The role of recovered thiols in venous endothel adaptation in case of autovenous reconstruction of lower limb arteries

Aim. To evaluate the role of thiol status and its correlative relationship with the level of nitric oxide metabolites and vascular endothelial growth factor A (VEGF-A) in the blood plasma of patients with critical lower limb ischemia (CLI) after autovenous reconstructions of femoropopliteal segment’s arteries in the setting of venous endothelium of the arterial bed. Materials and methods. 54 patients with critical lower limb ischemia had been examined and divided into groups: synthetic prosthesis, in situ autovenous bypass procedure and reversed vein autovenous bypass procedure. Peripheral venous blood was taken on the 1 st and 10th days, and in 1, 3 and 6 months after the operation. Nitric oxide metabolites level was examined with photocolorimetric method by reaction with Griess reagent on a microplate analyzer (Awareness Technology, USA). VEGF-A number estimation was done by ELISA test (PersonalLab, Italy) with the use of Human VEGF-A Platinum ELISA. The level of thiol (SH-) groups was estimated with the use of Ellman’s reagent (SERVA, Germany) on spectrophotometer (Saint-Petersburg, Russian Federation). Results. The concentration of VEGF-A and the level of SH-groups increase on the 10th day and after 1 month in the group of patients operated on using a synthetic prosthesis. The level of NO metabolites, the concentration of VEGF-A, and the content of SH-groups increase statistically significantly and then decrease to the initial values in the group of patients operated on by reversed vein autovenous bypass procedure. In the group of patients operated on by the in situ method, the level of nitric oxide metabolites increases, the concentration of VEGF-A increases on the 10th day, the level of SH-groups increases, and a positive correlation was found between the content of SH-groups and the concentration of VEGF-A. Conclusion. NO metabolites contribute to the build-up of SH groups and VEGF-A in patients operated by the “reversed vein” method, and in patients in the in situ group, the concentration of VEGF-A and the level of SH groups are not affected, which may be of clinical importance when prescribing NO donors. The revealed patterns of change in the level of recovered thiols, nitrogen oxide metabolites, VEGF-A in combination with analysis of early and late postoperative complications make it possible to conclude the advantage of autovenous reconstruction of the femoropopliteal segment due to functional adaptation of the venous endothelium compared to the group of patients, operated on by the method of “synthetic prosthesis”. Method in situ on biochemical and angiological indicators proved to be more favorable in terms of the clinical course, than the “reversed vein” method. of the manuscript for publication. Abalenikhina Pshennikov Vinogradov S.A. – conception and design of the study, analysis and interpretation of data, drafting of the article. Abalenikhina – carrying out of biochemical tests. Pshennikov Vinogradov – supervision of patients in the Department of Vascular Surgery of the Regional Clinical Hospital of Ryazan, treatment and surgical correction of the magistral blood flow, sampling of biological material from patients.


INTRODUCTION
The use of an autovenous conduit from the great saphenous vein (GSV) remains an acceptable method of open reconstructive vascular surgery. Currently, autovenous bypass is performed using the reverse vein method and the in situ method. The discussion about the advantages and disadvantages of both methods has not stopped for more than half a century: will the transformation of the autovein take the path of adaptation or maladjustment?
Published data describes a proposal on the possibility of considering the concentration of SH-groups as an indicator of the adaptive capabilities of the organism [1]. Since the 1960s, a large number of clinical studies have been performed in which a decrease in the concentration of SH-groups in the blood serum of patients with various diseases was obtained: cardiovascular disorders [2], diabetes mellitus [3], chronic renal failure [4], alcoholic cirrhosis [5] and other pathology [6]. The dynamics of changes in SH-groups against the background of autovenous reconstructions of the arteries of the lower extremities under conditions of reperfusion damage to the endothelium may turn out to be a marker for predicting the adaptive transformation of an arterialized vein.
