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From the * Boston University School of Medicine
and the Veterans Affairs Boston Healthcare
System, Boston, Massachusetts.
| Correspondence to: Dr Kazem Azadzoi, Urology Research (151), Boston VA Medical Center, 150 S Huntington Ave, Boston, MA 02130 (e-mail: kazadzoi{at}bu.edu). |
| Received for publication October 1, 2003; accepted for publication December 19, 2003. |
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Abstract |
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Key words: Erectile dysfunction, atherosclerosis, blood flow, oxygen tension
A close relationship between cardiovascular disease and erectile dysfunction (ED) has been reported (Kloner et al, 2003; Solomon et al, 2003). The authors found that nearly 75% of men with coronary artery disease also suffer from ED. Vascular and cavernosal smooth muscle dysfunction appear to share the same risk factors, such as smoking, hypercholesterolemia, atherosclerosis, and hypertension (Saenz de Tejada et al, 1989; Grein and Schubert, 2002). Additionally, ED may be an early signal of impending coronary artery disease. Arterial occlusive disease is recognized as the most common cause of organic ED (Krane et al, 1989). Damage to the pudendal-cavernous-helicine arterial tree can be caused by either atherosclerosis or trauma to the pelvic region (Levine et al, 1990; Grein and Schubert, 2002). This results in a decrease in blood flow to the corpus cavernosum and an inability to achieve or maintain an erection.
Nitric oxide (NO) is a key modulator of cavernosal smooth muscle relaxation and vasodilation as well as the resulting penile erection (Ignarro et al, 1990; Kim et al, 1991). Released by cavernous nerves and the endothelium, NO activates guanylate cyclase and catalyzes the formation of cyclic guanosine-3',5'-monophosphate (cGMP) from guanosine-5'-triphosphate. The increased levels of cGMP initiate a cascade of intracellular events, which, in turn, lead to smooth muscle relaxation (Ignarro et al, 1990; Kim et al, 1991). NO is derived from L-arginine and molecular oxygen, a reaction that is catalyzed by NO synthase (NOS). NOS exists in Ca2+-dependent constitutive neuronal (nNOS) and endothelial (eNOS) forms, and a Ca2+-independent inducible (iNOS) form. Basal production of NO is regulated by constitutive NOS (cNOS) and contributes to the physiology of cardiac and pulmonary perfusion, heart rate, myocardial contractility, vasodilation, and penile erection (Bredt and Snyder, 1994). NO generated by iNOS, however, is involved in pathophysiologic states such as oxidative stress and myocardial dysfunction (Wildhirt et al, 1997).
Previously, we reported that chronic cavernosal ischemia down-regulates cavernosal nNOS gene expression and impairs NO synthesis (Wang et al, in press). The goal of this study was to examine the effect of chronic ischemia on cavernosal nNOS, eNOS, and iNOS distribution and protein expression in relation to smooth muscle relaxation in a rabbit model of arteriogenic ED.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Measuring Intracavernosal Blood Flow and PO2
Systemic arterial pressure was measured with an angiocatheter positioned in
the auricular artery. Three methods of measurements were used to determine
arterial inflow to the penis: 1) iliac artery blood flow was measured with
perivascular flow sensors placed around the arteries and connected to an
ultrasonic flow meter; 2) intracavernosal blood flow was measured with a
23-gauge needle containing a laser Doppler flow probe inserted
intracavernosally and connected to a laser Doppler flow meter (Transonic
Systems Inc, Ithaca, NY); and 3) intracavernosal PO2 was
measured with a polarographic oxygen-sensing electrode that was positioned
within a 20-gauge needle inserted directly into the penile erectile tissue and
connected to a chemical microsensor (Diamond General, Ann Arbor, Mich).
Examining Erectile Function
Penile erection was examined by electrical stimulation (10 V, 8
milliseconds, 16 Hz for 30 seconds) of the cavernosal branch of the pelvic
nerve. Intracavernosal pressure was measured with a 23-gauge minicatheter
placed into the corpus cavernosum and connected to a transducer. Simultaneous
recordings of systemic blood pressure, iliac arterial blood flow,
intracavernosal blood flow, and intracavernosal PO2 were
obtained with an 8-channel recorder (Astro-Med, Inc, Warwick, RI). Animals
were then sacrificed, and cavernosal tissues were processed for organ bath,
Western blotting, and immunohistochemical studies.
