Secondary Logo

Journal Logo

Immunohistochemical expression of galectin-7 in hypertrophic scars and keloids

Mourad, Basmaa; Neinaa, Yomna M. El-Hamda; Shareef, Mohamad M.b

Journal of the Egyptian Women’s Dermatologic Society: September 2018 - Volume 15 - Issue 3 - p 151–157
doi: 10.1097/01.EWX.0000544114.72410.41
Original articles
Free

Background Cutaneous scarring is almost an inevitable end point of human wound healing. Galectin-7 is a prototype member of galectin family that is thought to play a role during wound healing.

Objective To study immunohistochemical expression of galectin-7 in hypertrophic scars and keloids to throw a light on its possible role in the pathogenesis of these scars.

Patients and methods This study was conducted on 40 patients with cutaneous scars (23 keloids and 17 hypertrophic scars) and 20 normal healthy controls. Skin biopsy was taken from each participant and examined for immunohistochemical expression of galectin-7.

Results Galectin-7 immunohistochemical expression was detected in epidermal keratinocytes and dermal fibroblasts of both hypertrophic scars and keloids. Its expression in epidermal keratinocytes of hypertrophic scars showed significant decrease in comparison with normal control skin, whereas its expression in the dermal fibroblasts of both hypertrophic scars and keloids showed significant increase in comparison with normal control skin. Additionally, there was a significant increase of galectin-7 expression in dermal fibroblasts of hypertrophic scars compared with keloids.

Conclusion Galectin-7 may participate in the pathogenesis of hypertrophic scars and keloids through controlling both epidermal keratinocytes and dermal fibroblasts. It could be a potential therapeutic target for the management of such scars.

Departments of aDermatology and Venereology

bPathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Correspondence to Yomna M. El-Hamd Neinaa, MD, Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt Tel: +20 100 533 3563; fax: +20 247 023 08; e-mail: yomnamazid@yahoo.com

Received December 25, 2017

Accepted July 7, 2018

Back to Top | Article Outline

Introduction

Cutaneous scarring is almost an inevitable end point of human wound healing that may be associated with significant morbidity, both physical and psychological. Excessive cutaneous scars can be particularly debilitating, with pain, pruritus, and a range of cosmetic deformities that may affect the patient’s quality of life 1–4.

Excessive cutaneous scarring has been identified into two types: hypertrophic scars and keloids 1. Hypertrophic scars are presented clinically as raised scars limited within the boundaries of the original wound that frequently regress spontaneously and do not recur after surgical excision. However, keloids present as raised red scars that grow beyond the boundaries of the original wound, rarely regress over time, and show high recurrence rate after surgical excision 1,2.

The pathogenesis of both hypertrophic scars and keloids involves complex pathways, and the exact mechanisms by which they are initiated, evolved, and regulated remain to be fully elucidated 5. They result from aberrations in the process of physiologic wound healing, most probably owing to an improper balance between deposition and degradation of extracellular components, notably collagen produced by malfunctioning fibroblasts 6.

There is a conflict regarding the precise histopathological criteria that distinguish keloids from hypertrophic scars, which arises from the fact that the pathological manifestations of these lesions overlap. Thus, the concepts that hypertrophic scars and keloids are characterized by ‘hyalinized collagen bundles’ have done little to promote their accurate clinicopathological diagnosis and subsequently effective therapeutic interventions 5,7.

Galectins are a family of 15 growth/adhesive-regulatory lectins with a unique carbohydrate recognition domain that binds to β-galactoside derivatives 8,9. Galectin-7 is a prototype member of galectin family that has been considered as a product of tumor suppressor gene p53 and also known as p53-induced gene 1 10. It has been proposed as a regulator of apoptosis, and its proapoptotic effects were noted in epidermal responses to a variety of stimuli 11,12. It is thought to play a role during wound healing, functioning as a regulator of keratinocyte proliferation and migration, which is central in maintaining and restoring epidermal homeostasis 13,14. So, it is worth to study galectin-7 expression in hypertrophic scars and keloids to throw light on its possible role in the formation and development of these scars.

