Assessment of tissue expression of matrix metalloproteinase-9 level in patients with vitiligo and its relation to disease pattern, activity, and severity

Samar M Mohammed; Moustafa M.K Eyada; Mohammed O Elokda; Ghada F Mohammed

doi:10.4103/ejdv.ejdv_37_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 42 | Issue : 3 | Page : 159-166

Assessment of tissue expression of matrix metalloproteinase-9 level in patients with vitiligo and its relation to disease pattern, activity, and severity

Samar M Mohammed¹, Moustafa M.K Eyada², Mohammed O Elokda³, Ghada F Mohammed²
¹ Department of Dermatology and Venereology, Ismailia general Hospital, Egypt
² Department of Dermatology and Venereology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
³ Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Date of Submission	10-Sep-2021
Date of Acceptance	22-Nov-2021
Date of Web Publication	01-Sep-2022

Correspondence Address:
MD Ghada F Mohammed
Department of Dermatology and Venereology, Faculty of Medicine, Suez Canal University, Ismailia 41511
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejdv.ejdv_37_21

Abstract

Background Vitiligo is a common acquired depigmentary disorder characterized by milky-white cutaneous macules devoid of identifiable functional melanocytes of unknown origin. Several hypotheses have been advanced to explain the link between matrix metalloproteinase-9 (MMP-9) and vitiligo.
Aim We aimed to compare the tissue expression of MMP-9 in vitiligo lesions with that of healthy controls.
Patients and methods A case–control study was performed to evaluate MMP-9 levels by punch biopsies using an immunohistochemistry technique in lesional and nonlesional tissues of the same patient with vitiligo and in comparison with healthy controls. The study included 20 patients (10 patients with vitiligo and 10 healthy controls). Patients included were 18 years of age and older of both sexes, any duration, and any subtype.
Results MMP-9 expression was found to be lower in the lesional skin of patients with vitiligo at the basal cell layer, follicular epithelium, and stromal cell layer when compared with nonlesional skin of the same patient with vitiligo and matched control individuals.
Conclusion Tissue MMP-9 imbalance has a role in the etiopathogenesis of vitiligo. The low MMP-9 tissue expression may not facilitate the migration of melanoblasts. Tissue MMP-9 is a significant predictor of severity of vitiligo.

Keywords: etiopathogenesis of vitiligo, matrixmetalloproteinase-9, vitiligo

How to cite this article:
Mohammed SM, Eyada MM, Elokda MO, Mohammed GF. Assessment of tissue expression of matrix metalloproteinase-9 level in patients with vitiligo and its relation to disease pattern, activity, and severity. Egypt J Dermatol Venerol 2022;42:159-66

How to cite this URL:
Mohammed SM, Eyada MM, Elokda MO, Mohammed GF. Assessment of tissue expression of matrix metalloproteinase-9 level in patients with vitiligo and its relation to disease pattern, activity, and severity. Egypt J Dermatol Venerol [serial online] 2022 [cited 2023 Mar 20];42:159-66. Available from: http://www.ejdv.eg.net/text.asp?2022/42/3/159/354679

Introduction

Melanocytes are a type of migrating cells that migrate from the neural crest to the epidermis during fetal development and from the infundibulum of the hair follicle to the depigmented epidermis during adulthood [1]. Such migratory cells must penetrate extracellular matrix (ECM); thus, timely degradation of ECM via activation of matrix metalloproteinases (MMPs), such as MMP-9, which is regulated by E-twenty-six-1, is critical [2],[3]. In vitiligo, repigmentation begins with the activation, proliferation, and migration of melanoblasts (repigmentation reservoir cells) from the outer root sheath of hair follicles or melanocytes from the border area of lesions into the depigmented epidermis. Cell migration entails changes in cell architecture, changes in cell adhesion, and ECM remodeling [4],[5]. The transcriptional activation of MMPs and their inhibitors is central to ECM remodeling and migration. MMPs (matrixins) degrade both matrix and nonmatrix proteins in the extracellular environment of cells [6],[7]. MMPs are typically expressed in very low levels, and their transcription is tightly regulated either positively or negatively by cytokines and growth factors such as interleukins (IL-1 and IL-6), epidermal growth factor, and tumor necrosis factor-α. Some of these regulatory molecules can be activated or deactivated proteolytically as a result of MMP feedback [8],[9]. The primary function of MMP-9 is to degrade proteins in the ECM. Decorin, elastin, fibrillin, laminin, gelatin (denatured collagen), and collagen types IV, V, XI, and XVI are proteolytically digested by MMP-9 [10]. As a result, MMP-9 plays an important role in paving a pathway in front of migratory melanocytes and acts as a pro-pigmenting factor [11].

