Abstract
Background
Although the endothelial dysfunction is considered to be implicated in the pathogenesis of chronic venous insufficiency (CVI) the endothelial status in patients with venous disorders is still not fully evaluated. Therefore the aim of the study was to measure the concentration of selected markers of endothelial dysfunction: von Willebrand factor (vWf), soluble P-selectin (sP-selectin), soluble thrombomodulin (sTM) and soluble VE-cadherin (sVE-cadherin) in CVI women who constitute the most numerous group of patients suffering from venous disease.
Materials and methods
Forty four women with CVI were involved in the study and divided into subgroups based on CEAP classification. Concentration of vWf, sP-selectin, sTM and sVE-cadherin were measured and compared with those obtained in 25 healthy age and sex-matched women.
Results
It was found that the concentration of sTM increased and sVEcadherin decreased along with disease severity in CVI women. A significant rise of sTM was observed especially in CVI women, with the highest inflammation status reflected by hsCRP or elastase concentration, and in CVI women with a high oxidative stress manifested by an increased plasma MDA. A significant fall of circulating sVE-cadherin was reported in CVI women with moderate to highest intensity of inflammation and oxidative stress. There was no change in vWF and sP-selectin concentration at any stage of CVI severity.
Conclusions
The results of the present study demonstrate the presence of endothelial dysfunction in women suffering from CVI which seems to progress with the disease severity and may be associated with inflammation and enhanced oxidative stress.
Introduction
Chronic venous insufficiency (CVI) is a condition widely prevalent in Western societies. Globally, CVI may affect more than 60% of the adult population [1], and its high costs, both at individual and societal levels, have been well documented [2]. The most common manifestation of chronic venous insufficiency are telangiectases, reticular veins, and varicose veins. More severe symptoms include soft tissue edema, dermatitis, hyperpigmentation, lipodermatosclerosis and ulceration The etiology and pathophysiology of CVI have been intensively studied in the past decades. Various epidemiological studies agree that women tend to suffer from CVI more frequently than men [3,4,5,6]. They experience varicose veins, the most common manifestation of CVI, three to four times more often than men [7]. Due to the high prevalence of venous diseases among women they are adequate subjects to be included in the study of mechanisms and the risk factors of CVI development.
One known hypothesis assumes that the inflammation plays a central role in the pathogenesis of CVI [8,9,10]. The inflammatory mechanisms accompanied by the liberation of cytokines, proteases and reactive oxygen species (ROS) contribute to endothelial damage and further pathological remodeling of the vein wall [11]. Endothelial cells undergoing injury release a variety of soluble particles known as biochemical markers of endothelial damage or dysfunction (ED) [12]. Their evaluation is a simple and non-invasive method of endothelial function measurement, therefore they are quantified and investigated in conditions associated with an increased vascular risk, such as peripheral and coronary atherosclerosis, aortic aneurysm, diabetes mellitus, or rheumatoid arthritis [13,14,15,16,17]. It is well known that non-invasive endothelial function assessment has predictive value for the occurrence of cardiovascular event in primary and secondary prevention settings [18,19]. Moreover the association of improvement in ED with risk reduction for future cardiovascular events has also been demonstrated [18,20]. At present flow-mediated vasodilatation (FMD) has become the most widely used non-invasive technique to measure endothelial function. This technique measures the ability of arteries to respond with endothelial NO release during reactive hyperemia after a 5-minute occlusion of the brachial artery with a blood pressure cuff. Although the principle of this technique seems to be simple, its application is technically challenging and requires an extensive training and standardization [21,22,23,24]. Moreover, in some situations, the degree of reactive hyperemia may vary even under the same stimulus. Also, changes in the structure of blood vessels and impaired dilation may be limiting factors during FMD assessment [22]. Therefore, a simpler and more reproducible parameter of ED is still being investigated and biochemical markers seem to be an adequate candidate. The advantages of using of serum biomarkers in the assessment of endothelial status are the simplicity of the procedure and the fact that venous blood samples are widely used in laboratory routine [22]. Moreover, biochemical markers demonstrate an excellent sensitivity in reflecting ED, comparable with biophysical measurements [25,26,27]. It is also highly probable that liberation of biochemical markers into circulation by injured endothelial cells may precede the alterations in their function measured by biophysical techniques, such as FMD. The use of these biochemical markers in the prognosis and/or diagnosis of vascular disease is most common at the initial stages, but this is still a study area with a great potential.
The fact that CVI may be another type of pathology associated with ED is confirmed by histological examination revealing alteration in morphology of endothelial cells and disruption in endothelial layer integrity [18]. However, the number of studies evaluating the markers of endothelial status and their potential impact on CVI diagnosis or prognosis is still limited [28,29,30,31]. There is also lack of data indicating the factors which may potentially participate in ED in CVI development. Therefore, researches conducted on ED and their markers may give not only insights into pathophysiology of CVI, but also possible clinical opportunity to detect early disease, prevent severe complications such as venous leg ulcers, and assess response to treatments.
The aim of this pilot study was to evaluate selected biochemical markers of ED, namely von Willebrand factor (vWf), soluble P-selectin (sP-selectin), soluble thrombomodulin (sTM) and soluble vascular endothelial cadherin (sVE-cadherin) in the blood of women affected by CVI. The association of markers of ED with disease severity, demographical data (BMI value, age) and parameters of inflammation and oxidative stress (hsCRP, elastase and MDA concentration) was also analyzed to find their potential contribution to endothelial pathology in CVI.
Materials and methods
Patients
The group of patients consisted of 44 women, aged 26–65 years (mean age: 45±11) with primary varicose vein (VV), who underwent lower extremity VV excision. Preoperative, lower-extremity venous color duplex ultrasound scanning was performed on all patients and both the superficial and the deep venous systems were studied. Venous reflux was defined as flow in the inverted direction for a period longer than 0.5 seconds. Partial and complete venous obstruction was assessed by the degree of compressibility of the venous walls, with normal defined as complete compressibility. Superficial venous functional disease (SFD) and deep venous functional disease (DFD) were defined as reflux on ultrasound or abnormal compression in superficial and deep veins, respectively. In all cases the patency of the deep vein system as well as the lack of thrombotic changes were confirmed. The patients with peripheral arterial occlusive disease (ankle-brachial index < 0.9) or any other conditions that might result in leukocyte activation, such as diabetes, cancer, connective tissue disorders or infection within the previous six weeks were not included into the study.
