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Effect of combination endothelial nitric oxide synthase gene
therapy and sildenafil on erectile function in diabetic rats
TJ Bivalacqua
1,2
, MF Usta
1
, HC Champion
3
, S Leungwattanakij
1
, PA Dabisch
2
, DB McNamara
2
,
PJ Kadowitz
2
and WJG Hellstrom
1
*
1
Department of Urology, Tulane University School of Medicine, New Orleans, Louisana, USA;
2
Department of
Pharmacology, Tulane University School of Medicine, New Orleans, Louisana, USA; and
3
Departtment of Internal
Medicine, Johns Hopkins Hospital, Baltimore, Maryland, USA
Erectile dysfunction associated with diabetes mellitus is caused in part by disordered endothelial
smooth muscle relaxation, neuropathy, and a decrease in cavernosal nitric oxide synthase (NOS)
activity. The purpose of this study was to determine whether a combination of sildenafil and
adenoviral gene transfer of endothelial NOS (eNOS) could enhance the erectile response in diabetic
rats. Five groups of animals were utilized: (1) age-matched control rats, (2) streptozotocin (STZ)-
induced diabetic rats (60 mg/kg i.p.), (3) STZ-rats þsildenafil (2 mg/kg i.v.), (4) STZ-rats transfected
with AdCMVbgal or AdCMVeNOS, and (5) STZ-rats transfected with AdCMVeNOS þsildenafil
(2 mg/kg i.v.). At 2 months after i.p. injection of STZ, groups 4 and 5 were transfected with the
adenoviruses and 1–2 days after transfection, all animals underwent cavernosal nerve stimulation
(CNS) to assess erectile function. Cyclic 30,50-guanosine monophosphate (cGMP) levels were
assessed in the cavernosal tissue. STZ-diabetic rats had a significant decrease in erectile function as
determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve;
AUC) after CNS when compared to control rats. STZ-diabetic rats þAdCMVeNOS had a peak ICP
and AUC, which were similar to control animals. STZ-diabetic rats administered sildenafil
demonstrated a significant increase in peak ICP at the 5 and 7.5 V settings, while the AUC was
significantly increased at all voltage (V) settings. The increase in both ICP and AUC of STZ-diabetic
rats transfected with AdCMVeNOS at all V settings was greater than STZ-diabetic rats transfected
with AdCMVbgal. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had
a significant increase in total ICP that was greater than eNOS gene therapy alone. Cavernosal cGMP
levels were significantly decreased in STZ-diabetic rats, but were increased after transfection with
AdCMVeNOS to values greater than control animals. In conclusion, overexpression of eNOS and
cGMP in combination with sildenafil significantly increased both the peak ICP and total ICP to CNS
in the STZ-diabetic rat, which was similar to the response observed in control rats. Moreover, the
total erectile response was greater in STZ-diabetic rats receiving eNOS gene therapy plus sildenafil
than STZ-rats receiving sildenafil or eNOS gene therapy alone.
International Journal of Impotence Research (2004) 16, 21–29. doi:10.1038/sj.ijir.3901054
Keywords: eNOS; gene therapy; sildenafil; diabetes; erectile dysfunction
Introduction
According to the American Diabetes Association,
diabetes mellitus affects approximately 16 million
individuals in the United States. A frequent com-
plication of diabetes is erectile dysfunction (ED),
with an estimated prevalence in diabetic men to be
as high as 50–75%.
1,2
The exact mechanism of ED
in diabetic patients is complex and can be caused
by several mechanisms including autonomic neuro-
pathy, endothelial dysfunction, and hormonal
imbalance.
Penile erection is a complex neurovascular phe-
nomenon that requires an increase in penile arterial
inflow, relaxation of cavernosal smooth muscle, and
restriction of venous outflow from the penis.