Objective

This is a case–control study to evaluate the relationship between MMP-9 levels and disease activity, duration, and severity of vitiligo using immunohistochemistry.

Patients and methods

Study design and patient population

This case–control study was approved by the Institutional Review Board and Ethical Committee of the Suez Canal University, Faculty of Medicine in Ismailia, Egypt, and was carried out in accordance with the Helsinki Declaration guidelines. Patients were recruited from the outpatient clinic of dermatology department at Suez Canal University Hospital. The following were inclusion criteria: patients with vitiligo 18 years of age and older, of any duration and subtype, both sexes. Patients were excluded if they had used topical or systemic treatment in the previous 2 months, had other autoimmune or inflammatory diseases, any chronic disease, pregnant or lactating, or refused to participate. All patients signed a written consent form, which included an agreement to participate in the study and allow a skin biopsy. The sample size was calculated to be 10 samples per group, for a total of 20 samples.

A dermatologist took a thorough history and examination of each patient to determine the pattern, activity, and severity of the vitiligo.

Assessment of disease pattern

Vitiligo is clinically classified into three types: localized, generalized, and universal vitiligo [12].

Assessment of the disease activity

The ‘vitiligo disease activity score (VIDA) score’ was used to assess disease activity. The VIDA score is a six-point scale used to assess vitiligo activity based on disease activity as measured by the expansion of existing lesions or the appearance of new lesions over a period of less than 6 weeks to 1 year. Only patients with VIDA scores of 1 or 0 are eligible for vitiligo surgery.

Assessment of disease severity

The vitiligo area scoring index (VASI) was used to evaluate the severity of the disease. It is a quantitative parametric score [13]. The total body VASI is calculated using a formula that includes contributions from all body regions. One hand unit, which includes the palm plus the volar surface of all the digits, is ∼1% of the total body surface area and is used as a guide to estimate the baseline percentage of vitiligo involvement in each body region [14]. VASI=all body sites (hand units)×(residual depigmentation) [15]. The VASI ranges from 0 to 100.

All patients signed a written consent form agreeing to participate in the study and allowing blood samples and a skin biopsy.

Assessment of matrix metalloproteinase-9 expression in vitiligo skin lesion and nonlesional skin by immunohistochemical staining

On 4 mm paraffin-embedded tissue sections, immunohistochemistry staining was performed. MMP-9 expression levels were measured using mouse monoclonal antibody MMP-9 (clone 15W2, Novocastra; Leica Biosystems, 1700 Leider Lane Buffalo Grove, IL, United States) as primary antibodies and goat polyclonal tissue inhibitor of MMP-1 (clone 6F6a, Novocastra; Leica Biosystems) as secondary antibodies.

The staining intensity score was used to evaluate the slides: no staining was assigned a score of 0, weak staining was assigned a score of 1, moderate staining was assigned a score of 2, and strong staining was assigned a score of 3.

The staining proportion corresponds to the quantity score, which is the percentage of immunoreactive cells. Quantity score was calculated as follows: no staining was scored as 0, 1–10% of cells with positive staining was scored as 1, more than 10–50% was scored as 2, more than 50–70% was scored as 3, and more than 70–100% was scored as 4. The final staining score was calculated by adding the intensity and proportion scores (the total immunohistochemical score that ranges from 0 to 12) (0–4=mild, 5–8=moderate, and 9–12=severe). Immunohistochemical scores ranging from 0 to 4 were considered low levels of expression, whereas scores ranging from more than 4 to 12 were considered high levels of expression.