All women had visible venous disease signs that corresponded to the Clinical Etiologic Anatomic Pathologic (CEAP) classification categories. There were 19 women with VV (class 2), 10 women with edema (class 3), 16 patients with pigmentation, lipodermatosclerosis, atrophie blanche, healed or active ulceration (class 4, 5 and 6). Therefore, the disease severity was the criterion for categorizing the women into two subgroups: with moderate CVI (included C2 and C3 classes) and severe CVI (included C4, C5 and C6 classes)
The control group consisted of 25 women, aged 36–60 (mean age: 42±7), all of whom were members of medical staff. They had no superficial functional disease (SFD) or deep functional disease (DFD) on ultrasound, no varicose veins, edema or trophic changes of the skin on physical exam and no reports of leg aching. The study procedure was approved by Bioethical Committee of the University of Medical Sciences in Poznan, and informed consent was obtained from all participants.
Sample collection
Blood samples were drawn preoperatively from the arms of CVI women on the day of surgery, in the recumbent position after 10 minutes of rest. Samples were collected in EDTA tube with anticoagulant for plasma and serum tubes. After 30 minutes, the tubes were centrifuged at 3.000 rpm for 15 minutes. Serum and plasma samples were stored at temperature of -80°C until all of assays were performed. The study procedure was approved by the Bioethical Committee of the University of Medical Sciences in Poznan and informed consent was obtained from all participants.
Laboratory analysis
The von Willebrand factor (vWf), soluble thrombomodulin (sTM), soluble P-selectin (sP-selectin), soluble VE-cadherin (sVE-cadherin), high sensitive C-reactive protein (hsCRP) and leukocyte elastase concentration were measured using enzyme-linked-immunosorbent assay (Abcam, UK; Gen-Probe Diaclone SAS, France; R&D System, USA; eBioscience, Austria; DRG International, USA; Hycult Biotech, The Netherlands). MDA concentration was measured using calorimetric assay kit (Abcam, UK).
Statistical analysis
The statistical analysis were conducted using GraphPad Prism software 6.0 (GraphPad Software, San Diego, CA). The normality of quantitative variables were tested using the Kolmogorov-Smirnow or Shapiro-Wilk test. Any parameter not following the normal distribution was presented as a median and interquartile ranges and analyzed using non-parametric Mann-Whitney test. Categorical data and proportions were compared using Chi-square or Fisher`s exact test, as appropriate. Normally distributed, continuous variables were presented as a mean and standard deviation and analyzed using the Student’s t test. Multiple group comparisons were performed by one-way analysis of variance or Kruskal-Wallis test, respectively. The Pearson or the Spearman correlation coefficient was used to test the strength of any association between different variables. In all cases, P value ≤ 0.05 was considered significant.
Results
After categorizing patients into appropriate subgroups according to CEAP classification, women with severe CVI showed to be older and had a higher BMI value (Table 1).
Table 1. Markers of endothelial dysfunction, inflammation and oxidative stress in women with moderate and severe CVI.
| Parameter | Control (n = 33) | Moderate CVI (n = 28) | Severe CVI (n = 16) | p-value |
|---|---|---|---|---|
| Age (years) | 45.18±10.53 | 41.72±9.15 | 51.44±10.22 | 0.001a |
| BMI (kg/m2) | 24.52±6.34 | 22.67±4.83 | 26.19±5.06 | 0.027a |
| hsCRP (mg/L) | 1.00 (0.12–1.86) | 0.98 (0.13–3.15) | 1.15 (0.56–2.84) | NSb |
| Elastase (ng/mL) | 41.50 (24.50–65.75) | 51.75 (19.00–81.25) | 59.00 (25–172) | NSb |
| MDA (μM) | 2.41 (2.03–4.06) | 3.93 (2.92–5.40) | 4.41 (3.47–5.93) | 0.001b |
| vWf (mU/mL) | 808 (723–957) | 890 (649–961) | 805 (627–997) | NSb |
| sTM (ng/mL) | 0.96 (0.86–1.63) | 1.23 (1.09–1.69) | 1.43 (1.10–2.25) | 0.027b |
| P-selectin (ng/mL) | 26.16 (11.45–45.82 | 19.64 (8–36.36) | 21.27 (10.27–52.50) | NSb |
| sVE-cadherin (ng/mL) | 39.30 (36.19–42.11) | 30.34 (23.36–34.44) | 31.56 (26.31–41.90) | 0.002b |
a Results shown as mean± standard deviation, one-way ANOVA test was used for comparison.
b Results shown as median and interquartile range, Kruskull-Wallis test was used for comparison.
NS—not statistically significant
There was no difference in hsCRP and elastase concentration between studied subgroups and control (Table 1). It was demonstrated that MDA concentration tends to increase together with the disease severity (Table 1). The same trend was observed for sTM concentration which rose reaching the highest value in women with severe CVI (Table 1). The level of sVE-cadherin was decreased in women with moderate as well as severe symptoms of CVI. There was no difference in the concentration of vWf and sP-selectin between subgroups and control. Significant positive correlation between sP-selectin and elastase was found (r = 0.419, P = 0.014) in the whole group of CVI women (Fig 1). Moreover, an elevated vWf values correlated with an increased hsCRP concentration (r = 0.373, P = 0.049) (Fig 2).
Fig 1. Correlation between sP-selectin and elastase.
(Spearman correlation coefficient r = 0.419, P = 0.014).
Fig 2. Correlation between vWF and hsCRP.
(Spearman correlation coefficient r = 0.373, P = 0.049).
To analyze the impact of inflammation and oxidative stress on endothelial function all examined CVI women have been divided into 3 groups using 25th and 75th percentiles of hsCRP, elastase and MDA concentration distribution, respectively, as cutoff points (group I—<25th percentile, group II - 25th-75th percentile, group III—>75th percentile). Then the endothelial markers were compared between quartiles and control group.
