Immunohistochemical expression of discoidin domain receptor 1 in keloid scar

Rehab M Samaka; Amr M El Meanawy; Mohammed A Basha; Shimaa H Abdel-Raouf

doi:10.4103/mmj.mmj_275_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 35 | Issue : 4 | Page : 1721-1725

Immunohistochemical expression of discoidin domain receptor 1 in keloid scar

Rehab M Samaka¹, Amr M El Meanawy², Mohammed A Basha³, Shimaa H Abdel-Raouf⁴
¹ Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
² Department of Plastic Surgery, Faculty of Medicine, Menoufia University, Menoufia, Egypt
³ Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Menoufia, Egypt
⁴ Department of Dermatology and Andrology, El Bagour Hospital, Menoufia, Egypt

Date of Submission	15-Aug-2022
Date of Decision	11-Sep-2022
Date of Acceptance	12-Sep-2022
Date of Web Publication	04-Mar-2023

Correspondence Address:
Shimaa H Abdel-Raouf
El Shahed Street, Garawan, El Bagour, Menoufia
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/mmj.mmj_275_22

Abstract

Objectives
To assess the role of discoidin domain receptor 1 (DDR1) in keloid scar.
Background
Keloid scars are formed of inert masses of collagen. Therefore, expression of collagen and DDR1 may determine the nature and extent of tissue scarring.
Patients and methods
This case–control study was carried out on 20 patients presented with keloid and 40 age-matched, sex-matched, and site-matched apparently normal skin from apparently healthy volunteers. Skin biopsies were sent to the Pathology Department of Faculty of Medicine, Menoufia University, for histopathological assessment and immunohistochemical staining of DDR1.
Results
Epidermal expression of DDR1 was positive in all keloid cases (lesional and perilesional). Its intensity was mild in all lesional biopsies and in only 20% of perilesional ones. Dermal expression was positive in all perilesional cases and in only 30% of lesional biopsies. A significant difference was found between lesional and perilesional keloid regarding DDR1 expression intensity in the epidermis and dermis (P < 0.001). A significant difference was found between lesional and perilesional keloid regarding DDR1 expression H-score in the epidermis and dermis (P < 0.001). A significant positive correlation was found between lesional epidermal and lesional dermal groups regarding keloid H-score (r = 0.552; P = 0.012). In control group, positive epidermal expression of DDR1 was found in all biopsies, with cytoplasmic localization and basal topography. Mild intensity was seen in 52.5% and moderate intensity was seen in 47.5%. The mean ± SD values of H-score were 81.50 ± 39.52. Positive dermal expression of DDR1 was found in all biopsies, with cytoplasmic localization and basal topography. Mild intensity was seen in 50% and moderate intensity was seen in 50%. The mean ± SD values of H-score were 82 ± 27.38.
Conclusion
DDR1 expression in keloid scar is suspected in early pathogenesis.

Keywords: discoidin domain receptor 1, expression, immunohistochemistry, keloid, scar

How to cite this article:
Samaka RM, El Meanawy AM, Basha MA, Abdel-Raouf SH. Immunohistochemical expression of discoidin domain receptor 1 in keloid scar. Menoufia Med J 2022;35:1721-5

How to cite this URL:
Samaka RM, El Meanawy AM, Basha MA, Abdel-Raouf SH. Immunohistochemical expression of discoidin domain receptor 1 in keloid scar. Menoufia Med J [serial online] 2022 [cited 2023 Jun 9];35:1721-5. Available from: http://www.mmj.eg.net/text.asp?2022/35/4/1721/371009

Introduction

Keloids are the result of aberrant tissue scarring, typically occurring in injured skin, and are caused by the overgrowth of granulation tissue or collagen type III during the healing process ^[1]. There is a genetic component, thus a predisposition can be genetically transmitted. Keloids are difficult to treat because of their post-excisional recurrence ^[2].

Keloids are of concern to patients as their deforming scars raise cosmetic issues and cause psychological distress, impairing quality of life ^[3]. Much information concerning keloid pathogenesis and risk factors has been unfolded over recent years. Several treatment alternatives are being investigated; however, more extensive studies are required to understand the most compelling alternatives to quantify their clinical efficacy and safety ^[4],[5].

Keloid pathogenesis is still complex, with both genetic and environmental factors involved ^[6]. Abnormal fibroblasts have been shown to play a key role, but new lines of research have driven attention to keratinocytes and altered signaling cross-talks ^[7],[8].

Discoidin domain receptors (DDRs), DDR1 and DDR2, lie at the intersection of two large receptor families, namely, the extracellular matrix and tyrosine kinase receptors. As such, DDRs are uniquely positioned to function as sensors for extracellular matrix and to regulate a wide range of cell functions ^[9],[10].

