In Vivo and In Vitro Assessment of Human Saphenous Vein Wall Changes

Full text

The Open Cardiovascular Medicine Journal, 2007, 1, 15-21 15
1874-1924/07 2007 Bentham Science Publishers Ltd.
In Vivo and In Vitro Assessment of Human Saphenous Vein Wall Changes
Akram M. Asbeutah*,a, Sami K. Asfarb, Hussain Safarb, Mabayoje A. Oriowoc, Ihab ElHagrassid,
Mona A. Abu-Assie, James D. Camerona and Barry P. McGratha
aDepartment of Medicine, Monash University and Department of Vascular Sciences in Southern Health, Dandenong
Hospital, Melbourne, Australia
bDepartment of Surgery, Faculty of Medicine, Kuwait University and Department of Surgery-Vascular Unit, Mubarak
Al-Kabeer Hospital, Kuwait
cDepartment of Pharmacology, Faculty of Medicine, Kuwait University, Kuwait
dDepartment of Cardiovascular Diseases, Chest Hospital, Kuwait
eDepartment of Science, School of Basic Education, Public Authority of Applied Education & Training, Kuwait
Abstract: Purpose: To investigate if noradrenaline (NA) and 5-hydroxyptamine (5-HT) drugs induce responses of iso-
lated control and varicose veins are altered by removal of the endothelium.
Subjects & Methods: Specimens of the great saphenous vein (GSV) were obtained from 12 subjects with primary varicose
veins and 12 subjects from donor vessels at cardiac surgery. A total of 10 normal healthy volunteers were selected for
comparison. The diameter changes of GSV during the resting phase, at the end of 5 minutes occlusion, and then every 30
seconds post deflation for five minutes were measured using B-mode ultrasound. Post-surgery the vein sample was col-
lected in a tube of Krebs-Henseleit solution.
Results: The repeated measure ANOVA test for the diameter, percent, and difference changes of GSV diameter from
maximum diameter at different time intervals showed significance difference within and between all groups. NA and 5-
HT produced concentration-dependent contractions of control and varicose saphenous vein segments. There was no sig-
nificant difference in the potency of NA and for 5-HT, but the maximum response, normalized for tissue weight, was less
in varicose vein segments. Removal of the endothelium had no effect on the potency of NA or 5-HT but significantly
(p<0.05) reduced the maximum response to NA and 5-HT in varicose vein segments but not to 5-HT in control veins.
Conclusion: The venous endothelial damage may cause vascular smooth muscle contractions dysfunction that favours
dilatation and secondary valvular insufficiency.
Keywords: Primary varicose veins, chronic venous insufficiency, noradrenaline, 5-hydroxyptamine, in vivo vein study, in vitro
vein study.
INTRODUCTION
There is some controversy regarding the primary pathol-
ogy that leads to the development of venous incompetence.
Various theories, the valvular hypothesis [1] and the vein
wall hypothesis [2], have been put forward to explain the
pathogenesis of varicose veins and chronic venous insuffi-
ciency (CVI). Although the pathophysiology of the disease is
largely unknown, abnormalities of the venous wall and the
valves are considered to play a major role [3]. The cause of
this abnormality has been assumed to be the result of either a
defective smooth muscle function or connective tissue dam-
age [4] resulting in distension, deficient leaflet apposition
and lack of closure rather than primary valve damage. A
general weakness of venous smooth muscle does not seem to
be involved since the defect is localized and proportional to
wall deformation [4]. However, Thulesius reviewed the four
*Address correspondence to this author at the Department of Radiologic
Sciences, Faculty of Allied Health Sciences, Kuwait University, P.O. Box
23924, Safat 13110, Kuwait; Tel: 00-965-6518535; Fax: 00-965-5350621;
E-mail: Asbeutah_akram@hotmail.com
most important etiological factors which can be involved in
the development of CVI [5].