Measuring Isometric Tension
Organ bath studies were performed as previously described
(Azadzoi et al, 1998). Corpus
cavernosum tissues were submerged in 25-mL organ chambers containing
physiologic solution at 37°C, pH 7.4. The solution was gassed with 95% air
and 5% CO2. The tissue was stretched incrementally until optimal
isometric tension was achieved. Tissue tension was measured with a force
transducer connected to an amplifier (Grass FT03, Quincy, Mass). Tissue
relaxation to electrical field stimulation was studied after contraction with
phenylephrine (2000 nM).
Western Blotting
Tissues were pulverized on dry ice and then homogenized in PIPES
(piperazine-1,4-bis(2-ethane) sulfonic acid) buffer (20 mM PIPES, 1 mM EDTA, 1
mM EGTA [ethyleneglycoltetraacetic acid], and 0.25 M sucrose, pH 7.4). The
homogenized mixture was then centrifuged, and the supernatant was collected.
After discarding the pellet, the protein concentration in the supernatant was
determined by the Lowry method (Bio-Rad, Richmond, Calif). Protein extracts
were diluted with buffer to ensure an equal amount of protein for each sample.
Equal amounts of protein were then mixed with the sodium dodecyl sulfate (SDS)
sample buffer and loaded into a 7.5% SDS-PAGE (polyacrylamide gel
electrophoresis) gel. The gel underwent electrophoresis for 16 hours at 15 mA.
The proteins on the gel were then electrotransferred onto nitrocellulose
membranes that were blocked with 5% nonfat milk in Buffer TBST (Tris-buffered
saline containing 0.05 Tween 20, pH 8.0). They were then incubated with the
primary antibody for 1 hour at 25°C with gentle shaking. After 3 washes,
the blots were incubated with the secondary antibody conjugated to alkaline
phosphatase for 1 hour at 25°C, washed 3 times, and incubated with the
substrate-color development reagents. The computer-stored images of the gels
were analyzed by densitometry.
Immunohistochemistry
Cross sections of penile tissue were fixed in formalin (10%) and then
processed for paraffin embedding. Five-micrometer cross sections were adhered
to glass slides and were then hydrated, incubated in
H2O2 for 5 minutes, and washed with deionized
H2O, followed by phosphate-buffered saline (PBS). Slides were
treated with 0.1% Triton X-100 for 20 minutes, washed in PBS for 5 minutes,
and then incubated with primary monoclonal antibody (anti-nNOS, eNOS, or
iNOS)at a dilution of 1:100 for 60 minutes. Then, samples were incubated with
biotinylated anti-mouse immunoglobulin G (IgG) secondary antibody, followed by
peroxidase-conjugated streptavidin at room temperature. Antigen visualization
was accomplished using diaminobenzidine substrate/chromagen. The counterstain
used was Harris hematoxylin. In the negative controls, 0.05 M Tris buffer at
pH 7.6 was used in an equivalent volume to replace the primary antibody. An
H2O2/methanol solution was used to block endogenous
peroxides.
Data Recording and Statistical Analysis
In vivo data were recorded on an 8-channel heat-writing physiologic
recorder (Astromed). Isometric tension was recorded by an 8-channel Grass 7D
Polygraph (Grass Division, Astromed). Data are expressed as the mean plus or
minus the standard error of the mean. Statistically significant differences in
treated groups that were compared with control groups were assessed by an
analysis of variance or an unpaired Student's t test, when
applicable, at the 95% confidence
level.
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Results |
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Changes in Erectile Response
At week 4, nerve-stimulated erection in the treated group was similar to
that of the age-matched control group. Significant impairment of erectile
function was evident 8 and 16 weeks after the induction of arterial occlusive
disease. This was characterized by a significant decrease in intracavernosal
pressure in response to electrical nerve stimulation (Table). Impairment of
erectile function at 8 and 16 weeks occurred simultaneously with the decrease
in intracavernosal blood flow and PO2 (Table).
