2. The Endothelial–Erectile Dysfunction Connection 2391
the complex mechanism of erection, exten- (EDHFs) [12]. These results in engorgement of
sive research has established an irrefutable link the sinusoidal spaces, increase in intracavernous
between impaired endothelial function and alter- pressure, lengthening and enlargement of the
ations in normal erectile capability [4–6]. This penis, and compression of the subtunical venules,
article will review basic and clinical evidence of allowing the complete occlusion of penile venous
impaired endothelial biological activities in erec- outflow (veno-occlusion) and trapping of blood
tile pathophysiology and the existing noninvasive within the corpus cavernosum (CC). Given that
tests that allow endothelial function assessment in penile endothelial health is essential for normal
erectile dysfunction (ED). erectile capability, any disruption on corporeal
ECs biological actions may affect the arterial
and/or veno-occlusive mechanisms, compromising
Vascular Endothelium and Penile Erection—A Brief the initiation and/or maintenance of an erection
Overview of the Nitric Oxide/Cyclic Guanosine
[13].
Monophosphate (NO/cGMP) and Adenylate
Cyclase/Cyclic Adenosine Monophosphate
(AC/cAMP) Signaling Mechanisms Penile Endothelial Cell Function and Dysfunction
The NO/cGMP and AC/cAMP Dependent Pathways Endothelium Physiology and Function
The penile circulation is composed of feeder arte- Vascular endothelium is a thin monocellular layer
rioles and helicine arteries that empty into a that covers all the inner surface of blood vessels,
network of connective tissue surrounding sinusoi- separating circulating blood from the tissues. For-
dal cavities lined by SM and ECs [4]. ECs function merly considered as a simple anatomic passive
dynamically to regulate the tone of the cavernous barrier, the endothelium is currently recognized
vasculature and subendothelial SM layer. During has a highly metabolically active organ that plays
sexual arousal or nocturnal tumescence, there is important autocrine, paracrine, and endocrine
production of neural NO (nNO) through the functions [14]. By the nature of its location, the
action of neuronal NO synthase in cavernosal endothelial monolayer regulates relevant biologi-
nerve terminals [7]. nNO is diffused into adjacent cal events, such as the maintenance of balanced
corporeal SM, binding soluble guanylate cyclase vascular pressure, patency and perfusion, inhibi-
and increasing intracellular levels of cGMP. tion of thrombosis, induction of fibrinolysis, regu-
cGMP interacts with subcellular effector proteins lation of inflammation and platelet aggregation,
such as ion channels and protein kinases, particu- and the behavior of the underlying vascular SM
larly protein kinase G-1, reducing SM calcium [15]. Under physiological conditions, to maintain
sensitivity to contractile proteins, which eventuate vascular homeostasis, the endothelium has the
in SMCs relaxation and in a nNO-mediated inflow capacity to respond to humoral, neuronal, and
of arterial blood [8,9]. Effective SMCs relaxation mechanical (in particular, shear stress) stimuli
may also be induced through the activation of a through the synthesis and release of a variety of
different cyclic nucleotide pathway, bypassing the endothelium-generated agonist and antagonist
nNO route. Different mediators, including neuro- molecules. These factors include procoagulants
peptides such as vasoactive intestinal peptide, and anticoagulants, inflammatory and anti-
calcitonin-related peptide, and prostaglandin E1 inflammatory, fibrinolytics and antifibrinolytics,
(PGE1), may react with SM membrane specific vasoconstrictors and vasodilators [14,16,17].
G-protein-coupled receptors, activating AC, Among those, the equilibrium between vasoactive
which converts adenosine triphosphate, in the mediators, such as eNO, prostacycline I2, and
second messenger cAMP [8,10,11]. Increased endothelium-derived EDHFs, and vasoconstric-
cAMP induces the relaxation of SM fibers through tors, such as endothelin-1, angiotensin II, prostag-
the activation of protein kinase A, resulting in a landin H2, and thromboxane A2, have revealed
decrease in intracellular free calcium, desensitiza- crucial on the regulation of vascular permeability,
tion of contractile mechanisms, allowing SMC inflammation and vascular tone [17,18]. Penile
relaxation and arterial blood inflow [10,11]. This endothelial bed is considered as a specialized
initial inflow of blood increases shear stress and extension of the peripheral vascular system,
stimulates the phosphorylation of phosphoi- responding similarly to diverse stimuli in order to
nositide 3-kinase/endothelial NOS (eNOS) and maintain homeostasis, and playing a particular
eNO production, and the release of prostanoids regulatory role on the modulation of vascular and
and endothelium-derived hyperpolarizing factors SM contractile tone, crucial for normal erectile
J Sex Med 2009;6:2390–2404
3. 2392 Costa and Virag
functionality. Considering the important role of in a synergistic fashion, contributing to the exac-
intact endothelium, any alterations impairing its erbation of penile and generalized vasculopathy
biological activities and disrupting its functional [27]. Considering vasculogenic ED as the “silent
integrity will alter endothelial ability to respond to tip of the iceberg” of a more generalized vascular
local and systemic changes, a condition referred to disorder may be relevant for the prevention of
as endothelial dysfunction [19]. cardiovascular events in patients with asymptom-
atic CAD [21].
