Expression of the ACTA1, PLXNA4, and SEMA3A genes in varicose veins of patients with different length of great saphenous vein reflux

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OBJECTIVE

Uncovering the molecular genetic causes of recurrence may contribute to the improvement of varicose vein disease (VVD) treatment. We aimed to study the expression of actin filament remodeling-associated genes ACTA1, PLXNA4, and SEMA3 in varicose (VVs) and non-varicose (NVs) vein segments of patients with different length of pathological GSV reflux.

MATERIAL AND METHODS

26 patients were divided into two groups: with the length of reflux up to the lower third of the thigh (13 patients) and up to the lower third of the calf (13 patients). After RNA isolation from paired (VV and NV, 26 pairs) segments of the GSV from each patient, an assessment at the mRNA level of the relative expression of the studied genes was made using real-time RT-PCR.

RESULTS

The difference in expression ratios (paired VV to NV) of the ACTA1 gene between the groups of patients with different lengths of reflux was confirmed (p=0.03). Differential expression of the PLXNA4 gene was found in VVs in the group of patients with the length of reflux up to the lower third of the thigh (p=0.04), as well as between the subgroups of NVs from the patients with the length of reflux up to the lower third of the thigh and NVs from the patients with reflux up to the lower third of the calf (p=0.03). Differential expression was shown in VVs (p=0.02) in the group of patients with reflux up to the lower third of the thigh for the SEMA3A gene, the product of which is the PLXNA4 receptor ligand.

CONCLUSION

The difference in the expression of the ACTA1, PLXNA4, and SEMA3 genes involved in the cytoskeleton remodeling processes and, subsequently, vein wall remodeling, was shown in the vein samples from patients with different length of GSV reflux. Thus, the length of GSV trunk reflux that determines the probability of reflux recurrence after ASVAL procedure is associated with vein wall remodeling. The identified genes can become promising therapeutic targets.

Keywords:

varicose veins

reflux recurrence

cytoskeleton

gene expression

Authors:

V.A. Korolenya

Institute of Chemical Biology and Fundamental Medicine;
Novosibirsk State University

ORCID: 0000-0002-1016-2156

K.A. Gavrilov

Novosibirsk State University;
Center of New Medical Technologies Institute of Chemical Biology and Fundamental Medicine

ORCID: 0000-0002-3839-3263

K.S. Sevostyanova

Novosibirsk State University;
Center of New Medical Technologies Institute of Chemical Biology and Fundamental Medicine

ORCID: 0000-0002-4110-8187

A.I. Shevela

Institute of Chemical Biology and Fundamental Medicine;
Novosibirsk State University;
Center of New Medical Technologies Institute of Chemical Biology and Fundamental Medicine

ORCID: 0000-0002-3164-9377

M.L. Filipenko

Institute of Chemical Biology and Fundamental Medicine

ORCID: 0000-0002-8950-5368

M.A. Smetanina

Institute of Chemical Biology and Fundamental Medicine;
Novosibirsk State University

ORCID: 0000-0001-6080-4615

Received:

16.08.2022

Accepted:

03.10.2022

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Introduction

Varicose vein disease (VVD) is a widespread vascular pathology [1], which can lead to a serious deterioration in the quality of life [2]. Improvement of minimally invasive methods of treatment of VVD following scientific and technological progress helps to reduce the number of postoperative complications, and also increases patients' confidence in treatment [3]. However, even the methods of hemodynamic correction that are gaining popularity by themselves do not always allow achieving the desired result, despite careful selection of patients. An approach based on a joint study of clinical and molecular genetic data is able to explain the cause of postoperative recurrences not associated with tactical or technical treatment errors [4]. The fact that researchers using the ASVAL procedure relate the length of reflux to the frequency of its postoperative recurrence [5], leads to the assumption that reflux-related pathological changes in the venous wall can reduce the chance of a successful outcome of treatment. The above makes it possible to use the reflux length as a sign that determines whether patients belong to one of the experimental groups.

