cadavers in 1668, providing early insight
tions and hormonal factors.
into the fluid dynamics and vascular basis of erections.
The historical basis for our current understanding of
[6] In 1863, the role of the nervous system in erectile
ED is rich and storied; with literary accounts of impo-
physiology was first discovered after Eckhard elicited
tence described in multiple ancient texts.[3,4] Indeed,
penile erections through the galvanic stimulation of
the erect penis symbolized power in ancient cultures,
pelvic nerves in dogs (nervi erigentes).[7] He further
with a loss of potency representing defeat and failure.
discovered arterial inflow as one of the principal
The extensive volumes of ancient materials dedicated
mechanisms of erection after observing the change
to remedying declining sexual ability have highlighted
from sluggish bleeding from a cut flaccid penis, to
the cultural importance of male potency. Suggestions
significant and rapid bleeding from the erectile bodies
ranging from reading tales of love and using erotic
after pelvic nerve stimulation.[8] Since this time, many
images to consuming herbal remedies and foods that
basic principles underlying erectile physiology have
resembled a phallus (i.e. leeks and asparagus) have
been substantiated by the use of animal models.

CONTACT Jason R. Kovac jkovac@urologyin.com Men’s Health Center, 8240 Naab Road, Suite 220, Indianapolis, IN 46260, USA.
© 2015 Taylor & Francis
 2 C. WU AND J. R. KOVAC
Article highlights
● Animal models have played a key role in understanding erectile
physiology. Over time, models have scaled from large animals
to the current gold standard of rodent models.
● Large animal models, such as non-human primates, are able to
translate findings to humans given similarities in both anatomy
and physiology. They provide easy tissue availability and larger
size but are hampered by increased husbandry costs and ethical
considerations.
● Initial studies involving the administration of intracavernosal
agents such as prostaglandin and sodium nitroprusside utilized
large animal models.
● Cat model, an example of a smaller animal model, was used to
study numerous vasoactive agents such as adrenomedullin and
potassium channel agonists; however, fundamental differences
in penile anatomy limit the translatability of this model.
● An intermediate-sized rabbit model provided valuable informa-
tion about erectile function in type 1 diabetes, hypercholester-
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olemia and metabolic syndrome (MetS).
● Currently, rodent models are the most commonly used animal
model in erectile dysfunction research. They are economical,
allow for transgenic manipulation and have well-established
protocols for many conditions associated with ED (i.e. type 1
and 2 diabetes, hypercholesterolemia, MetS) with excellent
reproducibility.
● Potential novel therapeutic targets investigated in the rodent
model includes the RhoA/Rho-kinase and Ninjurin-1 pathways
in diabetes, soluble guanylate cyclase enzyme activators in the
setting of cavernous nerve injury, and stem cell therapies.
Animal models were further utilized to study the
neurophysiological basis of erections, with the applica-
tion of selective lesioning of spinal tracts to understand
the role of the autonomic nerves and somatic nerves by
researchers. In one study, selective destruction at the T8
level of the spinal cord was performed in anesthetized
rats (via oscillating vibrotome). Dorsal to ventral trans-
verse cuts of varying depths were made thus lesioning
specific tracts within the spinal cord. Another study
using a rat model involved placing an intra-thecal can-
nula at different levels of the spinal cord and injecting
tetracaine resulting in a functional separation of the
brain.[9–13] Electrophysiological models similar to
those employed by early scientists are still used to this
day in modern research studies. Further models of
erectile stimulation include nerve stimulation, central
and peripheral pharmacologic stimulation, and stimula-
tion of the central brain nuclei postulated to be
involved with erectile pathways.
Animal models were also developed to objectively
monitor erectile physiology. Intra-cavernosal pressure
(ICP) measurement is one of the most prevalent and
reproducible methods to measure erectile response.
Lewis et al. first performed this type of experiment in
the bull in 1968.[14] Other methods include observation
and monitoring for erectile phenomena, penile
plethysmography, penile measurements, radionuclide
scintigraphy, bloodflow measurements by Doppler
flowmetry or a combination of these multiple
techniques.
Since the aforementioned early experiments in large
animals like dogs, animal models of ED have been
scaled down to primarily, sophisticated rodent models.
[15] These rodent models have now largely replaced
large animal models due to purchasing and mainte-
nance costs that were prohibitive. Moreover, the chan-
ging landscape of research ethics has also made the
availability of these large animals very difficult to justify.
