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ORIGINAL ARTICLE
Year : 2022  |  Volume : 25  |  Issue : 3  |  Page : 304-309

What is the relationship of varicose vein pathogenesis with collagen fibers?


1 Department of Pathology, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey
2 Department of Cardiovascular Surgery, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey
3 Department of Biostatistics, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey

Date of Submission10-May-2021
Date of Acceptance13-Sep-2021
Date of Web Publication16-Mar-2022

Correspondence Address:
Dr. S Kocarslan
Avsar Campus, Kahramanmaras Sutcu Imam University Hospital, Department of Pathology, 46050 - Onikisubat, Kahramanmaras
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_1505_21

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   Abstract 


Aims and Background: In this study, the densities of collagen 1 and collagen 4, which are an effective vascular component in the remodelling of varicose veins, were investigated. Materials and Methods: The study included primary varicose vein samples of 20 patients and vein samples of 20 healthy controls. Immunohistochemical staining was performed using collagen 1 and collagen 4 antibodies. Histochemical staining was performed using Masson Trichrome. Results: In the immunohistochemical analysis of varicose samples, collagen 1 immunostaining was negative in 17 cases (85%) and positive in 3 cases (15%). In healthy venous tissue samples, collagen 1 immunostaining was negative in 12 cases (60%) and positive in 8 cases (40%). There was no statistically significant difference between both groups concerning collagen 1 immunostaining (p > 0.05). In varicose samples, collagen 4 immunostaining was negative in 4 cases (20%) and positive in 16 cases (80%). In healthy venous tissue samples, collagen 4 immunostaining was negative in 13 cases (65%) and positive in 7 cases (35%). Statistical comparison of healthy veins and varicose veins concerning collagen 4 immunostaining showed a significant difference (p = 0.03). In the histochemical analysis of varicose samples, Masson Trichrome staining was negative in 4 cases (20%) and positive in 16 cases (80%). In healthy venous tissue samples, Masson Trichrome staining was negative in 18 cases (90%) and positive in 2 cases (10%). Statistical comparison of healthy veins and varicose veins concerning collagen 4 immunostaining showed a significant difference (p = 0.01). Conclusion: The change in the density of collagen types plays an important role in vein wall remodeling.

Keywords: Collagen1, collagen4, immunohistochemistry, varicose vein


How to cite this article:
Kocarslan S, Kocarslan A, Doganer A, Yasim A. What is the relationship of varicose vein pathogenesis with collagen fibers?. Niger J Clin Pract 2022;25:304-9

How to cite this URL:
Kocarslan S, Kocarslan A, Doganer A, Yasim A. What is the relationship of varicose vein pathogenesis with collagen fibers?. Niger J Clin Pract [serial online] 2022 [cited 2022 Dec 3];25:304-9. Available from: https://www.njcponline.com/text.asp?2022/25/3/304/339712




   Introduction Top


Varicose vein (VV) disease is a common pathology. It is characterized by insufficiency in superficial, perforating, and deep venous vessels. It affects adults and increases in incidence with age. While a cosmetic problem, it carries a high risk for superficial venous thrombosis and venous thromboembolic disease. Although VV is treated with a wide range of techniques, it has a recurrence rate of 26-62% after treatment.[1],[2] Identified risk factors include increased age, pregnancy, hormonal changes, excess weight, and prolonged standing.[3] However, it is also seen in young people who do not have risk factors, supporting genetic predisposition. But the lack of family history in 14% of individuals with no risk factors made VV pathogenesis even more obscure.[4],[5]

It affects the venous system of the lower extremities. It is very important to understand the pathogenesis of VV as it is widespread in society and the after treatment recurrence rate is high. Studies have suggested that various conditions, such as hypoxia, changes in neural innervation, and inflammation, initiate changes in the histomorphology of the vein wall by stimulating some molecular mechanisms.[6],[7] In vascular tissue, most of which consists of collagen and elastin, especially type 1, type 3, and type 4 collagens are the dominant component. In past studies, it has been mentioned that various collagen types in the VV wall increased during remodeling, while some collagen types decreased.[7],[8],[9],[10] For this reason, we wanted to investigate immunohistochemically collagen 1 and collagen 4 densities in saphenous vein specials excised due to VV in this study and the presence of fibrosis with Masson Trichrome histochemical stain.