Erectile capacity is dependent on the vascular tone
of the penis. Relaxation of corporal smooth muscle
is essential for normal erectile function and sub-
stantial evidence exists to implicate neuronal-
and endothelial-derived nitric oxide (NO) as the
principal mediator of cavernosal smooth muscle
Received 03 September 2002; revised 03 April 2003;
accepted 08 April 2003
*Correspondence: WJG Hellstrom, MD, Professor of
Urology, Tulane University School of Medicine, Depart-
ment of Urology SL–42, 1430 Tulane Avenue, New
Orleans, LA 70112, USA.
E-mail: whellst@tulane.edu
International Journal of Impotence Research (2004) 16, 21–29
&
2004 Nature Publishing Group All rights reserved 0955-9930/04 $25.00
www.nature.com/ijir
relaxation and penile erection.
3,4
NO released by the
endothelium that lines the corpus cavernosum and
penile arteries that supply the penis and NO from
nonadrenergic, noncholinergic (NANC) nerves bind
to the soluble form of guanylate cyclase to increase
cavernosal intracellular levels of cyclic 30,50-guano-
sine monophosphate (cGMP). The enzyme that
catalyzes this reaction in the endothelium and
NANC neurons is termed nitric oxide synthase
(NOS). The constitutive forms of the enzyme,
neuronal NOS (nNOS) and endothelial NOS (eNOS),
are the principal NOS isoforms involved in the
induction of penile erection.
5–7
Impairments in both
the neurogenic and endothelium-dependent caver-
nosal smooth muscle relaxation exist in diabetes
mellitus (DM).
8–11
It is well established that the
release of endothelium-derived NO and constitutive
NOS activity is reduced in diabetes.
1,10,12–14
Silde-
nafil (Viagra) is an orally active selective inhibitor of
the type 5 phosphodiesterase (PDE) and inhibits the
breakdown of intracellular cGMP.
15
Therefore, sil-
denafil facilitates NO-mediated corpus cavernosum
smooth muscle relaxation and thus enhances erec-
tile responses. However, the clinical efficacy of
sildenafil in diabetic patients is less than in patients
with vasculogenic or psychogenic ED.
16
Gene transfer approaches to the penis using
adenoviral vectors have successfully accomplished
sufficient transduction to enable gene expression
and functional activity. Recent reports have shown
that in vivo gene transfer with adenoviral vectors
encoding eNOS and nNOS can reverse age-related
ED in experimental animal models.
17–20
Addition-
ally, overexpression of eNOS in the aged penis
results in an increase in both cavernosal NOS
activity and cGMP formation.
17,18,21
In the present
study, we used the streptozotocin (STZ)-diabetic rat
model that represents a model of type I DM. In this
diabetic rat model, the penis exhibits both angio-
pathic vascular changes as well as neuropathic
changes. These biochemical alterations are similar
to molecular changes that occur in the human
diabetic penis.
8–11
Therefore, the aims of our study
were to determine whether sildenafil or eNOS gene
therapy could affect erectile dysfunction in STZ-
induced diabetic rats. Secondly, we sought to
determine whether a combination of eNOS gene
therapy and sildenafil could have a synergistic effect
on erectile function in the STZ-diabetic rat.
Materials and methods
Development of diabetes
Adult male CD rats (Harlan Sprague-Dawley, San
Diego, CA, USA) were divided into four groups: (1)
age-matched control rats receiving intraperitoneal
(i.p.) injection of citrate buffer (100 mM citric acid,
200 mM disodium phosphate, pH 7.0), (2) rats
receiving i.p. injection of STZ (Sigma Chemical
Company, St Louis, MO, USA) in a dose of 60 mg/kg,
(3) rats receiving i.p. injection of STZ (60 mg/kg) and
subsequently transfected with AdCMVbgal, and (4)
rats receiving i.p. injection of STZ (60 mg/kg) and
subsequently transfected with AdCMVeNOS. STZ-
diabetic rats (Groups 2 and 4) received one intrave-
nous (i.v.) administration of sildenafil (2 mg/kg i.v.)