Our preliminary studies [6] showed that in groups of patients with different length of pathological reflux in the great saphenous vein (GSV), genes related to the state of the cytoskeleton are differentially expressed: ACTA1 and PLXNA4. The participation of the cytoskeleton in the development of pathologies is explained by its functions associated not only with prop and shaping, but also with the provision of intracellular transport pathways, cell mobility and division. This structure is a complex of microtubules, actin and intermediate filaments, and proteins associated with them [7]. One of the most important structural proteins of the cytoskeleton is actin that is involved in vesicle transport, division, cell migration, and chromatin remodeling [8]. Actin is represented by several isoforms with unique cellular functions [9]. The main function of the alpha-1 skeletal actin gene (ACTA1) is to provide contraction of skeletal muscles [10], which determines its predominant expression in skeletal muscles. Mutations in ACTA1 can lead to nemaline myopathy [11], and knockout of this gene in mice is lethal [12]. It is interesting that disruption of the normal for the cell ratio of expression of actin isoforms is often found during malignant transformation of cells [8].

Plexin-A4 (PLXNA4) is a class 3 semaphorin coreceptor required for signal transduction with subsequent cytoskeletal remodeling [13]. It has been shown that PLXNA4 expression in vascular endothelium is decreased during inflammation, which may be one of the causes of atherosclerosis due to changes in the morphology of endotheliocytes and their barrier function [14]. Its most studied extracellular ligand is semaphorin-3A (SEMA3A), it binds to PLXNA4 in complex with neuropilin [15] that performs many ligand-mediated functions in vessels, neurons, and tumors [16]. It has been shown that the level of SEMA3A protein was elevated in the aqueous humor of patients with retinal vein occlusion [17].

The aim of this study is to investigate the expression at the mRNA level of the ACTA1, PLXNA4, and SEMA3A genes related to actin filament remodeling, in varicose (VVs) and non-varicose (NVs) vein walls of patients with different length of pathological GSV reflux. This work is a validation of our previous study (performed by mass RNA sequencing) [6] using a candidate gene approach performed by real-time PCR on an independent sample of patients.

Material and methods

Collection of samples of the venous walls and the formation of experimental groups

This study was approved by the Ethics Committee of the Center for New Medical Technologies of the Institute of Chemical Biology and Fundamental Medicine (CNMT ICBFM) of the Siberian Branch of the Russian Academy of Sciences (protocol No. 03 dated October 6, 2019) and carried out in accordance with the principles set forth in the Helsinki Declaration of the World Medical Association. The object of the study was frozen paired varicose- (VV) and non-varicose (NV) vein segments from patients with VVD. Vein samples, informed consents and patient questionnaires were collected on the basis of the CNMT ICBFM RAS from March to November 2021. The exclusion criteria for patients were thrombotic changes in the deep vein system and the absence of visible varicose veins. The characteristics of the patients participating in the study are shown in Table 1.

Table 1. Characteristics of the patient sample

Characteristics	Reflux up to L/3 of the thigh		Reflux up to L/3 of the calf		p-value¹
	Median	Range	Median	Range
Age, years	52.5	28—73	50	28—68	0.48
Age at the onset of VVD, years	40	20—40	30	20—40	0.22
VVD duration, years	19.5	3—33	21	3—30	0.91
Body mass index, kg/m²	28.65	18.06—43.06	26.30	18.52-39.26	0.28
Men, number of patients	3		4		0.69
Women, number of patients	10		9
C2², number of patients	7		7		0.76
C3², number of patients	2		4
C4², number of patients	4		2

Note. ¹ — According to the Mann—Whitney test; ² — Clinical class according to the CEAP classification system.

The principle of dividing patients into groups is based on a study [5], its authors relate the length of GSV reflux to the frequency of its recurrence after the ASVAL vein-preserving procedure. Groups of patients were formed on the basis of data on the length of pathological GSV reflux: patients with a reflux length up to the lower third of the thigh (up to L/3 of the thigh, n=13) and patients with a reflux length up to the lower third of the calf (up to L/3 of the calf, n=13) (Table 1). The reflux length was determined using USAS.

The presentation of the formation of groups/subgroups for statistical data processing is described in the paragraph «Statistical analysis» below.