The value of sTM was significantly increased in CVI women in the middle and upper quartiles of hsCRP in compared with control [1.23 (1.08–2.54) ng/mL vs. 0.96 (0.86–1.63) ng/mL P = 0.047; 1.40 (1.10–2.93) ng/mL vs. 0.96 (0.86–1.63) ng/mL P = 0.045] (Fig 3).
Fig 3. Concentration of sTM in CVI woman categorized into quartiles of plasma hsCRP.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * P≤0.05 was considered statistically significant.
The rise in sTM concentration was also noticed in CVI women in the highest quartile of elastase [1.41 (1.07–2.16) ng/mL vs 0.96 (0.86–1.63) ng/mL P = 0.049] and MDA concentrations [1.57 (1.15–2.57) ng/mL vs 0.96 (0.86–1.63) ng/mL P = 0.032], respectively (Figs 4 and 5).
Fig 4. Concentration of sTM in CVI woman categorized into quartiles of plasma elastase.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
Fig 5. Concentration of sTM in CVI woman categorized into quartiles of plasma MDA.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
The concentration of sVE cadherin was decreased in CVI women in the middle and highest quartiles of hsCRP [32.29 (23.36–36.14) ng/mL vs. 39.30 (36.19–42.11) ng/mL P = 0.002; 28.51 (27.41–47.44) ng/mL vs. 39.30 (36.19–42.11) ng/mL P = 0.043] and elastase concentration [32.23 (24.60–40.18) ng/mL vs. 39.30 (36.19–42.11) ng/mL P = 0.022; 30.26 (22.83–32.77) ng/mL vs. 39.30 (36.19–42.11) ng/mL P<0.001], respectively (Figs 6 and 7).
Fig 6. Concentration of sVE-cadherin in CVI woman categorized into quartiles of plasma hsCRP and control group.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
Fig 7. Concentration of sVE-cadherin in CVI woman categorized into quartiles of plasma elastase.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
A significant fall in sVE-cadherin concentration was also noticed in the group of CVI women with the middle values of MDA [28.51 (23.32–32.69) ng/mL vs 39.30 (36.19–42.11) ng/mL P<0.0001] (Fig 8).
Fig 8. Concentration of sVE-cadherin in CVI woman categorized into quartiles of plasma MDA.
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
Discussion
Numerous mechanisms have been proposed as the eatiology of CVI. One common hallmark of this disease is an elevated venous pressure and shift in the fluid shear stress [32]. These two conditions probably generate an abnormal biomechanical environment in the vein wall, which may initiate early activation of inflammatory cascade. Therefore, CVI is considered as a blood pressure-driven inflammatory disease, where inflammatory factors play a significant role [32]. Inflammation, accompanied by leukocyte activation and liberation of various cytokines, proteases, and reactive oxygen species (ROS) may affect morphology and function of endothelium of venules. The disruption of endothelial layer leads to the disturbance in specific vascular homeostasis, vascular tone, and permeability, resulting in the venous wall remodeling seen in all stages of CVI. The endothelial dysfunction is reflected either by the liberation of some specific molecules derived from damaged endothelial cells or the disruption in the concentration of factors naturally released by endothelium in physiological conditions. There is a lot of evidence which highlights the contribution of ED to the clinical status in cardiovascular diseases and shows a great potential of endothelial markers evaluation as a non-invasive method for predicting the disease progression and prognosis in patients with vascular disorders [33,34,35,36] Although ED is considered to be implicated in the pathogenesis of CVI, the number of studies assessing the endothelial status in patients with venous disease is still limited [28,29,30,31,37]. Little is known about the factors contributing to ED in CVI pathology, relationship of ED with the disease severity and the possible utility of endothelial markers in CVI diagnosis and monitoring. Due to the fact that CVI appears more commonly in women than in men [3,4,5,6], women seem to be an adequate model group for the investigation into the mechanisms and factors responsible for the disease occurrence and progression. Therefore the aim of the present study was to evaluate endothelial status in CVI women by measuring selected endothelial markers. The second purpose was to analyze the association of markers of ED with disease severity and factors potentially contributing to endothelial injury such as inflammation and oxidative stress.
In our study we chose four markers of endothelial dysfunction: vWf, sP-selectin, sTM and sVE-cadherin. The first marker mentioned, namely vWf, is a large glycoprotein that is required for normal hemostasis. It mediates in platelet aggregation and adhesion to the vascular wall, serves as a plasma carrier for factor VIII. vWf is produced and stored in Weibel-Palade bodies of endothelial cells. An increased liberation of vWf to circulation is followed by endothelial damage [38]. The elevated concentration of vWf has been found in diseases such as atherosclerosis, diabetes, rheumatoid vasculitis where damage to the endothelium is observed [39,40].
P-selectin is a cell adhesion molecule found alike vWf in the Weibel-Palade bodies of ECs. It can be rapidly released from the Weibal-Palade bodies of endothelial cells after appropriate activation by histamine, thrombin, complement, or ROS and mediate in leukocyte “rolling” on endothelium [41,42]. It has been suggested that plasma sP-selectin may reflect the functional status of ECs [43]. Its significant rise was demonstrated in stroke, acute myocardial infarction, coronary artery disease, and peripheral artery disease [44].
TM is one of the most popular indicators of endothelial injury, located on the vascular endothelium surface which functions as an anticoagulant. TM has an affinity for thrombin, forming a 1:1 thrombin-thrombomodulin complex that inhibits fibrin formation, platelet activation and protein S inactivation [45]. Apart from the transmembrane form, TM also occurs in soluble forms (sTM) in the plasma, which is probably the product of the cleaved transmembrane glycoprotein [46]. In vitro studies demonstrate that sTM is released from endothelial cells following cell membrane injury caused by the action of neutrophil derived proteases and oxygen radicals [47,48,49].