We will discuss the major animal models that had
been used, or are currently being used, to study ED
such as large animals, rabbits and rodents. This review
will also discuss major preclinical studies investigating
novel therapeutic targets in the treatment of ED.
2. Large-size animal models
Initial studies on ED were conducted in large animal
models such as dogs and non-human primates (NHPs).
Although rare, several landmark seminal simian studies
have explored the neuro-pharmacological mechanisms
involved in erections. In early studies, changes in the
ICP and penile diameter were measured in monkeys
after the intracavernosal injection of substances such
as prostaglandin (PG) E1. Nitric oxide donors such as
sodium nitroprusside and s-nitrocysteine illustrated the
roles of cyclic guanosine monophosphate (cGMP) and
cyclic adenosine monophosphate pathways in caver-
nous smooth muscle relaxation.[16,17] Lue and cow-
orkers further contributed to the understanding of
penile erection through electrophysiological and hemo-
dynamic studies on dogs and monkeys.[18,19] These
large animal models had very higher costs including
purchase price, animal husbandry and facilities charges
that limited their use. Initially, these models also had
limited research-specific methods and protocols.
Nonetheless, canine models contributed significantly
to the understanding of the signal transduction path-
ways of ATP, adenosine, vasoactive intestinal peptide
and acetylcholine (ACh) in erectile physiology.[20–22]
There are many similarities in the anatomy and phy-
siology of erection between humans and canines. For
example, the size of the penis allows for easy evaluation
of erectile function with canines having a similar vascular
and neural anatomy as well as a close approximation of
the collagen to smooth muscle ratio.[23] Dogs also have
well-studied mating anatomy, which allows for easy
observation of sexuality and arousal – factors that, like
humans, are based on sight, touch and smell.[24]
Limitations to the use of canine models in the study
of erectile physiology do exist. Canines, as well as some

NHPs, have an os penis (also known as a baculum): a
bony structure within the penis that aids in intromis-
sion. The os penis provides structural reinforcement to
the penis, whereas in humans, penile rigidity is solely a
hemodynamic process that is maintained by ICP alone.
Canines also have an intra-cavernosal septum that is
well developed. Its presence prevents communication
between the two corpora cavernosa, a situation not
fully observed in humans.[23] These important ana-
tomic differences may limit the applicability of canine
models to human studies. Canines are also not prone to
atherosclerotic disease, and, experimentally they
demonstrate an atypical pattern of cardiovascular dis-
ease. As such, extensive medial accumulation of plaque
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is traditionally observed in canines, unlike the severe
intimal response seen in humans. Thus, canine models
are not optimal to study atherosclerosis-related ED.[25]
Although NHP studies are uncommon, due to their
significant costs and ethical considerations, simian
models have contributed to our knowledge of erectile
physiology in several landmark studies. These include
investigations into the intracavernosal administration of
pharmacologic agents [26–28] and atherosclerosis-
induced ED.[29] There may still be significant utility of
NHP models in the study of atherosclerotic vascular
disease. Macaque monkeys have demonstrated pat-
terns of atherosclerosis (iliac, femoral and pudendal
artery plaques) and lesion characteristics (development
into complicated intimal atheromas with risks of plaque
rupture) that can lead to vascular complications such as
myocardial infarction and impaired vascular function
mirroring conditions seen in humans.[30,31]
Finally, large animal models provide easily observed
mating behaviors allowing for the measurement of
such parameters as erection quality and intromission.
[23] Lastly, these large animals provide significantly
more numerous and better quality tissues. This allows
researchers to employ more rigorous instrumentation
while improving the supply of tissues needed for
experiments consisting of multiple endpoints with vari-
able interventions.
3. Intermediate-size animal models
Over time, the expenses associated with the aforemen-
tioned large animals drove researchers into searching for
more scalable and affordable animal models. Cat models
were used in several landmark studies in the 1990s. Novel
vasoactive agents such as adrenomedullin, nociceptin
and potassium channel agonists were studied in these
models.[32–35] The limitations of the feline model
include several fundamental differences in anatomy com-
pared to humans. Similar to canines, feline penises have a
EXPERT OPINION ON DRUG DISCOVERY 3
baculum and the paired corpora cavernosa are located
ventral to the corpus spongiosum.[32] In addition to this,
there remains considerable expense in the facilities
required for feline models as well.
Due to these considerations, the rabbit then
emerged as a prime candidate for the study of erectile
physiology. Indeed, studies using the rabbit have had a
significant impact in improving our understanding of
erectile physiology and uncovering therapeutic targets.