RNA isolation and reverse transcription

To maintain the gene expression profile at the RNA level, vein samples were placed in liquid nitrogen immediately after surgical extraction. Until the moment of RNA isolation, the vein segments were stored at −80 °C. Total RNA was isolated from homogenized venous walls by the phenol-chloroform extraction method according to Chomczynski and Sacchi (2006) [18]. The quality and quantity of the isolated RNA was checked by agarose gel electrophoresis and spectrophotometrically.

To obtain cDNA (complementary DNA), 300 ng of RNA from each sample was subjected to a reverse transcription reaction according to the protocol presented in [19].

Real-time PCR

The expression level of target genes was determined using real-time PCR. For this purpose, forward and reverse primer systems were designed for target gene cDNA (Table 2) using the Oligo Analyzer and Annhyb222 programs and the BLAST tool.

Table 2. Primer systems for cDNA of the target genes

Gene symbol	Forward primer	Reverse primer
ACTA1	5’-CTACCCGCCCAGAAACTAGA-3’	5’-GAGCCATTGTCGCACAC-3’
PLXNA4	5’-GCAGCAGTGCGCTCTTAAC-3’	5’-AGACATATGCGATGACAGACGT-3’
SEMA3A	5’-ATGTTTATCGGAACAGATGTTG-3’	5’-CTTCCAGCAGAACCTCTTCTA-3’

Each reaction mixture, except for the PCR control (without template) and the reverse transcription control (without reverse transcriptase), contained cDNA in an amount corresponding to 12.5 ng of RNA. The composition of the reaction mixture corresponded to the protocol specified in [19].

Amplification was carried out in a CFX-96 thermal cycler (Bio-Rad, USA) as follows: cDNA denaturation at 96°C for 3 minutes, then 35 cycles in modes specific to primer systems (Table 3) with fluorescent signal collection in the SYBR channel.

Table 3. Temperature regime of amplification of PCR products with primer systems for cDNA of target genes

PCR steps	DNA melting	Primer annealing	Elongation	Signal detection
Temperature, °C	96	58—65*	72	75—82*
Time, sec	6	10	10	5

Note. * — Range reflects the spread of temperature values between different primer systems.

Each sample was presented in three technical replicates, with a standard deviation of the difference in the threshold cycles of triplicates >0.5, the knocked-out repeat was excluded from the analysis. A standard curve for each primer system was constructed using multiple dilutions of a cDNA sample prepared from a venous segment in the same parallel as the test samples.

The values of the mRNA amounts obtained by detecting the amounts of cDNA were normalized to the values of the mRNA amounts of the ACTB and GAPDH housekeeping genes [20] in the same samples. Thus, we obtained a normalized value of gene expression (NVGE), i.e. the relative value of the number of transcripts of the studied genes in each sample (VV or NV), obtained by constructing a calibration curve and normalized to the value of the number of transcripts of housekeeping genes (by the amount of cellular material) in the same samples. Such normalized values are a reflection of the relative expression level of the studied genes and, therefore, do not have units of measurement.

Statistical analysis

A total of 26 patients with VVD participated in the study, they were divided into 2 groups according to the length of GSV reflux. As a result of the sampling of varicose and non-varicose segments of the venous wall in each patient, subgroups of VV and NV were formed, respectively.

Using the qBase+, Microsoft Excel and STATISTICA software packages, we made the following comparisons:

1. Pairwise (26 pairs of VV-NV from 26 patients) comparison of NVGE in VV (case) versus NVGE in NV (control) from each patient in the entire sample of patients (Fig. 1a) was performed using a paired nonparametric Wilcoxon test. The data obtained are presented in the form of medians of the paired ratios of NVGE in VV to NV (Me(VV/NV)), as well as p-value (p) and 95% confidence interval (CI). This comparison was carried out as an additional one to identify the differential expression of genes in VV without taking into account the influence of the length of reflux.

2. Pairwise (13 pairs of VV-NV from 13 patients in each of the two groups) comparison of NVGE in VV (case) versus NVGE in NV (control) in each of the two groups of patients (Fig. 1b) was performed using a paired nonparametric Wilcoxon test. The data obtained are presented in the form of medians of paired ratios of NVGE in VV to NV (Me(VV/NV)), as well as p-value (p) and 95% confidence interval (CI) for each of the two groups of patients.