VE-cadherin is a novel marker of endothelial damage with a high specificity [50,51]. Contrary to the previously described molecules which could be liberated by other cell types, VE-cadherin is specifically expressed by endothelial cells [52]. VE-cadherin belongs to a large family of endothelial cadherin proteins. As a transmembrane molecule, it is located in the intracellular junctions where it regulates the barrier function of endothelium. Soluble VE-cadherin (sVE-cadherin) can be released into the blood after increased proteolytic activity mediated by metalloproteinases and other proteases [53]. Although sVE-cadherin has been evaluated in diseases associated with vascular endothelial injury [54,55,56] our study is the first to investigate the concentration of this protein in CVI.
Our study demonstrates a high concentration of sTM in CVI women which shows a significant trend to rise together with disease severity. This result suggests that the gradation of CVI severity might be connected with endothelial damage expansion. Moreover, we also observed that severe CVI is correlated with ageing and BMI value. These findings are in line with the results of Musil et al. who showed that age and BMI are significant predictors of clinical grade of venous disease according to the CEAP classification [57]. The authors observed that a greater age and elevated BMI were associated with an increased CEAP grade of visible disease and an increased risk of the clinical progression from varicose veins to trophic skin changes or venous ulcers. However, the predictive value of BMI was demonstrated only in women, which may suggest the existence of gender-specific factors predisposing to CVI development and progression. Due to the fact that increasing age and a high BMI value have been recognized as the factors contributing to ED [58,59,60], their influence on the endothelial abnormalities in CVI should also be taken into consideration.
Since it is known that sVE-cadherin may reflect endothelial barrier disruption we had expected to obtain an increased level of this molecule in our patients highly prone to endothelial injury. Surprisingly, both in moderate and severe CVI a decreased concentration of sVE-cadherin was observed. Although the rise of sVE-cadherin was noticed in patients with various vascular complications, there are some studies presenting results similar to ours. A decreased concentration of sVE-cadherin was demonstrated in patients infected by Shiga toxin 2 producing E. coli strain (STEC) and with the haemolytic ureamic syndrome (HUS), a complication caused by this bacteria and occurs via endothelial cell damage [61]. The study by Ebihara et al. shows that sepsis, a pathology accompanied by massive endothelial disruption and damage, is associated with decreased sVE-cadherin level [62]. Ostrowski et al., who investigated the markers of endothelial damage in patients with severe sepsis, obtained results identical to ours, namely a fall in sVE-cadherin concentration accompanied by the rise in sTM level [63]. One explanation of a decreased circulating level of sVE-cadherin suggested by the authors is an enhanced sequestration of this protein from the endothelial cell surface which may be induced by inflammatory mediators and ROS [64,65]. Sequestration of junctional VE-cadherin may decrease adhesive bonds between apposed endothelial cells and increase endothelial permeability. Therefore, a low concentration of circulating VE-cadherin presents either in moderate or severe stage of CVI may reflect a perturbation in endothelial barrier that occurs already at the initial stage of venous disease development.
In our study, there was no change in sWf and sP-selectin concentration at any stage of CVI severity. Our results are in contrast to the findings by Yasim et al. who observed an elevated concentration of vWf in patients with primary varicoce veins [66]. However, the authors included lower number of patients classified as C2 according to CEAP system. Bryan et al. revealed that a higher circulating sP-selectin is associated only with severe CVI and not with CVI overall [67]. However the correlation of sP-selectin and vWf with elastase and hsCRP, respectively, observed in the present study, suggests that they may be indicators of inflammation rather than ED. It is well-known that vWf is an acute phase reactant affected by inflammatory cytokines and as such, may be elevated even in the absence of ED [32]. The correlation of sP-selectin with platelet count demonstrated in previous studies [68,69] proves the utility of measuring this molecule as an inflammatory marker as well. sTM and sVE-cadherin seem to be less influenced by inflammation, and their concentrations remained constant regardless of hsCRP and elastase levels. This observation confirm a well-known fact that sTM, contrary to others endothelial markers, does not increase in an acute response to a variety of biological stimulations and, therefore, is the most likely a specific a marker of endothelial lesions and not of other cell types activation [45]. The sVE-cadherin seems to exhibit similar resistance to inflammatory stimuli. These findings suggest that these two particles may be markers of the true ED and that the concentration thereof, is dependent only on the actual endothelial status. Moreover, due to the fact that both sTM and sVE-cadherin already occur at an early stage of CVI, they may act as early markers of ED.
In our study the markers of inflammation and oxidative stress were used as the criteria to divide CVI women into appropriate groups with low, medium, and high inflammatory or oxidative stress status, respectively, in which endothelial parameters were evaluated and compared with healthy controls. This approach allowed to state which group of CVI women is particularly prone to endothelial injury. CVI women with the highest plasma hsCRP or elastase concentration, respectively, demonstrated an elevated value of sTM, which points to the inflammation as an important factor contributing to endothelial damage. A large body of evidence shows an increased level of oxidative stress parameters in the blood and varicose vein wall of CVI patients [70,71,72,73]. These findings were also confirmed by our study in which an elevated concentration of MDA was observed in plasma of CVI women. ROS liberated by inflammatory cells may have a direct effects on endothelial layer. Some experiments with monolayers of cultured endothelium demonstrated that ROS induce the cytolysis of endothelial cells and disruptions in endothelial cell adhesion [74,75]. Therefore, a simultaneous rise of sTM and MDA demonstrated in the present study may be the result of disruptive effects of enhanced oxidative stress on the endothelial layer, which escalate together with CVI severity. The contribution of ROS to endothelial perturbation is also confirmed by an increased concentration of sTM observed especially in CVI women with highest oxidative status. Moreover, the unique behavior of sVE-cadherin which starts to decrease in CVI women with the moderate plasma level of hsCRP, elastase and MDA respectively, suggests that not necessarily intense but even a mild inflammation or/and oxidative stress process may negatively influenced the endothelial barrier by decreasing the content of protein in endothelial junctions and increasing endothelial permeability.