Endothelial Dysfunction—Pathophysiological Effects
on Erection
Vascular Risk Factors: Impairing Penile Endothelial
The key feature of endothelial dysfunction is the
and Erectile Function
decreased responsiveness to vasodilator mediators
or the increased sensitivity to vasoconstrictor As reviewed in Figure 1, risk factor-associated cav-
molecules affecting the normal regulatory role of ernosal endothelial alterations are mostly induced
peripheral vascular endothelium, including caver- by unifying mechanisms, including oxidative stress
nosal arterial and venous systems. As consequence, and impaired eNOS/eNO functional activities,
the vasodilator potential is reduced, and vascular resulting in poor endothelium-dependent vascular
structures are unable to fully dilate in response and SM relaxation, presenting clinically as ED, the
to appropriate stimuli. Generally, this decrease particular difficulty in maintaining a firm erection.
in endothelial vasodilation is mostly caused by
a diminished synthesis and/or lost of eNO Diabetes Mellitus
bioavailability/bioactivity in the vasculature [20]. ED is a common complication in men with diabe-
Besides vasodilation, eNO-associated alterations tes, affecting up to 75% of all men with the disease
may also impair a series of relevant mechanisms, [28,29]. In diabetic men, ED occurs at an earlier
including anticoagulation and anti-inflammatory age than the general population, increasing with
activities, vascular growth, and remodeling capa- disease duration [29]. Hyperglycemia contributes
bility [17,20]. Overall, endothelial dysfunction is to metabolic derangements which promote endot-
defined by the pathologic impairment of eNO- helial dysfunction and vascular complications [30].
dependent vasodilatation and late structural vas- High glucose levels induce the formation of irre-
cular abnormalities, a condition considered as versible advanced glycation end products (AGEs),
common grounds of both cardiovascular disease affecting endothelial function by several mecha-
(CVD) and ED. Additionally, it was suggested that nisms, including targeting of penile eNO, genera-
ED may not only be a clinical manifestation of a tion of reactive oxygen species (ROS), and by
pathology affecting the penile circulation, but it affecting the expression of endothelial growth
may be a harbinger and a very early warning sign factors [31,32]. These alterations abrogate mul-
of a more generalized vascular systemic disorder tiple pathways promoting loss of penile endothe-
[21]. The relevance of adequate endothelial func- lium function and ED. AGEs are elevated in
tion in erection was recognized after the identifi- diabetic penile tissue and interfere with eNO pro-
cation of similar vascular risk factors (VRFs) for duction by directly inactivating the phosphoryla-
ED and coronary artery disease (CAD), conditions tion of eNOS [31]. ROS formation and increased
that are highly prevalent and frequently coexist. oxidative stress-associated to AGEs cause CC
Diabetes mellitus, hypertension, hypercholester- alterations, including an augment in lipid peroxi-
olemia, the more recently recognized metabolic dation, upregulation of superoxide anion (O2–),
syndrome (MetS), and aging have been identified and a decrease in antioxidants levels [33,34].
as key correlates of ED and CAD, having as Circulating monocytes, which are significantly
common denominator generalized endothelial increased in diabetic patients with ED, are also
dysfunction, which contributes to the develop- involved in the increased production of ROS [35].
ment of atherosclerosis [22–25]. The aforemen- Free radical O2- was shown to interfere with eNO
tioned risk factors are responsible for a variety of bioavailability, propagating endothelial dysfunc-
endothelial offending insults, numerous biochemi- tion and chronically impairing diabetic penile vas-
cal and metabolic alterations, which injure penile cular function [36]. The deleterious effects of ROS
lining ECs, constituting a relevant pathophysi- are supported by evidence that superoxide dismu-
ologic factor underlying ED [4,22,26]. It has also tase (SOD) gene transfer or treatment with anti-
been suggested that the cumulative effects of oxidants reduce superoxide production, increases
several VRFs may induce endothelial dysfunction eNO, and restores erectile function in diabetic-
J Sex Med 2009;6:2390–2404
4. The Endothelial–Erectile Dysfunction Connection 2393
Figure 1 Mechanisms underlying vascular risk factor-associated endothelial dysfunction. Left panel figures: terminal deoxy-
nucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay in human cavernosal tissue from a diabetic ED patient
and a control non-diabetic, non-ED individual. Labeled in green: cavernosal ECs in apoptosis detected by TUNEL assay [47];
labeled in blue: all cavernosal nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) [47]. ED = erectile dysfunction;
AGEs = advanced gycation end products; EC = endothelial cell; eNOS = endothelial nitric oxide synthase; SMCs = smooth
muscle cells; ROS = reactive oxygen species; VEGF = vascular endothelial growth factor. Scale bar = 100 mm.