10 min after the voltage-dependent erectile response
to cavernosal nerve stimulation (CNS) was deter-
mined 2 months after the induction of diabetes with
STZ. STZ-diabetic rats (Groups 3 and 4 received one
intracavernosal injection of the adenoviruses 2
months after i.p. injection of STZ. The total body
weight and blood glucose levels were determined
before and after i.p. injection of STZ with blood
glucose levels determined with an Accu-check
blood glucose meter (Roche Diagnostics, Indiana-
polis, IN). Animals were considered diabetic if their
blood glucose levels were greater than 200 mg/dl.
The blood glucose levels and total body weight of
the control and STZ-diabetic rats are summarized in
Table 1. These procedures have been described
previously.
22,23
All procedures were performed in
accordance with the NIH regulations and rats
were maintained under controlled temperature and
lighting.
Adenovirus vectors
Two replication-deficient recombinant adenoviruses
encoding nuclear-targeted b-galactosidase (Ad-
CMVbgal) and eNOS (AdCMVeNOS), both driven
by a cytomegalovirus (CMV) promoter, were gener-
ated by standard methods of the University of Iowa
Gene Transfer Vector Core Laboratory, as previously
described.
24
Briefly, bovine eNOS was cloned by
blunt-end ligation into pAdCMV4. The resultant
plasmid and adenovirus backbone sequences re-
stricted of E1 were transfected into HEK 293 cells,
and plaques were isolated and amplified for analysis
of eNOS expression. Recombinant adenoviruses
were triple plaque-purified to assure that viral
Table 1 Weight and blood glucose levels in control and STZ-
diabetic rats
Control rats Diabetic rats
Initial weight (g) 32976 32775
Final weight (g) 454713* 252713*
Initial blood glucose (mg/dl) 10575 10274
Final blood glucose (mg/dl) 9676 404712*
n
Po0.05 value is significantly different from that obtained at the
start of the study.
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
22
International Journal of Impotence Research
suspensions were free of wild-type virus, and virus
titers were determined by plaque assay on HEK 293
cells. After purification, the virus was suspended in
PBS with 3% sucrose and kept at 701C until use.
AdCMVbgal was used as a control virus in the
present study.
In vivo gene delivery to the corpora cavernosa
At 2 months after the rats developed diabetes, the
STZ-diabetic rats were anesthetized with sodium
pentobarbital (30 mg/kg i.p.) and placed in a supine
position on a thermoregulated surgical table. Using
sterile technique, the penis was exposed. A volume
of 20 ml of AdCMVbgal (1 10
8
parts/ml) or Ad-
CMVeNOS (1 10
8
parts/ml) was injected into the
corpus cavernosum with a 30-gauge needle attached
to a microliter syringe, as previously de-
scribed.
17,18,25
Rats did not show any overt signs of
systemic (fever, dyspnea, tachycardia) or local
(purulent discharge, erythema, edema) infection
when observed any day after transfection.
Measurement of cavernosal tissue cGMP levels
Cavernosal cGMP levels were measured in control
and STZ-diabetic rats 1–2 days after instillation of
AdCMVbgal and AdCMVeNOS into the corpus
cavernosum. Corpus cavernosal tissue was rinsed
with PBS, quick frozen in liquid nitrogen, and
stored at 701C until determination of cGMP levels.
The samples were assayed for cGMP using an
enzyme immunoassay kit (Cayman Chemical, Ann
Arbor, MI, USA), as previously described.
18
Caver-
nosal cGMP levels are expressed as picomoles per
milligram of protein.
Measurement of erectile responses
Control, STZ-diabetic, and STZ-diabetic rats 1–2
days after adenovirus administration were anesthe-
tized with thiopentobarbital (100 mg/kg i.p.) and
placed on a thermoregulated surgical table. The
trachea was cannulated (PE-240 polyethylene tub-
ing) to maintain a patent airway, and the animals
breathed room air enriched with 95% O
2
/5% CO
2
.