We are aware of the limitation of this pilot study. First, it can be agreed that the number of CVI female participants was small However, the present work calls attention to the important aspect of biochemical markers of ED in CVI prognosis or/and diagnosis that has not yet been evaluated. These preliminary observations are therefore a potential concept for future clinical studies involving a larger cohort of patients. Second, the use of MDA as an oxidative stress marker may be also controversial. Although MDA is a widely accepted assay for oxidative damage, the most common methods of MDA detection, TBA test, shows several pitfalls and has been criticized as being too unspecific and prone to artifacts. TBA can react with several compounds, including sugars, amino acids, bilirubin, and albumin, producing interferences in the measurement. MDA assay can be used in association with other indices of lipid peroxidation, such as 4-hydroxynnenal, conjugated dienes, ethane and pentane gases, and isoprostanes. Unluckily, these methods have limitations because they are either too expensive, too time consuming or their application needs specialized personnel [76]. Therefore one of the most important reason of choosing of MDA assay in the present study was its simplicity, rapidity and possibility of implementation in routine clinical testing.
Conclusions
The results of the present study demonstrates the presence of ED in women suffering from CVI which progresses with the disease severity and may be associated with inflammation and enhanced oxidative stress. Due to the fact that both sTM and sVE-cadherin already occur at an early stage of CVI, they may act as early markers of ED. Moreover, our findings, assumedly for the first time, demonstrate that endothelial perturbation may involve an increased endothelial permeability that can occur even at an early stage of CVI development.
Supporting information
(Spearman correlation coefficient r = 0.419, P = 0.014).
(PDF)
(Spearman correlation coefficient r = 0.373, P = 0.049).
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * P≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
(a)Results shown as mean± standard deviation, one-way ANOVA test was used for comparison.(b)Results shown as median and interquartile range, Kruskull-Wallis test was used for comparison.NS—not statistically significant.
(PDF)
Data Availability
All relevant data are within the paper and its Supporting Information files.
Funding Statement
The study was supported by the University of Medical Science in Poznan (Grant no. 501-01-02228370-03102-50564). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1.Rabe E, Guex JJ, Puskas A, Scuderi A, Fernandez Quesada F. Epidemiology of chronic venous disorders in geographically diverse populations: results from the Vein Consult Program. Int Angiol. 2012;31: 105–115. [PubMed] [Google Scholar]
- 2.Nicolaides AN, Allegra C, Bergan J, Perrin M, Nelzen O, Neglen P et al. American venous forum. Management of chronic venous disorders of the lower limbs: guidelines according to scientific evidence. Int Angiol. 2008;27: 1–59. [PubMed] [Google Scholar]
- 3.Canonico S, Gallo C, Paolliso G, Pacifico F, Signoriello G, Siaudone G et al. Prevalence of varicose veins in an Italian elderly population. Angiology 1998;49: 129–135. doi: 10.1177/000331979804900205 [DOI] [PubMed] [Google Scholar]
- 4.Capitao LM, Menezes JD, Gouveia-Oliveira A. The epidemiology of chronic venous insufficiency in Portugal. Acta Med Port. 1995;8: 485–491. [PubMed] [Google Scholar]
- 5.Laurikka J, Läärä E, Sisto T, Tarkka M, Auvinen O, Hakama M. Misclassification in a questionnaire survey of varicose veins. J Clin Epidemiol. 1995;48: 1175–1178. [DOI] [PubMed] [Google Scholar]
- 6.Komsuoğlu B, Göldeli O, Kulan K, Cetinarslan B, Komsuoğlu SS. Prevalence and risk factors of varicose veins in an elderly population. Gerontology 1994;40: 25–31. [DOI] [PubMed] [Google Scholar]
- 7.Staffa R. Chronic venous insufficiency—epidemiology. Bratisl Lek Listy. 2002;103: 166–168. [PubMed] [Google Scholar]
- 8.Pascarella L, Penn A, Schmid-Schönbein GW. Venous hypertension and the inflammatory cascade: major manifestations and trigger mechanisms. Angiology 2005;56: S3–S10. doi: 10.1177/00033197050560i102 [DOI] [PubMed] [Google Scholar]
- 9.Bergan J. Molecular mechanism in chronic venous insuffiency. Ann Vasc Surg. 2007;21: 260–266. doi: 10.1016/j.avsg.2007.03.011 [DOI] [PubMed] [Google Scholar]
- 10.Ojdana D, Safiejko K, Lipska A, Sacha P, Wieczorek P, Radziwon P et al. The inflammatory reaction during chronic venous disease of lower limbs. Folia Histochem Cytobiol. 2009;47: 185–189. doi: 10.2478/v10042-009-0029-8 [DOI] [PubMed] [Google Scholar]
- 11.Raffetto JD, Khalil RA. Mechanisms of varicose vein formation: valve dysfunction and wall dilation. Phlebology 2008;23: 85–98. doi: 10.1258/phleb.2007.007027 [DOI] [PubMed] [Google Scholar]
- 12.Constans J, Conri C. Circulating markers of endothelial function in cardiovascular disease. Clin Chim Acta. 2006;368: 33–47. doi: 10.1016/j.cca.2005.12.030 [DOI] [PubMed] [Google Scholar]
- 13.Gutiérrez E, Flammer AJ, Lerman LO, Elízaga J, Lerman A, Fernández-Avilés F. Endothelial dysfunction over the course of coronary artery disease. Eur Heart J. 2013;34: 3175–3181. doi: 10.1093/eurheartj/eht351 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Brevetti G, Silvestro A, Di Giacomo S, Bucur R, Di Donato A, Schiano V et al. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg. 2003;38: 374–379. [DOI] [PubMed] [Google Scholar]
