Primary hyperaldosteronism and endothelial ischemia-reperfusion injury

Primary hyperaldosteronism is a common cause of hypertension, with an estimated
prevalence of 10% in the hypertensive population. Importantly, patients with
PHA experience more cardiovascular events, including atrial fibrillation,
stroke, and myocardial infarction, compared to patients with primary
hypertension, independent of the blood pressure level. Also, in patients
without PHA, a high aldosterone level or high aldosterone-to-renin ratio (ARR)
is associated with an increased risk of cardiovascular events. In the setting
of acute myocardial ischemia and reperfusion (an acute myocardial infarction),
aldosterone levels are associated with cardiovascular death and heart failure,
even when aldosterone levels are within the normal range. Furthermore, in
patients with heart failure, aldosterone levels are increased and treatment
with MR antagonists improves outcome. Two large trials showed a reduced
morbidity and mortality in patients with heart failure during treatment with
the MR antagonists spironolactone and eplerenone.

Preclinical studies have provided data that aldosterone has direct adverse
cardiovascular effects and increases infarct size in animal models of
myocardial infarction, although this latter result has not been reported in
other studies. In addition, the administration of MR antagonists consistently
reduces myocardial infarct size in these animal models.
Based on these abovementioned preclinical and clinical findings, we hypothesize
that patients with PHA are more susceptible to ischemia-reperfusion (IR)-injury.

To study IR in humans in vivo, a safe and well-validated method is to examine
brachial artery flow mediated dilation (FMD) before and after forearm ischemia
and reperfusion. FMD is reduced after forearm IR, which is a measure of
endothelial IR-injury. We have recently used this approach to demonstrate that
prevention of IR-injury by ischemic preconditioning is impaired in elderly
patients. It has been shown previously in patients with hypertension that
brachial artery FMD is lower in patients with a high
aldosterone-to-renin-ratio, but the effect of IR on FMD has never been studied
in these patients.

The mechanism of the potentially deleterious effect of aldosterone on IR-injury
is still unknown. Recently, Schmidt et al. showed that the infarct
size-limiting effect of MR-antagonists was abolished by concomitant
administration of an adenosine receptor antagonist. In addition, the
cardioprotective effect was abolished in mice with a genetic deficiency of the
enzyme ecto-5*-nucleotidase (CD73; catalyses extracellular formation of
adenosine) and in adenosine A2B receptor knock-out mice. These observations
suggest that the formation of extracellular adenosine by CD73 is critical for
the cardioprotective role of MR antagonists.

Adenosine is an endogenous purine nucleoside, which is formed by intra-, and
extracellular degradation of adenosine monophosphate by CD73. Degradation of
adenosine occurs in the intracellular compartment. As a consequence,
facilitated diffusion of adenosine over the cellular membrane by the
equilibrative nucleoside transporter (ENT) is normally directed inwards.
Stimulation of membrane-bound adenosine receptors induces various effects,
including vasodilation, inhibition of inflammation, and protection against
IR-injury. Indeed, endogenous adenosine acts as a key mediator of the infarct
size-limiting effect of several pharmacological and non-pharmacological
strategies.

Based on the abovementioned preclinical data, we hypothesize that
susceptibility to IR is increased in patients with PHA due to reduced CD73
activity by aldosterone. To gain better insight into this hypothesis, we will
investigate endothelial IR-injury in patients with PHA and control patients
with essential hypertension. In addition, the expression and activity of CD73
will be measured on isolated mononuclear cells.

The results of these studies will provide a possible explanation for the
increased risk of cardiovascular events in patients with PHA. Our study is the
first to explore the hypothesis that patients suffering from PHA are more
vulnerable to IR injury, and that a reduced adenosine formation contributes to
this more vulnerable state. The finding that a reduced extracellular adenosine
concentration contributes to the accelerated rate of cardiovascular morbidity
and mortality in patients with PHA, would provide us novel pharmacological
targets for these patients.

In this clinical trial, we will test the following hypotheses:

1. The reduction in FMD by forearm IR will be greater in patients with PHA,
compared to matched control patients with primary hypertension.
2. CD73 activity is lower in patients with PHA
3. The circulating adenosine concentration is lower in patients with PHA.

Twenty patients (age 18-75 years) with PHA and without interfering
antihypertensive treatment will be asked to participate. Twenty matched
controls with primary hypertension will be sought. After signing for informed
consent, history taking, a physical examination, and electrocardiography will
be performed.

