Serum concentration of procoagulant,endothelial and oxidative stress markers in early primary varicose veins

Abstract

Objective

Endothelial disorders of the veins are considered to play a role in the pathogenesis of primary varicose veins and oxidative stress is implicated in these disorders. With this aim, we investigated vascular endothelial marker levels and the effect of endothelial damage on coagulation parameters and vasodilator substances to determine metabolic markers of oxidative stress in patients with varicose veins and vascular endothelial damage caused by oxidative stress.

Methods

We investigated the levels of malondialdehyde (MDA), protein C, protein S, fibrinogen, von Willebrand factor (vWf), tissue plasminogen activator (tPA), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-12 (IL-12) and nitric oxide (NO) in the blood taken from the brachial vein of 25 patients with primary varicose veins (study group) and compared the results with 25 healthy volunteers (control group). We also investigated the urinary levels of prostaglandin I₂ (PGI₂) in the study group and compared the results with the control group.

Results

There was no statistically significant difference between the study group and the control group with respect to the plasma levels of MDA, protein C, fibrinogen, tPA, IL-6, NO and urinary levels of PGI₂. But plasma levels of protein S, vWf, VEGF and IL-12 were found to be statistically significantly higher in the study group than in the control group.

Conclusions

Although the systemically measured levels of protein S, vWf, VEGF and IL-12 were found to be higher in patients of primary varicose veins, levels of protein C, fibrinogen, homocysteine and PGI₂ were not found to be statistically significant. No systemic increased oxidative stress seems to be related to the early stages of chronic venous insufficiency. Further studies are warranted for understanding the role of molecules such as NO, oxidative stress parameters and cytokines.

Keywords

Varicose veins inflammation vascular endothelial growth factor

Introduction

Incidence of varicose veins in adult population has been shown to vary among populations (between 10% and 60%) and to increase by age in various studies.^1–6 The main factors in the aetiology of varicose vein are venous dilation and valvular insufficiency that are started by unknown factors.^1–3,7,8 It is reported that mediators secreted from endothelium, such as nitric oxide (NO), prostaglandins and cytokines, have been thought to play a role in vasodilatation and vascular permeability in recent years.^2,5,9,10

Vasodilation and venous stasis cause relative hypoxia and hyperviscosity. Hypoxia and the decrease in pH cause leucocytes to be activated. Activated leucocytes secrete toxic metabolites, proteolytic enzymes and reactive oxygen metabolites.^6,7,9–11 These secreted mediators have toxic effects on vessel walls and surrounding tissue, hence, the capillary permeability increases and progressive cell and tissue destruction occurs.

A tendency to thrombogenicity occurs as a result of the endothelial damage. It is known that thrombosis occurs at the capillary level in the development of leg ulcers, a late stage of venous insufficiency.^3,10 No study has been performed to investigate thrombosis development in earlier stages. In addition, whether there is a tendency to coagulate or not in patients with clinical, aetiological, anatomical and pathological elements (CEAP) stages II and III, or whether varicose vein development increases or not in the pathologies causing hypercoagulation is not yet known. Furthermore, the endothelial damage seen in patients with varicose veins can contribute to thrombosis formation. With this aim, we wanted to study the coagulation state by measured levels of protein C, protein S, fibrinogen and homocysteine in patients with early varicose veins. In addition, we investigated vascular endothelial damage by measuring the levels of von Willebrand factor (vWf), tissue plasminogen activator (tPA), vascular endothelial growth factor (VEGF) in the blood taken from arm vein.

NO and prostaglandin I₂ (PGI₂), which also affect vascular system and control local venous tonus, are strong vasodilator substances.² An increase in NO and PGI₂ levels is expected in patients with varicose veins. However, studies performed up to now have given quite contrary results,^12,13 and free radicals have been implicated in this. Therefore, the role of free radicals and vasodilator substances in patients with varicose veins should be investigated again. It has also been reported that changes in the varicose vein occur by the effects of cytokines secreted from smooth muscle cells.¹³ In this study, we aimed to investigate cytokine levels and vasodilator mediators by measuring VEGF, interleukin-6 (IL-6), interleukin-12 (IL-12), NO, PGI₂ and malondialdehyde (MDA) in the blood taken from the brachial vein in patients with varicose veins (study group) and by comparing the results with the control group.

