| Abstract|| |
Background and Aims: Varicose vein (VV) is an important cause of morbidity in the young and elderly population. Many studies of the Western country suggest that matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs have a crucial role in the pathogenesis of VV, but limited work has been done in Indian population. The aim of this study is to study detailed histology of VV and to see the expression of MMP-1, MMP-9 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Materials and Methods: A total of 63 cases of VV and 10 control leg veins were included in this prospective study. Paraffin sections of VV were prepared. Hematoxylin and eosin (H and E), Masson trichrome and Verhoeff's staining were performed. Immunohistochemistry of VV was done with MMP-1, MMP-9, and TIMP-1 antibodies. Cytoplasmic expression of MMP-1, MMP-9 and TIMP-1 were graded as intense positive (++), weak/slight positive (+), and absent (−). Results: Focal intimal thickening (47.6%), increased medial thickening (73%) and fragmentation of elastin fibers (84.1%) were the major histological changes noted in H and E and special stained sections. MMP-1 expression increased in all layers of VV in 58 cases (92.1%) as compared to control veins. As compared to the control veins, intimal and adventitial expression of MMP-9 were increased in 31 (49.2%) and 40 (63.5%) cases, respectively. Expression of TIMP-1 was absent in both the varicose and the control veins. Conclusion: Increased expression of MMP-1 and MMP-9 suggests they have an important role in the pathogenesis of VV.
Keywords: Collagen, elastin fiber, matrix metalloproteinases, smooth muscle, tissue inhibitor of matrix metalloproteinase, varicose vein
|How to cite this article:|
Naik B, Kumar M, Khanna AK, Suman PK. Clinico-histopathological study of varicose vein and role of matrix metalloproteinases-1, matrix metalloproteinases-9 and tissue inhibitor of matrix metalloproteinase-1 in varicose vein formation. Indian J Pathol Microbiol 2016;59:25-30
|How to cite this URL:|
Naik B, Kumar M, Khanna AK, Suman PK. Clinico-histopathological study of varicose vein and role of matrix metalloproteinases-1, matrix metalloproteinases-9 and tissue inhibitor of matrix metalloproteinase-1 in varicose vein formation. Indian J Pathol Microbiol [serial online] 2016 [cited 2020 Apr 7];59:25-30. Available from: http://www.ijpmonline.org/text.asp?2016/59/1/25/178217
| Introduction|| |
The prevalence of varicose veins (VV) varies widely from 2% to 56% in men and 1-73% in women.  Despite the major medical and socioeconomical consequences of VV, the pathophysiological mechanisms involved are not fully understood. Matrix metalloproteinases (MMPs) are proteolytic enzymes that have been identified in many tissues and organs including the venous system. MMPs play a major role in tissue remodeling and the continuous turnover of collagen, elastin and other proteins of the extracellular matrix (ECM), and have been implicated in cardiovascular remodeling and vascular diseases. In addition to their proteolytic properties on ECM, MMPs may have early effects on other cellular components of the vein wall including endothelial cells and vascular smooth muscle. 
The concept of venous dilation secondary to valvular incompetence, with subsequent venous hypertension, as the major cause of VVs has been challenged. Loss of tone due to the structural weakness of the venous wall resulting from an imbalance in synthesis and degradation of matrix proteins has received increasing attention.  Because MMPs and their inhibitors, tissue inhibitor of matrix metalloproteinase (TIMP), are important in synthesis and degradation of ECM,  we sought to determine the expression of MMP-1, MMP-9 and TIMP-1 in normal and VVs.
| Materials and methods|| |
The present prospective study was based on the findings of VV specimens, obtained from 63 patients with a clinico-radiological diagnosis of varicosity, that were submitted in the Department of Pathology in collaboration with Department of Surgery from July 2012 to May 2014. Informed written consent was taken from all the patients. Ten control veins were obtained from patient undergone amputation of legs for road traffic accident or any nonvaricose diseases. Out of the 10 controls, seven were male and three were female. The mean age of the controls was 37.5 years (range 21-67 years).
Patients with lower limb venous disease belonging to the clinical, etiological, anatomical, pathophysiological (CEAP) classes of C1, C2, C3, C4, C5, C6; Ec, Ep, Es; As, Ap, Ad; Pr, Po, Pro were included in this study.
Patients of peripheral artery diseases, malignancy, pregnancy, musculoskeletal debilitating diseases, and deep vein thrombosis (DVT) were excluded from the study.
A detailed history related to symptoms and their duration, past history of DVT or any vascular disease, family history of VVs and occupation were noted. Personal history of smoking, alcohol intake, and oral contraceptive use in females were noted.