Rabbits are currently the most commonly used non-
rodent animal models with well-established models
existing for both atherosclerosis and diabetes.
Although more expensive than rodents, many institu-
tions already have established facilities that house rab-
bits. Moreover, the costs of care and husbandry are
many-fold less than that of large animals.
The erectile physiology of rabbits is well understood
with the anatomic structure of the rabbit penis being
similar to human. Indeed, many studies have shown
similarities in the factors modulating the tone of the
corpora cavernosa between humans and rabbits.[36–
38] Rabbit models have also been pivotal in identifying
potential targets for therapy including the ACh and NO
pathways, endothelin receptors, prostanoids and the
rho kinase pathways.[39–43] Compared to rodents, the
larger penis size of rabbits allows for easier study of the
pathophysiological effects of ischemia, atherosclerosis
and oxidative stress.
The Watanabe rabbit model has provided an invalu-
able model of human familial hypercholesterolemia,
with a mutant low-density lipoprotein (LDL) receptor
resulting in very little functional receptor reaching the
cell surface.[44] This results in the accumulation of lipo-
proteins including LDL and very low-density lipopro-
teins (VLDL) that contribute to fatty streak and
atheroma formation, similar to rabbits being fed high
cholesterol diets.[44] This model has led to insight into
both the pathophysiology of hypercholesterolemia and
to the development of ED.[45] The endothelium-depen-
dent, NO-mediated relaxation in response to ACh was
impaired in hyper-cholesterolemic rabbits with concur-
rently decreased expression of vascular endothelial
growth factor (VEGF) found in the cavernosal tissues.
[46] This has led to the identification of potential ther-
apeutic targets including basic fibroblast growth factor
and VEGF.[47,48]
Although well established, there are important dif-
ferences between hyperlipidemic rabbits and humans.
There is a higher component of VLDL in rabbits than
humans, and the atheromas tend to be more macro-
phage rich and less sclerotic.[46] The atheromas are
also more centrally distributed in the major conduit
arteries such as the aorta than in humans, which may
 4 C. WU AND J. R. KOVAC
affect generalizability.[45] The atheromas are also not
occlusive in the purely hyperlipidemic model, thus a
model of chronic cavernosal ischemia (CCI) was created
involving both a hyper-cholesterolemic diet and bal-
loon de-endothelization of the iliac arteries.[49] This
model had markedly decreased iliac artery blood flow
secondary to significant atherosclerotic occlusive dis-
ease. Both endothelium-dependent and neurogenic
NO-mediated relaxation to ACh was encountered in
the CCI model, suggesting that ischemic insult affects
both endothelium-dependent and neurogenic isoforms
of NO synthase (NOS), thus leading to reduced synth-
esis of downstream cGMP. These studies further con-
tributed to the hypothesis that cGMP-specific
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phosphodiesterase type 5 inhibitors (PDE5i) may be
able to restore intracellular levels of cGMP, leading to
improved cavernosal smooth muscle relaxation.
The alloxan-induced diabetic rabbit model has been
used to model DM-induced ED. This model has pro-
vided insight into the neurological and vascular path-
ways that contribute to the multifactorial
pathophysiology of DM-associated ED. Endothelial dys-
function was discovered to be a major contributor to
ED in DM, with decreased formation of NO and prosta-
cyclin (PGI2) in cavernosal tissues isolated from rabbits
with DM.[50] Diminished NO-dependent vasodilatation
was found to be associated with the up-regulation of
reactive oxygen species (ROS). ROS are known to inter-
act with NO, thus inhibiting its biological activity to
produce peroxynitrite.[51] Peroxynitrite in turn has
been found to damage nucleic acids and mitochondria
and has been implicated in apoptotic and necrotic cell
death.[51] The application of superoxide dismutase, an
enzyme that catalyzes the breakdown of the superoxise
anion to water, to diabetic rabbit caverosal tissue sig-
nificantly reversed impaired relaxation.[52] The interac-
tion of NO with guanylate cyclase to convert guanosine
triphosphate into cGMP then led to confirmatory stu-
dies in diabetic rabbits that the use of PDE inhibitors
enhanced NO-induced relaxation in cavernosal tissue.
[53] Although the alloxan-induced DM rabbit model has
significantly contributed to our knowledge in the
mechanisms of endothelial dysfunction found in DM-
associated ED, there are limitations to this model.