Materials and methods

Patients

Twenty-five patients were enrolled in this study. The patients considered for the study were those attending Kahramanmaras Sutcu Imam University Hospital for the management of venous disease. The diagnosis of primary varicose vein was performed by the combination of clinical examination and duplex scanning examination. Patients were C2 according to the CEAP classification. The exclusion criteria for the study were deep vein thrombosis, post-thrombophlebitic syndrome, deep venous reflux, skin ulcers, recent infection, alcoholism, smoking and those receiving medication. Patients with vascular, metabolic, neoplastic and inflammatory diseases were also excluded from the study, because oxidative–antioxidative balance is known to be disturbed in such patients.⁹ In addition, these conditions severely decrease vascular PGI₂ production.⁶

Twenty-five normal, healthy volunteers acted as control subjects. They had no symptoms or signs of arterial or venous disease in either limb. In addition, the control subjects did not have any disease established by careful history, examination and routine laboratory test. All individuals enrolled in this study were non-smokers and were receiving no medication.

Informed written consent was obtained from all patients and control volunteers fulfilling the entry criteria. This study protocol was approved by the Ethics Committee of Kahramanmaras Sutcu Imam University Faculty of Medicine.

Methods

The study was conducted in an environmental chamber at 22°C. All the patients and control subjects were acclimatized for 10–20 min. Blood samples were taken from the antecubital vein by atraumatic puncture. Blood samples were collected into citrate tubes (for protein C, protein S, fibrinogen, homocysteine, tPA, vWf, VEGF and MDA) and plain tubes (for NO, IL-6 and IL-12), and centrifuged at 4000 rpm (revolutions per minute) for 4 min to obtain plasma and serum fractions. Samples were then stored at −20°C until assayed. Urine samples were collected in plain tubes (for PGI₂) and were frozen at −20°C before analysis. Before assay, the samples were thawed at room temperature.

MDA

The MDA method was based on the measurement of the absorbance of thiobarbituric acid–MDA. The basis of the method used is the reaction of MDA with thiobarbituric acid to form a coloured complex, which can be quantified spectrophotometrically from its visible absorbance (E _max = 532 nm). MDA formed by lipid peroxidation was measured after incubation at 95°C with thiobarbituric acid under aerobic conditions (pH 3.4). The pink colour produced by these reactions was measured spectrophotometrically at 532 nm. Plasma MDA level was expressed as nmol/mL.

Proteins C and S

The assays were performed using an STA Compact coagulation analyser (Diagnostica Stago Inc., Asnières, France) with Diagnostica Stago test kits (STA-CLOT^®, Diagnostica Stago Inc.). Quantitative determination of functional protein C depended on the prolongation of activated partial thromboplastin time. Protein S was determined in accordance with the inhibition of activated factor V. The normal ranges of protein C and S antigens in plasma were 70–130% and 65–140%, respectively.

Fibrinogen

The fibrinogen concentrations were determined according to the Clauss method,¹⁴ using the STA Compact automatic coagulation analyzer with STA Fibrinogen 2 reagent (Diagnostica Stago Inc.). The test is based on clot formation of citrated plasma after the addition of excess thrombin. The normal range of fibrinogen level was 200–400 mg/dL.

Homocysteine

Serum total homocysteine levels were determined by chemiluminescence immunoassay.

vWf, tPA and VEGF

The vWf, tPA and VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercial reagents (for vWf and tPA: Diagnostica Stago Inc.; and for VEGF: R&D Systems Inc., MN, USA). The vWf, tPA and VEGF levels were presented as %, ng/mL and pg/mL, respectively.

IL-6 and IL-12

The IL-6 and IL-12 levels were measured by ELISA using commercial reagents (IL-12+p40 and IL-6 Immunoassay kits, BioSource Europe S.A., Nivelles, Belgium). The serum levels were presented as pg/mL.

PGI₂

ELISA was used to determine the urinary levels of PGI₂. Commercial kits were used according to the manufacturer's instructions (R&D Systems Inc.). The normal range of PGI₂ level was 107–171 ng/g.