A carotid artery was cannulated (PE-50 tubing) for
the measurement of mean systemic arterial pressure
(MAP). Systemic arterial pressure was measured
continuously with a Viggo-Spectramed transducer
(Viggo Spectramed, Oxnard, CA, USA), which was
attached to a data acquisition system (Biopac
Systems, Santa Barbara, CA, USA), and connected
to a computer simultaneously recording MAP. The
left jugular vein was cannulated (PE-50 tubing) for
the administration of fluids. The shaft of the penis
was freed of skin and fascia, and by removing part of
the overlying ischiocavernous muscle exposure of
the right crus was performed. A 25-gauge needle
filled with 250 U/ml of heparin and connected to
PE-50 tubing was inserted into the right crura and
connected to a pressure transducer to permit
continuous measurement of intracavernosal pres-
sure (ICP). The bladder and prostate were exposed
through a midline abdominal incision. The right
major pelvic ganglion and cavernosal nerve were
identified posterolateral to the prostate on one side,
and an electrical stimulator with a stainless-steel
bipolar hook was placed around the cavernosal
nerve. MAP and ICP were measured with a pressure
transducer connected to a data acquisition system
(Biopac Systems, Santa Barbara, CA, USA) for
continuous measurement of MAP and ICP pressures.
The cavernosal nerve was stimulated with a square
pulse stimulator (Grass Instruments, Quincy, MA,
USA). Each rat underwent CNS at a frequency of
15 Hz and pulse width of 30 s. The application of
2.5, 5, and 7.5 V was used in the current protocol to
achieve a significant and consistent erectile re-
sponse. The duration of stimulation was 1 min with
rest periods of 2–3 min between subsequent stimu-
lations. The total erectile response or total ICP was
determined by the area under the erectile curve
(AUC; mmHg s) from the beginning of CNS until the
ICP returned to baseline or prestimulation pres-
sures. The ratio between the maximal ICP and MAP
obtained at the peak of erectile response was
calculated to normalize for variations in systemic
blood pressure. These procedures have been de-
scribed previously.
18,23
The Tulane University
School of Medicine Animal Care and Use Commit-
tee has approved all procedures used in the present
study.
Drug preparation
Sildenafil (Stanford Research Institute, Menlo Park,
CA, USA) was dissolved in 0.9% saline with
sonication and stored at 201C in brown amber
bottles.
Statistics
All hemodynamic data are expressed as mean7
s.e.m. and were analyzed using a one-way analysis
of variance (ANOVA) with repeated measures and
Neumann-Keuls post hoc test for multiple group
comparisons (Statview, Abacus Concepts, Inc., Ber-
keley, CA, USA). A P-value of less than 0.05 was
used as the criterion for statistical significance.
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
23
International Journal of Impotence Research
Results
Effect of diabetes on the erectile response
At 2 months after the induction of diabetes, there
was a significant decrease (Po0.05) in erectile
function in STZ-diabetic rats (n¼10; Figure 1). We
elicited penile erection in control and STZ-diabetic
rats by stimulating the cavernosal nerve for 1 min.
The magnitude of the increase in ICP after caverno-
sal nerve stimulation in the STZ-diabetic rats was
significantly lower (Po0.05) than the age-matched
control animals at all levels of stimulation (Figure 1).
The total ICP (AUC) of penile erection, as measured
from the start of CNS until the ICP returned to
baseline levels, was significantly lower (Po0.05) in
STZ-diabetic rats at all voltage settings (Figure 1).
Influence of sildenafil on erectile responses in
diabetes
The effects of sildenafil on erectile responses to CNS
were investigated in STZ-diabetic rats and these
data are summarized in Figures 2 and 3. At 2 months
after the induction of diabetes with streptozotocin
(60 mg/kg i.p.), there was a significantly lower
(Po0.05) voltage-dependent cavernosal nerve-in-
Figure 1 Bar graph depicting the voltage-dependent erectile
response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for
1 min in age-matched control and STZ (60 mg/kg i.p.) diabetic
rats. nindicates the number of experiments; *Po0.05, response
significantly different from control rats.
Figure 2 Bar graph depicting the voltage-dependent erectile
response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for
1 min in control rats and before and after administration of the
type 5 cGMP-specific PDE inhibitor sildenafil in a dose of 2 mg/kg
i.v. in STZ-diabetic rats. nindicates the number of experiments;
*Po0.05, response significantly different from control rats;
**Po0.05, response significantly different from STZ-diabetic
rats.