Changes in Smooth Muscle Relaxation
No change in smooth muscle relaxation was noted among the control groups at
4, 8, and 16 weeks (Figure 1).
In the treated group, smooth muscle relaxation 4 weeks after the induction of
cavernosal ischemia was similar to that in the control group. At 8 and 16
weeks, however, a significant decrease in smooth muscle relaxation was noted
when compared with the age-matched controls
(Figure 1).
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Changes in NOS Protein Levels
Western blotting showed no significant difference in the expression of eNOS
or nNOS protein in the control groups between weeks 4, 8, and 16. In the
treated group, nNOS and eNOS protein levels at week 4 were similar to those in
the control group. At 8 and 16 weeks, nNOS and eNOS proteins significantly
decreased in comparison to controls (Figure
2). In contrast to nNOS and eNOS, the iNOS protein level
progressively increased between weeks 4 and 16 in the treated group
(Figure 2).
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Changes in NOS Expression
Immunohistochemistry of control penile tissue showed dense eNOS staining on
the lacunar spaces of the endothelium and nNOS-positive stains along penile
cavernosal and dorsal nerves (Figure
3). Sporadic iNOS was evident throughout the erectile tissue.
After the induction of cavernosal ischemia, there was no significant change in
the expression of either eNOS or nNOS at 4 weeks. However, a dramatic decrease
in both was observed 8 and 16 weeks after the induction of cavernosal
ischemia. Alternatively, iNOS expression seemed to increase during the course
of ischemia between weeks 4 and 16 (Figure
3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In our model, the induced iliac arterial injury results in diffuse atherosclerotic occlusive disease and a significant decrease in both iliac arterial and intracavernosal blood flow as well as intracavernosal PO2 (Azadzoi and Goldstein, 1992). The development of cavernosal ischemia and hypoxia took place in a progressive manner during several weeks' time, with the most dramatic changes occurring between 4 and 16 weeks after arterial injury. This implies that the model did in fact create a state of chronic ischemia and hypoxia and progressive impairment of erectile tissue function.
In the early stages of arteriogenic ED, the reduced erectile capacity may be solely a hemodynamic phenomenon resulting from reduced intracavernosal blood inflow (Wang et al, in press). In the later stages, however, the erectile tissue appears to have lost its capability to generate the amount of NO sufficient for smooth muscle relaxation. In a previous study, we found that the ischemic erectile tissue lacks sufficient NO synthesis but is capable of relaxing to NO donors such as sodium nitroprusside (Azadzoi et al, 1998). This suggests that the impairment of smooth muscle relaxation relates, to a greater extent, to ischemic functional alterations than to ischemic structural damage.
The mechanism of ischemic erectile tissue dysfunction may involve multiple mechanisms, including chronic lack of nutrients, chronic exposure to hypoxia, and lack of metabolic waste clearance. These conditions are likely either to directly interfere with NO production or to inactivate NO function due to cytotoxicity. Another possibility may be elevated levels of NO inhibitors or antagonists in the chronically ischemic tissue. In addition to impairing smooth muscle relaxation, ischemia has been shown to increase erectile tissue contraction (Azadzoi et al, 1999). The mechanism of ischemia-induced increased cavernosal smooth muscle contraction seems to involve an increased output of constrictor eicosanoids (Azadzoi et al, 1999). Another study suggests that an accumulation of endogenous NOS inhibitors in the corpus cavernosum is involved in ischemic functional damage (Masuda et al, 2002).
Hypoxia has profound effects on erectile tissue structure and function (Aasebo et al, 1993). The mechanism involves, among other factors, an impairment of NO synthesis and an increased production of growth factors and eicosanoids (Kourembanas et al, 1997). In both rabbit and human cavernosal tissue, hypoxia significantly diminishes NO-mediated neurogenic and endothelial-mediated relaxation (Kim et al, 1993). The authors reported that human corpus cavernosum smooth muscle cells, when exposed to PO2 values of 100 mm Hg, significantly increase prostaglandin E2 synthesis. These data support a role of oxygen in regulating and augmenting smooth muscle relaxation via prostaglandin E2 synthesis. It has been proposed that physiologic levels of oxygen modulate penile erection by regulating NO synthesis in erectile tissue (Kim et al, 1993). A role for hypoxia in cavernosal fibrosis via transforming growth factor beta has also been proposed (Moreland et al, 1995).