Patients with varicose veins exhibit increased threshold
autonomic nerve function to temperature and vibration stim-
uli [6]. Reduced contractile responses to noradrenaline [7]
and imbalance between endothelial cell-derived contractile
and vasodilator responses [8] have been described for vari-
cose veins. Hypothermia cause vasoconstirction of normal
veins while varicose veins are not affected [9]. Also Thule-
sius found in normal veins no endothelial-derived nitrous
oxide (NO) relaxation upon acetylcholine stimulation,
whereas NO was released from the endothelium of varicose
veins [7]. However, capacitance veins have been shown to
both release basal and stimulated NO [10]. Moreover, the
reduced tone in varicose veins and loss of endothelial de-
rived contracting factor (EDCF) may be related to a primary
endothelial abnormality since ultra-structural studies have
shown endothelial damage in varicose veins [11, 12].
Investigators have concentrated on the collagen, elastic,
and muscle fiber components of the vein wall and their effect
on the overall physical properties of the conduit [13]. Ab-
normalities in these components are certainly important but

16 The Open Cardiovascular Medicine Journal, 2007, Volume 1 Asbeutah et al.
the endothelial lining of the vein wall and flow properties of
the blood within these have received much less attention [14-
16].
The aim of the in vivo phase of the study was to examine
vein function by measuring diameter changes of the great
saphenous vein (GSV) using duplex ultrasound (DUS) be-
fore, during, and after venous occlusion in control, normal,
and varicose veins subjects. In the second in vitro phase of
the study the aim was to investigate if noradrenaline (NA)
and 5-hydroxytryptamin (5-HT) induce responses of isolated
control and varicose veins that were altered by removal of
the endothelium.
METHODS
Subjects
Subjects were recruited from the general population prior
to varicose vein surgery (varicose vein group) and undergo-
ing coronary artery bypass graft (CABG) but without vari-
cose veins (control group). A total of 24 venous segments
specimens were studied. Varicose vein subjects undergoing
surgery for stripping the great saphenous vein were asked to
be off all venotropic drugs including Daflon 500 mg. Control
subjects undergoing CABG were required to have no vari-
cose vein or any evidence of venous insufficiency. Ten nor-
mal volunteers without any history of diseases or medica-
tions were matched for age and sex for comparison.
Subjects were asked to complete a questionnaire adminis-
tered by the author that included personal and occupational
details, relevant medical and family history, and possible risk
factors for venous disease. They underwent duplex ultra-
sound to diagnose venous reflux and the great saphenous
vein, above knee, was assessed for diameter changes in re-
sponse to occlusion-release stimulation, and was marked one
day before surgery by permanent pen, so that the vein seg-
ment could be harvested for in vitro part of the study. The
study was approved by the Human Research and Ethics
Committee from Kuwait University, Faculty of Allied Health
Sciences and Ministry of Health, Mubarak Al-Kabeer Hospi-
tal at Kuwait. All subjects were asked to sign the consent
form.
Duplex Ultrasound Scanning (DUS) Techniques- In Vivo
Part
All subjects were assessed in temperature-controlled
(22±2°C) environment using a Philips HDI 5000 ATL (Phil-
ips Medical Systems, Bothell, WA) ultrasound machine
equipped with linear 7-4 MHz transducer. Venous reflux was
elucidated by manual compression distal to venous segments
under examination. Venous reflux duration more than one
second is considered abnormal [17]. Firstly, venous reflux
was assessed in all subjects in standing position while they
were holding to the edge of a couch or frame and the weight
distributed in the other leg. Measurements were made along
the deep and superficial veins of leg with varicose veins
(varicose vein group), veins of subject legs without varicose
veins prior to GABG (control group), and normal legs (nor-
mal group). The venous reflux procedure was similar to the
previously described method [17, 18].
Subject was then placed in a supine position with the leg
to be investigated elevated 30cm at the heel. The knee was
slightly flexed and externally rotated while ensuring ade-
quate relaxation of the leg. After 20 minutes rest the diame-
ter of the great saphenous vein 4 cm above the kneecap was
measured and then venous occlusion was induced by infla-
tion of a pneumatic cuff (17 cm) placed around the upper-
mid thigh to a pressure of 80 mmHg until the maximum di-
ameter is achieved. When there was no further changes in the
diameter (approximately 5 minutes), the cuff was deflated.