It is known that the most important and early marker of intimal damage is endothelial dysfunction associated, first of all, with the inhibition of the production of nitric oxide (NO), the most important regulator of vascular tone [7,8]. Nitric oxide, due to its functions, is a universal angioprotective substance on which structural changes in the vascular wall depend [9,10]. It has been proven that NO helps to slow down the formation of neointima in the area of surgical anastomoses of an artery with a synthetic prosthesis in patients after surgery [11], therefore, it was suggested that treatment with drugs that stimulate the production of NO may have an inhibitory effect on the development of restenosis after surgery [12], acting as one of the mechanisms of long-term compensation of blood circulation. Since NO is a "shortlived" molecule, many of its protective properties are associated not only with direct action, but also with the endogenous protective systems activated by it, in Для цитирования: Калинин Р.Е., Абаленихина Ю.В., Пшенников А.С., Виноградов С.А. Роль восстановленных тиолов в адаптации венозного эндотелия при аутовенозной реконструкции артерий нижних конечностей. Бюллетень сибирской медицины. 2020; 19 (4): 86-93. https://doi.org/10.20538/1682-0363-2020-4-86-93. __________________________ particular with the activation of vasculoendothelial growth factor (VEGF) [13].
In this regard, it is relevant to study the thiol status and identify correlations between the content of SH-groups and the level of metabolites of nitric oxide, VEGF-A in blood plasma in combination with the analysis of early and late complications in patients with critical lower limb ischemia (CLI) after autovenous reconstruction of the arteries of the femoral-popliteal segment to assess the degree of adaptation of the venous endothelium in the settings of the arterial bed.

MATERIALS AND METHODS
The study included 54 patients with CLI (III-IV stage according to the Pokrovskiy -Fontaine classification), of whom there were 48 men (88.9%) and 6 women (11.1%). The average age was (64.6 ± 6.6) years. All patients gave informed consent to participate in the study and to use their bioassay. The patients were divided into three groups depending on the type of graft used for revascularization of the arteries of the femoral-popliteal segment: group A -synthetic prosthesis, group B -reversed vein and group C -in situ autovenous bypass grafting. Conical prostheses made of 4/8 mm polytetrafluoroethylene were used as a synthetic graft in all cases. In patients with autovenous revascularization, the ipsilateral great saphenous vein was used in all cases. Clinical groups were matched by gender, age, stage of the disease, comorbidity, initial minimum diameter of GSV, Rutherford outflow score (Table 1).
Post-operative peripheral venous blood withdrawal for evaluation of the examined biochemical indicators had been done on days 1 and 10, and on months 1, 3 and 6.
The level of nitric oxide metabolites (the total concentration of nitrates and nitrites) was determined by the photocolorimetric method by the development of color in the diazotization reaction with sulfanilamide nitrate, which is part of the Griess reagent (NevaReaktiv, Russia). Nitrites diazotize sulfanilamide, and the resulting substance reacts with azo coupling with naphthylethylenediamine to form a pink compound, the color intensity of which is proportional to the total concentration of nitrites and nitrates in the sample [14].
The color intensity was assessed in the visible spectral region with registration on a StatFax 3200 microplate analyzer (Awareness Technology, USA) at a wavelength of 540 nm and expressed in nmol/mg protein.
Determination of the active form of human vasculoendothelial growth factor A (VEGF-A) was carried out by enzyme immunoassay on Personal Lab units (Italy) using Human VEGF-A Platinum ELISA human VEGF-A (BMS277/2) (BioChimMac, Moscow, Russia). The intensity of the color measured at 450 nm is directly proportional to the concentration of VEGF-A present in the samples. The VEGF-A concentration in the samples was determined using a standard curve and expressed in pg/ml.
The level of intracellular and extracellular reducing agents was assessed by the change in the content of thiol (SH-) groups, which were determined using Ellman's reagent on an SF-2000 spectrophotometer (St. Petersburg, Russia) [15]. The reaction of sulfhydryl (thiol) groups with Ellman's reagent (5,5'dithiobis-2-nitrobenzoic acid, SERVA, Germany) breaks the disulfide bond in the reagent and forms 2-nitro-5-thiobenzoic acid, which at alkaline pH in water turns into a quinoid form and has a bright yellow color. The resulting thionitrophilic anion is quantified on a spectrophotometer at 412 nm. To calculate the level of reduced thiols in blood plasma (in μmol/ml), the molar extinction coefficient of the Ellman reagent was taken as 14150 M -1 cm -1 .