induced animals [33,34,37,38]. Similarly, adenovi- ernosal tissue of diabetic patients with ED have
ral gene transfer of eNOS was demonstrated to increased endothelial apoptotic cell density (ACD)
improve erectile responses in a diabetic model as compared with nondiabetic non-ED individu-
[39]. In diabetic-associated ED, eNO production als. Further, we demonstrated that ACD correlates
is also affected by the RhoA/Rho-kinase signaling with endothelial function assessed in a preopera-
system. The vasoconstrictor protein Rho-kinase tive stage by Penile NO Release Test (PNORT)
is increasingly expressed in experimental diabetic and duplex scan ecography, and established an
CC and downregulates eNOS activity, contribut- important threshold between in situ ACD values
ing to cavernosal endothelial dysfunction and ED and cavernosal endothelial functionality [47].
[40]. Penile vasculopathy in diabetics is also asso-
ciated to a reduction in the expression of vascular Hypertension
endothelial growth factor (VEGF), a pleiotropic Hypertensive patients have a higher prevalence of
molecule essential for endothelium homeostasis ED than the general population [48]. Despite the
[41]. VEGF expression is diminished in diabetic epidemiologic link between hypertension and ED,
corporeal tissue, altering VEGF-mediated intrac- basic science evidence is limited, and the mecha-
ellular signaling mechanisms, and leading to nisms leading to penile endothelial dysfunction are
decreased eNOS activation and EC viability [32]. under investigation. Studies in hypertensive pre-
Intracavernous therapies with VEGF were clinical models have suggested that high blood
referred to improve erectile function in diabetic pressure causes morphological changes in penile
models through the restoration of the insulin-like vascular bed, contributing to erectile failure
growth factor system and by the amelioration of [49,50]. Further, hypertension-induced EC
apoptosis in diabetic CC [42,43]. In fact, scarce mechanical injury and increased oxidative stress
data has suggested that apoptosis may be an impor- are thought to promote endothelial dysfunction
tant mechanism in diabetic ED [44–46]. Our by deleteriously affecting penile ECs-specific
group has recently shown for the first time how organelles, such as the endoplasmatic reticulum
programmed cell death affects human diabetic cor- and mitochondria [51]. Oxidative stress and ROS
poreal endothelial function. We reported that cav- production are also involved in impaired
J Sex Med 2009;6:2390–2404
5. 2394 Costa and Virag
endothelium-dependent vasodilation in hyperten- reported that ED significantly rises with the
sion. In a hypertensive model, increased CC levels increased number of metabolic risk factors present
of thiobarbituric acid-reactive substances were concomitantly in a patient [66]. In fact, the accu-
detected, and involved in the reduction of eNO- mulation of VRFs, independently recognized as
dependent functions, impairing both endothelial detrimental on loss of penile endothelial function,
and erectile function [52]. The sources and mecha- may contribute synergistically to the severity of
nisms responsible for ROS formation in hyperten- cavernosal vascular degeneration and ED [27].
sion remain mostly unknown; however, it was On a mechanistic level, VRFs’ common grounds
suggested that ROS increase might be mediated by leading to endothelial dysfunction involve an
its enzymatic production by adenine dinucleotide augment in oxidative stress and decreased eNOS/
phosphate (NADPH) oxidase and by alterations in eNO activation/bioavailability, as aforementioned.