NO

Samples were initially deproteinized with Somogyi reagent. Plasma NO was measured in terms of its products, such as nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻), using the Griess reaction by copperized cadmium granules. NO products were presented as μmol/dL plasma.

Statistical analysis

Differences between the groups were evaluated by Student's t-test and were considered statistically significant at P < 0.05. Results were reported as mean ± SEM.

Results

The ages of the subjects with varicose veins included in our study (study group) ranged between 19 and 55 years (mean 38.3 ± 10.7 years) and the ages of the subjects in the control group ranged between 22 and 50 years (mean 33.1 ± 7.9 years); there was no statistically significant difference between the ages of both the groups. The women:men ratio was 11:14 in the study group and 10:15 in the control group. No statistically significant difference was observed between both the groups with respect to haematological and biochemical parameters, including whole blood count, erythrocyte sedimentation rate, prothrombin time, partial thromboplastin time, C-reactive protein level, glucose level, cholesterol profile, electrolyte levels, liver and kidney function tests, and protein levels (Table 1). These parameters were within normal ranges in both the groups.

Table 1

The comparison of haematological and biochemical parameters in patient and control groups

Parameters	Patients group	Control group
Haemoglobin (g/dL)	13.8 ± 1.4	13.5 ± 1.3
Haemotocrit (%)	41.7 ± 4.6	41.0 ± 4.0
White blood cell (K/μL)	5773 ± 1947	5491 ± 1696
Platelet (K/μL)	269,261 ± 56,998	257,916 ± 51,744
Sedimentation rate (mm/h)	16.7 ± 13.5	18.0 ± 11.7
INR	1.1 ± 0.1	1.0 ± 0.1
aPTT (s)	27.5 ± 2.9	28.7 ± 2.4
Creatinine (mg/dL)	0.8 ± 0.1	0.8 ± 0.2
Total cholesterol (mg/dL)	180.8 ± 33.2	177.6 ± 18.9
Triglyceride (mg/dL)	120.5 ± 61.4	101.5 ± 46.2
HDL cholesterol (mg/dL)	36.5 ± 8.8	44.5 ± 9.1
LDL cholesterol (mg/dL)	120.2 ± 20.6	116.4 ± 20.3
AST (IU/L)	21.4 ± 5.7	22.1 ± 2.4
ALT (IU/L)	43.7 ± 12.0	40.7 ± 6.9
Total protein (g/dL)	7.3 ± 0.5	7.2 ± 0.2
Albumin (g/dL)	4.2 ± 0.4	4.2 ± 0.2
Sodium (mmol/L)	139.4 ± 3.3	140.7 ± 2.3
Potassium (mmol/L)	4.4 ± 0.3	4.3 ± 0.3
Chlorine (mmol/L)	103.0 ± 3.3	102.8 ± 1.6
C-reactive protein (mg/L)	5.7 ± 4.5	5.8 ± 6.4

HDL – high-density lipoprotein; LDL – low-density lipoprotein; AST –aspartate aminotransferase; ALT – alanine aminotransaminase

INR – international normalized ratio

aPTT – activated partial thromboplastin time

No statistically significant difference was observed between both the groups with respect to the levels of protein C, fibrinogen, homocysteine, PGI₂, tPA, MDA, NO and IL-6, which were the most important parameters of study (Table 2). While the average plasma level of protein S was 102.9 ± 35.4% in the study group, it was 72.2 ± 31.4% in the control group, and this difference was statistically significant (P < 0.05). While the average level of plasma vWf was 131.8 ± 50.2% in the study group, it was 73.0 ± 55.5% in the control group, and this difference was also statistically significant (P < 0.01). The plasma levels of VEGF were statistically significantly higher in the study group than in the control group (258.5 ± 139.7 pg/mL in the study group, 49.5 ± 23.7 pg/mL in the control group, P < 0.01). Although the average plasma level of IL-12 was 25.2 ± 15.2 pg/mL in the study group, it was statistically significantly lower in the control group (5.8 ± 5.9 pg/mL, P < 0.01). Results of our study are summarized in Table 2.