- 15.Budzyń M, Gryszczyńska B, Majewski W, Krasiński Z, Kasprzak MP, Formanowicz D, Strzyżewski KW, Iskra M. The association of serum thrombomodulin with endothelial injuring factors in abdominal aortic aneurysm. Biomed Res Int. 2017;2017: 2791082 doi: 10.1155/2017/2791082 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Hadi HA, Suwaidi JA. Endothelial dysfunction in diabetes mellitus. Vasc Health Risk Manag. 2007;3: 853–876. [PMC free article] [PubMed] [Google Scholar]
- 17.Foster W, Shantsila E, Carruthers D, Lip GY, Blann AD. Circulating endothelial cells and rheumatoid arthritis: relationship with plasma markers of endothelial damage/dysfunction. Rheumatology (Oxford). 2009;48: 285–288. [DOI] [PubMed] [Google Scholar]
- 18.Kitta Y, Obata JE, Nakamura T, Hirano M, Kodama Y, Fujioka D et al. Persistent impairment of endothelial vasomotor function has a negative impact on outcome in patients with coronary artery disease. Journal Am Coll Cardiol. 2009;53: 323–330 [DOI] [PubMed] [Google Scholar]
- 19.Xu Y, Arora RC, Hiebert BM, Lerner B, Szwajcer A, McDonald K et al. Non-invasive endothelial function testing and the risk of adverse outcomes: a systematic review and meta-analysis. Eur Heart J Cardiovasc Imaging. 2014; 15:736–746. doi: 10.1093/ehjci/jet256 [DOI] [PubMed] [Google Scholar]
- 20.Modena MG, Bonetti L, Coppi F, Bursi F, Rossi R. Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women. Journal Am Coll Cardiol. 2002;40: 505–510. [DOI] [PubMed] [Google Scholar]
- 21.Arrebola-Moreno AL, Laclaustra M, Kaski JC. Noninvasive assessment of endothelial function in clinical practice. Rev Esp Cardiol (Engl Ed). 2012;65: 80–90. [DOI] [PubMed] [Google Scholar]
- 22.Sampaio Storch A, Dario de Mattos J, Alves R, dos Santos Galdino I, Naly Miguens Rocha H. Methods of endothelial function assessment: description and application. Int J Cardiovasc Sci. 2017;30:262–273. [Google Scholar]
- 23.Flammer AJ, Anderson T, Celermajer DS, Creager MA, Deanfield J, Ganz P et al. The assessment of endothelial function- from research into clinical practice. Circulation 2012;126: 753–767. doi: 10.1161/CIRCULATIONAHA.112.093245 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Al-Qaisi M, Kharbanda RK, Mittal TK, Donald AE. Measurment of endothelial function and its clinical utility for cardiovascular risk. Vasc Health Risk Manag. 2008; 4:647–652. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Elhadd TA, Abdu TA, Oxtoby J, Kennedy G, McLaren M, Neary R et al. Biochemical and biophysical markers of endothelial dysfunction in adults with hypopituitarism and severe GH deficiency. J Clin Endocrinol Metab. 2001;86: 4223–4232. doi: 10.1210/jcem.86.9.7813 [DOI] [PubMed] [Google Scholar]
- 26.Andor M, Suciu M, Cristescu C, Dragan L, Vlaia L, Vlaia V et al. Correlation analysis between biochemical, functional and structural markers of endothelium damage in patients with essential hypertension. Atherosclerosis 2014;235: e136. [Google Scholar]
- 27.Gungor ZB, Sipahioglu N, Sonmez H, Ekmekci H, Toprak S, Ayaz G et al. Endothelial dysfunction markers in low cardiovascular risk individuals: comparison of males and females. J Med Biochem. 2017;36: 62–72. doi: 10.1515/jomb-2016-0030 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Carrasco OF, Ranero A, Hong E, Vidrio H. Endothelial function impairment in chronic venous insufficiency: effect of some cardiovascular protecting agents. Angiology 2009;60: 763–771. doi: 10.1177/0003319709332108 [DOI] [PubMed] [Google Scholar]
- 29.Komarów W, Hawro P, Lekston A, Urbanek T, Zagrodzki P. Endothelial dysfunction in patients with chronic venous disease: an evaluation based on the flow-mediated dilatation test. Int Angiol. 2015;34: 36–42. [PubMed] [Google Scholar]
- 30.Tisato V, Zauli G, Voltan R, Gianesini S, di Iasio MG, Volpi I et al. Endothelial cells obtained from patients affected by chronic venous disease exhibit a pro-inflammatory phenotype. PLoS One 2012;7: e39543 doi: 10.1371/journal.pone.0039543 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Georgescu A, Alexandru N, Popov D, Amuzescu M, Andrei E, Zamfir C et al. Chronic venous insufficiency is associated with elevated level of circulating microparticles. J Thromb Haemost. 2009;7:1566–1575. doi: 10.1111/j.1538-7836.2009.03525.x [DOI] [PubMed] [Google Scholar]
- 32.Pocock ES, Alsaigh T, Mazor R, Schmid-Schönbein GW. Cellular and molecular basis of venous insufficiency. Vasc Cell. 2014;6: 24 doi: 10.1186/s13221-014-0024-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Hadi HA, Carr CS, Al Suwaidi J. Endothelial dysfunction: cardiovascular risk factors, therapy, and outcome. Vasc Health Risk Manag. 2005;1: 183–198. [PMC free article] [PubMed] [Google Scholar]
- 34.Thorand B, Baumert J, Chambless L, Meisinger C, Kolb H, Döring A et al. Elevated markers of endothelial dysfunction predict type 2 diabetes mellitus in middle-aged men and women from the general population. Arterioscler Thromb Vasc Biol. 2006;26: 398–405. doi: 10.1161/01.ATV.0000198392.05307.aa [DOI] [PubMed] [Google Scholar]