The statistical analysis of the results of the experimental study was carried out using the Statistica 10.0 pro-gram. The normal distribution of data was checked using the Shapiro -Wilk test (W test). The results were presented as Ме [Q 1 ; Q 3 ], where Ме is the median, Q 1 is the first quartile (25%), Q 3 is the third quartile (75%), arithmetic mean and its error M ± m. To analyze the statistical significance of differences between independent samples, the Mann -Whitney rank test (U test) was used. To check the equality of the medians of several samples, the Kruskall -Wallis test was used. Spearman's coefficient (R) was used to assess the rank correlation. The critical level of statistical significance of the differences in the null hypothesis (p) was taken equal to 0.05.

RESULTS AND DISCUSSION
NO metabolites level does not change significantly in Group A and increases in post-operation period in groups B and C.
In groups B and C NO metabolites level significantly grows on days 1 and 10, in the period from month 1 to month 6 NO level is stable but statistically higher than before operations (Fig. 1).
Any operative intervention on arteries leads to endothelium damage which causes inflammation with adhesion and thrombocyte aggregation, proliferation and migration of smooth muscle cells into intima. Endothelium damage induces production of NO synthase-2 (NOS-2) in smooth muscle cells and endothelium [16,17], which can explain the results reached in groups B and C. Venous conduit from great saphenous vein has functional endothelial lining and reacts on local and systemic molecular and hemodynamic stimulants and regulates vascular tone and homeostasis which is not the case of synthetic prosthesis due to absence of endothelium in it and that is why NO metabolites level does not change in group A (Fig. 1). Along with changes in the level of nitric oxide metabolites, vascular endothelial growth factor VEGF is one of the well-studied indicators of angiogenesis, indicating the severity of the lesion [18]. It has been proven that VEGF ensures normal growth and development of the body, wound healing. However, high levels of VEGF were found in the development of cancerous tumors and rheumatoid arthritis [19]. equality of the medians of several samples, the Kruskall -Wallis test was used. Spearman's coefficient (R) was used to assess the rank correlation. The critical level of statistical significance of the differences in the null hypothesis (p) was taken equal to 0.05.

NO metabolites level does no't change significantly in Group A and increases in post-
operation period in groups B and C.
In groups B and C NO metabolites level significantly grows on days 1 and 10, in the period from month 1 to month 6 NO level is stable but statistically higher than before operations (Figure 1).  It has been proven that VEGF ensures normal growth and development of the body, wound healing.
However, high levels of VEGF were found in the development of cancerous tumors and rheumatoid arthritis [19].
The concentration of the active form of human VEGF-A in experimental group A increases statistically significantly from the 1st day to 6 months, reaching a maximum on the 10th day. In experimental group B, the amount of VEGF-A statistically significantly increased after 1 month relative to the values before surgery. In group C, it statistically significantly increases on the 10th day after surgery and decreases in the period from 1 to 6 months relative to the values before surgery.
* significant differences in data between pre-operation and post-operation (p < 0.05)  The concentration of the active form of human VEGF-A in experimental group A increases statistically significantly from the 1st day to 6 months, reaching a maximum on the 10th day. In experimental group B, the amount of VEGF-A statistically significantly increased after 1 month relative to the values before surgery. In group C, it statistically significantly increases on the 10th day after surgery and decreases in the period from 1 to 6 months relative to the values before surgery.
It is known that the survival rate of patients with a high level of VEGF is significantly lower than that of patients with a low expression of VEGF-A [18], therefore, the results obtained indicate a positive trend in autovenous bypass grafting using the in situ technique.
After surgery, the restoration of the body is accompanied by adaptation to new conditions. Currently, thiol-containing compounds (molecules containing-SH groups) are considered not only as a new marker of oxidative stress, but also as an indicator of homeostasis and adaptation under pathophysiological conditions [20].