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
24
International Journal of Impotence Research
duced erectile response (ICP/MAP and total ICP) in
STZ-diabetic animals when compared to the age-
matched control rats (n¼9; Figure 2).
After voltage-dependent (2.5, 5 and 7.5 V) erectile
responses were determined, sildenafil was adminis-
tered intravenously at a dose of 2 mg/kg. Intravenous
administration of sildenafil (2 mg/kg) resulted in a
significant decrease (Po0.05) in MAP to
7276 mmHg from a baseline level of 8877 mmHg
in the STZ-diabetic rats. The decrease in systemic
arterial pressure in response to sildenafil was rapid
in onset, with the MAP returning to preinjection
baseline within 15–20 min. At 10 min after the
administration of sildenafil, the magnitude of the
increased ICP/MAP in response to cavernosal nerve
stimulation was increased significantly (Po0.05) at
the 5 and 7.5 V settings (Figure 2). The total ICP
(AUC) was significantly increased at all voltage
settings (Figure 2). However, the erectile responses
after sildenafil treatment were still significantly
lower than the erectile responses in the control rats
(Figure 2). A representative intracavernosal tracing
after stimulation of the cavernosal nerve at 5 V for
1 min in control (a), STZ-diabetic (b), and STZ-
diabetic rats after the administration of sildenafil (c;
2 mg/kg i.v.) is depicted in Figure 3. Age-matched
control animals had a rapid increase in ICP after
cavernosal nerve stimulation that reached a plateau
within 5–10 s, while the STZ-diabetic rats stimula-
tion curve had a significantly lower erectile re-
sponse with regard to the duration of the curve that
was shorter (Figure 3). After the administration of
sildenafil (2 mg/kg i.v.), STZ-diabetic rats’ stimula-
tion curve approached that of the control animals
and the duration of the erectile response was longer
than the STZ-diabetic rats (Figure 3).
Influence of eNOS gene therapy and sildenafil
on erectile responses in diabetes
The effect of overexpression of eNOS via adenoviral
gene transfer of eNOS to the corpus cavernosum of
STZ-diabetic rats in addition to the i.v. administra-
tion of the type 5 cGMP-specific PDE inhibitor
sildenafil was investigated 2 months after induction
of diabetes with streptozotocin (60 mg/kg i.p.). At
1–2 days after transfection with AdCMVbgal or
AdCMVeNOS, STZ-diabetic rats’ erectile responses
to CNS were investigated and these data are
summarized in Figures 4 and 5. There was a
significantly lower (Po0.05) voltage-dependent ca-
vernosal nerve-induced erectile response (ICP/MAP
and total ICP) in STZ-diabetic rats transfected with
AdCMVbgal when compared to the control rats
(n¼9; Figure 4). The magnitude of the increase in
ICP (ICP/MAP) in response to CNS in the STZ-
diabetic rats transfected with AdCMVbgal was
significantly lower (Po0.05) than the control rats,
whereas rats transfected with AdCMVeNOS had a
greater response to CNS that was similar to the
response obtained in the control rats (Figure 4).
However, the total ICP in the AdCMVeNOS-trans-
fected STZ-diabetic rats was still significantly lower
than the control rats. There was no statistical
difference between STZ-diabetic rats and STZ-
diabetic rats transfected with AdCMVbgal erectile
responses (data not shown).
After voltage-dependent (2.5, 5, and 7.5 V) erectile
responses were determined in the STZ-diabetic rats
transfected with AdCMVeNOS, sildenafil was ad-
ministered intravenously at a dose of 2 mg/kg.
Intravenous administration of sildenafil (2 mg/kg)
resulted in a significant decrease (Po0.05) in MAP
to 6774 mmHg from a baseline level of 8375 mmHg
in the AdCMVeNOS-transfected STZ-diabetic rats.
After the administration of sildenafil, there was no
change in the magnitude of the increase ICP/MAP in
response to CNS at all voltage settings (Figure 4).