As explained in detail the research protocol, most patients will at the time of
inclusion use calciumchannel blockers and/or alpha-adrenergic-receptor
antagonists and/or hydralazine as antihypertensive drugs, since only these
drugs are allowed during the diagnostic workup for primary hyperaldosteronism
because they do not (significantly) affect the renin/aldosteron plasma
concentration. To ensure a similar use of antihypertensive drugs in both
groups, we will change the drugs into diltiazem with or without hydralazine
(dependent on the blood pressure level and the number of antihypertensive
drugs) one week before the experiment. This will be done in consultation with
the treating physician. Patients with primary hyperaldosteronism often have a
low serum potassium. Therefore, many patients will use potassium suppletion at
the time of the diagnostic work up for PHA. If the plasma potassium
concentration was <3.5 mmol/l upon inclusion, the potassium suppletion will be
increased to ensure a plasma potassium level of >3.5 mmol/l at the moment of
the experiment. Finally, when patients use statins for cholesterol lowering,
these drugs will be stopped one week before the experiment.

At least one week after changing the medication (whenever necessary), the
volunteers will participate in the following experiment. Because all
antihypertensive drugs can potentially affect the tolerance against
ischemia-reperfusion, the patients will not take the antihypertensive drugs at
the morning of the experiment.

Ultrasonographic measurement of brachial artery Flow Mediated Dilation (FMD)
Brachial artery FMD will be measured before and after 20 minutes of forearm
ischemia and 20 minutes of reperfusion. This protocol of IR, will result in an
immediate decrease in brachial artery FMD, which is believed to reflect
IR-induced endothelial dysfunction. In previous studies with this experimental
design, 20 minutes of ischemia and 15 minutes of reperfusion was used. However,
in our recent study with this method, we observed that the FMD had not yet
completely returned to baseline values after 15 minutes of reperfusion.
Therefore, we have chosen to use 20 minutes of reperfusion in the current
study.
All measurements will be performed in a temperature-controlled room (22.5 °C)
and using recent guidelines of FMD. The patients will rest in a supine position
with both arms extended and immobilized, supported at an angle of ~80°
abduction from the torso. Heart rate and mean arterial pressure will be
determined. For the assessment of FMD, a rapid inflation/deflation pneumatic
cuff will be placed distal to the olecranon process to provide an ischemic
stimulus distal from the brachial artery to provoke vasodilation and subsequent
shear stress. The brachial artery will be imaged in the distal third of the
upper arm. We will simultaneously obtain a continuous Doppler velocity
assessment, and data will be collected using the lowest possible insonation
angle (always <60°), which does not vary during each study.
After a resting period of at least 15 minutes, 1 minute of baseline recording
of the arterial diameter and velocity will be performed. This is followed by
inflation of a pneumatic cuff around the forearm for 5 minutes. The arterial
diameter and velocity recordings will be restarted at least 30 seconds before
cuff deflation and continued for at least three minutes after deflation. We
will record peak arterial diameter and flow, and the time to reach this peak
after cuff deflation.
Subsequently, a rapid inflation/deflation cuff will be positioned around the
upper arm, so that the brachial artery will be within the ischemic zone. The
cuff will be inflated for 20 minutes to 220 mmHg, which will be followed by 20
minutes of reperfusion. Afterwards, the same FMD measurement is performed as
described above.
CD73 activity on mononuclear cells
Before start of the FMD experiment, blood will be drawn to isolate mononuclear
cells. We will determine the activity of CD73 exposed on the surface of intact
mononuclear cells, by quantifying the conversion of 1,N6-ethenoadenosine 5*-
monophosphate to 1,N6-ethenoadenosine in the presence and absence of the
CD73-specific inhibitor α,β-methyleneadenosine 5*-diphosphate.

Circulating adenosine concentration
It is tempting to measure the circulation adenosine concentration because of
its very short half life of <1 second. We have recently published a method to
determine the plasma adenosine concentration using a purpose-built syringe.
Using this syringe, the blood mixes immediately at the end of the needle with a
solution containing pharmacological blockers of the proteins involved in
adenosine formation, transport, and degradation. Before the start of the
experiment, 3 ml of blood will be drawn with this syringe system to measure the
plasma adenosine concentration.

Additional blood drawing
Before start of the experiment 3 mL of blood will be drawn to determine
caffeine concentrations. Subjects with a circulating caffeine concentration >
1.0 mg/l will be excluded from analyses, since caffeine is a potent adenosine
receptor antagonist. Also 16 ml of blood will be drawn for the determination of
the plasma glucose, creatinine, sodium, potassium, and cholesterol
concentration (to evaluate in- and exclusion criteria; these factors could
modify the effect of IR on the endothelium) and the renin and aldosterone
concentration

Brachial artery FMD will be measured in all patients before and after 20
minutes of forearm ischemia and 20 minutes of reperfusion. This can cause mild
discomfort, but is otherwise without any side-effects.
Blood drawing (during screening and before the experiment) can cause a
hematoma.

To participate in the study the patients will have to come to the research
centre during half a day and this will come along with extra costs (of
travelling).