Increased blood pressure in the varicose veins (VV) can contribute to the overexpression of matrix metalloproteinases (MMPs), affecting the endothelium, smooth muscle, and extracellular matrix of the vein wall. Gelatinases (MMP-2 and MMP-9), hypoxia, and inflammation occurring in the VV wall contribute to the increased expression of vascular endothelial growth factor (VEGF).
Our objective was to analyze the concentration of gelatinases and VEGF in the great saphenous VV wall and plasma of patients.
In total, 65 patients (2nd degree according to clinical state classification, etiology, anatomy, and pathophysiology—CEAP classification) aged 22 to 70 were enrolled. Control veins (
A significant increase in MMP-9 (11.2 vs. 9.98 ng/mg of protein) and VEGF (41.06 vs. 26 ng/g of protein) concentration in VV wall compared with control veins was observed. A positive correlation between VEGF versus MMP-2 (
The results of the present study confirm that VV’s patients have altered expression of MMPs and VEGF. Overexpression of MMP-9 and VEGF in the VV wall may contribute to the spreading of inflammatory process and suggests the intense remodeling of extracellular tissue within the VV wall.
Chronic venous insufficiency (CVI) is an important medical problem in developed countries. Increased blood pressure in the varicose veins (VV) can contribute to the overexpression of selected matrix metalloproteinases (MMPs), affecting the endothelium, smooth muscle, and extracellular matrix proteins of the vein wall [
Vascular endothelial growth factor (VEGF) stimulates the synthesis of MMPs, especially MMP-9 [
As the mechanisms leading to the formation of the VV are still not fully understood, the objective of our study was to analyze the concentration of gelatinases and VEGF in the VV wall and in the plasma of patients with VV as the potential agents involved in VV pathogenesis.
Sixty-five patients (♀49, ♂16) aged 22 to 70 were enrolled (Table Characteristics of the study group Age (years) Mean age ± SD (years) 22 to 70 60 ± 11.002* 50 to 75 62 ± 8.3 29 to 62 56 ± 10.2 *Difference between either VV wall group vs. control 1 or control 2VV wall Control 1 Control 2 65 (♀49, ♂16) 10 (♀8, ♂2) 20 (♀11, ♂9) NA BMI 22.4 ± 3.1* 23.1 ± 2.3 22.9 ± 2.9 Hypertension (yes/no) 12/53 4/6 7/13
The material of VV patients was collected from femoral segment of varicose great saphenous vein using Babcock method and patients’ blood was collected from the antecubital vein during surgery. Control groups consist of two subgroups. The first one includes control group no. 1 (
Blood sample (VV and control no. 2) was collected from the antecubital vein into tubes with lithium heparin as an anticoagulant. Then it was centrifuged at 3000 rpm to obtain plasma, in which gelatinases and VEGF concentration was determined. Next 0.5 g of venous walls (VV and control no. 1) was homogenized in 5 ml of buffer containing 0.1 M Tris–HCl pH 7.4 and centrifuged for 15 min at 3000 rpm. The prepared material was stored at temperature − 70 °C. Gelatinases and VEGF levels were measured in plasma and vein walls of VV patients and both control individuals (control no. 1 and control no. 2).
Commercially available diagnostic kits, Human MMP-2 Quantikine ELISA Kit and Human MMP-9 Quantikine Immunoassay (R&D System, Abingdon, UK), were applied to gelatin concentration evaluation. The gelatinase levels were expressed in nanograms per milliliter in plasma and in nanograms per milligram of protein in vein wall homogenates. VEGF concentration was determined using diagnostic kit Human VEGF Quantikine Immunoassay (R&D System, Abingdon, UK) and expressed in picograms per milliliter in plasma, and nanograms per gram and nanograms per milligram of protein in tissue. Protein level in tissue samples was estimated with the usage of commercially available Bradford reagent (BIO-RAD Protein Assay, Hercules, USA). The assays were performed with the usage of Thermoshaker DTS-4 (ELMI, Calabasas, North America) and Microplate Reader Model 680 (BIO-RAD, Hercules, USA) with software Microplate Manager version 5.2.1 (BIO-RAD, Hercules, USA). All measurements were performed according to manufacturers’ manuals.
A significant increase in MMP-9 (11.2 vs. 9.98 ng/mg of protein) and VEGF (41.06 vs. 26 ng/g of protein) concentration in VV wall compared with control no. 1 was observed (Fig. Changes in concentration of gelatinases and VEGF in plasma and great saphenous veins of VV patients. Data are means (SD) or median (1st–3rd quartile); *** A positive correlation between the concentration of VEGF vs. MMP-2 in the VV wall (Spearman correlation coefficient
Changes in the activity of MMPs and VEGF were observed in many diseases of the circulatory system [
The previous results related to gelatinase activity in VV were ambiguous. The activity of MMP-2 was found to be decreased [
MMP-9 is important for maintaining the proper tension of blood vessel wall [
VEGF is considered to be the most potent stimulator of angiogenesis [
However, to the best of our knowledge, this is the first study evaluating the correlation between the concentration of gelatinases and VEGF in the VV wall. Wojcik et al. showed a significant correlation between the level of VEGF and MMP-9 in plasma of patients with small cell lung cancer that is associated with increased angiogenesis [
In conclusion, the results of the present study confirm that VV patients have altered expression of MMP-9 and VEGF. Overexpression of MMP-9 and VEGF in the VV wall may contribute to the spreading of inflammatory process and suggests the intense remodeling of extracellular tissue within the VV wall. The conducted study shows the coexistence of VV with elevated concentrations of VEGF and MMP-9 in the VV wall. The research does not determine whether it was the primary cause of VV or the increase in MMP-9 and VEGF.
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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by A.Horecka, A.Hordyjewska, J.B., W.D., T.Z., A.M., I.M., and J.K. The first draft of the manuscript was written by A.Horecka and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
The study was supported by Medical University of Lublin, Poland.
The authors declare that they have no competing interests.
The study protocol was approved by the Ethical Committee at the Medical University of Lublin (Poland), acceptance KE-0254/222/2009.
All subjects enrolled into the study voluntarily agreed to participate in the study and signed an informed consent form before any study procedure in compliance with the Declaration of Helsinki.