However, the total ICP (AUC) was significantly
increased at the 5 and 7.5 V settings (Figure 4).
A representative intracavernosal tracing after
stimulation of the cavernosal nerve at 5 V for
1 min in control (a), STZ-diabetic rats transfected
with AdCMVbgal (b), and STZ-diabetic rats trans-
fected with AdCMVeNOS before (c) and after (d)
Figure 3 Representative ICP tracing after CNS at the 5 V setting
for 1 min in (a) control rats, (b) STZ-diabetic rats, and (c) STZ-
diabetic rats treated with sildenafil (2 mg/kg i.v.).
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
25
International Journal of Impotence Research
administration of sildenafil (2 mg/kg i.v.) is depicted
in Figure 5. STZ-diabetic rats transfected with
AdCMVeNOS had an increase in ICP after CNS that
was similar to the responses exhibited in control
rats. However, after the administration of sildenafil
(2 mg/kg i.v.), AdCMVeNOS-transfected STZ-dia-
betic rats’ AUC or total ICP was significantly longer
(Po0.05) than the AdCMVeNOS-transfected STZ-
diabetic rats before treatment with sildenafil
(Figure 5).
Cavernosal cGMP levels
Cavernosal tissue concentrations of cGMP were
measured in control and in STZ-diabetic rats 1–2
days after transfection with AdCMVbgal and Ad-
CMVeNOS, and these data are summarized in
Figure 6. Cavernosal cGMP levels were significantly
lower (Po0.05) in the STZ-diabetic rats when
compared to control rats (n¼4; Figure 6). Gene
transfer of eNOS to the diabetic penis resulted in
cavernosal cGMP concentrations that were signifi-
cantly higher (Po0.05) when compared to STZ-
diabetic rats transfected with AdCMVbgal (n¼4;
Figure 6). Cavernosal cGMP levels were similar in
STZ-diabetic rat cavernosal tissue and STZ-diabetic
rat corpus cavernosum transfected with AdCMVbgal
(data not shown).
Figure 4 Bar graph depicting the voltage-dependent erectile
response (ICP/MAP) and total ICP (AUC; mmHg s) after CNS for
1 min in control rats, STZ-diabetic rats transfected with Ad-
CMVbgal, and AdCMVeNOS before and after administration of
the type 5 cGMP-specific PDE inhibitor sildenafil at a dose of
2 mg/kg i.v. In vivo erection experiments were conducted 1–2
days after transfection with adenoviruses. nindicates the number
of experiments; *Po0.05 response significantly different com-
pared to control rats; **Po0.05 response significantly different
compared to STZ-diabetic rats transfected with AdCMVbgal; r
indicates Po0.05, response significantly different compared to
STZ-diabetic rats transfected with AdCMVeNOS.
Figure 5 Representative ICP tracing after CNS at the 5 V setting
for 1 min in (a) control rats, (b) STZ-diabetic rats transfected with
AdCMVbgal, (c) STZ-diabetic rats transfected with AdCMVeNOS,
and (d) STZ-diabetic rats transfected with AdCMVeNOS and
treated with sildenafil (2 mg/kg i.v.).
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
26
International Journal of Impotence Research
Discussion
The results of the present study demonstrate, for the
first time, that sildenafil increases both the peak ICP
and total ICP erectile responses in STZ-diabetic rats
in vivo. Additionally, this study demonstrates that
adenoviral-mediated gene transfer of eNOS to the
STZ-diabetic rat penis increases erectile responses
in vivo as a result of an increase in cGMP formation.
Moreover, the combination of eNOS gene therapy
and sildenafil in STZ-diabetic rats resulted in a
synergistic erectile response that was greater than
either of these therapies alone.
Cavernosal cellular responses are regulated by
cyclic 30,50-adenosine monophosphate (cAMP) and
cGMP. The cellular levels of cAMP and cGMP are
determined by the relative synthetic activities of
adenylate and guanylate cyclase and the degradative
activities of the cyclic nucleotide PDEs in caverno-
sal tissue.