intracellular antioxidant enzymes, including SOD However, other pathologic elements of the syn-
[53,54]. Accordingly, diminished SOD activity has drome may deleteriously affect additional path-
been observed in hypertensive rat corporeal tissue, ways of the vascular biology of the penis. A recent
implicating this mechanism in ROS formation study using a strain of obese-diabetic rats, mimick-
associated to hypertensive ED [54]. ing MetS phenotype and metabolic alterations, has
attempted to elucidate how the combination of
Hypercholesterolemia several VRFs contributes to cavernosal endothelial
Hypercholesterolemia is considered an indepen- dysfunction. This report has shown that in MetS
dent risk factor for ED development, contributing animals, there is also an increase in endothelium-
to the degeneration of the penile vascular bed [55]. produced Rho-kinase protein, which enhances
Studies demonstrated that hypercholesterolemia vasoconstriction mechanisms [67]. Although the
causes a reduction in normal CC vasoreactivity, epidemiological link between MetS and ED is
mostly by decreasing local eNOS/eNO activity irrefutable, further cellular and molecular studies
and bioavailability [56,57]. Impaired eNOS are required to unveil additional endothelial-
function/eNO production in the hypercholester- associated pathways impaired and involved in ED.
olemic cavernosal vasculature has been mostly
attributed to O2- production, potentially mediated Aging
by increased NADPH oxidase expression/activity It is established that the incidence of ED increases
[58]. Oxidized low-density lipoprotein has also with the advancement of age [68]. Vasculogenic
been implicated in ED, since its presence has been erectile impairment with aging was suggested to
identified in corporeal structures, including ECs, involve penile vascular structural alterations
and considered a causative factor for impaired CC [69,70]. Additionally, loss of endothelial function
relaxation responses [59]. Hypercholesterolemia has been reported in elderly CC due to alterations
may also affect penile endothelial function by in eNO bioavailability, increased oxidative stress,
altering the homeostatic expression of vascular altered vascular growth factor expression, and
growth factors. Studies in hypercholesterole- increase in activity of the RhoA/Rho-kinase
mic models have demonstrated that VEGF, pathway [71–74]. The association between oxida-
angiopoietin-1, and -2 were downregulated in cor- tive stress and age-related ED was established by
poreal tissue [56,60]. Further, single or combined the observation that experimental aging cavernosal
intracavernous therapies with endothelial growth endothelium produces high levels of O2-. Accord-
factors resulted in increased vasoreactivity in ingly, intracavernous SOD gene transfer in aged
hypercholesterolemic cavernosal tissue and animals reduces O2- formation, restoring erec-
improvement of erectile function [61–63]. tile function [72]. Similarly, impaired eNOS
expression/activity in the aged penis may be
Metabolic Syndrome (MetS) improved by local eNOS therapy, augmenting
MetS is a highly prevalent condition in industrial- erectile responses [71]. In fact, downregulation of
ized countries, manifested by the collective com- eNO in elderly CC has been thoroughly reported
bination of several VRFs for CVD and ED, and related to a decrease in VEGF expression [74],
including obesity, hyperglycemia, hypertension, an increase in endothelial activity of arginase,
and lipidic alterations [64]. Clinical studies have the enzyme that competes with eNOS for the
established an association between MetS and ED, common substrate L-arginine (L-Arg) [75], and
being both conditions linked by abnormal endot- with a deregulation in eNOS activation due to a
helial function [65,66]. Additionally, it was also decreased phosphorylation of its positive regula-
J Sex Med 2009;6:2390–2404
6. The Endothelial–Erectile Dysfunction Connection 2395
tory site (Ser-1177) and an increased phosphory- evaluate endothelium-dependent, as well as
lation of its negative regulatory site (Thr-495) endothelium-independent vasodilation. This
[76]. In addition, eNOS physiology may be method involves the ultrasonographic measure-
affected by an increased release/activity of Rho- ment of the brachial artery diameter before and
kinase in aged penis, a fact corroborated by the after a 5-minute occlusion of the forearm blood
improvement of erectile function through the flow induced by inflation and deflation of a proxi-
inhibition of the RhoA/Rho-kinase pathway [73]. mal upper arm cuff [78]. The result is increased
Besides the aforementioned mechanisms associat- blood flow, shear stress-mediated NO release, and
ing endothelial dysfunction with aged ED, we endothelium-dependent vasodilation, generally
must also have into account that with the advance- determined as the percentage change from the
ment of age, there is an accumulation of VRFs, baseline measurement to the artery diameter at
which might exacerbate penile endothelial dys- 40–60 seconds after cuff deflation. Additionally,
function and age-related ED. FMD endothelium-independent SMC-mediated
Efforts to disclose basic mechanisms involved in vasodilation can be assessed by sublingual admin-
cavernosal endothelial dysfunction allied to the istration of nitroglycerin (NTG) and measuring
abovementioned conditions is essential in order to the diameter of the artery three times at 5 minutes
propose novel tailored adjuvant therapies. Addi- post-NTG intake. SM-modulated vasodilation is
tionally, it is essential to verify in vivo the effects of defined as the percentage change from resting
impaired endothelial mediators and intracellular baseline artery diameter to the average of the
pathways in EC function in men with ED. The three post-NTG diameters [79]. Both FMD tests
basis underlying this important evaluation is the are valuable and used to specifically identify
recognition of endothelial dysfunction as a biom- endothelial-associated vasodilation dysfunction,
arker of atherosclerosis and generalized vascular SM relaxation alterations, and vasodilation im-
disease, being ED the first clinical manifestation. pairment. To evaluate solely vascular reactivity
As so, it is crucial to determine in ED patients through an endothelial response, it is commonly
systemic and local endothelial functional abnor- used the endothelium-dependent FMD analysis.