DDRs play an important role in modulating collagen synthesis and organization during scar formation ^[11]. As collagen directly bound to the receptor ectodomain results in phosphorylation of the tyrosine kinase domain, which in turn leads to a downstream cascade signaling activity, so expression of collagen and DDRs may determine the nature and extent of tissue scarring ^[12].

The aim of the study was to assess the role of DDR1 in the pathogenesis of keloid through its immunohistochemical expression.

Patients and methods

This case–control study was carried out on 20 patients who presented with scars and 40 age-matched, sex-matched, and site-matched apparently healthy individuals as a control group. Patients were selected from the Plastic Surgery Department of Menoufia University Hospital spanning the period between January 2020 and January 2021. A written consent form approved by the Local Ethics Research Committee of Menoufia Faculty of Medicine was obtained from every participant before the study initiation. All specimens were collected and sent to the Pathology Department of Faculty of Medicine, Menoufia University, for preparation of formalin-fixed paraffin-embedded tissues for preparation of H and E-stained slides.

Complete history was taken from the studied groups, general examination was done, and dermatological assessment of the lesion was done according to Manchester, Vancouver, and Stony Brook Clinical Assessment Scales. In Manchester and Vancouver scales, high score means bad outcome, whereas in Stony Brook scale, high score means good outcome ^[13].

Sections were cut at 5 μm and stained as follows: they were dewaxed and rehydrated in graded alcohol solutions. For heat retrieval, the sections were placed in citrate buffer (1: 10 dilution, pH: 7.2) and heated at 120°C for 3–5 min. Endogenous peroxidase activity was neutralized using a peroxidase block for 5 min. Nonspecific binding was blocked by preincubation with a protein block for 5 min at room temperature. Slides were then incubated for 1 h at room temperature with the primary diluted antibodies, rabbit polyclonal antibodies raised against DDR1 [name of kit: DDR1 (c20), sc-532] (Santa Cruz Biotechnology Inc., Dallas, Texas, United States) at a dilution of 1/100. The slides were washed with PBS (2–5 min) and then incubated with secondary antibody for 10 min. The color reaction was developed in diaminobenzidine for 5 min. Sections were then counterstained with hematoxylin, dehydrated, and mounted. Positive control slides were prepared of formalin-fixed tissues of breast carcinoma. Negative control slides were prepared by omitting the primary antibodies from the staining procedure ^[14].

Statistical analysis

Data were collected, tabulated, and statistically analyzed using a personal computer with the 'Statistical Package for the Social Sciences (SPSS) version 22' program (IBM Company, Chicago, Illinois, USA). Data were presented as arithmetic mean, SD, percentage, median, and range. Difference between the groups was analyzed using χ² test and Kruskal–Wallis test. P value less than or equal to 0.05 was set to be significant, and P value less than 0.01 was highly significant.

Results

This case–control study was conducted on 20 patients with keloid and 40 age-matched, sex-matched, and site-matched healthy participants as a control group.

The mean ± SD of age of the keloid cases was 22.50 ± 8.73 years. Males were 20% of cases and 80% were females, with 4: 16 as male to female ratio. Overall, 45% of cases had a positive family history of keloid. The mean ± SD values of duration of the disease were 15.05 ± 7.67 years. Progressive course was seen in 100% of cases. Onset was accidental in 20% and gradual in 80% of cases. Clinical assessment of scars was done by Manchester scale, where total score ranged from 15 to 18, with a mean ± SD of 16 ± 1.03; Vancouver scale, where total score ranged from 11 to 13, with a mean ± SD of 11.80 ± 0.77; and Stony Brook scale, where total score ranged from 1 to 3, with a mean ± SD of 1.95 ± 0.76 [Table 1].

Table 1: Clinical data of keloid cases

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Lesional keloid group: positive epidermal expression of DDR1 was found in all lesional biopsies, with cytoplasmic localization, basal topography, and mild intensity. The mean ± SD values of H-score of DDR1 expression were 27.50 ± 8.51. Negative dermal expression was found in 70%. All cases had cytoplasmic localization and mild intensity. The mean ± SD values of H-score of DDR1 expression were 38.50 ± 8.51 [Table 2].

Table 2: Comparison between keloid cases (lesional and perilesional) and control group regarding epidermal and dermal expression of discoidin domain receptor 1

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In perilesional keloid groups, positive epidermal expression of DDR1 was found in all biopsies with cytoplasmic localization. Basal topography was found in 70% of cases. Mild intensity was seen in 20% and moderate in 80% of cases. The mean ± SD values of H-score of DDR1 expression were 125.50 ± 48.07. Positive dermal expression was found in all biopsies with cytoplasmic localization. Intensity was mild in 40%, moderate in 40%, and strong in 20%. The mean ± SD values of H-score of DDR1 expression were 117 ± 56.11 [Table 2].