Alloxan-induced disease causes severe dehydration,
which results in significant morbidity and mortality in
a large percentage of rabbits.[54]
Metabolic syndrome (MetS) has also been linked to
both hypogonadism and ED and is a constellation of
clinical risk factors comprising cardiovascular disease,
stroke, T2DM and kidney disease.[55] MetS is a signifi-
cant threat to public health, with prevalence estimates
in adults from the National Health and Nutrition
Examination Survey in 2010 of 22.9%.[56] Studies sug-
gest that MetS is associated with ED that is unrespon-
sive to PDE5i.[57] A rabbit model of MetS was created
through feeding adult male rabbits a high fat diet for 12
weeks. In the rabbit MetS model, NO-mediated relaxa-
tion of cavernosal tissue is blunted to both ACh and
non-adrenergic non-cholinergic (NANC) nerve stimula-
tion. Through this model, the roles of the farnesoid X
receptor (FXR) and TNFα in modulating ED were dis-
covered. FXR is a nuclear receptor that functions as a
sensor for bile acids. It has also been implicated in
numerous metabolic pathways by exerting an anti-
inflammatory effect, suppressing hepatic fatty acid
and triglyceride synthesis, and also affecting vascular
homeostasis via eNOS production.[58] Administration of
the potent and selective FXR agonist obeticholic acid in
the rabbit MetS model restored responsiveness to both
ACh and NANC stimulation.[59] Further study sug-
gested that FXR activation increased not only NO-
mediated relaxation but also interfered with pro-con-
tractile RhoA/RhoKinase (ROCK) signaling, thus normal-
izing erectile function.[59] FXR activation also
normalized visceral adiposity and glucose intolerance
[59] making it a promising new therapeutic target.
The MetS rabbit model was also found to demonstrate
histological findings of non-alcoholic steatohepatitis
(NASH), brought on by liver injury and the generation of
a pro-inflammatory state through generation of TNFα.
Treatment by infliximab, an anti-TNFα monoclonal anti-
body, fully restored cavernosal tissue responsiveness to
Ach via increased eNOS while concurrently reducing visc-
eral fat accumulation.[60] Further research into the mod-
ulation of TNFα levels may be another promising direction
for therapeutic discovery in the treatment of ED.
In summary, the rabbit as an animal model repre-
sents an intermediate-sized animal model with a cost
and applicability lying somewhere between the large
animal models and the smaller rodent model.
4. Small-animal models
Rodents, the most common example of a small-animal
model, are currently the most common animal models
for the investigation of ED. This will likely remain the
case in the near future given their multiple advantages
and availability. These models have contributed signifi-
cantly to the current fund of knowledge and have been
critical to the discovery of many of the mechanisms
modulating erectile function. We will describe only a
small portion of the findings in this article.
One of the pioneering studies using the rat as a
model for the study of penile erection was by Quinlan
et al. in 1989.[61] In this work, the authors

demonstrated that electrical stimulation of the caver-
nous nerve resulted in a reproducible repetitive tumes-
cence of the corporal bodies in 40 Sprague-Dawley rats.
Furthermore, following surgical ablation of the caver-
nous nerves, no erections were obtained.[61] This study
firmly established rats as a viable model for understand-
ing penile physiology. Martinez-Pineiro et al. further
optimized the electro-stimulatory parameters to obtain
maximal ICP in the rat model, as well as an understand-
ing of physiologic responses of intracavernosal admin-
istration of agents such as phenylephrine, papaverine
and phentolamine.[62]
There are numerous advantages to using rat models.
They are economical to work with and their small size
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allows for convenience in terms of handling and hous-
ing. There are also various well-defined methods for
evaluating erectile function, including peripheral elec-
trical field stimulation of pelvic nerves or cavernous
nerves, apomorphine-induced central nervous system
stimulation or the introduction of vasoactive agents
by local, or systemic means. Techniques for the surgical
preparation and dissection of the cavernous nerves in
the rodent model are also well established. Moreover,
the rodent model has durability with research out-
comes from therapeutic trials being consistently repro-
duced. Rats also have a short lifespan, between 1 and
3 years, which permits longitudinal studies to be easily
performed, and allows for the study of the effects of
aging on erectile function.[63]
Since these initial studies, many well-established pro-
tocols for known risk factors for ED have been
described. These models include cavernous nerve injury
(CNI), diabetes, hypercholesterolemia, hypertension and
arterial insufficiency models (among others).
CNI models in the rat have been described using
various methods, including using crush injury,[64] dis-
section,[65] transection,[66] and excision.[67] These stu-
dies identified numerous findings including lower ICP
and decreased neuronal nitric oxide synthase (nNOS) in
both cavernous nerves and pelvic ganglia following CN
injury. The results of these works have been the com-
mon outcome measures used in a majority of CN
studies.