   Subjects and Methods Top


Power analysis was used to determine the number of samples. α:0.01 at the first type error level, β: 0.10 at the second type error level, 0.90 at the strength of the test, the statistical parameters in the prolidase immunostaining variable in the reference study of Kocarslan and Koçarslan[11] (group patient: P = 0.80 group control = 0.133) were determined as n: 17 for each group. A total of N: 40 individuals, including N: 20, were included in each group, taking into account possible data losses [Table 1]. Between April 2016 and June 2020, 20 saphenous vein specimens excised due to VV disease were included in the study at the Department of Cardiovascular Surgery of the Faculty of Medicine, Kahramanmaras Sütçü Imam University. 20 special samples of saphenous vein samples that were routinely removed in coronary bypass operations as a control group and used as autologous anastomosis graft in the by-pass area were included in the study. Clinical and demographic information of patients was obtained from the hospital information management system. Tissue samples were examined in the Department of Medical Pathology of the Faculty of Medicine, Kahramanmaras Sütçü Imam University. 20 saphenous vein tissue samples used as autologous grafts in coronary by-pass and 20 saphenous vein samples excised due to VV were taken into slides. Collagen 1 and collagen 4 immunohistochemical stain (mouse IgG1 monoclonal to collagen I and IV; Thermo Fisher Scientific, MA, USA) in the Roche Ventana model, fully automatic immunohistochemical dye device available in our laboratory and used in routine, diluted 1/500, painted automatically. Histochemical staining was performed using Masson Trichrome histochemical stain (Thermo Fisher Scientific, MA, USA) This study was approved by the Kahramanmaras Sutcu Imam University local ethics committee for clinic investigations on 05.08.2020 and 286 protocol number.[12]
Table 1: Power analysis table

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Evaluation of immunohistochemical and histochemical staining

The immunohistochemical and histochemical evaluation was analyzed according to Kocarslan and Koçarslan's[11] study. To detect staining prevalences, the reactions of the tested antibody (5 slides for each stage) were observed by 2 blinded examiners using an Olympus microscope (BX51). In immunohistochemical staining, the cytoplasmic and nuclear stainings in endothelial, intimal, and muscular cells of varicose veins were considered immunohistochemically positive. Immunohistochemical expressions of collagen 1 and 4 were assessed using a semi-quantitative scoring system for staining presence. Collagen 1 and 4 immunostaining and Masson Trichrome staining were negatively scored as 0 and positively scored as 1.

Statistical analysis

Statistical data were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). In reporting statistical analyses, normally distributed continuous variables are given as the mean ± standard deviation, abnormally distributed continuous variables are given as the median values, and categorical variables are given as percentage value. The distribution of the data was tested by using a Kolmogorov-Smirnov test. In addition, comparisons between the two groups were performed with an unpaired two-tailed t-test for the normally distributed continuous variables, while the Mann-Whitney U test was used for those that were abnormally distributed. Pearson's Chi-square test or Fischer's exact test was used for categorical variables. A value of P < 0.05 was considered statistically significant.


   Results Top


There was no statistically significant difference between both groups in terms of demographic data (P > 0.05) [Table 2]. Histomorphological examination of healthy veins has regular histologic formation [Figure 1]. Histomorphological examination of varicose veins showed signs of neointima formation, thickening of the media layer, hypertrophic and irregular smooth muscle cells [Figure 2]. In the immunohistochemical analysis of varicose samples, collagen 1 immunostaining was negative in 17 cases (85%) and positive in 3 cases (15%) [Figure 3]. In healthy venous tissue samples, collagen 1 immunostaining was negative in 12 cases (60%) and positive in 8 cases (40%). There was no statistically significant difference between both groups concerning collagen 1 immunostaining (p > 0.05). In varicose samples, collagen 4 immunostaining was negative in 4 cases (20%) and positive in 16 cases (80%) [Figure 4]. In healthy venous tissue samples, collagen 4 immunostaining was negative in 13 cases (65%) and positive in 7 cases (35%). Statistical comparison of healthy veins and varicose veins concerning collagen 4 immunostaining showed a significant difference (p = 0.03). In the histochemical analysis of varicose samples, Masson Trichrome staining was negative in 4 cases (20%) and positive in 16 cases (80%) [Figure 5]. In healthy venous tissue samples, Masson Trichrome staining was negative in 18 cases (90%) and positive in 2 cases (10%). Statistical comparison of healthy veins and varicose veins concerning collagen 4 immunostaining showed a significant difference (p = 0.01) [Table 2].
Figure 1: A regular histological structure is observed in the cross--section of the healthy saphenous vein (Hematoxylene & eosin × 100 magnification)