The level of reduced thiols in the blood plasma of patients in groups A and B increases on the 10th day and after 1 month, and in group C it statistically significantly increases from the 10th day to the 6th month (Fig. 3).
The degree of adaptation directly depends on the concentration of thiols: the higher the degree of adaptation, the higher the level of SH-groups in the blood serum. It follows from this that the functional adaptation of the venous endothelium is higher in patients operated on by the in situ method, compared with the reverse vein method.
Thiols are able to form reversible mixed disulfide bonds between protein and low molecular weight SH groups. The results obtained in group C indicate that oxidative stress decreases, disulfide bonds are restored to thiol groups, and thiol-disulfide homeostasis is maintained, and the body's resistance to reconstructive surgery increases. Abnormalities in thiol-disulfide homeostasis can play an essential role as a biomarker in the development of pathophysiological conditions. In patients in groups A and B, after mobilization of the body's defense reactions, which are expressed in an increase in the level of thiols, their decrease to the initial level is observed, which indicates adaptation or compensation without depletion.
The obtained biochemical results confirm the clinical observations given in Table. 2.
Бюллетень сибирской медицины. 2020; 19 (4): 86-93 It is known that the survival rate of patients with a high level of VEGF is significantly lower than that of patients with a low expression of VEGF-A [18], therefore, the results obtained indicate a positive trend in autovenous bypass grafting using the in situ technique.
After surgery, the restoration of the body is accompanied by adaptation to new conditions. Currently, thiol-containing compounds (molecules containing -SH groups) are considered not only as a new marker of oxidative stress, but also as an indicator of homeostasis and adaptation under pathophysiological conditions [20].
The level of reduced thiols in the blood plasma of patients in groups A and B increases on the 10th day and after 1 month, and in group C -it statistically significantly increases from the 10th day to the 6th month (Fig. 3). * significant differences in data between pre-operation and post-operation (p < 0.05) The degree of adaptation directly depends on the concentration of thiols: the higher the degree of adaptation, the higher the level of SH-groups in the blood serum. It follows from this that the functional adaptation of the venous endothelium is higher in patients operated on by the in situ method, compared with the reverse vein method.
Thiols are able to form reversible mixed disulfide bonds between protein and low molecular weight SH groups. The results obtained in group C indicate that oxidative stress decreases, disulfide bonds are restored to thiol groups, and thiol-disulfide homeostasis is maintained, and the body's resistance to reconstructive surgery increases.
Abnormalities in thiol-disulfide homeostasis can play an essential role as a biomarker in the development of pathophysiological conditions. In patients in groups A and B, after mobilization of the body's defense reactions, which are expressed in an increase in the level of thiols, their decrease to the initial level is observed, which indicates adaptation or compensation without depletion.  From the perspective of early and late postoperative complications, patients in group C have a more favorable clinical picture, as indicated by a high percentage of primary and secondary patency of bypasses, a decrease in mortality, and an increase in the degree of limb preservation. The most unfavorable postoperative period according to the list of declared angiological signs is in patients in group A (see Table 2).
Interestingly, in terms of biochemical parameters in group B, positive correlations were revealed: the higher the level of nitric oxide metabolites, the higher the number of VEGF-A and SH-groups. Patients of group C showed a positive correlation between the amount of VEGF-A and free thiols, regardless of the level of nitric oxide metabolites (Table 3). Thus, in group B, NO metabolites promote the growth of SHgroups and VEGF-A, and in group C, they do not affect the concentration of VEGF-A and the level of SHgroups, which may be of clinical significance when assigning NO donors.

CONCLUSION
The obtained patterns of changes in the level of reduced thiols, nitric oxide metabolites, VEGF-A in patients with CLI indicate adaptation of the venous endothelium in the settings of the arterial bed during autovenous reconstruction of the femoropopliteal segment in comparison with the group of patients operated on using a synthetic prosthesis, which is confirmed by the analysis of early and late postoperative complications. The in situ method demonstrates the development of a more favorable clinical picture in terms of biochemical and angiological parameters than the reverse vein method.