Further investigations in unilateral versus bilateral CNI
models found increased nNOS and ICP expression in rats
with unilateral injury compared to bilateral injury in
cases where a complete loss of erectile function per-
sisted at 6 months.[68] These findings were consistent
with human patients who developed ED following non-
nerve sparing radical prostatectomy.[69] Histological sec-
tioning of the bilateral CNI rat specimens identified
apoptosis of the cavernous smooth muscle cells (SMCs)
and endothelial cells.[70] Further study of these models
EXPERT OPINION ON DRUG DISCOVERY 5
revealed the role of the sonic hedgehog (SHH) protein as
a critical regulator in both endothelial and cavernosal
SMCs. When SHH protein was inhibited, a 12-fold
increase in the apoptosis of cavernosal SMCs occurred
leading to a fourfold decrease in ICP and resultant ED.
[71] This is another promising potential therapeutic
direction, with studies showing that exogenous admin-
istration of SHH is neuroprotective, and can induce VEGF
and NOS, stimulating CN regeneration and improved
erectile function after CN injury.[72]
The molecular mechanisms underlying use of
vacuum erectile devices (VED) were also investigated
in the rat model. The use of VED after bilateral CNI
increased the oxygenation of the penile tissue, prevent-
ing apoptosis and collagen deposition compared to a
control group.[73]
Rat models have also been used to simulate the
condition of penile vascular insufficiency resulting
from either traumatic or atherosclerotic occlusive
vascular disease; a common cause of ED. Alterations of
the inflow from occlusion of the iliac, pudendal or
cavernosal arteries decrease perfusion pressures in the
penis, leading to ED.[15] Traumatic arterial insufficiency
models have also been created through bilateral liga-
tion of either the iliac arteries or pudendal arteries,
resulting in the immediate decline in ICP following
electrostimulation. Histologic findings from these trau-
matic, arteriogenic ED models included degeneration of
dorsal nerve fibers, decreased NOS-containing nerve
fibers, and collapse of sinusoids, loss of fibroblasts,
intracellular fat deposition and fatty degeneration.[74]
Unilateral ligation models showed inconsistent out-
comes as there was a compensatory increase in arterial
flow in the contralateral artery.[75] Chronic occlusion of
the penile vessels in other models has shown minimal
effect on erections by neurostimulation due to the
extensive collaterals to the penis.[76]
ED is commonly associated with DM, with preva-
lence rates of up to 51% in patients with type 1 DM
and 37% with type 2 DM in one large Italian cohort
study of 10,000 patients.[77] The pathophysiology of ED
in DM is multimodal, with decreased NO synthesis,
decreased cavernosal smooth muscle relaxation,
increased free radical formation, upregulation of the
ROCK pathway, hyperglycemia, cGMP pathway dysfunc-
tion and hormonal imbalances.[78] The streptozocin
(STZ)-induced rat is a well-established animal model of
type-1 DM. Based on multiple studies, the STZ-induced
rat model has impaired parasympathetic relaxation of
the cavernosal smooth muscle as well as decreased
smooth muscle and endothelial cell density.[79]
Furthermore, there was degeneration of the nitrinergic
nerves, leading to decreased nNOS expression.[80]
 6 C. WU AND J. R. KOVAC
There were also higher levels of oxidative stress, with
higher levels of ROS measured.[81] A promising study in
the STZ rat provided molecular and functional evidence
that the upregulation of the ROCK pathway played a
major role in the pathogenesis of ED in DM-induced ED.
Smooth muscle contraction is mediated by calcium/
calmodulin-dependent activation of myosin light chain
(MLC) kinase and subsequent phosphorylation of MLC,
leading to myosin/actin complex formation.[82] Smooth
muscle relaxation is facilitated by the dephosphoryla-
tion of MLC by MLC phosphatase.[83] RhoA activation
of Rho-Kinase results in the attenuation of MLC phos-
phatase activity, and is thought to play an important
role in the regulation of penile smooth muscle tone and
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detumescence.[84] The ROCK pathway was found to be
significantly higher in STZ rats than controls, and STZ
rats had significantly lower constitutive NOS activity
and cGMP concentrations.[85] Treatment of the STZ-
rats with either in vivo gene delivery of an inhibitor of
RhoA through an adeno-associated virus or through
direct intra-cavernosal injection of an experimental
Rho-kinase inhibitor led to enhancement of penile
NOS expression, NOS activity and cGMP levels, thus
restoring erectile function.[85] This established the
ROCK pathway as an alternative potential therapeutic
target for the treatment of DM-related ED.
