Chronic venous insufficiency (CVI) is a multifactorial disease, commonly caused by valvular incompetence (clinically diagnosed by venous reflux) and venous hypertension. The incidence of these factors clearly increases with patient age, and aging is one of the risk factors involved. The activity of the PI3K/Akt/mTOR pathway is considered fundamental in vascular pathologies, and understanding its involvement would help in the development of possible therapeutic targets. This is an observational, analytical, and prospective cohort study that reviewed 110 patients with CVI scheduled to undergo stratified saphenectomy. They were distributed according to the presence (
Venous pathology develops when venous pressure increases and blood return is disrupted. The peripheral venous system functions as a conduit that returns blood to the heart. Proper functioning of this system depends on the venous valves and the muscular pump [
PI3K/Akt/mTOR pathway activity is reported by numerous researchers to be fundamental in vascular pathologies [
Varicose veins develop through a series of gradual stages; it is possible to consider the development of venous wall insufficiency as an aging process. Generally, this condition proceeds according to the age of the individual, especially after the fifth decade of life [
This is an observational, analytical, and prospective cohort study that reviewed patients with CVI scheduled to undergo stratified saphenectomy and divided them according to age (cutoff point of 50 years). The research was developed through a collaboration between the Service of Angiology and Vascular Surgery of the Ruber International Hospital and the Department of Medicine and Medical Specialties of the University of Alcalá. The study cohort was selected according to the following criteria. Inclusion criteria are as follows: women and men diagnosed with CVI, with and without venous reflux in the great saphenous vein;
Each patient underwent exploration with the aid of an M-Turbo Eco-Doppler (SonoSite) transducer at 7.5 MHz. The examination of the lower limbs was performed in a standing position with the leg that was explored maintained in external rotation and supported by the contralateral leg; the study included the great saphenous axis from the inguinal region to the ankle and femoral vein. A study of the small saphenous vein and popliteal vein was also performed standing, with the back to the examiner and the body weight resting on the examined leg. A distal compression maneuver was performed. In this study, Valsalva maneuvers were performed, which when producing a proximal circulatory stop will allow exploration of venous insufficiency proximal to the detection point, as well as the identification of leakage points (it evaluates the absence of reflux in the femoral-iliac and saphenous-femoral union). The distal compression and decompression maneuver was also performed to assess the direction of the truncal venous flow, although it was not a physiological maneuver. Pathological reflux was considered when this was greater than or equal to 0.5 sec.
The present study was conducted in accordance with the basic ethical principles (autonomy, beneficence, nonmaleficence, and distributive justice), and its development followed Good Clinical Practice standards and the principles set forth in the last Declaration of Helsinki (2013) and the Convention of Oviedo (1997). The patients were duly informed, and each was asked to provide written informed consent. The project was approved by the Clinical Research Ethics Committee of the Ruber International Hospital.
Once saphenectomy was performed, the entirety of the great saphenous vein was extracted. These fragments were introduced into two different sterile tubes, one containing MEM (minimum essential medium) with 1% antibiotic/antimycotic (both from Thermo Fisher Scientific, Waltham, MA, USA) and another containing RNAlater® Solution (Ambion, Austin, TX, USA). All samples are transferred refrigerated to the Department of Medicine and Medical Specialties (Faculty of Medicine and Health Sciences, University of Alcalá) for processing. In all cases, the transfer was made within four hours after the sample was taken.
The samples were processed in a Telstar AV 30/70 Müller class II laminar flow hood 220 V 50 MHz (Telstar SA Group, Terrassa, Spain), thus allowing an environment of sterility. Samples conserved in RNAlater remained in 1 mL of the same solution at −80°C until further processing for analysis of gene expression. The samples conserved in MEM were destined for histological studies of venous tissue. The samples were washed/hydrated several times with MEM without antibiotic to remove blood cells and then cut into fragments that were kept in different fixatives: F13 (60% ethanol, 20% methanol, 7% polyethylene glycol, and 13% distilled H2O). After the necessary fixing time for each fixative solution, the samples were dehydrated. At the end of the inclusion, paraffin blocks were made using molds. Once the paraffin solidified, an HM 350 S rotation microtome (Thermo Fisher Scientific) was used to obtain 5
Toluidine blue staining was conducted as follows: (1) staining with toluidine blue in 0.03% aqueous solution for 15 minutes, (2) washing with running water for 10 minutes, (3) dehydration in 96% alcohol for 5 minutes, (4) dehydration in 100% alcohol for 5 minutes, (5) clarification in xylol for 5 minutes, and (6) assembly with Cytoseal™ balsam. Toluidine blue is often used to identify mast cells, by virtue of the heparin present in their granules.