3. Comparison of the ratios of NVGE in VV to NVGE in NV (within a pair from each patient) between two groups of patients with different length of reflux (Fig. 1c) was carried out using an unpaired Mann—Whitney test (13 values of the ratios from one group versus 13 from the other). The data obtained is presented in the form of p-value (p).

4. An additional study of the difference in the expression of the PLXNA4 gene between the two groups of patients separately in non-varicose veins, as well as in varicose ones, in order to identify patterns of changes in the expression level of this gene in each of the subgroups (VV or NV) depending on the length of pathological reflux (Fig. 1d). The additional analysis was carried out because of the assumption of a possible role of changes in the expression of PLXNA4 in NV at different lengths of reflux, based on the data of the above comparisons. Comparison of NVGE in NV (or VV) between two groups of patients was carried out using an unpaired Mann—Whitney test (13 NV (or VV) NVGEs from one group versus 13 NV (or VV) NVGEs from the other). The data obtained is presented in the form of p-value (p).

Fig. 1. Representation of the formation of groups/subgroups for statistical processing of normalized values of expression of the target genes.

Up to L/3 of the thigh (calf) — a group of patients with the length of pathological reflux up to the lower third of the thigh (calf); VV_i (NV_i) — the normalized value of gene expression (NVGE) in the varicose (non-varicose) vein segment of the venous wall of the i-th patient; VV_i/NV_i — the NVGE ratio in the varicose segment of the venous wall to that in the non-varicose segment in the i-th patient; * — according to the paired Wilcoxon test; ** — according to the unpaired Mann—Whitney test.

The critical significance level was taken as p<0.05.

Results

Skeletal actin alpha-1

1. Differential expression of the ACTA1 gene in VV compared to NV has not been identified in the entire sample of patients: Me(VV/NV)=1.3 (p=0.37; 95% CI: 0.65—2.60).

2. In groups of patients with reflux length up to L/3 of the thigh and up to L/3 of the calf, the result of the analysis showed the following values: Me(VV/NV)=0.87 (p=0.38; 95% CI: 0.66—1.15) and Me(VV/NV)=1.94 (p=0.13; 95% CI: 0.47—8.12), respectively (Fig. 2).

Fig. 2. The normalized values of the ACTA1 gene expression in the groups of patients with different length of pathological GSV reflux.

The graphs were obtained using the qBase+ software package: the columns show the median of the ACTA1 gene expression values normalized to the ACTB and GAPDH gene expression values; «whiskers» show the spread of values; p* — according to the Mann-Whitney test; NVGE — normalized value of gene expression; VV — varicose vein segments; NV — non-varicose vein segments; VV/NV — the ACTA1 NGVE ratios in VVs to NVs (within a pair from each patient); L/3 — lower third (thigh/calf), designation of the length of pathological reflux in the GSV.

3. When comparing the ratio of ACTA1 NVGE in VV to NV between groups of patients, we identify a difference: in patients with reflux up to L/3 of the lower calf, the ratio of NVGE in VV to NV was greater compared to those in patients with reflux up to L/3 of the thigh (p=0.03).

Thus, a difference was shown in relation to the expression of the ACTA1 gene in the VV and NV between groups of patients with different length of GSV reflux.

Plexin-A4

1. No differential expression of the PLXNA4 gene in the VV when comparing paired VV to NV was shown in the entire sample of patients (Me(VV/NV)=1.3; p=0.18; 95% CI: 0.94—1.81).

2. When dividing patients into the groups according to the length of venous reflux, differential expression of the PLXNA4 was shown in the VVs (it turned out to be higher, Fig. 3) in the group of patients with reflux up to L/3 of the thigh (Me(VV/NV)=1.76; p=0.04; 95% CI: 1.04—2.97). In the group of patients with reflux up to L/3 of the calf, no difference was shown between VVs and NVs: the ratio of NVGE in VV to NV was 1.01 (p=0.84; 95% CI: 0.65—1.56).

Fig. 3. The normalized values of the PLXNA4 gene expression in the groups of patients with different length of pathological GSV reflux.