malities in the vascular bed using, developing, and FMD has become a standard test to assess the
further improving noninvasive clinical methods. interplay between VRFs, endothelial function and
CVD, and, more recently, to evaluate ED patients
[6,80,81]. Studies have suggested that endothelial
Clinical Evaluation of Endothelial (Dys)Function in
function detected on the brachial artery correlates
ED Patients
with function in conduit coronary arteries, estab-
When clinically evaluating endothelial function in lishing a link between endothelial-dependent
ED patients, there are two inherent challenges: FMD and CAD [82]. Given that VRFs are
one involves the assessment of general endothelial common grounds for the development of both
function, linking ED with a more generalized CAD and ED, it was not surprising that several
process of vascular deterioration, and the second is studies have related impaired FMD and vasculo-
to study local endothelium injury by assessing genic ED in populations with CVD and/or one or
penile endothelial dysfunction. For both purposes, several risk factors, confirming our early pioneer-
there are several hemodynamic and biological ing statement considering erectile failure mostly as
evaluation tools, with advantages and limitations a vascular disease [6,26,83]. Concordantly, patients
that will be further discussed. with vasculogenic ED were shown to present more
coronary atherosclerosis than control individuals
Hemodynamic Evaluation of Systemic [84]. More interesting are reports demonstrating
Endothelial Dysfunction that FMD detected endothelial dysfunction in
Flow-Mediated Dilation (FMD) men with ED, but without the presence of any
Since the early nineties, it has been demonstrated clinically established vascular disease. This has
that postischemic vasodilatation in the medium raised the main interest of systemic endothelial
large arteries is dependent largely on an function measurements in ED patients: the diag-
endothelium-derived NO response [77]. Based on nosis of preatherosclerotic states [79,84]. In addi-
this concept, vascular reactivity tests were devel- tion to an endothelial FMD dysfunction, the same
oped and established for clinical assessment of study has also showed that SM-induced vasodila-
endothelial function. FMD of the brachial artery is tion was impaired. Nonetheless, the relevance of
the most widely used noninvasive technique to this systemic endothelium-independent alteration
J Sex Med 2009;6:2390–2404
7. 2396 Costa and Virag
on erectile capability should be further assessed Biological Evaluation of Endothelial Function in
specifically and thoroughly in the penis. The FMD Peripheral Blood
technique, although reliable, has reproducibility
problems and some limitations particularly related Serum Markers
to the mandatory ultrasonografic expertise, the To date, many circulating biomarkers have been
significant day-to-day variability (approximately proposed for the evaluation of endothelial func-
25%) due to the biological circadian rhythm, tion; however, none have been considered the ideal
variations on the baseline diameter of the artery, or more specific, and most are unavailable for
and postprandial opposed to fasting state [78,85]. current practice [82]. Serum markers of inflamma-
tion and cellular adhesion, underlying the impor-
Peripheral Arterial Tonometry (PAT) tance of these processes in early atherosclerosis,
This new noninvasive technique is based on bilat- are the most commonly used to assess endothelial
eral comparative digital plethysmography. It dysfunction. Among the inflammatory markers,
involves the plethysmographic recording of the C-reactive protein (CRP) and endothelin-1 (ET-1)
arterial pulse wave amplitude at both index finger- are used to determine loss of EC function. Accord-
tips using stiff finger cuffs with air-inflatable pres- ingly, elevated levels of CRP have been associated
sure chambers. The PAT signal is recorded in one with impaired endothelial function, cardiovascular
index finger after postocclusive reactive hyperemia events, and ED [89]. Increased ET-1 has also been
(RHI), induced by a cuff on the upper arm, and linked to endothelial dysfunction manifested by
compared with values obtained on other hand at the presence of ED [90]. Additionally, circulating
the equivalent finger. PAT signals are processed levels of cellular adhesion molecules, such as selec-
using specific software, and the results are ex- tins, intercellular adhesion molecule-1, vascular
pressed as index of RHI (EndoPat; Itamar Medical cell adhesion molecule-1 (VCAM-1), have also
Ltd, Caesarea, Israel) [86]. Accordingly, endothe- been associated with endothelial dysfunction and
lial dysfunction is ruled out when RHI values are increased risk of CAD and ED [91]. The assess-