Antigen-antibody detection was achieved using the ABC (avidin-biotin complex) method with chromogenic peroxidase or alkaline phosphatase according to the following protocol: (1) washing the samples with PBS 1x, three passes of 5 minutes. (2) Blocking of nonspecific binding sites with BSA (bovine serum albumin) at 3% in PBS for 30 minutes at room temperature. (3) Incubation with the primary antibody (Table
Primary antibodies that were used and their dilutions.
Antigen | Species | Clone | Dilution | Provider | Protocol specifications |
---|---|---|---|---|---|
Akt | Rabbit | Polyclonal | 1 : 1000 | Abcam (ab8805) | — |
CD4 | Rabbit | Monoclonal | 1 : 50 | Abcam (ab133616) | EDTA |
CD8 | Rabbit | Polyclonal | 1 : 25 | Abcam (ab4055) | EDTA |
CD19 | Mouse | Monoclonal | 1 : 200 | Abcam (ab31947) | — |
HIF-1 |
Mouse | Monoclonal | 1 : 800 | Abcam (ab16066) | EDTA |
HIF-2 |
Mouse | Monoclonal | 1 : 2000 | Abcam (ab8365) | EDTA |
PI3K | Mouse | Monoclonal | 1 : 500 | Abcam (ab86714) | — |
mTOR | Rabbit | Polyclonal | 1 : 500 | Abcam (ab1093) | — |
Secondary antibodies that were used and their dilutions.
Antigen | Species | Clone | Dilution | Provider |
---|---|---|---|---|
IgG (mouse) | Goat | Polyclonal | 1/300 | Sigma |
IgG (rabbit) | Mouse | RG-96 | 1/1000 | Sigma |
Through real-time polymerase chain reaction (qPCR), the amount of cDNA in each sample of the gene of interest was quantified (Table
Primers used for RT-qPCR: sequences and binding temperatures (Temp).
Gene | Sequence fwd (5 |
Sequence rev (5 |
Temp (°C) |
---|---|---|---|
GAPDH | GGA AGG TGA AGG TCG GAG TCA | GTC ATT GAT GGC AAC AAT ATC CAC T | 60 |
Akt | TGT CTC GTG AGC GCG TGT TTT | CCG TTA TCT TGA TGT GCC CGT C | 60 |
HIF-1 |
ACG TGT TAT CTG TCG CTT TGA G | ATC GTC TGG CTG CTG TAA TAA TG | 59 |
HIF-2 |
ACC CAG TAC CAG GAC TAC AGC | GGC ACG TTC ACC TCA CAG TC | 61 |
PI3K | CTT GCC TCC ATT CAC CAC CTC T | GCC TCT AAT CTT CTC CCT CTC CTT C | 60 |
mTOR | ATC CAG ACC CTG ACC CAA AC | TCC ACC CAC TTC CTC ATC TC | 60 |
For statistical analysis, the GraphPad Prism® 6.0 program was used to apply the Mann–Whitney
For each of the patients in the established groups, five sections and 10 fields per section were randomly selected and examined. The patients were described as positive when the average of the analysis of the labeled sample for each study subject was greater than or equal to 5% of the total, following the anatomopathological protocol of Ortega et al. [
For the present study, a total of 110 patients were contacted. These patients were classified according to the absence of venous reflux (NR;
There were no significant differences in the hemogram or in the general biochemistry (data not shown). This nonsignificant relationship was maintained when the absence or presence of venous reflux was considered, as well as when age was considered. No significant differences were found in the clinical history of the patients when studying demographic factors (data not shown).
The expression of the PI3K/Akt/mTOR pathway was revealed using protein and relative quantity mRNA detection techniques.
