Diagnostic Center of Altay Region, 

Barnaul, Russia

 

Varicose disease (VD) is diagnosed in 80 % of general population and is the leading cause of chronic venous insufficiency of lower extremities. The disease pathogenesis is complex and is not well studied. The main mechanisms of varicose veins are conditioned by rebuilding of endothelial and connective tissue structures of the wall and its valves, with significant relation to endogenous changes and genetic predisposition [1, 2]. The indicated factors pass into the disease in conditions of distal orthostatic flebohypertension and hypertension in lower extremities [3, 4, 5]. The pathobiochemical and cellular background of these disorders in varicose disease are not studied to the full extent. During the last decades the phenomenon of vascular remodeling has been found in pathogenesis of the main cardiovascular diseases. It is associated with uncontrolled proliferation of smooth muscle cells, elastic fibers of vascular wall, resulting in lengthening, tortuosity, and presenting the adaptive response to regionary hypertension and hypoxia, with key role of oxyradical-induced endothelial hyperproduction of mitogenic factors [6, 7, 8, 9]. The similar metabolic disorders are noted in varicose disease [10, 11, 12, 13]. However, the disease mechanisms are more precisely testified by changes in the region of affected extremities.

 

Objective – to specify the role of endothelial dysfunction in pathogenesis of varicose disease on the basis of the comparative study of the level of systemic and regionary mitogenic factors of venous wall.              

 

MATERIALS AND METHODS

The examination included 69 patients with varicose disease (35 women, 34 men, age of 18-69, mean age of 37 ± 1.8). The duration of the disease was from 1 year till 36 years (10.6 ± 1.2 at average). Concurrent diseases were found in 29 patients (42 %). According to CEAP classification, 63 patients had C2 and C3 types of the disease (table 1). Therefore, most examined patients demonstrated some clinical signs of the disease in view of varicose veins and edema in legs and the feet.

Table 1
Distribution of patients into clinical stages

35 patients with VD and 22 volunteers (age of 25-40) received examination of oxidative-antioxidant status on the basis of blood plasma examination of total oxidant activity (TOA), levels of thiobarbiturate-reactive products (TBRP) measured with spectrophotometric method, and total antioxidant activity of plasma and red blood cells (TAA), activity of the main antioxidative enzymes in erythrocytic hemolysate – superoxide dismutase (SOD) and catalase (CAT).

34 patients were examined for blood plasma levels of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor (FGF basic) and endothelin by means of immunoenzymometric analysis with Multiskan EX, wave length of 450 nm. In 32 cases these peptides were studied in systemic blood flow (v. mediana cubiti), in 21 cases – in the blood (great saphenous vein) flowing from affected extremities (the blood was taken during surgery). Blood samples were incubated at room temperature during 30-60 min. Plasma was separated and stored in a refrigerating chamber at temperature of -20⁰Ñ. The results were compared to the examination data of systemic blood flow from 11 clinically healthy individuals (5 men and 6 women), age of 22-45, mean age of 26.4  ± 1.3.

The exclusion criteria were severe arterial hypertonia, chronic circulatory inefficiency of 2^nd functional class, diabetes mellitus, acute liver (increasing transaminase) and renal diseases, hypothyroidism, inflammatory diseases, neoplasms, hemostasis disorders, surgical interventions during last 3 months.

All invasive studies were carried out after written informed consent from the patients.                              

9 patients with VD (4 women, 5 men, mean age of 31.1 ± 1.5, 8 patients with C2 and C3 clinical signs) were examined according to dynamics of endothelial peptides in system blood flow before and after two-week administration of  phlebotropic antioxidative drug Antistax (Boehringer Ingelheim), 720 mg in the morning. Estimation of clinical efficiency of drug therapy was performed on the basis of calculation of Venous Clinical Severity Score, measurement of leg circumference, ultrasound measurement of diameter and maximal lineal rate of blood flow in magistral veins of the lower extremities with Medison Sonoase 8000, the transducer of 7.5 MHz, and photoplethysmography with doppler analyzer Smartdop 30EX with PPG probe.          

The statistical analysis was performed with Excel Medcalc. The main quantitative characteristics of sampled population are presented in tables as Ì ± m (mean arithmetic and error in mean). The hypothesis of equality in two mean independent groups was performed with two-sample parametric Student’s test for samples with normal distribution. The level of threshold statistical significance was 0.05 in comparison of quantitative parameters.

 

RESULTS

The study showed that the examined patients demonstrated 70 % increase in plasma levels of free radicals (TOA) and 13 % increase in end products of peroxile lipid oxidation (TBRP) (table 2). The patients demonstrated significantly decreased level of TAA (40 % decrease compared to the controls) and decreasing activity of the main antioxidative enzymes of red blood cells – SOD (13 %) and CAT (40 %).

Table 2
The values of oxidative plasma system and antioxidative system in red blood cells in varicose disease (Ì ± m)

Note: P - reliability of differences with control; n – number of observations; M – mean value; m – error in mean.

Therefore, the results confirm oxidative stress in the patients with VD; this stress is conditioned by insufficient neutralizing efficiency of intracellular antioxidative enzymes.

The examination of plasma levels of mitogenic peptides identified their increase in the patients with VD compared to healthy individuals (table 3).

Table 3
The level of mitogenic peptides in the blood of patients with varicose disease  (Ì ± m)

Note: P  – reliability of differences in healthy and ill persons; P₁  – reliability of differences in systemic and regionary blood flow; n  – amount of observations; M  – mean; m  – error in mean. 