Back to Top | Article Outline

Patients and methods

This study was conducted on 40 patients with excessive cutaneous scars (23 with keloids and 17 with hypertrophic scars) and 20 normal control skin specimens from age-matched and sex-matched healthy individuals with no past history of scarring. Patients were selected from the Outpatient Clinic of Dermatology and Venereology Department, Tanta University Hospitals. Normal control skin specimens were obtained from Plastic Surgery Department, Tanta University Hospitals. Ethical approval was obtained from Ethical Committee before the commencement of the study. Inclusion criteria were patients with hypertrophic scars or keloids, duration not less than 6 months; patients who did not receive any medication for at least 2 months before biopsy taking; and those who agreed to join the study and signed written consent. Exclusion criteria were patients with any other dermatological or systemic disease, and pregnant and lactating women.

All participants were subjected to full history taking, thorough general and dermatological examinations, and routine laboratory investigations. After taking written informed consent, 4-mm punch skin biopsy specimens were taken from the edge of the scar in each patient under local anesthesia and complete aseptic technique. In addition, normal control skin specimens were obtained during plastic operations. All were fixed in neutral formalin 10% and processed for paraffin blocks. Two sections (each 4 µm thick) were cut from each block and were stained with the following:

  • Hematoxylin and eosin staining for confirmation of the clinical diagnosis.
  • Galectin-7 immunohistochemical staining using the standard streptavidin-peroxidase complex method. Primary antibody used was rabbit polyclonal anti-galectin-7 antibody (catalogue no. ab10482, 50 μg at concentration 1 mg/ml; Abcam Inc., Cambridge, Massachusetts, USA) and diluted by phosphate buffer saline in a dilution 1 : 500. Secondary antibody (Universal Kit, BioLegend Inc., USA) was biotinylated anti-rabbit polyclonal antibody and was used in accordance with the manufacturer’s instructions. The slides were submitted to subsequent steps of deparaffinization and rehydration. Antigen retrieval was done by boiling in citrate buffer saline (pH 6) followed by cooling at room temperature. The primary antibodies were incubated overnight at room temperature, and then the secondary antibody was applied with diaminobenzidine as a chromogen substrate and Mayer hematoxylin as a counter stain. Each slide was evaluated by light microscopy at the same magnification for the following:
    • Staining pattern: for each specimen, three to five separate fields of view were evaluated and analyzed in both epidermis and dermis. The expression of galectin-7 was detected as brownish staining in positive cells. Only the nuclear staining was taken into consideration.
    • Score of intensity of galectin-7 expression 15: quantitative evaluation of galectin-7 immunohistochemical expression was performed in both the epidermis and dermis of each specimen (by image analysis; Leica Q Win, Bensheim, Germany), and the number of positive cells was expressed as the percentage of the total number of cells in the examined field and the mean was calculated from the measurement of four fields at magnification ×400. Then, the score of intensity of galectin-7 immunohistochemical expression was graded from 0 to +3 as follow: 0 (negative expression), no staining; +1 (mild expression), less than 25%; +2 (moderate expression), 25–50%; and +3 (strong expression), more than 50%.
Back to Top | Article Outline

Statistical analysis

Data were fed to the computer and analyzed using IBM SPSS software package, version 20.0 (IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percent. Quantitative data were described using range (minimum and maximum) and mean±SD. Comparison between different groups regarding categorical variables was tested using χ2 test. For normally distributed data, comparison between two independent populations was done using independent t test. For abnormally distributed data, Kruskal–Wallis test was used. Correlations between two quantitative variables were assessed using Spearman’s coefficient. A significant difference was considered at P value less than or equal to 0.05.

Back to Top | Article Outline

Results

Clinical results

The clinical characteristics of the studied groups are summarized in Table 1. There was a statistically significant increase in spontaneous occurrence of keloids than hypertrophic scars. In addition, there was a significant familial association and past history of development of similar scars in keloids than hypertrophic scars.