General physical examination and systemic examinations were performed. Patient's lower extremity was examined for telangiectasias, reticular veins, VVs, edema, lipodermatosclerosis, pigmentation, eczema, and ulceration. Clinical tests were done to locate the site of incompetence of superficial veins and perforators. Clinical stage ("C") was ascertained along with clinically appreciable anatomical sites determination ("A") as per CEAP classification. Duplex assessment of lower limb venous system was done to detect competence of saphenofemoral and saphenopopliteal valves and perforator veins.
The samples of VVs collected from patients were routinely fixed in 10% formalin and grossed by pathologists to obtain representative 3-4 mm thick tissue sections, which were processed routinely for embedding in paraffin wax. 3 μ thin sections were cut from the paraffin embedded blocks and placed on albumin-coated glass slides for hematoxylin and eosin (H and E) staining. The H and E stained slides of normal veins from control subjects were also prepared. All the original H and E stained slides of VVs were evaluated to see the changes in tunica intima, tunica media, and tunica adventitia. All the observed changes were compared with the histomorphology of control leg veins.
Verhoeff's staining method was used to detect the distribution and morphological changes of elastin fibers in the varicose and control veins. Masson trichrome stain was used for delineating collagen and smooth muscle in the wall of varicose and control veins.
The appropriate tissue blocks were chosen for immunohistochemical examination. Rabbit anti-human polyclonal antibodies against MMP-1, and MMP-9 and mouse anti-human monoclonal antibody against TIMP-1 were used along with the secondary detection kit obtained from Lab Vision corporation, USA through a proper agency. All the antibodies were prediluted and ready to use.
2-3 μ thin sections were placed on 1% poly-l-lysine coated slides. Sections were deparaffinzed in xylene followed by hydration in three changes of 95%, 80%, and 70% alcohol, for 2 min each. Antigen retrieval was done in citrate buffer (pH 6.0) by EZ retriever system v. 2.1 (BioGenex laboratories, San Raman, California, USA) for two cycles. The first cycle was performed at 95±C for 10 min followed by the second cycle at 97±C for 10 min. Endogenous peroxidase was blocked by UltraVision peroxidase block for 10 min at room temperature. Slides were washed with Tris buffer 3 times for 5 min each, followed by Ultra Vision protein block for 5 min at room temperature. Again, slides were washed thrice with Tris buffer. Sections were covered with primary antibody solution at room temperature for 30 min. After washing with Tris buffer, sections were covered with primary antibody amplifier quanto and incubated in a humid chamber for 10 min, followed by three washes of Tris buffer. Secondary antibody was added and incubated in a humid chamber for 10 min, and then washed with Tris buffer thrice. Diaminobenzidine substrate was applied to sections for 5 min until brown color developed in a humid chamber. Sections were counterstained with Harris' hematoxylin, dehydrated with graded alcohol and cleared in xylene and mounted with DPX.
Evaluation of immunoreactivity
The evaluation of all immunostained slides of both VV and a normal vein was performed. The slides of breast carcinoma were taken as the positive controls for MMP-1, MMP-9 and TIMP-1. The quantitative evaluation of immunostaining for three antibodies was done by assessing the extent of cytoplasmic positivity in the intima, media and adventitia of VVs and normal veins. The intensity of staining was scored as follows: Intense positive (++), weak/slight positive (+), and absent (–).
| Results|| |
In 63 patients, 63 limbs were examined, and 10 normal leg veins were taken as controls. In our study, VV was more common in younger age group (21-30 years) as compared to older age groups. Body mass index (BMI) of 32 cases (50.8%) was in normal range (18.5-24.99). Distribution of cases according to age groups and BMI are shown in [Table 1] and [Table 2]. VV was more common in male (88.9%, 54 cases) as compared to female (11.1%, 7 cases). Eighteen cases (28.6%) were in occupations involving prolonged standing. Three patients (4.7%) were referred from the medical board. History of the long duration of standing was present in 39 (61.9%) cases. The family history of VV was present in one patient (1.6%). Eight patients had a history of smoking, and eight patients (12.7%) were addicted to alcohol. Twenty-three (36.5%) patients had received previous medical treatment for VV and two patients (3.2%) had a history of surgery for varicosity. Out of 7 female patients, 6 (85.7%) were multipara and 2 (28.6%) had a history of taking oral contraceptive pills.
