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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 2, March/April 2020
AFRICA
59
dysfunction and promotion of atherosclerosis in HIV-infected
individuals is complex but is thought to be related to a number
of factors including direct involvement of HIV, endothelial
activation and vascular inflammation.
13-15
Endothelial dysfunction is regarded as a link between
infection, inflammation and atherosclerosis, and there are recent
data suggesting the presence of endothelial dysfunction in
untreated HIV-positive patients.
16
Furthermore, following cART,
it has been shown that there is a fall in markers of endothelial
activation such as monocyte chemo-attractant protein, P-selectin
and VCAM-1.
12
Although endothelial function has been studied in numerous
studies in the past, there is a paucity of data on endothelial
function in HIV-positive patients presenting with ACS.
17
Therefore we aimed, firstly, to assess the presence and degree of
endothelial dysfunction in HIV-positive patients presenting with
ACS by using flow-mediated dilatation (FMD) and endothelial
biomarkers. Our secondary aim was to assess carotid intima–
media thickness (CIMT) in these patients as a surrogate marker
of atherosclerosis.
Methods
This was a prospective study of 60 patients at a large urban
public hospital in Johannesburg, South Africa, recruited over a
three-year period (July 2012 to July 2015). Twenty HIV-positive
patients presenting with ACS (HIV+/ACS) were compared
to 20 HIV-negative patients with ACS (HIV-/ACS) and 20
HIV-positive patients without ACS (HIV+/no ACS).
Inclusion criteria included age ≥ 18 years and HIV-positive
patients presenting with ACS. Risk factors for coronary artery
disease (CAD) that were studied included age, smoking,
hypertension, diabetes and family history of premature CAD
(men ≥ 55 years, women ≥ 65 years of age). Exclusion criteria
included prior myocardial infarction, life-threatening disease
that prevented a two-year follow up, and known carotid artery
disease. Ethical approval for the study was obtained from the
local institutional body (M111143).
The HIV+/ACS and HIV-/ACS patients were matched for age
and gender. The HIV+/no ACS patients could only be matched
for gender as cART-naïve patients presenting at our HIV clinic
were found to be much younger patients. CIMT thickness
was measured in all three groups using an 11-Mhz transducer
(Phillips iE33 ultrasound machine) with the patient in the supine
position with the head tilted 30 degrees to the left for the right
carotid artery assessment and then 30 degrees to the right for the
left carotid artery. The carotid artery bifurcation was visualised
and the software automatically measured carotid artery intima
thickness of the distal wall 10 mm from the bifurcation bulb.
Endothelial function was measured non-invasively using
brachial FMD according to standardised guidelines.
18
The
maximum diameter of the brachial artery was measured at rest.
A blood pressure cuff was then inflated to at least 50 mmHg
above the systolic pressure to occlude arterial inflow for five
minutes. The brachial artery diameter was again measured two
minutes after cuff release when the maximum dilation of the
vessel usually occurs.
Applanation tonometry of the radial artery converts the
radial pulse wave into an aortic pulse wave. Pulse-wave velocity
(PWV) was measured from sequential waveform measurements
at the carotid and femoral sites. The distance that the pulse wave
travels was determined as the difference between the distance
from the femoral sampling site to the suprasternal notch, and the
distance from the carotid sampling site to the suprasternal notch.
Endothelial biomarkers were measured in all 60 patients and
included interleukin (IL)-1
β
, IL-1Ra, IL-6, tumour necrosis
factor alpha (TNF-
α
), monocyte chemotactic protein-1 (MCP-1),
plasminogen activator inhibitor-1 (PAI-1), E-selectin, P-selectin,
ICAM-1 and VCAM-1. Following blood collection in EDTA
tubes, specimens were centrifuged for 20 minutes at 4°C and 1
000 ×
g
. The plasma was decanted into 1.5-ml microcentrifuge
tubes and stored at –70°C for later analysis.
IL-1
β
, IL-1Ra, IL-6, MCP-1 and TNF-
α
concentrations were
quantified using the Bio-Plex Pro™ human cytokine standard 27
(Plex Bio-Rad Laboratories, Hercules, CA) as per manufacturer’s
instructions. The plasma was diluted four-fold for these assays.
E-selectin and P-selectin plasma levels were measured using the
Human Magnetic Luminex assay (R&D Systems, Minneapolis,
MN) as per manufacturer’s recommendations. Plasma was diluted
two-fold to measure E-selectin and P-selectin levels. The serum
concentrations for VCAM-1, ICAM-1 and PAI-1 were analysed
in sera diluted 40-fold using the Milliplex human sepsis magnetic
panel 1 (Merck Millipore, Billerica, MA) as per manufacturer’s
instructions. Samples were analysed using the Bio-Plex 200
system (Bio-Rad) and concentrations were determined using the
5-PL method using Bio-Plex Manager 5.0 software.
Statistical analysis
The
χ
2
test was used to assess the relationship between categorical
variables and groups. The Fisher’s exact test was used where the
requirements for the
χ
2
test could not be met. The relationship
between continuous variables and groups was assessed with
one-way analysis of variance (ANOVA) for the three groups and
the unpaired
t
-test for two groups.
Post hoc
tests for ANOVA
were conducted using the Tukey–Kramer adjustment for multiple
comparisons. Where the data did not meet the assumptions of
these tests, a non-parametric alternative, the Kruskal–Wallis test
was used for three groups, and the Wilcoxon rank sum test for
two groups. Paired comparisons between continuous variables
were carried out with the paired
t
-test or the Wilcoxon matched-
pairs test. Data analyses were carried out using SAS version 9.4
for Windows. A 5% level of significance was used.
Results
The HIV+/ACS patients had a mean age of 51.1 years (
±
8.1)
and 13 were male (65%). The mean age of 36.0 years (
±
6.8) in
the HIV+/no ACS group was significantly lower than that of the
HIV+/ACS group [51.1 years (
±
8.1)] and the HIV-/ACS group
[52.3 years (
±
9.0)] (
p
< 0.0001). The proportion of males in each
group ranged between 50 and 80%, but the differences were not
statistically significant (
p
=
0.14) (Table 1).
Ten (50%) of the HIV+/ACS group were on cART and none
was on protease inhibitors. Seven (35%) of the patients in the
HIV+/ACS group were newly diagnosed with HIV. There were 15
(75%) hospital admissions with ST-segment elevation myocardial
infarction (STEMI) (eight anterior, seven inferior), three (15%)
with non-ST segment elevation myocardial infarction and two
patients with unstable angina (10%). The typical presentation