Type 2 DM was investigated in rodents using models
such as the obese zucker rat and the leptin receptor
mutated db/db mouse. These models exhibit hyperin-
sulinemia, hyperglycemia, and obesity. Studies have
shown decreased eNOS production, corporal fibrosis
and corporal veno-occlusive dysfunction (CVOD).[86–
88] Cavernosal smooth muscle tone was also found to
be elevated in T2 DM rodents, leading to CVOD. This
suggests that a greater vasodilatory stimulus is required
to achieve erection.
Another novel class of therapeutics targeting the
soluble guanylate cyclase (sGC) enzyme has shown pro-
mising results in pre-clinical rat model studies. In patho-
physiologic conditions such as DM and CNI, resistance
to PDE5i can arise from impaired NO formation and
increased ROS production, leading to inhibition of
sGC. BAY 60-2770 is a sGC activator that has shown
very potent erectile activity after intra-cavernosal
administration despite CNI or treatment with a NOS
inhibitor.[89] Agents in this emerging class may prove
to be useful in the treatment of ED from which NO
production and availability are diminished – such as in
DM or in ED resulting from cavernous nerve damage
post-prostatectomy.
Following fibrosis and/or apoptosis of corporal SMCs,
the subsequent impaired tissue compliance results in
veno-occlusive dysfunction following cavernosal nerve
injury post-RP. As such, ED is frequently resistant to on-
demand oral PDE5 inhibitor therapy.[90] Studies on
pharmacologic therapies using long-term continuous
daily administration of PDE5i have shown promise of
potentially exploiting an anti-fibrotic mechanisms,
resulting in an improvement over on-demand PDE5i
interventions.[91–93] Unfortunately, these studies have
shown little translatable relevance to humans, with
inconclusive results being documented.[94,95] Due to
the cellular damage that occurs in CNI, alternative
approaches to post-RP ED such as stem cell (SC) therapy
are now being examined and show promising results in
preclinical animal models.
SCs can be isolated from numerous tissues includ-
ing adipose tissue, skeletal muscle, umbilical cord
blood, bone marrow and embryonic stem cells (ESCs).
[96] They have the capacity to develop into various
cell types based on signals they receive. They also
have the capacity and ability to self-renew. Currently,
two major sources of SCs are ESC and mesenchymal
stem cells (MSC), or adult SCs.[97] ESCs are derived
from the blastocyst of the embryo and are pleuripo-
tent meaning that they are able to differentiate into
almost any tissue. In contrast, MSCs are derived from
various tissues of a developed adult, and are multi-
potent with the ability to differentiate into most types
of its tissue origin.[97] ESCs are superior to MSCs in
terms of differentiation potential, but unfortunately,
research on ESCs has largely been hampered by ethi-
cal and governmental restrictions, thus, SC research
has primarily involved MSCs. SC therapy for ED was
founded on the hypothesis that these cells could
replenish endothelial cells and cavernosal SMCs that
were lost or damaged in the disease processes asso-
ciated with ED. These transplanted SCs could also
provide paracrine actions as well as promote cellular
differentiation and maturation in the host’s own
endothelial and cavernosal SMCs. Multiple preclinical
studies in rodent models have shown promise with
bone marrow derived SCs, muscle-derived SCs and
adipose tissue-derived SCs.[98–103]
In the context of SCs, CNI and DM-associated ED are
the most commonly tested disease models. The cells
can be transplanted via an intra-cavernosal injection,
though studies have now shown that this technique is
similar to intravenous administration since most of the
SCs exit the corpora within a day of injection and target
largely to the bone marrow and major pelvic ganglion
(MPG).[104,105] The cells that then populate in the
bone marrow allowing for sustained reservoirs with
regenerative activity. Thus, cells targeting to the MPG
may provide the therapeutic efficacy seen in CNI. SCs
may hold great promise through the treatment of

various urologic conditions as the field of regenerative
medicine continues to develop.