The diameter of the vein was measured every 30 seconds for
the first two minutes and then every one minute for five
minutes. The great saphenous vein diameter was measured
with B-mode ultrasound during quite respiration. In applying
the transducer against the cutaneous surface, minimal pres-
sure was used to guarantee satisfactory saphenous vein imag-
ing, while avoiding undesirable compression that could have
modified vein diameter assessment. This was easily obtained
in every case, since vein compression is clearly visible on
the ultrasound image. In measuring vein diameter the axis
parallel to the skin was preferred as it depends more on the
flattening of the saphenous vein [19]. Venous diameters
were measured from wall to wall in longitudinal section 4
cm above the kneecap base using the electronic callipers
incorporated in the duplex ultrasound machine software.
Once satisfactory position was found, the investigator re-
quested the subject to maintain that same position throughout
the study. Diameters were determined where the walls were
best visualized, ignoring luminal irregularities.
Following studies of in vivo venous responses to occlu-
sion and reperfusion, surface skin markings were made 4 cm
about the knee in 12 of the legs exhibiting varicose veins and
in 12 of the normal legs of control subjects awaiting CABG
surgery. At surgery the marked great saphenous vein seg-
ments were harvested for subsequent in vitro pharmacologi-
cal studies.
Vein Harvesting & Pharmacological Study- In Vitro Part
The specimens were harvested at operations for radical
removal of varicose veins under pentobarbital-halothane or
spinal anesthesia. Moreover, the anesthesia for control: di-
azepam 100 μ/kg, papaveretum 120 μg/kg, fentanyl 40 μg/kg
and pancuronium bromide 100 μg/kg.
After excision the vein samples were put into cool Krebs-
Henseleit solution and send for testing immediately or kept
in a fridge at 4ºC for testing within 1-4 hours of sample col-
lection. The specimen was cleaned of connective tissue and
fat and cut into two rings, each 4 mm in leng th. During this
procedure great care was taken to avoid unintentional rub-
bing of the intimal surface so as to maintain the integrity of
the endothelial layer and meanwhile not damaging the mus-
cular layer of the vessel. One ring served as control and the
other was de-endothelialized by inserting the tip of a small
forceps and gently rolling the ring for 15 seconds according
to the method of De May and Vanhoutte [20]. The prepara-
tions were vertically mounted in 25 ml organ bath filled with
Krebs-Henseleit solution (NaCl 113 mmol/L; KCl 4.6
mmol/L; CaCl2 2.5 mmol/L; MgSo4 1.2 mmol/L; NaHCO3
22.1 mmol/L; KH2PO4 1.1 mmol/L and glucose 11.0
mmol/L) maintained at pH 7.4, at a temperature 37ºC and
gassed with 95% oxygen and 5% carbon dioxide.
The vein rings were attached to the bottom of the organ
bath and the upper end connected to dynamometer UF-1

Varicose Veins Wall Changes The Open Cardiovascular Medicine Journal, 2007, Volume 1 17
force transducer. Isometric contractions were continuously
recorded on a Lectromed 4-channel polygraph (MultiTrace
4P). Before the experiments were begun a pretension of 2
gram was applied to the preparation by adjustment of a mi-
crometer screw. The system was allowed to equilibrate for
30-40 minutes. After the period of equilibration, KCl (80
mM) was added to the bath to test for tissue viability. This
concentration of KCl was repeated after 30 minutes. The
tissues were then washed repeatedly over the next 30 min-
utes period. Thereafter, agonist (NA or 5-HT) concentration-
response curves were established by adding, cumulatively,
increasing concentrations (0.5 log unit increments) of each
agonist to the bath and allowing the response to each concen-
tration reach a peak before adding the next concentration. In
all cases, two consecutive concentration-response curves
were separated determined by a rest period of 60 minutes.
The second concentration-response curve was used as the
control (Fig. 1). Agonist potencies were expressed as pD2
values, where pD2 is the negative logarithm of the agonist
concentration producing 50% (EC50) of the maximum re-
sponse. EC50 and maximal contraction were determined
from dose-response curves.
Rubbed and non-rubbed rings from the same vessel were
subjected to identical testing steps which were run and re-
corded simultaneously in two organ baths. Similarly, vaso-
constrictor drug effect in control veins was tested with
rubbed and non-rubbed endothelium. The experiments were
repeated using the drug 5-HT on varicose and control vein
segments from different subject. At the end of the experi-
ments, the venous segments were blotted and weighed.
Maximum response was expressed as mg/mg tissue weight.