Statistical data analysis

Data were fed into the computer and analyzed with the IBM SPSS software package, version 20.0 (IBM Corp., Armonk, New York, USA). The significance of the obtained results was set at 5%.

Results

There were 20 patients in this study. Ten patients with vitiligo were paired with 10 healthy individuals. The age of patients in the vitiligo group was 19.66±41.10 years, compared with 18.60±30.30 years in the healthy individual group; the mean age of onset of vitiligo was 20.27±34.20 years. Mean duration of the disease was 8.35±6.90 years. Overall, 60% of the patients had an active course of disease, 30% had a positive family history of vitiligo, and 50% of patients had generalized vitiligo ([Table 1]).

Table 1 Comparison between the two studied groups according to demographic data

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The vitiligo activity was +4 in 70% of patients (activity of 6 weeks or less periods), +3 in 10%, +2 in 10%, and 0 in 10%). In 70% of patients, the mean severity of vitiligo was 6.20±10.07.

There was a significant difference in MMP-9 expression between lesional and nonlesional skin biopsy from the patient and control groups at the basal cell layer, follicular epithelium, and stromal cell layer. In patients with vitiligo, MMP-9 expression was found to be lower in the lesional skin at the basal cell layer and follicular levels when compared with nonlesional skin, whereas MMP-9 expression was higher in healthy controls. In patients with vitiligo, MMP-9 expression in lesional and nonlesional skin ranged from negative to mild, moderate, and intense. Lesional skin had 90% negative expression, whereas nonlesional skin had 50% moderate expression in the basal cell layer. MMP-9 expression was found to be lower in the lesional skin of patients with vitiligo at the follicular epithelium when compared with nonlesional skin, whereas MMP-9 expression was higher in healthy controls. MMP-9 moderate expression was found in the lesional skin of 40% of patients with vitiligo. The nonlesional skin of patients with vitiligo exhibited 60% moderate expression. The lesional skin of patients with vitiligo exhibited 40% mild MMP-9 expression in the stromal cell layer. The nonlesional skin of patients with vitiligo showed 60% intense expression compared with 50% intense expression in the healthy control group ([Table 2] and [Figure 1],[Figure 2],[Figure 3],[Figure 4]).

Table 2 Comparison between the different studied groups according to expression of matrix metalloproteinase-9 at the level of basal cell layer (N=10)

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Figure 1 Lesional skin biopsy form one of patient group. (A) H&E staining; absent epidermal melanin and perivascular mononucleated cells. (B) MMP-9immunostaining; Basal cell layer (−v), outer follicle cell layer (+2), perifollicular stromal cell staining (+2) and reticular dermal stromal cells & mononuclear cells (+2). H&E, hematoxylin and eosin; MMP-9, matrix metalloproteinase-9.

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Figure 2 Non-lesional skin biopsy from the same patient. (A) H&E staining; Melanocytes identified in basal epidermis (Black arrows), no epidermal vacuolization and no perivascular dermal inflammation. (B) MMP-9 immunostaining; epidermal cells mainly (Black arrows) and dermal cells (Red arrows) (+3). H&E, hematoxylin and eosin; MMP-9, matrix metalloproteinase-9.

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Figure 3 MMP-9 immunostaining for skin biopsy form one of patient group. (A) lesional; Epidermal cells (Black arrows) and dermal cells (Red arrows) (+2). (B) Non lesional; Epidermal cells mainly (Black arrows) and dermal cells (Red arrows) (+3). MMP-9, matrix metalloproteinase-9.

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Figure 4 MMP-9 immunostaining for skin biopsy form one of control group. (A) H&E staining; Melanocytes identified (Black arrows), no epidermal vacuolization or dermal inflammation. (B) MMP-9 immunostaining; Expression in epidermal cells (Black arrows) and few stromal dermal cells (Red arrows) (+1). H&E, hematoxylin and eosin; MMP-9, matrix metalloproteinase-9.