above 2.07, and considered if RHI is below 1.67. ment of asymetric dimethylarginine (ADMA)
In between these two values lies a gray area, which blood levels is also a used method. Increased levels
represents a sensitive zone predisposing to future of ADMA, which is a competitive inhibitor of vas-
development of endothelial dysfunction. In addi- cular eNOS, have been associated to the patho-
tion, besides assessing endothelial function, the genesis of vascular endothelial dysfunction, and
PAT evaluation software can also be used for spe- established a close link between CAD and ED
cific measurements of arterial stiffness [86]. PAT [92,93]. ADMA concentrations seemed directly
has been used to evaluate early and clinically rel- correlated to other methods of endothelial
evant CAD, and the results showed a correlation dysfunction evaluation, such as endothelium-
with coronary endothelial function [87]. A recent dependent FMD [94]. Additionally, the assessment
study from the Framingham heart study group has of the L-Arg/ADMA ratio might also be a valuable
related the measurements by this technique to the tool for the evaluation of specific alterations on
presence of VRFs [88]. In fact, PAT can be a very endothelial function [92]. Recent studies have also
useful method in the evaluation of conditions as suggested that increasing levels of homocysteine
diabetes or the MetS, given that similarly to seem related to a decrease in eNO synthesis and to
endothelium-dependent FMD, it seems to repre- a decrease in erectile function [95]. Nonetheless,
sent a true physiological reflection of peripheral and besides being a minimal invasive test, using
EC function. This relevant tool has recently peripheral blood, which is easily drawn, there are
started to be used for the evaluation of patients some setbacks in the clinical usability of these cir-
with ED [26], and is currently being applied for culating biomarkers. For instance, the systemic
those purposes in our institution. The advantage increase in proinflammatory and cell adhesion
of this procedure when compared with FMD is molecules might be an acute and transient event
that it requires less specialized training, and the induced by other pathophysiological processes,
obtained results are automatic and totally operator and their correlation with endothelial dysfunction
independent. However, PAT accuracy may lead to status has to be carefully established. In addition,
conflicting results, particularly in patients with low the discovery of other markers more sophisticated
pressure and low flow states, and therefore further and used under various regimens and treatments,
additional studies are required to validate the clini- have been reported, but not directly associated
cal use of this methodology. with other endothelial function measurements,
J Sex Med 2009;6:2390–2404
8. The Endothelial–Erectile Dysfunction Connection 2397
Figure 2 Cavernosal artery endothelium-dependent FMD assessed by PNORT in a non-ED individual. (A) Preocclusion
(basal artery diameter measurement = 0.6 mm); (B) postocclusion endothelial response (average diameter measure-
ment = 0.92 mm). The difference between pre- and postocclusion is 0.32 mm, and the percentage increase is 53% (normal
response). FMD = flow-mediated dilation; PNORT = Penile Nitric Oxide Release Test; ED = erectile dysfunction.
particularly with brachial FMD, in ED individuals Hemodynamic Evaluation of Penile
[96]. Endothelial Function
PNORT
Cellular Markers Penile anatomical and vascular characteristics led
In case of vascular endothelium damage, either us to establish a standardized test, the PNORT,
mechanically or by the noxious action of VRFs, which could specifically measure in cavernosal
there is a rapid need to reestablish endothelial arteries the endothelium-dependent capacity to
integrity and vascular homeostasis. Efficient vasodilate, giving an estimation of the arterial
endothelium repair involves the activation of the response mediated by locally produced eNO.
vasculogenic mechanism, by which bone marrow- Based on the same principles as the endothelium-
derived endothelial progenitor cells (EPCs) are dependent FMD in the forearm artery, this non-
mobilized and recruited to the peripheral circula- invasive technique consists on the ultrasonographic
tion, and to the site of injury where they differen- measurement of the pre- and postocclusive diam-
tiate into mature ECs, helping to regenerate the eter of one of the cavernous arteries. Similarly,
affected monolayer [97]. However, the vasculo- cavernosal artery diameter is measured before and
genesis process was shown to be impaired in CVD, after a 5-minute occlusion of penile blood flow
as reduced levels of circulating EPCs (cEPCs) induced by a specific cuff (Figure 2) [102,103].