- 35.Widmer RJ, Lerman A. Endothelial dysfunction and cardiovascular disease. Glob Cardiol Sci Pract. 2014;2014: 291–308. doi: 10.5339/gcsp.2014.43 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Poggesi A, Pasi M, Pescini F, Pantoni L, Inzitari D. Circulating biologic markers of endothelial dysfunction in cerebral small vessel disease: A review. J Cereb Blood Flow Metab. 2016;36: 72–94. doi: 10.1038/jcbfm.2015.116 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Saharay M, Shields DA, Georgiannos SN, Porter JB, Scurr JH, Coleridge Smith PD. Endothelial activation in patients with chronic venous disease. Eur J Vasc Endovasc Surg. 1998. April;15: 342–9. [DOI] [PubMed] [Google Scholar]
- 38.Horvath B, Hegedus D, Szapary L, Marton Z, Alexy T Koltai K et al. Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases. Exp Clin Cardiol. 2004:9; 31–34. [PMC free article] [PubMed] [Google Scholar]
- 39.Lip GYH, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders?. Cardiovasc Res. 1997;34: 255–265. [DOI] [PubMed] [Google Scholar]
- 40.Kloczko J, Kuryliszyn-Moskal A, Bernacka K, Bielawiec M, Cylwik B, Radziwon P. von Willebrand factor antigen in assessment of vasculitis in patients with connective tissue diseases. Clin Rheumatol. 1994;13: 34–38. [DOI] [PubMed] [Google Scholar]
- 41.Kansas GS. Selectins and their ligands: current concepts and controversies. Blood 1996;88: 3259–3287. [PubMed] [Google Scholar]
- 42.Rivera-Chavez F, Toledo-Pereyra LH, Nora DT, Bachulis B, Ilgenfritz F, Dean RE. P-selectin blockade is beneficial after uncontrolled hemorrhagic shock. J Trauma. 1998;45: 440–445. [DOI] [PubMed] [Google Scholar]
- 43.Kaikita K, Ogawa H, Yasue H, Sakamoto T, Suefuji H, Sumida H et al. Soluble P-selectin is released into the coronary circulation after coronary spasm. Circulation. 1995;92: 1726–1730. [DOI] [PubMed] [Google Scholar]
- 44.Merten M, Thiagarajan P. P-selectin in arterial thrombosis. Z Kardiol. 2004;93: 855–863. doi: 10.1007/s00392-004-0146-5 [DOI] [PubMed] [Google Scholar]
- 45.Chong AY, Blann AD, Lip GY. Assessment of endothelial damage and dysfunction: observations in relation to heart failure. QJM 2003;96: 253–267. [DOI] [PubMed] [Google Scholar]
- 46.Blann AD. Taberner DA. A reliable marker of endothelial cell dysfunction: does it exist?. British J Haematol Oncol. 1993;15: 338–342. [DOI] [PubMed] [Google Scholar]
- 47.Boehme MW, Deng Y, Reath U, Bierhaus A, Ziegler R, Stremmel W et al. Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies. Immunology 1996; 87: 134–140. [PMC free article] [PubMed] [Google Scholar]
- 48.Boehme MW, Raeth U, Scherbaum WA, Galle PR, Stremmel W. Interaction of endothelial cells and neutrophils in vitro: kinetics of thrombomodulin, intracellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1): Implication for relevance as serological disease activity markers in vasculitides. Clin Exp Immunol. 2000;119: 250–254. doi: 10.1046/j.1365-2249.2000.01108.x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Boehme MW, Galle P, Stremmel W. Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals. Immunology 2002;107: 340–349. doi: 10.1046/j.1365-2567.2002.01469.x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Sidibé A, Polena H, Mannic T, Stidder B, Bouillet L, Vilgrain I. Soluble vascular endothelial (VE)-cadherin: toward a marker of endothelial dysfunction In: Jean-Jacques Feige, Gilles Pagès, Fabrice Soncin, editors. Molecular Mechanisms of Angiogenesis—From Ontogenesis to Oncogenesis. Springer; Paris; 2014. pp. 461–478. [Google Scholar]
- 51.Flemming S, Burkard N, Renschler M, Vielmuth F, Meir M, Schick MA et al. Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis. Cardiovasc Res. 2015;107: 32–44. doi: 10.1093/cvr/cvv144 [DOI] [PubMed] [Google Scholar]
- 52.Dejana E, Corada M, Lampugnani MG. Endothelial cell-to-cell junctions. FASEB J. 1995;9: 910–918. [PubMed] [Google Scholar]
- 53.Hermant B, Bibert S, Concord E, Dublet B, Weidenhaupt M, Vernet T et al. Identification of proteases involved in the proteolysis of vascular endothelium cadherin during neutrophil transmigration. J Biol Chem. 2003; 278: 14002–14012. doi: 10.1074/jbc.M300351200 [DOI] [PubMed] [Google Scholar]
- 54.Chen T, Guo ZP, Cao N, Qin S, Li MM, Jia RZ. Increased serum levels of soluble vascular endothelial-cadherin in patients with systemic vasculitis. Rheumatol Int. 2014;34: 1139–1143. doi: 10.1007/s00296-014-2949-7 [DOI] [PubMed] [Google Scholar]
- 55.Soeki T, Tamura Y, Shinohara H, Sakabe K, Onose Y, Fukuda N. Elevated concentration of soluble vascular endothelial cadherin is associated with coronary atherosclerosis. Circ J. 2004; 68: 1–5. [DOI] [PubMed] [Google Scholar]
- 56.Koga H, Sugiyama S, Kugiyama K, Watanabe K, Fukushima H, Tanaka T et al. Elevated levels of VE-cadherin-positive endothelial microparticles in patients with type 2 diabetes mellitus and coronary artery disease. J Am Coll Cardiol. 2005;45: 1622–30. doi: 10.1016/j.jacc.2005.02.047 [DOI] [PubMed] [Google Scholar]
- 57.Musil D, Kaletova M, Herman J. Age, body mass index and severity of primary chronic venous disease. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155: 367–371. doi: 10.5507/bp.2011.054 [DOI] [PubMed] [Google Scholar]
- 58.Katusic ZS. Mechanisms of endothelial dysfunction induced by aging, role of arginase I. Circ Res. 2007;101: 640–641. doi: 10.1161/CIRCRESAHA.107.162701 [DOI] [PubMed] [Google Scholar]
- 59.Herrera MD, Mingorance C, Rodrίguez- Rodrίguez R, Alvarez de Sotomayor M. Endothelial dysfunction and aging: an update. Ageing Res Rev. 2010;9: 142–152. doi: 10.1016/j.arr.2009.07.002 [DOI] [PubMed] [Google Scholar]
- 60.Pulerwitz T, Grahame-Clarke C, Rodriguez CJ, Miyake Y, Sciacca RR, Hirata K et al. Association of increased body mass index and impaired endothelial function among Hispanic women. Am J Cardiol. 2006;97: 68–70. doi: 10.1016/j.amjcard.2005.07.125 [DOI] [PubMed] [Google Scholar]
