The role of TGFb in the pathobiology of atheroscle- rosis is being increasingly recognised. Its functional
regulation of the vessel wall is by its direct effect on the vascular smooth muscle cells VSMCs, endothelial cells
and extracellular matrix ECM. In addition, TGFb has a number of other functions including suppression of
immune responses [12] and reduction of superoxide anions [13], which are considered to be important in
atherogenesis. TGFb1 and TGFb3, although share 70 homology, exhibit distinct functions in a range of
pathobiological situations, such as wound healing, radi- ation-induced tissue damage and tumour development
[14 – 16]. What functional role the two isoforms play in coronary artery disease CAD is not known.
Previously two independent studies have reported that CD105 is raised in patients with peripheral vascu-
lar disease [17] and TGFb1 levels are depressed in patients with atherosclerosis [18]. However, no informa-
tion is available in the same cohort of patients about the possible relationship of soluble CD105, TGFb and
the CD105TGFb complexes. VSMCs participate in the development and progression of atherosclerosis through
their migration, proliferation and secretion of ECM. Each of these aspects of VSMC behaviour can be
modified by TGFb. A recent finding, which demon- strated the existence of CD105 mRNA and protein in
human VSMCs [19], leads to the speculation that in addition to endothelial cells, it may modulate effects of
TGFb on VSMCs. Based on these findings, we hypoth- esised that the interaction of CD105 with TGFb might
affect the bioavailability of TGFb and hence contribute to the progression of CAD. To examine this hypothesis,
in this study we have examined circulating levels of CD105, TGFb and the CD105TGFb complexes in
healthy individuals, in patients with established triple vessel coronary artery disease and a group with normal
angiograms but with continued chest pain and positive exercise electrocardiogram.
2. Materials and methods
2
.
1
. Subjects Two groups of patients were recruited for this study
which had ethics committee approval. One group of patients had significant coronary artery disease in all
three coronary arteries on coronary angiograms TVD. Significant coronary artery disease was defined as a
reduction of more than 50 in the intraluminal diame- ter of the coronary artery. The second group were
patients who had continued chest pain suggestive of angina pectoris, a positive exercise electrocardiogram
but normal coronary angiograms NCA. Blood sam- ples were withdrawn through the needle inserted into
the femoral artery for angiography before heparin ad- ministration. Control samples were obtained from age
matched healthy hospital staff and all were asymp- tomatic for vascular disease. Blood was allowed to clot
in plastic tubes for 2 h at room temperature, and the serum was collected after centrifugation. All measure-
ments were performed on aliquots of sera stored at −
70°C.
2
.
2
. Detection of acti6e TGFb
1
The specific measurement for active TGFb1 by ELISA was defined by the nature of the antibodies used
in the assay. Both the coating antibody mouse mono- clonal anti-TGFb antibody, Genzyme, MA and the
detecting antibody chicken polyclonal anti-TGFb1 an- tibody, RD Systems, Abingdon, UK recognise ma-
ture active TGFb1 rather than latent forms. The validity of ELISA for active TGFb1 was further ver-
ified by substituting the active TGFb1 with the latent TGFb1 both from RD Systems, indicating that the
cross-reactivity with latent TGFb1 was less than 1 Fig. 1. The assay was therefore considered to detect
active TGFb1 in serum and the details of procedure are as follows.
A total of 96-well white plates Dynatech Microfluor, VA were coated at 4°C overnight with 100 mlwell
mouse monoclonal antibody against TGFb Genzyme, MA at 1mgml in PBS. After blocking with 1 BSA in
0.1 M PBS and 0.1 Tween 20 PBS-Tween for 2 h at room temperature, plates were washed three times with
PBS-Tween and 100 ml serum was added to each well. A standard curve was generated using purified recombi-
nant human TGFb1 RD Systems. The plate was left at 4°C overnight in a humidified box. Subsequently, the
wells were incubated with 100 ml polyclonal chicken anti-TGFb1 antibody RD Systems, diluted 11000
1 mgml in PBS-Tween, for 3 h at 4°C. Three washes
Fig. 1. Specificity of the ELISA for active TGFb1. To evaluate the specificity of the ELISA for active TGFb1, the active TGFb1 was
substituted with latent TGFb1, wherein the signal was barely de- tectable. The calculated cross-reactivity with the latent TGFb1 was
less than 1.
with PBS-Tween were given between each procedure. After washing, the plates were incubated on a shaker
with 100 ml per well horseradish peroxidase conjugated rabbit anti-chicken IgG Jackson ImmunoResearch
Laboratories Inc. PA, at 12000 dilution 0.2 mgml in 1 BSA and PBS-Tween, for 30 min at room tempera-
ture. Finally, the plates were rinsed 3 times, 100 ml Amerlite signal reagent Amersham UK was added to
each well and the plate was read immediately in an Amerlite plate reader Kodak Clinical Diagnostics. All
the samples were run in duplicate and the measured values of light emission at 420 nm were converted into
absolute TGFb1 concentrations by reference to the standard curve.
2
.
3
. Measurement of a
+
l TGFb
1
Serum was treated using pH 2.0 buffer to release the mature TGFb1 as described by Grainger et al. [20]. The
TGFb1 determined under this condition represents the active plus the acid-activatable latent TGFb1 referred
to as a + lTGFb1. For the activation of the samples, 50 ml per well, test serum was added to a 96-well plate
followed by addition of 3 ml 150 mM sodium phosphate buffer at pH 2.0. The plate was incubated for 20 min at
room temperature with agitation, then the mixture was neutralised by addition of 3 ml 150 mM sodium phos-
phate buffer at pH 12.0. Forty-four microlitres of PBS- Tween was added to each well. Thus the sample was
diluted 12. The acid-activated samples were transferred to the coated plate to measure TGFb1 using the same
procedures as for the active TGFb1.