The graphs were obtained using the qBase+ software package: the columns show the median of the PLXNA4 gene expression values normalized to the ACTB and GAPDH gene expression values; «whiskers» show the spread of values; p* — according to the paired Wilcoxon test, p** — according to the Mann-Whitney test; NVGE — normalized value of gene expression; VV — varicose vein segments; NV — non-varicose vein segments; L/3 — lower third (thigh/calf), designation of the length of pathological reflux in the GSV.

3. When comparing the ratio of PLXNA4 NVGE in VV to NV between the groups of patients with different length of reflux, we did not detect any difference (p=0.43).

4. When comparing the groups of patients with different length of reflux, we observed a difference in the PLXNA4 expression specifically for NV. Fig. 3 shows a 1.64-fold increase in the PLXNA4 expression in NV in the group of patients with reflux up to L/3 of the calf compared to NV in the group of patients with reflux up to L/3 of the thigh (p=0.03). At the same time, the comparison of VV with VV in different groups of patients did not show a significant difference in the expression of this gene (p=0.41).

Semaphorin-3A

Since the SEMA3A gene product is an extracellularly secreted protein that binds to PLXNA4, it is appropriate to study its gene expression at the protein level. However, in this study, we decided to first analyze the expression of the SEMA3A gene at the mRNA level.

1. No different in the expression of the SEMA3A has been shown between paired VV and NV in the entire sample of patients (Me(VV/NV)=1.54; p=0.49; 95% CI: 0.65—3.33).

2. In the group of patients with reflux up to L/3 of the thigh when comparing paired VV to NV, differential expression of SEMA3A in VV was shown (Me(VV/NV)=1.58; p=0.02; 95% CI: 0.65—3.33). In the group of patients with reflux up to L/3 of the calf, differential expression of this gene was not detected (Me(VV/NV)=0.80; p=0.60; 95% CI: 0.21—2.02).

3. When comparing the ratios of the SEMA3A NVGE in VV to NV between the groups of patients with different length of reflux, we did not detect any difference (p=0.18).

A summary of the obtained data on the expression of the ACTA1, PLXNA4, and SEMA3A genes in different groups of patients is presented in Table 4.

Table 4. Tabular presentation of the data analyzed

Gene symbol	The entire sample		Reflux up to L/3 of the thigh		Reflux up to L/3 of the calf
	Me(VV/NV)¹ (95% CI)	p²	Me(VV/NV)¹(95% CI)	p²	Me(VV/NV)¹(95% CI)	p²
ACTA1	1.30 (0.65—2.60)	0.37	0.87 (0.66—1.15)	0.38	1.94 (0.47—8.12)	0.13
			p-value³: 0.03
PLXNA4	1.30 (0.94—1.81)	0.18	1.76 (1.04—2.97)	0.04	1.01 (0.65—1.56)	0.84
			p³=0.43
			NV (L/3 of the thigh) vs. NV (L/3 of the calf): p⁴=0.03 VV (L/3of the thigh) vs. VV (L/3 of the calf): p⁴=0.41
SEMA3A	1.54 (0.65—3.33)	0.49	1.58 (0.65—3.33)	0.02	0.80 (0.21—2.02)	0.60
			p³=0.18

Note. ¹ — Designation of the median paired ratios of the NVGE in VV to NV from each patient. The value reflects the direction of regulation of the expression in VV (case) vs. NV (control): >1 means increased gene expression in VV vs. NV, <1 — reduced; ² — According to the paired Wilcoxon test. The paired NVGEs in different groups of patients were compared; ³ — According to the unpaired Mann—Whitney test. The NVGE ratios in VV to NV (within a pair from each patient) were compared between the groups of patients with different reflux length; ⁴ — According to the unpaired Mann—Whitney test. The NVGEs in NVs (or VVs) between different groups of patients were compared.

Discussion

Identification of the causes of postoperative recurrence can help to adjust treatment tactics to increase the chances of favorable treatment outcomes. Early studies based on comparison of the expression in varicose and non-varicose vein segments [21] were carried out without taking into account the influence of pathological reflux length. We suggest a possible relationship between the length of reflux in the GSV and pathological molecular processes, such as remodeling of the cytoskeleton of cells in the venous wall and, as a consequence, its remodeling.