In the control group, positive epidermal expression of DDR1 was found in all biopsies, with cytoplasmic localization and basal topography. Mild intensity was seen in 52.5%, and moderate intensity was seen in 47.5%. The mean ± SD values of H-score were 81.50 ± 39.52. Positive dermal expression of DDR1 was found in all biopsies, with cytoplasmic localization and basal topography. Mild intensity was seen in 50% and moderate intensity was seen in 50%. The mean ± SD values of H-score were 82 ± 27.38 [Table 2].

There was a significant difference between lesional, perilesional keloid, and control groups regarding epidermal DDR1 expression topography (P = 0.008). Significant differences were detected among lesional, perilesional keloid, and control group regarding DDR1 expression intensity and H-score values in epidermis and dermis (P < 0.001 for all). Moreover, a significant difference was found among lesional, perilesional keloid, and control group regarding dermal DDR1 expression status (P < 0.001) [Table 2].

A significant positive correlation was found between lesional epidermal and lesional dermal groups regarding keloid H-score (r = 0.552; P = 0.012) [Table 3] and [Figure 1] and [Figure 2].

Table 3: H-score in keloid

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Figure 1: (a) Epidermis of perilesional keloid showed positive cytoplasmic DDR1 expression with moderate intensity and whole thickness distribution. (b) Dermis of perilesional keloid showed preserved adnexa (hair follicle and eccrine sweat gland ducts) exhibiting moderate DDR1 expression; however, mild DDR1 cytoplasmic expression was noted in stromal cells (DDR1 immunohistochemical ×200 for both). DDR1, discoidin domain receptor 1.

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Figure 2: (a) Lesional keloid skin showed positive mild cytoplasmic DDR1 expression in basal cell layer of epidermis. Dermis showed scar tissue (black arrow). (b) Dermis showed: positive mild cytoplasmic DDR1 expression in fibroblasts/myofibroblasts (DDR1 immunohistochemical ×40 for a and ×400 for b). DDR1, discoidin domain receptor 1.

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Discussion

There is scarce data about the role of DDR1 in keloid; therefore, the aim of this study was to investigate the possible role of DDR1 in the pathogenesis of keloid through its immunohistochemical expression and to correlate its expression with clinicopathological parameters.

DDR1 binds to and organizes type I collagen fibers and activates a series of signals that regulate diverse cell behaviors from matrix remodeling, proliferation, migration, to differentiation. Herein, we demonstrate that DDR1 is significantly upregulated in mesenchymal stem cells encapsulated within a 3D type I collagen microenvironment and through crosstalk with traditional integrin-mediated signaling, which upregulates the osteogenic potential of mesenchymal stem cells ^[15].

In the current work, DDR1 overexpression in perilesional area than lesional area in keloid scar could be considered the main player in early pathogenesis of scar. This showed that the expression of DDR1 was related to keloid infiltration; this may explain keloid progression and growth beyond the boundaries of the original wound, and this was in accordance with Samaka et al.^[16], who reported that histopathological changes of perilesional areas in different types of scar throw the light on dynamic processes. These areas could be responsible for progression or regression of scar. Therefore, perilesional area is a shadow area and may be the main player in pathogenesis of scar, and this could open the gate for new target therapy for this disease. Further studies are required to establish these findings ^[16].

Our finding was matched with Russell et al. ^[17], who reported that higher DDR1 expression associated with ECM remodeling. The expression of DDR1 was also higher in perilesional keloid skin than the normal healthy skin; when the cell culture was done from the tissue explants, the keloid fibroblasts were more easily cultured from the keloid margin.

Syed et al.^[18] demonstrated that biopsies taken from the growing margin (perilesional) of keloid scars show a greater collagen production than those taken from different sites from the same keloid lesion, and this is in line with our study result.

Our study matched with Ashcroft et al.^[19], who demonstrated that perilesional and intralesional keloid fibroblast conditioned media induce significantly elevated metabolic viability and proliferation in normal scar and skin fibroblasts, So perilesional area of keloid may be responsible for its progression and growth beyond the boundaries of the original wound ^[19].

Our results are consistent with the results of Coelho et al.^[12], who reported that mechanical inhibition of wound contraction induces maintenance of both DDR1 and myofibroblasts in wounds together with an increase in collagen alignment. Dysregulation of normal cutaneous wound healing may lead to the formation of keloids. Keloids are due to the accumulation of extracellular matrix components, especially thick collagen bundles ^[12].

Our study is in agreement with Ongusaha et al.^[20], who demonstrated that some DDR1 can exert regulatory effect on the collagen structure. These findings reveal that DDR1 can sense the quality of collagen, activate the signals, and regulate cellular behavior, thus modulating collagen proliferation, production, and organization ^[20].

Conclusion

Overexpression of DDR1 in perilesional skin of keloid could be contributed in early pathogenesis. Downregulation of DDR1 expression in keloid could be accused in scar pathogenesis (perilesional expression positivity and H-score are more than that seen in lesional).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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