The ultrasound measurements were performed by the
author. Data was stored into the hard disks of the ultrasound
machine and any data from any former ultrasound measure-
ment was not examined prior to the next diameter measure-
ment. The pharmacological tests were performed with the
help and supervision of one pharmacology technologist.
Statistical Analysis
Data was entered and analyzed using the SPSS version
12 for Windows. Data are reported as mean±SD. The multi-
ple repeated measurement (ANOVA) was used to determine
the significance of difference between control, normal, and
varicose veins (percent diameter changes from maximum
diameter at occlusion and difference diameter changes from
maximum at different time intervals). Cross-tabulation
analysis (Chi-Square test) was used to test for the signifi-
cance of the differences among groups in return of vein di-
ameters to < 10% of resting baseline.
Data was recorded as mean of ‘n’ experiments where n is
the number of samples from ‘N’ subjects. Graphs were plot-
ted and analyzed using Graphpad Prism software. Differ-
ences between mean values were tested for significance us-
ing paired Student's t-test. The difference was assumed to be
significant when p<0.05. The relationship between duplex
ultrasound and pharmacological parameters were carried out
using Pearson correlation coefficients test.
RESULTS
Subject Characteristics
Specimens of the great saphenous vein were obtained
from 12 subjects with primary varicose veins above knee (6
females and 6 males, aged 41.4±8.5 (mean±SD year), and
with body mass index (BMI) 27.1±4.5 (mean±SD kg/m).
Control great saphenous vein specimens were obtained from
donor vessels of the great saphenous vein above knee at car-
diac surgery from 12 subjects (6 females and 6 males, aged
50.2±8.3, (mean±SD years) and with BMI 29.9±4.5
(mean±SD kg/m). Table 1 summarize population character-
istics. Table 2 summarizes subject's medical conditions and
medication history.
In Vivo Duplex Ultrasound Findings
Table 3 presents the mean, percent, and difference of
diameter changes of great saphenous vein from maximum
Fig. (1). Shown here are contractile responses of two rings of a varicosed human saphenous vein preparation recorded simultaneously. One
rings with endothelium (I) and the other de-endothelialized (II). First part: washing and stabilizing with 80 mM KCL solution; and second
part: dose-response to increasing contractions of NA.

18 The Open Cardiovascular Medicine Journal, 2007, Volume 1 Asbeutah et al.
Table 1. Subject Characteristics
Characteristic Control Normal Varicose Veins
Subject & Limb
numbers 12 10 12
Age (mean±SD, years) 50.2±8.3 38.7±7.7 41.4±8.5
Sex (Number, M/F) 6/6 5/5 6/6
Body Mass Index
(mean±SD, kg/m) 29.9±4.5 28.1±1.8 27.1±4.5
Table 2. Subject Medical Conditions and Medications
Characteristic Control Veins (N)
Varicose Veins (N)
Medical Conditions
DM, HT, HL, & MI 5 3
DM, HT, & MI 3 0
DM, HL, & MI 3 0
DM & HT 4 0
HT 0 2
HT & HL 4 0
HT, MI, & CVA 2 0
HT & MI 3 0
DM & MI 1 0
Medications
Aspirin 24 3
Isosorbide dinitrate 17 3
Atenolol 10 2
Metroprolol 10 2
Propranolol 1 0
Amlodipine 5 3
Diltiazem 3 0
Captopril 4 0
Lisinopril 2 0
Frusemide 4 0
Atorvastatin 6 0
Simvastatin 4 0
Micronized flavonoid
(diosmin 90% and hes-
peridin 10%) 0 2
DM= diabetes mellitus; HT= hypertensive; HL= hyperlipidemic; MI= myocardial
infarction; CVA= cerebrovascular accident; and N= number of subjects.
diameter during the resting phase (pre-occlusion), at the end
of 5 minutes occlusion, and then every 30 seconds post de-
flation for the first two minutes, and every 1 minutes until
five minutes post deflation for 12 control, 10 normal, and 12
varicose vein segments.