Table 2

The comparison of study parameters in patient and control groups

	Patient group	Control group	P
MDA	4.2 ± 4.6	4.4 ± 4.5	NS
Protein C (%)	119.1 ± 34.5	108.5 ± 46.1	NS
Protein S (%)	102.9 ± 35.4	72.2 ± 31.4	<0.05
Fibrinogen (mg/dL)	296.3 ± 81.7	297.6 ± 83.3	NS
Homocysteine (μmol/L)	12.7 ± 2.8	14.9 ± 7.3	NS
vWf (%)	131.8 ± 50.2	73.0 ± 55.5	<0.01
tPA (ng/mL)	22.4 ± 3.3	21.7 ± 2.3	NS
VEGF (pg/mL)	258.5 ± 139.7	49.5 ± 23.7	<0.01
IL-6 (pg/mL)	3.5 ± 4.7	1.3 ± 1.4	NS
IL-12 (pg/mL)	25.2 ± 15.2	5.8 ± 5.9	<0.01
PGI₂ (ng/g)	126.2 ± 50.8	128.1 ± 77.3	NS
Nitric oxide (μmol/dL)	10.4 ± 8.7	12.2 ± 7.9	NS

NS: not significant

Discussion

With respect to MDA levels, we did not find a statistically significant difference between patients with varicose veins (study group) and the control group in blood samples taken from the brachial vein. MDA is an aldehyde end-product that is produced in highest amount when the lipid peroxidation is generated by free oxygen radicals. The levels of free oxygen radicals can be expected to increase in plasma of patients with varicose veins because tissue ischaemia and the decrease in pH that occur as a result of venous hypertension cause the secretion of large amounts of free oxygen radicals and pro-inflammatory cytokines.^7,10,12 Concordant with our results, Jacob et al. ¹¹ have also not found a statistically significant difference between the blood samples taken from arms and legs of patients with varicose veins with respect to MDA levels and they have shown that experimentally formed stasis did not affect MDA levels. Flore et al. ¹⁵ have investigated the levels of free oxygen radicals in patients with chronic venous insufficiency and have found that the levels of free oxygen radicals in blood samples taken from a foot vein were higher in patients with varicose veins than in the control group and they have reported that these levels returned to normal after saphenous vein stripping. Our finding that the levels of MDA in arm veins were similar in patients in both the study and control groups shows that the disturbance in oxidative–antioxidative balance occurring locally in legs does not affect the systemic circulation. But it is also known that the high levels of free oxygen radicals in foot veins locally damage the vessel. Glowinsky and Glowinsky⁷ have shown that the levels of free oxygen radicals increased in the walls of varicose veins, especially after superficial thrombophlebitis. Our study does not assess increased oxidative stress in early varicose veins, whereas in more advanced stages, with increased iron deposition, several authors described such a finding alongside a relationship with iron stores outside the venous wall.¹⁶ Also, leucocytes and platelets are activated as a result of stasis and hypoxia. It is known that there is thrombosis at the capillary level in patients with venous ulcers. It has been reported that erythrocyte aggregation and plasma levels of fibrinogen increase in chronic venous diseases.¹³ But, in our study, there was no significant difference between the study group and the control group with respect to plasma levels of fibrinogen. We also did not find significant difference between both groups with respect to the levels of other coagulation parameters such as homocysteine and protein C. But, interestingly, although the levels of protein S were found to be in normal ranges in both groups, they were higher in the study group than in the control group, and the difference was statistically significant (P < 0.05). It is not possible to explain the exact reason. Perhaps the levels of protein S, an anticoagulant protein, may have been increased as a compensation mechanism of the body's tendency to thrombogenicity that occurs as a result of the endothelial damage.

Endothelium is the starting point of cell damage in patients with varicose veins. We measured the levels of vWf and tPA to evaluate the endothelial damage, as tPA is generated by endothelial cells and its levels change with localization of endothelial cells.⁴ In view of this, we thought that there could be a change in the plasma levels of tPA due to endothelial damage in patients with varicose veins. However, we did not find a significant difference between the study group and the control group with respect to the plasma levels of tPA. On the other hand, the levels of vWf were significantly higher in the study group than in the control group (P < 0.01). This result shows that the endothelial damage that occurs in the early stage in patients with CEAP stage C2 can be clearly evaluated by measuring plasma vWf levels.