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Figure 2: Neointima formation, hypertrophic and irregular smooth muscle cells and thickening in the media layer is observed in the cross-section of the varicose saphenous vein (Hematoxylene & eosin × 100 magnification)

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Figure 3: Collagene 1 immunostaining is considered as negative in this varicose vein sample (Collagene 1 immunostaining × 100 magnification)

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Figure 4: Collagene 4 immunostaining is considered as positive in this varicose vein sample (Collagene 4 immunostaining × 100 magnification)

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Figure 5: Masson Trichrome histochemical staining is considered as positive in this varicose vein sample (Masson Trichrome histochemical staining × 100 magnification)

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Table 2: Demographic, immunohistochemical and histochemical data of patients

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   Discussion Top


In this study, we immunohistochemically investigated the densities of collagen 1 and collagen 4, which are the effective vascular component in the dilation and distortion of VVs. We also histochemically investigated fibrosis in the vein walls. We observed that the density of collagen 4 increased in VVs compared to healthy veins, the density of collagen 1 decreased, and fibrosis increased in vein walls.

Despite the high prevalence of VV disease, the biological mechanisms underlying its pathogenesis have not been fully clarified. In pathogenesis, it is obvious that venous hypertension, valvular insufficiency, and structural changes in the vein wall are intertwined. Venous hypertension, which forces and damages the vein wall, elicits ischemic and inflammatory responses with dilation and valvular insufficiency in the vein wall. In response to this situation, various molecular mechanisms are activated by stimulation. As the disease progresses, molecular mechanisms initiate changes in vein wall histomorphology.[6],[7] There are numerous studies on molecular mechanisms and the resulting histopathological changes and various results are obtained. Surendran et al.[13] found that there was an increase in hair/enhancer-of-split related with YRPW motif protein2 (Hey2) and Delta-like ligand4 (DLL4) as a result of FOXC2 gene polymorphism in VVs. As a result, it has been argued that VVs are thicker compared to normal veins due to neointima development and media fragmentation with structural remodeling. Li et al.[14] found that long noncoding RNAs (lncRNA), effective in the proliferation and migration of saphenous vein smooth muscle cells, overexpress in VVs and therefore proliferate venous smooth muscle cells. Lim et al.[15] showed that levels of HIF-1α, HIF-2α, Hypoxia-inducible factors (HIFs), which regulate the expression of genes responsible for oxygen homeostasis in the case of hypoxia in VVs and target genes GLUT1, CA9, VEGF, and BNIP 3, were upregulated compared to the control veins. They blamed hypoxic-ischemic mechanisms for structural changes in the VV Wall. Raffetto et al.[16] investigated angiotensin2-mediated contraction in VV and healthy veins. They showed that Angiotensin2-mediated contraction decreased in VVs compared to a healthy vein, but angiotensin receptor protein expression was the same in VV and normal saphenous vein. A study conducted by Agu found that effective endothelin-1 receptor binding in vascular contraction decreased in VVs and argued that a decrease in endothelin-1 receptor binding may be responsible for decreased contractile function in VVs.[17] Barber, on the other hand, found that the number of endothelin-1 receptors in VVs is less than that of healthy veins.[18] Pascual et al.[19] investigated TGF-β and mast cell population, which are effective factors in tissue repair and remodeling, in VVs and the control saphenous vein. They observed increased levels of TGF-β correlated with mast cell degranulation with age in patients with VV. They blamed TGF-β for fibrotic remodeling and venous insufficiency in the VV wall. Jacob et al.[20] observed increased expression of TGF-β and nitrous oxide in VVs compared to the control group. Metcalfe et al.[21] investigated purinergic signaling, which mediates trophic changes in blood vessels in the cardiovascular system and is important in the control of tone. They found decreased contraction response to purinergic signal molecules in VVs compared to the control group. Aunapuu and Arend[22] in their study, found that smooth muscle cells in the VV media layer were increased and were in irregular morphology and that the surrounding extracellular matrix was disorganized and the elastic layer was novelized. They also found increased expression of laminin, ICAM-1, and VCAM-1 in the VV segment. Hollingsworth et al.