Among the 63 limbs examined, swelling of vein (98.4%, 62 cases) was the most common complaint of patients, followed by pain (55.5%, 35 cases) and tiredness (34.9%, 22 cases). Varicosity-C2 (95.2%) [Figure 1]a] and reticular veins-C1 (65%) were the most common clinical grades, and active ulceration-C6 (6.3%) [Figure 1]b] was the least common clinical grade. Distribution of patients according to clinical grade (C) is shown in [Table 3]. The majority of the cases (98.9%, 62 cases) were of primary VV and only one case (1.1%) was of congenital VV. Calf perforators (93.6%) were the most common site of incompetence on duplex examination followed by saphenofemoral incompetence (50.7%). Pathophysiology involved in all cases (100%) was reflux. None of the cases had obstruction of VV. In our study, most commonly performed invasive intervention was radiofrequency ablation + phlebectomy + sclerotherapy, which was done in 22 patients (34.9%), followed by ligation of only perforators in 19 patients (30.1%) and radiofrequency ablation + phlebectomy in 11 patients (17.4%). Compression therapy was usually used as an adjunct measure with the other treatment modalities in 88.8% of cases.
|Figure 1: (a) Varicosities of leg vein (clinical grade C2). (b) Active ulceration of varicose vein (clinical grade C6)|
Click here to view
|Table 3: Distribution of cases according to clinical (C) criteria of CEAP |
Click here to view
Histopathological changes seen in VV on H and E-stained and special stained sections are shown in [Table 4], [Table 5] and [Figure 2]a-d. The majority of cases (92.1%, 58 cases) showed intense staining of MMP-1 in all the layers of VV. Six (7.9%) cases showed slight staining of MMP-1. Expression of MMP-1 in control veins and VVs is shown in [Table 6] and [Figure 3]a-b. Staining of MMP-9 was intense in the adventitia in 40 cases (63.5%) [Figure 3]c. Thirty-one cases (49.2%) showed intense endothelial staining of MMP-9 [Figure 3]d. Only 10 cases showed intense staining of MMP-9 in the tunica media. In controls, nine cases showed slight staining and one case showed absent expression of MMP-9 in the adventitia [Table 7]. Expression of TIMP-1 was absent in all cases of VV and controls. Positive control of TIMP-1 showed adequate staining.
|Figure 2: (a) Medial thickening of varicose vein (H and E, ×100). (b) Proliferation of smooth muscle and deposition of collagen in the tunica media of varicose vein (Masson trichrome, ×100). (c) Regular distribution of elastin fibers in control vein (Verhoeff's, ×200). (d) Fragmented elastin fiber in the wall of varicose vein (Verhoeff's, ×200)|
Click here to view
|Figure 3: (a) Weak cytoplasmic positivity (+) of matrix metalloproteinases-1 in control leg vein (immunohistochemistry, ×200). (b) Intense cytoplasmic positivity (++) matrix metalloproteinases-1 in varicose vein (immunohistochemistry, ×200). (c) Intense cytoplasmic positivity (++) of matrix metalloproteinases-9 in the adventitia of varicose vein (immunohistochemistry, ×100). (d) Intense positivity (++) of matrix metalloproteinases-9 in the endothelium of varicose vein (immunohistochemistry, ×400)|
Click here to view
|Table 4: Histopathological changes seen in varicose vein in H and E stained section |
Click here to view
|Table 5: Histological changes seen in special stain in cases of varicose vein |
Click here to view
|Table 6: Expression of MMP-1 in varicose vein wall in cases and controls |
Click here to view
|Table 7: Expression of MMP-9 in varicose vein wall in cases and controls |
Click here to view
| Discussion|| |
Our study shows that VV was more common in male (56 out of 63; 88.9%) as compared to female (7 out of 63; 11.1%). This is in contrast to previous studies that reported prevalence rates of 25-33% in women as compared to 10-20% among men.  The Edinburgh Vein Study found an overall prevalence of 40% in men and 32% in women.  Others have suggested that although reticular veins may be more common in women, the gender distribution of more severe VV is approximately equal. 
Our study shows that majority of the VV were seen in the young age group. Twenty-seven cases (42.9%) were in the age group of 21-30 years. This is in contrast to most of the studies that have shown a steep increase in the prevalence of VV with age. , In a previous study, the prevalence of VV between the age group of 20-29 years was 1% in men and 8% women, which was increased to 43% and 72% respectively in the seventh decade.  The increase in prevalence with age may therefore simply reflect an accumulation of cases rather than an increased propensity to develop VV with aging. Since VV is a chronic condition, there occurs an accumulation of cases with increasing age group. Our study is a hospital based study rather than a population-based study, so the incidence of VV is higher in young age group patients.
Among the 7 female cases studied, 6 (85.7%) female were multiparous. Two cases were taking an oral contraceptive, and none of the patients had an abnormal menstrual cycle. One study supported our finding that risk of VV formation increases with parity. 