The control venous segments average resting diameter
was 0.42±0.06, for normal venous segment was 0.4±0.06,
Table 3. Mean Vein Diameter (M), Percent (%) Diameter
Changes from Maximum, and Difference (D) Diame-
ter Changes from Maximum Data for Control,
Normal, and Varicose Vein Segments
Time Control Veins
Normal Veins
Varicose Veins
Resting M 0.42±0.06
0.40±0.06
0.42±0.11
% 76±5
77±6
72±11.5
D -0.13±0.03
-0.12±0.03
-0.16±0.08
Maximum M 0.55±0.06
0.52±0.06
0.58±0.12
At 300s % 100
100
100
Occlusion D 0
0
0
30s post M 0.44±0.06
0.43± 0.05
0.48±0.11
Deflation % 80±5.5
82±4
82±8
D -0.11±0.03
-0.09±0.02
-0.10±0.06
60s post M 0.44±0.06
0.41± 0.05
0.48±0.11
Deflation % 80±5.2
79±6.7
82±9
D -0.11±0.03
-0.11±0.04
-0.10±0.06
90s post M 0.43±0.06
0.41± 0.05
0.46±0.11
Deflation % 78±5.3
79±7
79±9
D -0.12±0.03
-0.11±0.04
-0.12±0.06
120s post M 0.43±0.06
0.40± 0.05
0.46±0.11
Deflation % 78±5.3
77±7
79±9
D -0.13±0.03
-0.12±0.04
-0.12±0.06
180s post M 0.43±0.06
0.40± 0.05
0.45±0.11
Deflation % 78±5.7
77±6.3
77±9.5
D -0.13±0.03
-0.12±0.04
-0.13±0.07
240s post M 0.43±0.06
0.40± 0.05
0.45±0.11
Deflation % 78±5.4
77±6.3
77±9.5
D -0.13±0.03
-0.12±0.04
-0.13±0.07
300s post M 0.43±0.06
0.40± 0.05
0.45±0.11
Deflation % 78±5
77±6.3
77±10
D -0.13±0.03
-0.12±0.04
-0.13±0.07
Values are mean±SD of diameter for all subject groups. ANOVA test showed signifi-
cance within and between all groups at different time interval (p<0.05).
and for varicose veins was 0.42±0.11. Average maximum
dilation diameter after 5 minutes of occlusion was 0.55±0.06
in control, 0.52±0.06 in normal, and 0.58±0.12 in varicose
vein segments, and diameter alteration had returned to 10%
original resting diameter in 9 out of 12 control veins, 10 out
of 10 normal veins, and 4 out of 12 varicose veins segments
within 30 seconds of deflation but the remaining 3 control
and 8 varicose vein segments the diameter alteration had still
not returned to resting within more than 60 second of defla-
tion (Cross-tabulation analysis). In addition, the cross-
tabulation analysis were carried out for 20% and 30% and
found to be significant but not at 30% for all groups. It is
apparent that the varicose vein segments dilate more and
return more slowly to original resting diameter than the con-
trol and normal venous segments.

Varicose Veins Wall Changes The Open Cardiovascular Medicine Journal, 2007, Volume 1 19
The repeated measure ANOVA test for the diameter,
percentage, and difference changes of great saphenous vein
diameter from maximum diameter at different time interval
showed significance difference (p <0.05) within and between
control, normal and varicose vein venous segments. Data are
represented in Table 3.
In Vitro Pharmacological Findings
NA and 5-HT produced concentration-dependent con-
tractions of control and varicose saphenous vein segments.
There was no significant difference in the potency of NA
(log EC50 values were -5.55±0.06 and -5.58±0.10, n =6, in
control and varicose vein segments, respectively) and for 5-
HT (log EC50 values were -6.44±0.08 and -6.66±0.09, n=5,
in control and varicose vein segments, respectively) between
the two groups but the maximum response, normalized for
tissue weight, was less in varicose vein segments for both
NA and 5-HT. Results are represented in Fig. (2a,b) and
Table 4.
a
b
Fig. (2). Bar graphs of human great saphenous vein rings to increas-
ing concentration of (a) NA and (b) 5-HT for control and varicose
vein segments. Endothelium (+) and rubbed (-) in each control (Cv)
and varicose veins (Vv) specimens.
*p<0.05 Cv+ compared to Vv+ and # p<0.05 Vv+ compared to Vv-
using Student's paired t-test.
Removal of the endothelium significantly (p<0.05) re-
duced the maximum response to NA in control veins. The
maximum response to NA in varicose vein segments was not
statistically significant after endothelial denudation. Re-
moval of the endothelium had no effect on the potency of 5-
HT in control vein segments but significantly (p<0.05) re-
duced the potency of 5-HT in varicose vein segments (log
EC50 values were -6.66±0.09 and -6.13±0.10, n=5, in vein
segments with and without the endothelium, respectively).