26,27
PDE enzymes are responsible for the
hydrolysis of cAMP and cGMP, which in their active
state induce smooth muscle relaxation. PDE inhibi-
tors increase intracellular levels of cAMP and cGMP
by hindering the hydrolytic activity of PDE, thus
maintaining the vasodilator activity of cAMP and
cGMP. Sildenafil, a selective type 5 PDE inhibitor,
inhibits the hydrolysis of cGMP in the corpus
cavernosum, thereby increasing cavernosal smooth
muscle relaxation and prolonging penile erection.
28
Sildenafil has been shown to enhance NO-mediated
corpus cavernosum smooth muscle relaxation and
penile erection both in vitro and in vivo.
29–32
The
effect of sildenafil on normal erectile responses to
CNS has been previously studied in the penile
vasculature of the rat and, in that study, sildenafil
was found to prolong the decay period of erections
but did not increase the amplitude.
33
In the present
study, we found in the STZ-diabetic rats both an
increase in the total ICP or decay period of erections
as well as an increase in the amplitude (ICP/MAP) of
the erectile response to CNS, suggesting that
sildenafil therapy improves both erectile parameters
in STZ-diabetic rats.
The process of penile erection is contingent on an
increase in arterial inflow and restricted venous
outflow from the penis, coordinated by relaxation of
the penile corpus cavernosum. The principal med-
iator responsible for the relaxation of the corpus
cavernosum is NO, which is produced in the
endothelium and NANC nerves by eNOS and nNOS,
respectively. Manifestation of ED may result from a
number of vascular, neurological, and hormonal
complications. The prevalence of ED in diabetic
patients has been reported to be as high as 50–75%.
1
Since NO plays a dominant role in erectile physiol-
ogy, most studies propose that diabetic-related ED is
a result of disordered endothelial smooth muscle
relaxation and NANC-related neuronal defects in
the corpus cavernosum of the penis.
8,23
Recently,
cGMP formation in the corpus cavernosum of
diabetic rabbits was found to be significantly
reduced.
34
This finding is consistent with the
reduction in cGMP levels found in the STZ-diabetic
rats 2 months after induction of diabetes in the
present study.
Sildenafil is an effective and well-tolerated treat-
ment for ED in men with diabetes.
15,35,36
However,
sildenafil does not have the same efficacy in
improving erectile function as measured by the
International Index of Erectile Function in diabetic
patients when compared to other ED etiologies.
16
Sildenafil requires at least partial function of NANC
penile nerves to be effective, and, therefore, men
who have DM or have undergone non-nerve-sparing
radical prostatectomy are less likely to respond.
37
Recently, sildenafil significantly enhanced sodium
nitroprusside- and electrical field stimulation-
mediated corpus cavernosum smooth muscle relaxa-
tion in rabbit diabetic corpus cavernosum in vitro,
suggesting that sildenafil can have beneficial effects
on impaired diabetic cavernosal smooth muscle
relaxation.
34
In the present study, we examined the effect of
sildenafil on erectile function in STZ-diabetic rats in
vivo. STZ-diabetic rats had a significant reduction in
erectile function 2 months after i.p. injection of STZ.
After a voltage-dependent (2.5, 5, and 7.5 V) erectile
response was determined, sildenafil was adminis-
tered at a dose of 2 mg/kg intravenously and the
voltage series was repeated. Sildenafil increased the
peak ICP at the 5 and 7.5 V settings and the total ICP
at all voltage settings studied in the STZ-diabetic rat.
However, this increase was still statistically differ-
ent from the age-matched control rat’s erectile
Figure 6 Bar graph demonstrating cGMP levels in cavernosal
tissue of control and STZ-diabetic rats transfected with Ad-
CMVbgal or AdCMVeNOS. nindicates the number of experi-
ments; *Po0.05 indicates that cGMP levels are significantly
different when compared to control rats. **Po0.05 indicates that
cGMP levels are significantly different when compared to STZ-
diabetic rats transfected with AdCMVbgal.
eNOS gene therapy and sildenafil in diabetes
TJ Bivalacqua et al
27
International Journal of Impotence Research
response. Our findings demonstrate that inhibition
of the type 5 PDE enzymes can partially normalize
the diminished erectile response in the STZ-diabetic
rat and suggests that this pharmacological drug can
partially compensate for the diabetes-induced re-
duction in cavernosal cGMP. To our knowledge, this
is the first report demonstrating that sildenafil can
improve diabetic erectile dysfunction in vivo.