In our model, functional changes in the ischemic erectile tissue coincide with dramatic changes in the expression of NOS isoforms. Previously, we found that ischemia caused an initial increase in nNOS gene expression between 4 and 6 weeks after arterial injury in the rabbit (Wang et al, in press). This was followed by a marked decrease in nNOS expression at weeks 8 and 16. Western blotting data in the present study showed that the nNOS protein was unchanged at week 4, suggesting that, under the ischemic conditions, increased nNOS transcript levels are not capable of stimulating nNOS protein synthesis. One possible explanation is that, during the early stages of ischemia, although nNOS gene expression is up-regulated, a feasible environment for stimulating nNOS protein expression is lacking. During the later stages of ischemia (8 and 16 weeks), however, decreased transcript levels appear to correlate with reduced nNOS protein levels. Western blotting showed that both nNOS and eNOS protein levels dramatically decreased 8 and 16 weeks after the induction of cavernosal ischemia. In previous studies, we found that, despite moderate fibrosis, no apparent differences existed in the number of cavernosal endothelial cells between the control and the chronically ischemic erectile tissues (Wang et al, in press). This supports the hypothesis that a reduced eNOS level results from ischemic down-regulation of its gene and protein and not from ischemic endothelial atrophy.
Immunohistochemical staining showed that chronic ischemia led to disproportionate changes in NOS expression, favoring iNOS over nNOS and eNOS. It appears that as nNOS and eNOS expression decreases during the course of cavernosal ischemia, iNOS expression increases. Our data suggest that while cNOS is more abundant in healthy erectile tissue, iNOS dominates the erectile tissue under ischemic/hypoxic conditions, suggesting its pathophysiologic role in ED. The precise mechanism by which ischemia inhibits the cNOS while up-regulating the inducible form is not known. Studies of other organs have shown that iNOS is induced in response to a variety of cytokines, including hypoxia inducible factor 1 (HIF-1), interleukins IL1-B and IL-6, and tumor necrosis factor alpha (Kinugawa et al, 1994; Oddis et al, 1994). Further, Palmer et al (1998) and Jung et al (2000) have shown that the vascular cells of mice release IL-6 in response to hypoxia and thereby up-regulate iNOS expression. They have also reported that hypoxia activates HIF-1, which binds the promoter region of the iNOS gene and stimulates its expression. Additionally, increased iNOS levels in vascular tissues may down-regulate eNOS and thus inhibit NO production and smooth muscle relaxation. It has been shown that inhibition of iNOS expression augments smooth muscle relaxation in proinflammatory mediator-induced blood vessels.
In diabetic patients, Seftel et al (1997) have shown that inhibition of iNOS significantly increases relaxation of erectile tissue. The authors found that increased iNOS levels in diabetic erectile tissue were associated with marked down-regulation of eNOS. It is speculated that such a disproportionate change in iNOS and eNOS expression plays a role in the pathophysiology of diabetic ED. Another study showed that aging is accompanied by increased iNOS expression in rat erectile tissue (Ferrini et al, 2001). This is believed to play a role in collagen accumulation and apoptosis of erectile tissue. Studies with a rat model of Peyronie disease showed that up-regulation of iNOS was accompanied by down-regulation of cNOS and ED (Bivalacqua et al, 2000). The authors showed that the inhibition of iNOS reduced ED in this model.
Our studies with the rabbit model suggest that the cellular and molecular reactions of erectile tissue to early-stage arterial disease are different from those seen after prolonged arterial occlusive disease (Wang et al, in press). The early stage of arteriogenic ED appears to involve a solely vasculopathic mechanism, involving reduced inflow and perfusion pressure. In the later stages of arterial disease, however, the mechanism of ED appears to be more complex, involving arterial insufficiency, hemodynamic impairment, and functional disability of erectile tissue. The down-regulation of cNOS, along with the up-regulation of iNOS that is seen at the late stages of arteriogenic ED, may play an important role in ischemic erectile smooth muscle dysfunction. Ischemically altered NOS isoforms may be of great importance in the pathophysiology of arteriogenic ED.
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Footnotes |
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References |
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