Results are represented in Fig. (2a,b) and Table 4.
Table 4. Contractile Effect (Mean±SEM) of NA and 5-HT on
Control and Varicose Vein Segments (+) with Endo-
thelium; (-) Without Endothelium; (Cv) Control
Vein Specimens; (Vv) varicose vein specimens; N =
Number of Specimens; EC50 = 50% Effective Con-
centration; Max Cont = Maximum Contraction
mg/mg Tissue Weight; * p<0.05 Cv+ Compared to
Vv+; # p<0.05 Vv+ Compared to Vv- Using Paired
Students' t-Test
Noradrenaline 5-Hydroxytryptamine
log EC50 Max Cont N
log EC50 Max Cont N
Cv+
-5.55±0.06
80.62±15 6 -6.44±0.08 67.63±16.41 6
Cv- -5.71±0.08
45.42±11.4
6 -6.30±0.14 94.87±34.26 6
Vv+
-5.58±0.10
44.4±19.2*
7 -6.66±0.09 26.88±5.32 5
Vv- -5.58±0.08
26.9±9.3 6 -6.13±0.10# 8.08±4.33# 5
The relationships between in vivo and in vitro responses
were examined and correlations are shown in Figs. (3a,b)
and (4). Whilst there was a trend for an inverse relationships
between the maximum contraction responses to NA, 5-HT
and the rate of contraction/return to resting diameters of the
same venous segments in vivo, the results did not achieve
statistical significance. The (r) values for 5HT responses and
combined 5HT and NA responses were -0.32 and -0.40
respectively.
DISCUSSION
The main findings of the ultrasound component of the
study were that the control and normal group showed almost
identical responses. This suggests that the venous wall was
functioning normally in the control group. In contrast, in the
varicose vein group the vein dilated more and returned more
slowly to its original diameter. A weaker maximal contrac-
tion in varicose veins when compared to control veins was
expected and this was confirmed in this study.
The findings of the present study must be taken in con-
text with the subjects investigated and the methods of inves-
tigations. The former is important because the control group
subjects had underlying risks for cardiovascular diseases as
from Table 2 it is clear that the majority of subjects from
which the veins were taken had hyperlipidemia, diabetes or
both. Furthermore they were, as would be expected, on mul-
tiple drugs including a large number on nitrates which could
clearly have an effect on their responses but, more impor-
tantly, they did not have any history of CVI as investigated
clinically and by ultrasound. To overcome this problem,
healthy volunteers without any venous diseases or any medi-

20 The Open Cardiovascular Medicine Journal, 2007, Volume 1 Asbeutah et al.
cations history were entered to our present study to correlate
the diameter changes by ultrasound to control group versus
the varicose vein group. The method of investigation is also
important because it may affect the sensitivity of the out-
come. The ultrasound diameter measurements in all subject
groups were assessed by the same experienced vascular
technologist (A.A) in a room at constant temperature
(22±2°C).
a
b
Fig. (3). Correlations of the maximum contraction responses of
varicose and control vein segments as measured in vitro for (a) NA;
and (b) 5-HT to time to return to 10% of resting diameter as meas-
ured in vivo.
The pharmacological study confirmed the findings of
Thulesius [7] in that there was contractile reduction in the
maximal contractile force of varicose vein preparations to
NA and 5-HT compared to controls. This was more evident
when the endothelium was denuded. The veins were de-
endothelialised by using the standard technique of De May
and Vanhoutte [20]. Moreover, endothelial denudation in-
creased the maximum response to 5-HT in control vein seg-
ments but this was not statistically significant. This data
could be explained by a lack of release of endothelial derived
relaxing factor(s) (EDRFs), in particular NO as was sug-
gested by Datte and colleagues in studies of isolated rat por-
tal vein segments [21]. However, the results must be inter-
preted with caution given the large variation in contractile
responses of the vein segments, possibly influenced by
stretch or damage to the tissue during surgery.
Fig. (4). Relationship between maximum contraction responses of
varicose and control vein segments as measured in vitro with NA or
5-HT and time to return to 10% of resting diameter as measured in
vivo.