On classifying the VV limbs according to the clinical grade of CEAP classification, it was found that C2 (95.2%) and C1 (65%) were the most common clinical classes. Other studies have shown the prevalence of C2 from 14.3% to 46.3%. , These population-based studies showed that the most common clinical grade was C1. However, the majority of C1 patients do not seek treatment. Since our study is a hospital-based study, we found the majority of patients of C2 clinical grade.
When classified according to the etiological class of CEAP grading, 98.9% of the limbs were in the primary class, and 1.1% cases were in the congenital class. This shows that the majority of the limbs suffering from VV did not have a definite underlying etiology.
In H and E stained sections, focal intimal thickening was seen in 30 cases (47.6%). The most common histological change observed was a loss of demarcation between circular and longitudinal muscle layer (48 cases, 76.2%), and followed by increased thickening of tunica media (73%). The loss of demarcation between two muscle layers may be due to abnormal proliferation of smooth muscle cells and increased deposition of collagen fibers in the tunica media. Adventitia showed an increase in collagen deposition in 19 cases (30.1%) and vasa vasora was absent in 13 cases (20.6%). The results of few studies were consistent with the above histological changes. ,, Two histological changes, subendothelial fibrosis and collagen plaque deposition underneath the endothelium that were described in earlier studies were not found in any of the cases of our study. 
Verhoeff's stain for elastin fibers showed fragmentation of fibers in 54 cases (84.1%). Elastin fibers shortened in length. The decrease in elastin content was described in some studies. , The finding of our study suggests a structural alteration of elastin fibers have a definite role in the formation of VV. In earlier studies, it was found that thickening of media is due to the proliferation of smooth muscle and deposition of collagen. , In our study, we found that medial thickening was due to both proliferation of smooth muscle and collagen deposition (41 cases, 65.1%) approximately in equal proportions. The predominant proliferation of smooth muscle was noted in 15 cases (23.9%) and 7 cases (11.1%) showed marked collagen deposition.
In normal leg vein expression of MMP-1 was noted in endothelial and smooth muscle cells of all layers of the venous wall, while VV showed MMP-1 expression in endothelial cells, fibroblast and smooth muscle cells.  In our study, 58 cases (92.1%) showed increased expression of MMP-1 in all the layers of venous wall and in 5 cases (7.9%) expression was not much different from control leg veins. The above findings suggest that MMP-1 is over expressed in smooth muscle cells, endothelial cells and fibroblasts of VV. MMP-1 may have a role in the degradation of ECM material which leads to VV formation. Increased expression of MMP-1 was found in all eight cases of VV in the earlier study done by Sansilvestri-Morel et al.  In a previous study, Western blot analysis was performed in 15 patients of VV and all the cases showed increased MMP-1 level in the venous wall.  The study done by Kowalewski et al. showed that there was not much difference in the expression of MMP-1 between the normal vein and VV.  In contrast to our finding, decreased expression of MMP-1 was found in all the 18 cases of VV in the study performed by Ishikawa et al.
In 31 cases (49.2%) endothelial cells of intima layer showed increased expression of MMP-9 as compared to control veins. Tunica adventitia also displayed increased expression of MMP-9 in 40 patients (63.5%). Increased medial expression of MMP-9 was found only in 10 cases (15.9%). One of the previous studies showed increased expression of MMP-9 in smooth muscle cells of tunica media.  According to Kowalewski et al., there was no difference in expression of MMP-9 in VV as compared to the normal vein.  In contrast to our findings, a study done by Woodside et al. by zymography method showed normal vein demonstrates more MMP-9 activity than VVs.  Endothelial injury is one of the important factors in the pathogenesis of VV. Increased endothelial expression of MMP-9 suggests it may have a role in endothelial injury which eventually leads to VV formation. Fragmented elastin fibers and increased adventitial expression of MMP-9, both were found in 40 cases (63.5%). This finding suggested that MMP-9 enzymatically degrades the elastin fibers, which leads to shortening of the fibers.
TIMP-1 expression was absent in all VV specimens. The control veins also lacked the expression of TIMP-1.The results of few studies matched with our results, , while in few other studies, an increase in TIMP-1 expression was found. ,
| Conclusion|| |
To conclude, increased expression of MMP-1 and MMP-9 suggests that they have a key role in the pathogenesis of VVs. TIMP-1 is less likely to be involved in the formation of VV. Structural changes in ECM materials like elastin fibers and collagen, and altered smooth muscle proliferation in media are important determinants that lead to varicosity. Further studies are required to detect the role of TIMP-1, TIMP-2, other MMPs like MMP-2, MMP-3, MMP-7, relative ratio of MMPs and TIMPs in the formation of varicosity.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Beebe-Dimmer JL, Pfeifer JR, Engle JS, Schottenfeld D. The epidemiology of chronic venous insufficiency and varicose veins. Ann Epidemiol 2005;15:175-84.