The relative quantity mRNA of PI3K showed a significant increase in patients with R in comparison with the NR group (
(a) Significant levels of mRNA for PI3K quantified by RT-qPCR in R and
The percentage of patients with positive protein expression based on immunohistochemical studies of PI3K expression was higher (75.31%) in the group of R individuals. Notably,
Patients with venous reflux exhibited differential expression depending on their age. In the
mRNA expression did not reveal any significant differences between the study groups (NR versus R). The distribution by age showed a significant increase in
(a) Significant levels of mRNA for Akt quantified by RT-qPCR. In
The percentage of Akt protein expression was not different between NR (79.31%) and R (75.31%) individuals. Remarkably,
Microscopic observation revealed that the
A relative amount mRNA of mTOR was higher in the R patients versus the NR group, establishing a statistically significant difference (
(a) Significant levels of mRNA for mTOR quantified by RT-qPCR in R and
The percentage of positive mTOR expression was higher in patients with venous reflux (R), with 62.07% in the NR and 77.78% in the R patients. Consideration of the age factor showed that the percentage was more elevated in the
The microscopic observation showed that mTOR was distributed differently in the different study groups. The
The
(a) Histological images for protein expression of mTOR in the different tunicae of the venous wall in
The study of the hypoxic component was performed by relative quantity mRNA and protein detection of hypoxia-inducible factors (HIFs) based on the expression of the subunits 1 alpha (HIF-1
A relative amount of mRNA of HIF-1
(a) Significant levels of mRNA for HIF-1
When studying HIF-1
A relative quantity of mRNA of HIF-2
(a) Significant levels of mRNA for HIF-2
The study of HIF-2
Protein expression was present in the nuclei of smooth muscle fibers (Figure
In terms of quantification between the NR and R patient groups, no significant differences were observed. By considering age, we found significant differences. The highest number of CD4
(a) Positive cell quantification of CD4+, CD8+, CD19+, and mast cells in the three tunicae of the vein wall for patients without reflux (NR) and with reflux (R), as well as by the ages of the same.
Quantification of the number of CD8+ cells did not show significant differences between the NR and R patients. Including age in the analysis did not reveal differences between the different patient groups but showed a slight tendency toward an increase similar to that observed in the CD4+ cells (Figure
The presence of B lymphocytes was evidenced by immunodetection of CD19+ cells, and an increasing tendency in the R patients with respect to the NR patients was found. In terms of age, statistically significant differences were established between the
The presence of mast cells in the vein wall was detected with toluidine blue. The results revealed a significant increase in the number of mastocytes in the R patients with respect to the NR patients (
Despite scientific and technological advances, the failure of the venous wall responsible for CVI still has no clear etiology. Therefore, multiple factors have been proposed to contribute to venous wall failure and to potentially determine the failure either by distension of the wall, valvular failure, or valvular agenesis in some key places [
Previous works have shown that of the multiple noxae that affect the wall and venous functioning, the very process of life (aging) is one contributor to venous failure [
One of the events observed by our research group is the gene and protein expression of different components of the PI3K/Akt/mTOR cellular transduction pathway. Our results have shown that patients with venous reflux, especially young individuals, have a significant increase in vein activation. Yuan et al. [
At another point, hypoxia caused by venous hypertension sets in motion molecular pathways involved in the cellular response to the lack of oxygen, such as PI3K/Akt/mTOR and consequently the HIF transcription factors. Research in the field of these factors suggests that HIF-2
Interestingly, in our study, we observed that reflux becomes a point of inflection for adaptation relative to the age profile of the patients studied, especially in relation to mTOR expression. All these findings are compatible with the process of the hypertrophy-atrophy sequence that an insufficient venous wall undergoes during this process, which is aggravated in venous reflux due to valve incompetence, as shown by Buján et al. [
The presence of CD4+, CD8+, and CD19+ cells suggests the existence of an inflammatory process and stress in relation to the above. A variety of studies have shown an increase in degranulation and extravasation of leukocytes in patients with venous hypertension. Saharay et al. [
In relation to the above, it should be noted that mast cells can perform a wide variety of functions that induce the inflammatory cascade via mediators derived from their granules in endocytosis. Pascual et al. [
Current research suggests that the PI3K/Akt/mTOR pathway plays an important role in cellular metabolism regulation and in the functions of the immune system, two subjects that are closely related to cell and organ functionality [
Our results help demonstrate the notion that among several possible mechanisms of activation, venous reflux in CVI is to a considerable degree an inflammatory disease induced by blood pressure. The venous pressure elevation (change in the type of pressure) and the displacement of the shear stress generate an abnormal biomechanical environment in the venules, in their walls, and in the valves, which can initiate early activation of enzymatic activity and in turn set in motion a cascade of cellular transduction, leading to cytoarchitectonic changes to compensate for these alterations.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
Miguel A. Ortega and Ángel Asúnsolo contributed equally to this work.
This work was supported by grants from the National Institute of Health Carlos III (FIS-PI13/01513) and Province of Madrid (B2017/BMD-3804 MITIC-CM).