The reason for reduced contraction of varicose veins may
involve disordered smooth muscle responsiveness and/or
endothelial dysfunction. The reduced contractile responses to
NA suggest the former; the responses of GSV segments from
subjects with varicose veins to 5-HT, showing a further de-
crease in maximum contraction after endothelial denudation,
in contrast to the control vein responses, suggest the latter. It
is possible that the significant fall in 5-HT induced maxi-
mum contraction of the varicose vein rings after endothelial
denudation may reflect an endogenous endothelial-dependent
venoconstrictor influence in these rings, perhaps due to en-
dothelin.
By correlating the ultrasound findings with the pharma-
cological findings, it might be considered that dilatation of
the varicose vein segments with slow return to initial resting
diameter represents a contraction dysfunction of the venous
wall initiated most probably by the endothelium due to defi-
cient EDCF or due to a release of EDRF in a healthy vascu-
lar smooth muscle fibres in the venous wall of the varicose
vein group. The results of a previous study [22] clearly dem-
onstrated intimal hypertrophy and hyperplasia of the suben-
dothelial smooth muscle cells in saphenous varicose vein
samples. Also they reported that the changes in the subendo-
thelial smooth muscle cells did not correlate with any
changes in the endothelial cells of the varicose veins studied.
They suggested that the endothelial cells do not go through
the same cascade of events that the smooth muscle cells un-
dergo. Despite the fact that endothelial cells not only act as
barriers but also as regulators of vascular tone [23]. Never-
theless, it has been reported that a significant increase in
endothelial adhesion molecule ICAM-1, associated with
more thromboxane A2 and prostaglandin E2 synthesis, and
with an increased interaction among leukocytes, platelets and
endothelial cells, is observed in varicose veins [24, 25]. This
could imply that although the morphology of endothelial
cells may not change in varicosis, the overall function of

Varicose Veins Wall Changes The Open Cardiovascular Medicine Journal, 2007, Volume 1 21
such cells may. In the present study, the combination of
structural and functional differences between normal and
varicose GSVs defined by ultrasound, with differences in in
vitro contractility of the same segments, suggest that endo-
thelial dysfunction may be an important aetiological factor in
the pathogenesis of primary varicose veins and CVI devel-
opment.
Whilst there was a trend for the maximum in vitro veno-
constrictor responses to be inversely related to the in vivo
functional responses (time of return of vein within 10% of its
resting diameter), with (r) value of -0.32 for all data and -
0.40 for 5-HT responses, these do not reach statistical sig-
nificance. The significant variability of responses suggests
that greater numbers need to be studied to further explore
this relationship.
Finally, in order to come up with a proper management
of varicosis, it is essential to understand the basic mecha-
nisms leading to the development of such a disease in people
at various ages. The prevailing view of the aetiology of vari-
cose veins revolves around the trigger(s), as well as the cas-
cade of events. A better understanding of the nature of the
triggers(s) and of the progressive changes occurring in the
vessel wall of the varicose vein, obtained by combining
structural and functional studies, including electron micros-
copy as well as pharmacological techniques, may lead to not
only a better treatment, but also prevention of the disease.
Also studies are necessary to explore haematological as well
as biochemical markers in varicose veins subjects, and how
these change with disease development. In view of that en-
dothelial damage may cause vascular smooth muscle dys-
function that favours dilatation and secondary valvular insuf-
ficiency, it could equally well be that valvular insufficiency
leads to dilatation which causes endothelial damage. Our
present studies do not allow differentiation between these
two possibilities and further studies are needed to resolve
such issue. In addition the role of calcium storage in vascular
smooth muscle in the wall of the veins warrant more study.
CONCLUSION
Varicose venous segments dilated more during occlusion
hyperemia and returned slowly to their original resting di-
ameter when compared to control and normal venous seg-
ments. This might be attributed to venous wall dysfunction.
Venous endothelial damage may cause vascular smooth
muscle contractile dysfunction that favours dilatation and
secondary valvular insufficiency with deficient cusps apposi-
tion or valvular insufficiency leads to dilatation which causes
endothelial damage. This may be the primary factor in the
development of varicose veins and CVI.
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Received: Jul 5, 2007 Revised: July 13, 2007 Accepted: July 14, 2007