However, there are limitations to rat models that
may affect their human relevance. Although rat and
human penile anatomy and physiology have been
well described and show similarities, there are funda-
mental differences in sexual behavior between the spe-
cies.[106] Rats are stimulated by smell as well as
behavior and touch, whereas humans are stimulated
by visual stimulus as well as behavior and touch.[107]
There are also differences in the distribution of athero-
sclerotic plaques in rat models, where the patterns are
more centrally distributed versus in humans where they
are peripherally distributed.[108] There are also funda-
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mental differences in lipoprotein metabolism between
humans and rats.[109] Also, vascular rat models for ED
such as arterial ligation may not be translatable to
human vascular ED as peripheral vascular disease rarely
leads to complete occlusion of the pelvic vessels in
humans.[110]
The growing use of genetically modified mice has
greatly enhanced our knowledge of the pathophysiology
of a wide variety of diseases, and because of this, there
has been a more recent shift toward mouse models for
the study of penile erection.[15] Initial challenges with
mouse models were mainly technical; since the animals
were much smaller, it made the introduction of monitor-
ing equipment (i.e. pressure transduction catheters) into
vessels quite morbid and challenging. As such, many of
the initial mouse studies were in vitro investigations of
erectile mechanisms.[111,112] With the refinement of
multiple in vitro techniques and improved monitoring
protocols, the mouse model has risen to prominence.
The anatomy and physiology of penile erection in mice
has been well established,[113] and interestingly, the
mechanisms leading to erection are similar between
mice and humans. Also, many mouse models have
been created including CNI models, STZ-induced DM
models, and diet-induced DM models, thus making the
mouse an attractive model for studying ED.
The use of transgenic mice has provided a powerful
tool to study the mechanisms involved in erectile phy-
siology and specific types of ED. One model, the apoli-
poprotein E knockout (ApoE KO) mouse, is a model of
murine hyper-cholesterolemia that has been well stu-
died.[114] Unlike the rat models, this mouse version has
demonstrated atherosclerotic lesions that have great
similarity to humans with similar vascular endothelial
dysfunction.[114–116] Xie and colleagues further
validated an ApoE KO mouse model of ED, with
abnormalities seen in endothelium-dependent and
independent vaso-reactivities as well as in eNOS,
nNOS and cGMP expression.[117]
EXPERT OPINION ON DRUG DISCOVERY 7
Further studies in the STZ-induced mouse model
have shed light on new therapeutic targets for ED in
the setting of DM. With the severe endothelial dysfunc-
tion and peripheral neuropathy that accompanies DM,
many patients respond poorly to oral PDE5i, as these
depend on endogenous NO formation. A promising
potential therapeutic target is the nerve injury-induced
protein 1 (Ninjurin 1), a cell surface protein implicated
in the initiation and progression of multiple sclerosis,
and was noted to be upregulated in nerve transection
studies.[118,119] It has also been shown to play an
important role in vascular homeostasis in general and
vascular regression of capillaries in the ocular lens in
particular.[120] Initial studies in diabetic mice revealed
upregulation of Ninjurin 1 protein in the penile tissues,
with markedly increased levels in the dorsal nerve bun-
dle and cavernous endothelial cells.[121] A promising
study by Yin and colleagues found inhibition of Ninjurin
1 using a neutralizing antibody directly injected into
the penis restored erectile function, induced prolifera-
tion of cavernous endothelial cells, deceased cavernous
endothelial cell apoptosis, increased cavernous angio-
genesis, decreased cavernous ROS formation and pro-
moted the secretion of neurotrophic factors in both
cavernous tissues and the dorsal nerve bundle of a
STZ-mouse model of DM.[122] The authors further con-
firmed the importance of Ninjurin 1 in the pathogenesis
of DM-induced ED using a Ninjurin-1 knockout mouse
model created with siRNA, with preserved erectile func-
tion as well as endothelial and neuronal cell contents.
[122,123] The targeted depletion of Ninjurin-1 in this
preclinical study showed restored erectile function via
both angiogenic and neurotrophic effects, suggesting
that neural regeneration and therapeutic angiogenesis
may be a promising direction for therapeutic discovery
in the treatment of DM-induced ED.
5. Conclusions
Animal models have played a crucial role in the under-
standing of the pathophysiology of ED. Initial studies
began in large animals such as canines and advanced
the field by providing the basis of our understanding of
the nervous systems role in erectile physiology. NHP
large animal models have had great value in under-
standing the physiology behind the administration of
intracavernosal agents and vascular ED due to simila-
rities in the patterns of atherosclerosis, penile anatomy
and mating behavior. The economic and ethical con-
siderations have led to a decrease in the use of large
animal models. Medium-size animal models such as
rabbits have also had significant impact on the study
of erectile function. Through early models of MetS,
 8 C. WU AND J. R. KOVAC
hypercholesterolemia and DM, they have greatly con-
tributed to identifying promising therapeutic targets
such as the FXR and TNFα. In the past decade, small
animal rodent models have become the predominant
model for experimentation. Various rat and mouse
models have been developed to replicate specific per-
turbations that are known to commonly lead to ED in
humans. With increasingly sophisticated experimental
techniques such as knockout models and SC therapy,
further insights into the molecular biology of the
mechanisms contributing to ED have led to the discov-
ery and testing of many novel therapeutic agents.