[23] found that the VEGF messenger RNA (mRNA) expression and its associated receptor increased in VV compared to control and showed overexpression in cases with saphenofemoral junction failure compared to cases without failure. Raffetto and Khalil[24] in the experimental rat model, investigated MMP expression in response to elongated vein wall contraction. They suggested that elongated vein wall contraction is accompanied by positive expression of MMP2 and MMP9, which leads to a decrease in vein wall contraction. On the other hand, Woodside suggested that there were no differences between VV and the control group in terms of activity and expression of MMP2, MMP9, and MMP12.[25] Koçarslan and Koçarslan[11] found increased expression of the enzyme, prolidase—a member of the MMP Family—in VVs. They suggested that increased prolidase enzyme activity in VVs correlates with increased extracellular matrix turnover, which contributes to varicose dilation progression. Ducasse et al. in two different studies, investigated the presence of apoptosis with collagen and elastin levels in VVs and the control vein. They found that collagen levels increased in VVs, elastin levels decreased, and apoptosis decreased in the medial layer.[26],[27] Xiao et al.[28] compared smooth muscle cells derived from VVs and healthy veins in a cell culture environment. They found that smooth muscle cells cultured from VVs have an excess proliferation and migration capacity compared to the control group and store more collagen. They also found that these cells are dedifferentiated by reducing normal phenotypic markers of smooth novelization. Jeanneret et al.[29] in their study, found that the content of adventitial elastin and medial collagen 3 decreased in VVs. They suggested that adventitial elastin reduction was responsible for the increase in VV diameter at rest and medial collagen 3 reductions were responsible for the increase in VV dilation during the Valsalva maneuvre. Travers et al.[10] in their study, reported that there was a greater amount of collagen, elastin, and smooth muscle in the VV wall compared to the normal vein, and therefore its wall was thicker. In the Venturini study, they found that the collagen content was the same compared to VV and control saphenous veins, but that the elastin content in VVs was very low, arguing that elastin metabolism was effective in varicose veins pathogenesis.[30] Because of these controversial results in the density of collagen fibers, which are the structural component of vascular tissues, we also investigated the density of collagen 1 and 4 in VVs. The significant increase in the amount of collagen 4 shows compatibility with the increases in collagen found in previous studies. Hypoxic-ischemic damage, inflammatory response, and secondary reparative process begin due to stasis and pressure increase in the VV wall. The density of collagen also increases due to the extracellular matrix produced in excess during the remodeling process. But the reduction in the amount of collagen 1 supports Jeanneret's work.[28] We assume that conflicting results about collagen fiber densities may be due to different stages of VV progression or sampling from different areas of the skip varicose segments. In the early stages of VV disease, we predict that collagen densities will be low. Collagen density will increase significantly as the disease progresses. Therefore, the collagen densities of VVs sampled from different segments at different times may also be different.

Limitations

There are several limiting factors in our study. The limited number of VV samples, only collagen 1 and collagen 4 investigation taking samples from a single segment of VVs, limits statistical value and advanced interpretation. At patient groups with a large number of VV samples, we believe that the investigation of vascular components, including elastic fiber and all collagen types, in various vascular segments will further clarify the pathophysiology of VV.

Author contributions

All the authors contributed throughout the study, especially in data collection/analysis, writing, and revising the manuscript till the final approval of the version to be published. We all agree to be accountable for all the technical and moral aspects of the work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]


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