were detected and related to endothelial dysfunc- Determination of the arterial endothelial response
tion and to future cardiovascular events [98]. capacity is calculated as for brachial or radial
Additionally, low numbers of cEPCs were found in FMD. The FMD in cavernous arteries is stronger
CAD patients and correlated with erectile function than in the forearm arteries, and, accordingly,
in the same population [99]. Other studies confirm non-ED and nonorganic ED subjects were shown
the presence of lower cEPCs in patients with ED, to have an average increase of 68% in the diameter
confirming the existence of an endothelial dys- of the cavernosal arteries as compared with a
function and supplying evidence that ED may 5–10% augment in the radial arteries [79,102]. In
be the first symptom of a systemic endothelial opposition to the significant increase of PNORT
damage [100]. A recent report demonstrated that in healthy penile vascular beds, organic diseased
cEPCs levels were reduced in overweight men cavernosal endothelium presents a lower response
with ED and correlated with the severity of erec- to this test. This may be explained in certain cases
tile failure [101]. The direct quantification of by the histological detection of a higher endothe-
cEPCs may represent a novel noninvasive surro- lial apoptotic rate [47]. Interestingly, it was also
gate marker for vascular homeostasis/dysfunction, observed that postradical prostatectomy ED
ED and CVD. However, the determination of patients presented a severe decreased response to
cEPCs performed by flow cytometry only indi- PNORT, which was comparable or even lower to
cates the peripheral circulating number of this the PNORT detected on the most diseased vascu-
progenitor cell population, and additional studies logenic ED patients [104]. This suggests that the
are required for further information on impaired NO pathways after radical prostatectomy are
cEPCs function. impaired or abrogated, affecting several erectile
J Sex Med 2009;6:2390–2404
9. 2398 Costa and Virag
cellular and molecular mechanisms, including penile diagnosis. Additionally, the arterial stiffness
endothelial function. Another valuable finding is can also be studied by further ultrasound evalua-
the significant difference observed in PNORT tion of the cavernous arteries after intracavernous
results in different groups of patients such as, injection of vasoactive medications (i.e., PGE1).
non-ED or nonvascular ED individuals with or
without VRFs [103]. Moreover, some ED patients Penile Veno-Occlusive Plethysmography
considered nonorganic with normal arterial, neu- In a recent publication, changes in blood flow in
rologic, and hormonal evaluation, seem to present both arms and penis were evaluated by veno-
a low response to PNORT, indicator of a previ- occlusive plethysmography. The authors sug-
ously undiscovered early sign of systemic endothe- gested the use of postischemic changes in blood
lial dysfunction (R Virag, unpublished data). In the flow in both arms and penis to assess endothelial
future, comparative measurements of systemic function using essentially the calculation of the
endothelial function by FMD or PAT and penile area under the flow-time curve. They observed a
endothelial function by PNORT should identify if significant difference between ED and non-ED
both values are strictly correlated, and/or answer patients at the penile level, but not in the forearm
to the question: does specific penile endothelial [105]. Albeit this is, at first, promising data, it
dysfunction exist? A recent work using matching appears noticeable that their ED and non-ED
systemic and penile veno-occlusive plethysmogra- patient groups were not age matched, and no
phy corroborates this idea [105], however, previ- information was disclosed concerning their vascu-
ous data has suggested some discrepancies [106]. lar characteristics. The penile flow measurements
Larger series comparing RHI and PNORT in ED with veno-occlusive plethysmography might be
and non-ED patients are necessary to understand somewhat cumbersome due to the fact that veno-
if a specific or earlier specific penile endothe- occlusion in the resting penis is not easy. Actually,
lial dysfunction exists. Our previous findings the basal blood flow in the penis is different in
[102,103] and ongoing studies (R Virag, unpub- both study groups, raising questions of a bias in the
lished data) have encouraged us to recommend this real meaning of the amplitude of the flow postoc-
local hemodynamic test as a first-line evaluation clusive decrease. Further and larger studies in ED
tool for endothelial function in any individual are needed to evaluate the accuracy of this tech-
presenting ED symptoms. In patients presenting nique, which is already in use for neuroanatomical
one or more VRFs, the decrease of the PNORT studies of volume increase evaluation under visual
response seems to have a prognostic value, and in sexual stimulation [107].
those free of VRFs, the discovery of a low PNORT
response should lead to a more complete and
Therapeutic Relevance of Endothelial Dysfunction
detailed evaluation. PNORT can be easily per-
in ED Patients
formed in conjunction with PAT measurements,
and it might also become a good means for the The strong link established between endothelial
follow-up of protective or repairing treatments for dysfunction and ED suggests that the use of
endothelial dysfunction. Although PNORT pre- certain medications and supplements may improve
sents the same bias drawback as the systemic FMD concomitantly endothelial and erectile function.