The disease phenotype of vein wall cells in VVD, which is determined by cell shape and migration, is associated, among other things, with the processes of actin cytoskeleton remodeling [8]. The researchers found the effect of compensating for the shift in the expression of one actin isoform by another, which keeps the total amount of actin in the cell at the same level [22]. Thus, when the expression of one actin isoform changes, compensatory mechanisms shift the expression of other isoforms, which can lead to a ratio of actin filament isomers atypical for the cell. Previously, the researchers have already shown an increase in the expression of the cardiac actin gene — ACTC1 — in VVD [20, 23], which can serve as a compensation for ACTA1 disorders [24].

In the present study, we have shown that the ACTA1 expression ratios in the VVs to NVs of patients with VVD differ in the groups of patients with different reflux length. As can be seen in Fig. 2, in the group of patients with a reflux length of up to L/3 of the thigh, there is a tendency to a lower level of the ACTA1 expression in the VVs compared to the NVs, and in the group with a reflux of up to L/3 of the calf, on the contrary, there is a tendency to an increased expression of this gene in VV, which may indicate a possible divergence in the expression. The data obtained indicate the processes of remodeling of the actin cytoskeleton, which are associated with the length of reflux. It is still difficult to say exactly how these events are related to each other, however, it can be assumed that there is a relationship between the dynamics of changes in the isomeric composition of the actin filament and regulation via signals from the PLXNA4 receptor [25]. The functions of its gene are associated with the development of the nervous system [26], as well as with the state of endotheliocytes and monocytes, the violation of which contributes to atherosclerosis [14]. In this study, we have shown that in case of a longer pathological reflux, the level of the PLXNA4 expression is increased in non-varicose vein segments. With a longer reflux, the level of the PLXNA4 expression in the NV increases almost to the values in the VV (Me(VV/NV)=1.1. The results may indicate that despite the absence of morphological changes in the NV, the processes associated with signaling from PLXNA4 are more pronounced in patients with longer reflux. This may determine the chance of reflux recurrence after ASVAL [5, 27]. In a preliminary study [6], no difference was found in the expression at the mRNA level of neuropilins that form complexes with PLXNA4 [15], however, it is possible that with a different approach it will be possible to identify some patterns. These complexes bind to semaphorins that play a role in inflammatory and autoimmune processes; in particular, SEMA3A is thought to contribute to microcirculatory disorder. Semaphorins and plexins are promising drug targets for the prevention and treatment of various diseases [28]. In this study, we showed differential expression at the mRNA level of the SEMA3A gene in VVs in a group of patients with a reflux length of up to L/3 of the thigh. Since the product of this gene is a protein secreted into the extracellular space, analysis at the protein level is necessary for a clearer picture. However, this work points to a role for semaphorins in the pathogenesis of VVD and a potential for further research.

Interestingly, without dividing patients into the groups according to the length of GSV reflux (in the entire sample of patients), the genes we studied did not show differences in their expression between varicose and non-varicose segments of veins, which suggests that the length of reflux is associated with the expression of the ACTA1, PLXNA4, and SEMA3A genes.

Conclusion

The difference in the expression of the ACTA1, PLXNA4, and SEMA3 genes involved in the cytoskeleton remodeling processes and, subsequently, vein wall remodeling, was shown in the vein samples from patients with different length of pathological GSV reflux. Thus, the length of pathological GSV trunk reflux that determines the probability of reflux recurrence after ASVAL procedure, is associated with vein wall remodeling. The identified genes can become promising therapeutic targets.

This work was supported by the Program of Fundamental Scientific Research of the Russian Federation (PFSR RF 0245-2021-0006) within the framework of a scientific project No. 121031300045-2 “Fundamental Basics of Health Preservation”.

Author contributions:

Study concept and design —M.A. Smetanina, V.A. Korolenya

Collection and processing of material — K.A. Gavrilov, K.S. Sevostyanova, M.A. Smetanina, V.A. Korolenya

Statistical data processing —V.A. Korolenya, M.A. Smetanina

Writing the text —V.A. Korolenya, M.A. Smetanina

Editing —M.A. Smetanina

Provision of research materials, administrative and technical support —A.I. Shevela, M.L. Filipenko

We express our gratitude to Zolotukhin I.A. for his ideological inspiration.

The authors declare no conflict of interest.