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MMP-9 lesional expression at the follicular epithelium level ranged from negative to mild to moderate. Overall, 75% of moderate expression was generalized, with a ‘+4’ VIDA score and an average VASI score of 8.16. MMP-9 nonlesional expression at the follicular epithelium level ranged from negative to mild. MMP-9 expression was mild in 60% of the cases ([Table 3]).

Table 3 Correlation between follicular epithelium and different parameters in diseased group (N=10)

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The lesional expression of MMP-9 in the basal cell layer ranged from negative to mild. Overall, 90% of all vitiligo patterns had negative expression, 66.7% had a ‘+4’ VIDA score, and 6.67 had a VASI score. MMP-9 nonlesional expression at the basal cell layer level ranged from negative to mild to moderate. Overall, 50% had moderate MMP-9 expression, 80% had a ‘+4’ VIDA score, and the mean VASI score was 5.30 ([Table 4]).

Table 4 Correlation between basal cell layer and different parameters in cases group (N=10)

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MMP-9 lesional expression at the stromal cell level ranged between mild and moderate. Overall, 60% of moderate expression was generalized with a ‘+4’ VIDA score and a VASI score of 11.9 on average. The nonlesional expression of MMP-9 at the stromal cell level ranged from moderate to intense. MMP-9 was found to be highly expressed in 60% of the samples. Overall, 33.3% were acrofacial, 33.3% were generalized, 66.7% had a ‘+4’ VIDA score, and the mean VASI score was 9.05 ([Table 5]).

Table 5 Relation between stromal cell and different parameters in the case group (N=10)

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Discussion

The cause of vitiligo has long been a source of contention. The exact cause of vitiligo is unknown, and the pathogenesis is complex, involving the interaction of a number of variables [12]. MMP-9 is involved in the degradation of ECM and non-ECM in tissue remodeling, affects immune cell function, and is upregulated during inflammatory processes such as arthritis and diabetes [16]. MMP expression is markedly increased during cell migration, and induction of MMP-9 is associated with KC migration [17]. Thus, MMP-9 plays an important role in paving a pathway in front of migratory melanocytes and represents a pro-pigmenting factor [11]. The purpose of our case–control study was to determine whether vitiligo is associated with changes in the tissue expression of MMP-9 in cases with vitiligo lesions using immunohistochemistry assessment of the tissues, as well as to evaluate the relationship between MMP-9 levels and disease activity and duration as well as severity of vitiligo. In the current study, a lower expression of MMPs-9 was found in all vitiligo lesions compared with perilesional areas, indicating a possible defect in melanocyte motility and migration and possibly representing one of the challenges of repigmentation. It is well understood that in vitiligo, the migration of melanocytes from hair follicles or the margins of vitiligo lesions into clinically depigmented epidermis is critical to the repigmentation of vitiliginous skin. This necessitates the activation of MMPs to penetrate ECM tissue barriers in vivo. Our findings support the findings of Kumar et al. [5], who discovered lower expression of MMPs-9 in vitiligo melanocytes compared with controls, and El Mofty et al. [18], who discovered the absence of E-twenty-six-1 expression in vitiligo melanocytes, which could potentially decrease MMP-2 and MMP-9 expression by ELISA. Such decreased MMP expression could be attributed to a defect in the cells of origin in the vitiligo skin, such as melanocytes, fibroblasts, and keratinocytes. In line with our findings, Esmat et al. [19] discovered low significant expression of MMP-1, MMP-2, and MMP-9 in all acral and nonacral vitiligo lesions when compared with perilesional areas and control healthy individuals using immunohistochemistry. To the best of our knowledge, there are no results that disagree with our results.

Conclusion

Tissue MMP-9 imbalance has a role in the etiopathogenesis of vitiligo. The low MMP-9 tissue expression may not facilitate the migration of melanoblasts. Tissue MMP-9 is a significant predictor of severity of vitiligo.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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