Systemic blood flow showed 40 % increase in fibroblast growth factor in comparison with the controls (p < 0.01). The level of endothelial growth factor, the marker of endothelial injury, increased by 12 % (p > 0.05). The level of endothelin did not differ from control values in the blood flow of the patients with VD (p > 0.2). 

As for the regionary blood flow of the patients with VD, the same table demonstrates that the level of endothelial cytokines was higher than in the cubital vein. The level of endothelial growth factor increased by 18 % (p < 0.02), fibroblast growth factor – by 13 % (p > 0.05). Such pattern was observed in 2/3 of the patients with VD. The plasma level of vasoconstrictive peptide endothelin exceeded the similar value by 33 % in total blood flow (p < 0.02).

The comparative analysis of changes in endothelial peptides at different stages of the disease showed that the blood from the great saphenous vein was associated with dominating level of endothelial growth factor (at primary stage of the disease [C2, C3], 22 % increase in comparison with systemic blood flow; p < 0.01), but simultaneously the level of endothelin decreased (p < 0.01). In case of decompensated disorders of venous return, at the background of local microcirculatory changes and pigmentation (C4), such patterns were not detected.

Therefore, the patients with VD demonstrated increased levels of mitogenic peptides (endothelial growth factor and fibroblast growth factor) in the blood flowing from the affected extremities in comparison with total blood flow; one can observe it during the period of disease initiation, and it indicates presence of endothelial dysfunction in the veins of lower extremities and allows tracing the relationship between the above-mentioned disorders and varicose transformation of subcutaneous veins.

After antioxidative phlebotropic therapy with antistax the patients with varicose disease showed some biochemical changes in systemic blood flow (table 4). There were no significant changes in levels of fibroblast growth factor and endothelial growth factor in systemic blood flow. At the same time, one could observe three-fold increase in endothelin, which is characterized with vasoconstrictive effect.

Table 4
Dynamics of endothelial markers in blood plasma in patients with varicose disease during therapy with Antistax

Note: P – reliability of differences before and after treatment; n – number of observations; M – mean; m – error in mean.

The physical examination showed decreasing clinical manifestation of venous insufficiency. The heaviness in the legs and intensity of varicose veins decreased in case C2 clinical stage. As for the patients with C3 stage, the success was associated with decreasing or disappearing edema in the legs and pain feelings. In the patient with C4 signs of the disease the local dermatitis of the leg was jugulated. During treatment period the index of Clinical Scale of Disease Severity decreased from 8.9 ± 1.0 till 6.4 ± 1.0 (p > 0.05). Photoplethysmography showed reducing duration of venous return from 18 ± 4.9 s to 16.8 ±  3.7 s (p > 0.1). Ultrasonic angioscanning of the veins of the same patients did not show any significant changes in return in the magistral veins of the lower extremities.

 

DISCUSSION

The performed examinations found presence of systemic oxidative-antioxidative disbalance in the patients with varicose disease. It manifest itself as significant increase in plasma levels of oxyradical molecules and products of peroxide oxidation of membrane lipids, with simultaneous decrease in detoxicating abilities of intracellular antioxidative protection suggesting the disbalance in the systems of generation and neutralization of reactive oxygen species (superoxide, nitrogen oxide, hydroperoxide) in the vascular wall.         

The indicated changes can be considered as genetic determined disorder of endothelial function of veins in view of insufficient substrate inducibility of the main antioxidative enzymes.

Excessive free radicals, especially superoxide, stimulates synthesis of mitogenic factors by endotheliocytes of the vascular wall, particularly, synthesis of endothelial growth factor; this fact has been supported by the studies of patients with varicose disease. As it turned out, at the primary stages of the disease the increased generation of vascular growth factor is observed mainly in the great saphenous vein blood flowing from lower extremities.      

The identified biochemical features are associated with local hemodynamic factors, namely with distal orthostatic phlebohypertension of lower extremities and tissue hypoxia. Moreover, physiologic dynamic loads to lower extremities determine increasing endothelial production of signal oxyradicals for initiation of adequate blood return; accumulation of these radicals, in addition to regulatory effect, leads to remodeling influence on the vascular wall. As result, adaptive hyperplasia, hypertrophy of venous wall elements and capillary angiogenesis develop.   

Therefore, the results of the study determine the role of endothelial dysfunction in varicose transformation of superficial veins in the lower extremities [14].

Special secondary drug prevention of varicose disease and treatment are required for disordered adaptation to orthostatic phlebohypertension in the lower extremities in some individuals. The researches have shown the efficiency of antioxidative therapy for VD with use of the modern phlebotropic drug Antistax, which decreases clinical manifestations of the disease and venous insufficiency and simultaneously increases endothelin synthesis.

 

CONCLUSION

1. The patients with VD showed systemic oxidative stress conditioned by insufficiency of intracellular antioxidative systems which activates synthesis of mitogenic factors of the vascular wall.
2. Dysfunction of endothelium in the veins of the lower extremities is determined by decreasing adaptation to orthostatic dynamic loads, and it stimulates remodeling the venous wall and can be considered as one of the factors of varicose transformation.
3. Antioxidative therapy for varicose disease with use of the modern bioflavonoid drug Antistax results in decreasing clinical signs of the disease and of chronic venous insufficiency at the background of three-fold increase in plasma levels of vasoconstrictive peptide endothelin.