measurements, specially the correlation between Statins, intracavernous injections of vasoactive
the basal diameter of the cavernous artery and its agents and phosphodiesterase 5 inhibitors
percentage of increase, it is a reliable and repro- (PDE5I) are the most frequently cited for this
ducible technique, being an excellent noninvasive effect [108–110]. Additionally, we have recently
tool to evaluate penile endothelial function at early reported [111] an increase of endothelial function,
stages of vascular disease. Additionally, it allows to assessed by PNORT, in a series of patients after 2
assess the degree of severity of diseased endothe- months intake of a mixture of L-Arg, SOD and
lium and to diagnose an initial stage of systemic grape extract (Elliovir; Laboratoire Ellios Bio Tek,
endothelial dysfunction, manifested as prema- Paris, France). Studies showed that chronic intake
ture ED with deleterious endothelium function. of the PDE5I tadalafil significantly reduced serum
The use of PNORT to assess endothelium- levels of CRP, ET-1, and VCAM-1, and improved
independent FMD in cavernous arteries might both ED and endothelium-dependent FMD mea-
also be an additional testing essential to evaluate surements of penile arteries [112]. Additionally,
SMC functional capacity and complement the the same therapeutic regimen with tadalafil
endothelial analysis establishing a more accurate resulted in an increase in cEPCs mobilization and
J Sex Med 2009;6:2390–2404
10. The Endothelial–Erectile Dysfunction Connection 2399
ameliorated endothelial function, as assessed by be performed in conjunction with a thorough
FMD [113]. Another report demonstrated a posi- local penile vascular evaluation. Nonetheless,
tive correlation between PNORT increase and endothelial-erectile function seem tightly con-
erection improvement after vardenafil therapy nected, and there is a growing concern for an
[114]. Accordingly and considering our histologi- earlier protection of both penile and systemic
cal [47] and clinical studies [104], we assume that endothelium through the alteration of lifestyle
the response to PDE5I might be correlated to the behaviors, allowing the control of VRFs, comple-
PNORT. Below a certain PNORT value, indica- mented with specific endothelial-rehabilitating
tive of severe endothelium injury, the drugs are less compounds. Concerns to their sexual capability
prone to be effective. Most importantly, the iden- should be for many young men a strong motiva-
tification of endothelial dysfunction manifested tion to quit smoking, bad alimentary habits, and
earlier by ED is an alert of a potential development help to protect them from life-threatening vascu-
of systemic vascular disease and must be taken into lar diseases.
account. In populations at risk and when endothe-
lial dysfunction is detected by ED symptoms,
besides therapeutic intervention, it is crucial to Acknowledgments
establish early preventive measures, such as CC was supported by the Portuguese Foundation for
changes in lifestyle habits (smoking, alcohol, high Science and Technology (PTDC/SAU-OSM/65599/
caloric food). 2006).
Corresponding Author: Carla Costa, PhD, Faculty
Conclusions of Medicine of the University of Porto, Department
of Biochemistry (U38-FCT), Porto, Portugal. Tel:
Endothelium dysfunction and ED are definitely +351 225513654; Fax: +351 225513655; E-mail:
linked as Siamese twins. VRFs are implicated in carcosta@med.up.pt
endothelial deleterious alterations, affecting
primordially the eNO-dependent vasodilation Conflict of Interest: None.
pathway, which severely injure penile lining ECs,
disturbing also their important interaction with Statement of Authorship
other erectile components, such as the SM layer.
Importantly, endothelial dysfunction may be Category 1
manifested initially by ED, which is considered as (a) Conception and Design
an early-warning sign for the development of ath- Carla Costa; Ronald Virag
erosclerosis and CVD. The noninvasive assess- (b) Acquisition of Data
ment of peripheral and local endothelial function Carla Costa; Ronald Virag
in the office setting is currently available, allowing (c) Analysis and Interpretation of Data
Carla Costa; Ronald Virag
the diagnosis of endothelial dysfunction and vas-
culogenic ED. Although with advantages and limi-
tations, these evaluation tools have demonstrated Category 2
that early diagnosis of ED may have preventive (a) Drafting the Article
value on future cardiovascular events and can also Carla Costa; Ronald Virag
(b) Revising It for Intellectual Content
be used as predictive tests regarding therapeutic
Carla Costa; Ronald Virag
response. As so, we suggest PNORT as the
primary local exam to determine vasculogenic
ED, in conjunction with brachial endothelium-
Category 3
dependent FMD or digital PAT assessment of sys- (a) Final Approval of the Completed Article
Carla Costa; Ronald Virag
temic endothelial function. In addition, a complete
penile evaluation should be complemented with
the assessment of endothelium-independent, References
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