VGEF, which is another endothelial marker, is secreted as a response to local venous hypertension and is also very effective in vascular wall permeability. It plays an important role in increased vascular permeability, tissue oedema, fibrin deposition and lipodermatosclerosis. We found that plasma VGEF levels were statistically significantly higher in the study group than in the control group (P < 0.01). It has also been previously reported that VGEF levels increase in varicose veins.^12,17 Shoab et al.¹⁸ have reported that plasma VGEF levels were high in patients with CEAP stage C4 only, in contrast to our study, and these levels decreased with treatment. But they have not included a control group (of healthy individuals) in their study and, for this reason, they have not compared their results with healthy individuals. Howlader and Smith¹⁹ have shown that plasma VGEF levels were high in all stages, but this was significant in patients with varicose ulcers only. However, we have detected this difference in a significant manner in CEAP stage C2 patients. It is known that VEGF increases vascular permeability and causes growth in blood vessels.¹⁰ This increase in vascular permeability has a minor effect physiologically, but it has a rather major effect in pathological conditions.²⁰

We investigated the levels of cytokines, such as IL-6 and IL-12, in addition to the levels of VGEF. IL-6, a pro-inflammatory cytokine, plays a role in the intensification of inflammatory response. IL-12 strengthens the immunity response by stimulating the formation of γ interferon and activates neutrophils. Although the levels of both cytokines were found higher in patients with varicose veins than in the control group, this difference was significant for IL-12 only (P < 0.02). It is known that an inflammatory reaction occurs in the venous wall by the infiltration of monocytes and macrophages as a result of venous disease, and this causes the secretion of inflammation markers. Also, increased levels of inflammation markers such as IL-1a and TNF α have been determined in some varicose vein segments.²¹ It has been reported that most of the changes seen in the varicose vein wall can occur secondary to the effects of secreted cytokines.¹²

In addition to the effect of cytokines on the vessel wall, vasodilator prostacycline (PGI₂) and NO have been reported as the most effective compounds in the local control of the venous tonus.² PGI₂ is the major prostacycline generated from venous tissue. The level of PGI₂ is higher in veins than in arteries.² Although the level of PGI₂ is high in vein walls, we measured the levels of PGI₂ in urine in patients with varicose veins and did not find statistically significant differences between patients with varicose veins and the control group with respect to the levels of PGI₂. Moreover, this emphasizes that the levels of PGI₂ passing into systemic circulation in patients with varicose veins are not different from the control group. We also found that there was no significant differences between the patients with varicose veins and the control group with respect to NO levels in systemic circulation. But, as NO is a vasodilator and vasodilation occurred in patients with varicose veins, NO levels were theoretically expected to be higher in those patients with varicose veins. On the other hand, as endothelial damage occurs in patients with varicose veins, a decrease in the levels of NO that is secreted from endothelium can also be expected. But we did not find any difference in the levels of NO in our study. NO does not only regulate the venous tonus, but it also has various pathological functions, such as inhibition of the platelet adhesion/aggregation, neurotransmission and cytotoxicity. It also inhibits the growth of vascular smooth muscle and adhesion of inflammatory cells to endothelial surface.²²

Simultaneous measurements of the above-mentioned parameters in varicose vein wall and blood samples obtained from leg and arm may contribute more readily in recognizing the issue. It should be kept in mind that our findings are speculative, as they are more related to the further appearance of complication in varicose veins, rather than to their pathogenesis.

In conclusion, although systemically measured levels of protein S, vWf and VGEF were significantly different in patients with primary varicose veins than the control subjects, the levels of protein C, fibrinogen, homocysteine and PGI₂ were not found to be statistically significant.

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Serum concentration of procoagulant,endothelial and oxidative stress markers in early primary varicose veins

Abstract

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Patients

Methods

MDA

Proteins C and S

Fibrinogen

Homocysteine

vWf, tPA and VEGF

IL-6 and IL-12

PGI2

NO

Statistical analysis

Results

Discussion

References

PGI₂