Rodent models appear to be the animal model of
choice for ED research in the near future due to low
Downloaded by [University of California, San Diego] at 01:51 30 December 2015
costs, well established modeling protocols, and the
near limitless potential of genetic manipulation.
6. Expert opinion
Animal models have been critical in establishing the
foundations of ED. They have facilitated the study of
molecular pathways and have led to the development
of numerous therapeutic targets. Many of the pioneer-
ing studies in erectile physiology were conducted in
large and intermediate-size animal models since their
size and anatomic similarities with humans facilitated
evaluation and investigation. With further refinement of
experimental techniques, the costs and ethical consid-
erations of using these large and intermediate-size ani-
mal models drove researchers toward smaller animal
models.
Small animal rodent models are the currently the
most prevalent model used in ED research. Reasons
include lower costs, ease of husbandry and housing,
established protocols, and an improved ability to inves-
tigate and manipulate the genome. They provide a
valid and reproducible experimental platform with
which to investigate and test novel hypotheses.
Rodents allow interventions and multi-step experimen-
tal protocols to be conducted coupled with the ability
to perform washout periods and cross-over experi-
ments. The application of increasingly sophisticated
techniques such as transgenic rodent models, SC thera-
pies as well as refinements in proteomics, immunohis-
tochemistry and high-throughput microarray
technologies have greatly broadened our pathophysio-
logic knowledge of ED. Indeed, the future holds great
promise. Specifically, the rise of genomic experimenta-
tion has the potential to tailor treatment based on an
individual genetic profile. Such a treatment could hold
the potential for individual cures given the proper
identification of unique gene expression and profiles.
The biggest challenge in achieving this reality is the
identification of specific genes that result in ED.
Unfortunately, the greatest source of difficulty in this
task will be eliminating confounding data and sorting
through the massive amounts of data derived.
One area of particular interest is the impact of DM
and CNI on cavernosal tissues. This review has high-
lighted several promising new directions for novel ther-
apeutics based upon the preclinical animal model data.
For example, in cases of CNI, exogenous administration
of SHH can be neuroprotective, while SC therapies
might aid in neural regeneration and replacement of
endothelial and cavernosal SMCs. In animal models of
DM, inhibition of Ninjurin-1 has demonstrated
increased angiogenesis and neural regeneration, lead-
ing to the postulation of a novel pathway by which to
restore of erectile function. Furthermore, inhibition of
the ROCK pathway also resulted in increased NOS
expression and increased cGMP, and as such, novel
direct sGC activators could be theorized to demonstrate
potent erectile function despite nerve injury or NO-
inhibition. Such research opens up the potential for
further investigations into alternative NO-independent
pathways of erection.
In the current article, we have highlighted emerging
novel pathways in the pathophysiology of ED. There
remain multiple areas of weakness underscoring our
current level of understanding. Indeed, further experi-
ments are needed to determine whether differences in
rodent strains, age or type have an effect on erectile
physiology. Furthermore, many emerging novel thera-
peutics still empirically utilize oral or intra-cavernosal
drug delivery. This suggests that there is still ample
opportunity for further research into alternative
modes of administration. By moving agents to possible
topical forms of administration, local effects could be
emphasized over the systemic. Such a mechanism
would be advantageous to those who are unable to
take the currently available forms of medication.
There also exists further opportunity for study into
the molecular mechanisms of targeted therapeutic
delivery. This will become important as further studies
into treatments with potentially concerning off-target
effects, such as stem-cell therapies, are further explored.
Lastly, although the sexual physiology and anatomy
of each animal model has been well established, funda-
mental differences still separate all discussed models
from the human condition. While this unfortunately
limits direct translation to the human condition, the
principals and experimental protocols established in
the recent past herald exciting new drug discoveries
in the near future.
 EXPERT OPINION ON DRUG DISCOVERY 9

Declaration of interest
J Kovac is a paid speaker for AbbVie. The authors have no
other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript apart from those disclosed.
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