Raffetto JD, Barros YV, Wells AK, Khalil RA. MMP-2 induced vein relaxation via inhibition of [Ca2+]e-dependent mechanisms of venous smooth muscle contraction. Role of RGD peptides. J Surg Res 2010;159:755-64.
Badier-Commander C, Verbeuren T, Lebard C, Michel JB, Jacob MP. Increased TIMP/MMP ratio in varicose veins: A possible explanation for extracellular matrix accumulation. J Pathol 2000;192:105-12.
Borden P, Heller RA. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Expr 1997;7:159-78.
Evans CJ, Fowkes FG, Ruckley CV, Lee AJ. Prevalence of varicose veins and chronic venous insufficiency in men and women in the general population: Edinburgh Vein Study. J Epidemiol Community Health 1999;53:149-53.
Krijnen RM, de Boer EM, Bruynzeel DP. Epidemiology of venous disorders in the general and occupational populations. Epidemiol Rev 1997;19:294-309.
Bradbury A, Evans C, Allan P, Lee A, Ruckley CV, Fowkes FG. What are the symptoms of varicose veins? Edinburgh vein study cross sectional population survey. BMJ 1999;318:353-6.
Brand FN, Dannenberg AL, Abbott RD, Kannel WB. The epidemiology of varicose veins: The Framingham Study. Am J Prev Med 1988;4:96-101.
Sadick NS. Predisposing factors of varicose and telangiectatic leg veins. J Dermatol Surg Oncol 1992;18:883-6.
Lim CS, Davies AH. Pathogenesis of primary varicose veins. Br J Surg 2009;96:1231-42.
Rabe E, Pannier F. What have we learned from the Bonn Vein study? Phlebolymphology 2006;13:188-94.
Carpentier PH, Maricq HR, Biro C, Ponçot-Makinen CO, Franco A. Prevalence, risk factors, and clinical patterns of chronic venous disorders of lower limbs: A population-based study in France. J Vasc Surg 2004;40:650-9.
Wali MA, Dewan M, Eid RA. Histopathological changes in the wall of varicose veins. Int Angiol 2003;22:188-93.
Travers JP, Brookes CE, Evans J, Baker DM, Kent C, Makin GS, et al.
Assessment of wall structure and composition of varicose veins with reference to collagen, elastin and smooth muscle content. Eur J Vasc Endovasc Surg 1996;11:230-7.
Woodside KJ, Hu M, Burke A, Murakami M, Pounds LL, Killewich LA, et al.
Morphologic characteristics of varicose veins: Possible role of metalloproteinases. J Vasc Surg 2003;38:162-9.
Elsharawy MA, Naim MM, Abdelmaguid EM, Al-Mulhim AA. Role of saphenous vein wall in the pathogenesis of primary varicose veins. Interact Cardiovasc Thorac Surg 2007;6:219-24.
Gandhi RH, Irizarry E, Nackman GB, Halpern VJ, Mulcare RJ, Tilson MD. Analysis of the connective tissue matrix and proteolytic activity of primary varicose veins. J Vasc Surg 1993;18:814-20.
Sansilvestri-Morel P, Fioretti F, Rupin A, Senni K, Fabiani JN, Godeau G, et al.
Comparison of extracellular matrix in skin and saphenous veins from patients with varicose veins: Does the skin reflect venous matrix changes? Clin Sci (Lond) 2007;112:229-39.
Gillespie DL, Patel A, Fileta B, Chang A, Barnes S, Flagg A, et al.
Varicose veins possess greater quantities of MMP-1 than normal veins and demonstrate regional variation in MMP-1 and MMP-13. J Surg Res 2002;106:233-8.
Kowalewski R, Sobolewski K, Wolanska M, Gacko M. Matrix metalloproteinases in the vein wall. Int Angiol 2004;23:164-9.
Ishikawa Y, Asuwa N, Ishii T, Ito K, Akasaka Y, Masuda T, et al.
Collagen alteration in vascular remodeling by hemodynamic factors. Virchows Arch 2000;437:138-48.
Kosugi I, Urayama H, Kasashima F, Ohtake H, Watanabe Y. Matrix metalloproteinase-9 and urokinase-type plasminogen activator in varicose veins. Ann Vasc Surg 2003;17:234-8.
Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]