Alterations in the NO/cGMP system are also
present in the natural aging process and contribute
to age-associated alterations in erectile function.
Several groups have demonstrated that adenoviral
gene therapy can be used to restore NO production
in the rat corpus cavernosum to improve erectile
function.
18–20,38
Recently, adenovirus-mediated
gene transfer of eNOS to diabetic rabbit aorta
augmented vascular reactivity and partially restored
impaired smooth muscle relaxation.
39
Therefore, a
gene transfer approach can be used to restore
impaired cavernosal smooth muscle relaxation seen
in diabetic corpus cavernosum.
In this study, we have successfully transferred the
eNOS gene to STZ-diabetic rat penises, which
resulted in an increase in cGMP formation in the
corpus cavernosum. This increase in cGMP forma-
tion after adenoviral-mediated gene transfer of
eNOS resulted in an increase in ICP/MAP and total
ICP after CNS in STZ-diabetic rats to values that
were similar to the control animals. However, the
total ICP of erectile response in STZ-diabetic rats
transfected with AdCMVeNOS was still lower than
control animals. We next wanted to determine if the
administration of sildenafil to eNOS-transfected
STZ-diabetic rats could increase the total ICP. In
theory, this combination therapy should prove
beneficial because eNOS-transfected STZ-diabetic
rats have an increase in cavernosal cGMP, the
second messenger molecule sildenafil selectively
inhibits the catabolism in cavernosal smooth muscle
cells. Consequently, this combination should result
in an increase in the total duration of erectile
response. After the administration of sildenafil to
eNOS-transfected STZ-diabetic rats, the total ICP
was significantly greater than the total ICP before
treatment at the 5 and 7.5 V settings. Thus, these
data suggest that when cGMP is overexpressed by
eNOS gene therapy in cavernosal tissue and an
inhibitor of the PDE 5 enzyme is present in the
tissue, penile erectile response is significantly
enhanced due to a synergistic effect of this combina-
tion pharmacological and gene therapy.
In the present study, we have used an adenoviral
vector, which has a transduction efficiency of a
particular gene in the corpus cavernosum of the rat
for approximately 7–10 days.
38
This adenovirus has
a peak expression of the eNOS gene at 1–2 days;
therefore, we used this time point to determine all
biochemical and physiological changes in the STZ-
diabetic rat. Gene therapy trials using adenoviral
vectors have not been conducted for the treatment of
ED. However, the use of similar viral vectors such as
‘gutless’ adenoviral and adeno-associated viral
vectors, as well as ex vivo expanded marrow stromal
cells (stem cells) that have the potential to differ-
entiate into endothelial and smooth muscle cells,
are the focus of future experiments. Development of
better vectors to deliver specific genes to the penis,
which cause no local immune response and longer
durations of expression, will be the selected vectors
used in the application of gene therapy for the
treatment of ED.
Conclusion
In summary, the results of the present study
demonstrate for the first time that sildenafil can
improve erectile responses in STZ-diabetic rats in
vivo. Moreover, the combination of eNOS gene
therapy and sildenafil enhanced the total ICP
response to CNS in STZ-diabetic rats to a value
greater than either therapy alone. Therefore, it is
reasonable to conclude that using oral PDE 5
inhibitors in combination with eNOS gene therapy
could represent an exciting new form of therapy for
the treatment of diabetic ED.
Acknowledgements
We thank Drs Donald D Heistad and Beverley
Davidson and the University of Iowa Vector Core
Laboratory for preparation of the virus. This work
was supported in part by a Young Investigator
Award from the International Society of Impotence
Research and Pfizer Inc. and the American Medical
Association to TJ Bivalacqua.
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