2
.
4
. Detection of soluble CD
105
in serum CD105 was measured using an indirect sandwich
ELISA wherein purified Mab E9, which specifically reacts with CD105 [21], and biotinylated Mab E9, in
conjunction with streptavidin peroxidase, were used as capture and detection reagent [22], respectively. Briefly,
96-well white microtitre plates were coated with Mab E9 100 mlwell diluted 11000 1 mgml in 0.1 M PBS
and incubated in a damp box overnight at 4°C. The coated plates were blocked using 1 BSA and PBS-
Tween for 2 h at room temperature. Test samples diluted 12 in PBS-Tween were added to the plates in
duplicate. One serum sample with a high level of CD105 approximately 100 ngml was titrated to make
a standard curve in each plate. After incubation overnight, at 4°C, 100 mlwell, biotinylated Mab E9
12000 dilution; 0.2 mgml was added to the plates and incubated at 4°C in a humidified box for 3 h, followed
by addition of 100 mlwell horseradish peroxidase con- jugated avidin at 12000 dilution in PBS-Tween and 1
BSA, which was incubated, with shaking, at room temperature for 30 min. Three washes with PBS-Tween
were carried out between each of the procedures. Fi- nally, 100 mlwell, Amerlite signal reagent Amersham
was applied to each well and the light emission was immediately measured in an Amerlite plate reader.
2
.
5
. Immunoassay of CD
105
TGFb
1
complex The measurement of soluble CD105TGFb1 complex
was performed using untreated serum. The procedures were the same as described in the assay for active
TGFb1, with the exception that the coating anti-TGFb antibody was substituted by Mab E9 at 1 mgml in
order to capture the complex from the serum [23]. A serum sample with a high level of CD105TGFb1 com-
plex 100 arbitrary unitsml was serially diluted from 12 to 1512 to generate a standard curve on each plate.
The assay showed no cross-reaction with CD105 TGFb3 complex and exhibited a wide range of detec-
tion from 0.05 to 100 unitsml.
2
.
6
. Immunodetection of TGFb
3
in serum The procedure to measure serum levels of TGFb3
was as follows: 96-well white plates were coated with mouse monoclonal antibody against TGFb at 100 ml
well 1 mgml diluted in PBS, and incubated in a humidified box overnight at 4°C. The coated plates
were subsequently blocked using 1 BSA and PBS- Tween for 2 h at room temperature. Test samples
diluted 12 in PBS-Tween were added to the plates in duplicate. A standard curve was generated on each
plate
using purified
recombinant human
TGFb3 rhTGFb3; RD Systems. After incubation at 4°C,
100 mlwell, goat anti-TGFb3 antibody RD Systems 1 mgml diluted in PBS-Tween, were added to the
plates and incubated for 3 h at 4°C. After washing, the wells were incubated with agitation with 100 mlwell
horseradish peroxidase conjugated donkey anti-goat antibody Jackson ImmunoResearch Laboratories Inc.,
PA at 0.2 mgml diluted in 1 BSA in PBS-Tween for 30 min at room temperature. Following washing,
Amerlite signal reagent, was added to each well, 100 mlwell, and the light emission immediately measured in
an Amerlite plate reader. The measured values of light emission were converted into absolute concentrations
using a TGFb3 standard curve.
2
.
7
. Measurement of CD
105
TGFb
3
complex In the assay for CD105-TGFb3 complex, Mab E9
was used as coating antibody to capture the complex from the serum. The ELISA procedure was the same as
described in the assay for TGFb3, with the exception that mouse monoclonal antibody to TGFb was re-
placed by Mab E9 100 mlwell at 1 mgml diluted in PBS and subsequently detected using goat anti-TGFb3
Table 1 Some clinical characteristics of controls and patients with TVD and NCA
a
Parameters NCA
TVD Controls
10:20 31:5
20:11 Male: female
9 Smokers
7 12
56 9 9 63 9 7
56 9 7 Age years
Total cholesterol, mmoll 6.2 9 0.9
6.0 9 1.1 5.3 9 1.1
1.2 0.5–3.7 1.66 0.7–4.4
1.25 0.7–4.1 Triglycerides, mmoll
0.8 9 0.2 HDL cholesterol, mmoll
1.1 9 0.2 1.5 9 0.4
4.3 9 1.1 3.1 9 1.0
LDL cholesterol, mmoll 4.5 9 0.8
143 9 23 138 9 21
134 9 22 Systolic blood pressure
Diastolic blood pressure 78 9 11
74 9 15 78 9 9
a
Data represented as mean 9 SD or as median and range. Comparisons between groups were made by the Mann–Whitney U test. TVD, triple vessel disease; NCA, normal coronary angiogram; HDL, high density lipoprotein; LDL, low density lipoprotein.
Signifies P50.01.
antibody. Thus the molecules determined in this system contain both CD105 and its ligand TGFb3. The stan-
dard curve was generated using a serum sample con- taining 50 unitsml of the complex on each plate.
2
.
8
. Statistical analysis Data are presented either as mean when normally
distributed or median when skewed for the various parameters in each group. The logged data were first
analysed by the Kruskall – Wallis test. Further compari- sons between groups were conducted using the Mann –
Whitney U test. To determine associations between two variables, the Spearman’s correlation test was per-
formed. For all the tests, the significance level was set at P 5 0.05.
3. Results