- 61.Doulgere J, Otto B, Nassour M, Wolters-Eisfeld G, Rohde H, Magnus T et al. Soluble plasma VE-cadherin concentrations are elevated in patients with STEC infection and haemolytic uraemic syndrome: a case-control study. BMJ Open. 2015;5: e005659 doi: 10.1136/bmjopen-2014-005659 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Ebihara I, Hirayama K, Nagai M, Koda M, Gunji M, Okubo Y et al. Soluble vascular endothelial-cadherin levels in patients with sepsis treated with direct hemoperfusion with a polymyxin B-immobilized fiber column. Ther Apher Dial. 2014;18: 272–278. doi: 10.1111/1744-9987.12215 [DOI] [PubMed] [Google Scholar]
- 63.Ostrowski SR, Berg RM, Windeløv NA, Meyer MA, Plovsing RR, Møller K et al. Coagulopathy, catecholamines, and biomarkers of endothelial damage in experimental human endotoxemia and in patients with severe sepsis: a prospective study. J Crit Care. 2013;28: 586–96. doi: 10.1016/j.jcrc.2013.04.010 [DOI] [PubMed] [Google Scholar]
- 64.Alexander JS, Alexander BC, Eppihimer LA, Goodyear N, Haque R, Davis CP et al. Inflammatory mediators induce sequestration of VE-cadherin in cultured human endothelial cells. Inflammation. 2000;24: 99–113. [DOI] [PubMed] [Google Scholar]
- 65.Zhao X, Alexander S, Zhang S, Zhu Y, Sieber NJ, Aw TY et al. Redox regulation of endothelial barrier. Am J Physiol Lung Cell Mol Physiol 2001;281: L879–L886. doi: 10.1152/ajplung.2001.281.4.L879 [DOI] [PubMed] [Google Scholar]
- 66.Yasim A, Kilinc M, Aral M, Oksuz H, Kabalci M, Eroglu E et al. Serum concentration of procoagulant, endothelial and oxidative stress markers in early primary varicose veins. Phlebology. 2008;23: 15–20. doi: 10.1258/phleb.2007.007014 [DOI] [PubMed] [Google Scholar]
- 67.Bryan LJ, Callas PW, Criqui MH, Cushman M. Higher soluble P-selectin is associated with chronic venous insufficiency: the San Diego Population Study. Thromb Res. 2012;130: 716–719. doi: 10.1016/j.thromres.2012.07.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Semenov AV, Kogan-Ponomarev MIa, Ruda MIa, Komarov AL, Panchenko EP, Chazova IE, Mazurov AV. Soluble P-selectin—a marker of platelet activation and vessel wall injury: increase of soluble P-selectin in plasma of patients with myocardial infarction, massive atherosclerosis and primary pulmonary hypertension. Ter Arkh. 2000;72: 15–20. [PubMed] [Google Scholar]
- 69.McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF. GMP-140, a platelet alpha granule protein, is also synthesized by vascular endothelium and is localized in Weibel-Palade bodies. J Clin Invest. 1989;84: 92–99. doi: 10.1172/JCI114175 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Budzyń M, Iskra M, Krasiński Z, Dzieciuchowicz Ł, Kasprzak M, Gryszczyńska B. Serum iron concentration and plasma oxidant-antioxidant balance in patients with chronic venous insufficency. Med Sci Monit. 2011. D;17:CR719–CR727. doi: 10.12659/MSM.882132 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Condezo-Hoyos L, Rubio M, Arribas SM, España-Caparrós G, Rodríguez-Rodríguez P, Mujica-Pacheco E et al. A plasma oxidative stress global index in early stages of chronic venous insufficiency. J Vasc Surg. 2013;57: 205–213. doi: 10.1016/j.jvs.2012.05.085 [DOI] [PubMed] [Google Scholar]
- 72.Głowinski J, Głowinski S. Generation of reactive oxygen metabolites by the varicose vein wall. Eur J Vasc Endovasc. 2002;23: 550–555. [DOI] [PubMed] [Google Scholar]
- 73.Karatepe O, Unal O, Ugurlucan M, Kemik A, Karahan S, Aksoy M et al. The impact of valvular oxidative stress on the development of venous stasis, valvular oxidative stress and venous ulcers. Angiology 2010;61: 283–288. doi: 10.1177/0003319709343177 [DOI] [PubMed] [Google Scholar]
- 74.Thies RL, Autor AP. Reactive oxygen injury to cultured pulmonary artery endothelial cells: mediation by poly (ADP-ribose) polymerase activation causing NAD depletion and altered energy balance. Arch Biochem Biophys.1991;286: 353–363. [DOI] [PubMed] [Google Scholar]
- 75.Van Wetering S, Van Buul JD, Quik S, Mul FP, Anthony EC, ten Klooster JP et al. Reactive oxygen species mediate Rac—induced loss of cell—cell adhesion in primary human endothelial cells. J Cell Sci. 2002;115: 1837–1846. [DOI] [PubMed] [Google Scholar]
- 76.Grotto D, Santa Maria L, Valentini J, Paniz C, Schmitt G, Garcia SC et al. Importance of the lipid peroxidation biomarkers and methodological aspects for malondialdehyde quantification. Quimica Nova 2009;32: doi: 10.1590/S0100-40422009000100032 [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
(Spearman correlation coefficient r = 0.419, P = 0.014).
(PDF)
(Spearman correlation coefficient r = 0.373, P = 0.049).
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * P≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
Box and whisker plots show median (central line), upper and lower quartiles (box) and range excluding outliers (whiskers). Data were analyzed using Kruskal-Wallis test followed by the Dunn`s multiple comparison test. * p≤0.05 was considered statistically significant.
(PDF)
(a)Results shown as mean± standard deviation, one-way ANOVA test was used for comparison.(b)Results shown as median and interquartile range, Kruskull-Wallis test was used for comparison.NS—not statistically significant.
(PDF)
Data Availability Statement
All relevant data are within the paper and its Supporting Information files.