Table 1

Table 1

Back to Top | Article Outline

Histopathological results

Hematoxylin and eosin results

Hypertrophic scar specimens showed dermal proliferation of numerous fibroblasts and intertwining fine fibrillar collagen predominantly oriented parallel to the epidermis (Fig. 1a). However, keloids specimens showed thick hyalinized, haphazardly lying, nonoriented collagen bundles ‘keloid collagen,’ and relatively few fibroblasts (Fig. 3a).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Back to Top | Article Outline

Galectin-7 immunohistochemical results

  • Control group: galectin-7 was observed as uniformly positive nuclear and cytoplasmic immunostaining in both basal and suprabasal epidermal keratinocytes as well as follicular epithelium and dermal blood vessels in all specimens. On the contrary, no expression was observed in dermal fibroblasts in most specimens (85%). The intensity of its expression was illustrated in Table 2 and Fig. 2a, b.
  • Hypertrophic scars: galectin-7 was positively expressed in the epidermis of all specimens (100%) mainly along the basal keratinocytes. However, in the dermis, it was expressed in the fibroblasts in 88.2% of specimens. The intensity of its expression was illustrated in Table 2 and Fig. 1b–d. The intensity of overall galectin-7 expression in the epidermal keratinocytes of hypertrophic scars was significantly decreased in comparison with normal control skin (P1<0.001). On the contrary, its expression in the dermal fibroblasts showed significant increase in comparison with normal control skin (P1<0.001) (Table 2).
  • Keloids: galectin-7 was positively expressed in the epidermis of all specimens (100%) in both basal and suprabasal keratinocytes. However, in the dermis, it was expressed in fibroblasts in 91.2% of the specimens. The intensity of its expression is illustrated in Table 2 and Fig. 3b–d. The intensity of overall galectin-7 expression in the epidermal keratinocytes of keloids was higher than normal control skin but with no significant difference (P2=0.173, Table 2). However, in the dermal fibroblasts, its expression showed statistically significant increase in comparison with the normal control skin (P2<0.001, Table 2).
  • The intensity of overall galectin-7 expression in the epidermal keratinocytes of hypertrophic scars showed statistically significant decrease in comparison with its expression in keloids (P3<0.001). However, in the dermis, there was a statistically significant increase in galectin-7 expression in the dermal fibroblasts of hypertrophic scars than those in keloids (P3<0.04, Table 2).
Table 2

Table 2

Back to Top | Article Outline

Correlations between intensity of galectin-7 expression in hypertrophic scars and keloids, and duration of the scars

No statistically significant correlation was detected between galectin-7 expression in the epidermal keratinocytes of hypertrophic scars or keloids and duration of the scars (P>0.05). On the contrary, there was a statistically significant negative correlation between intensity of galectin-7 expression in the dermal fibroblasts of both hypertrophic scars and keloids, and duration of the scars, with P value less than 0.05 (Graph 1, Table 3).

Table 3

Table 3

Back to Top | Article Outline

Discussion

The current work detected galectin-7 expression in normal control skin as uniformly positive immunostaining in the cytoplasm and nuclei of both basal and suprabasal epidermal keratinocytes as well as follicular epithelium; however, no expression was observed in dermal fibroblasts in most specimens (85%). These findings are in agreement with Cho et al.16 who reported that galectin-7 is an abundant component of normal keratinocytes. Furthermore, Gendronneau et al.13 suggested that galectin-7 may present as a ‘sentinel molecule, constantly sensing the integrity of the skin in steady-state conditions while contributing to wound healing.’ They provided evidence that galectin-7 plays a critical role in the maintenance of epidermal homeostasis by modulating keratinocyte apoptosis and proliferation as well as participating in the process of cell migration.

Regarding hypertrophic scars, galectin-7 expression in this work was significantly reduced in epidermal keratinocytes compared with normal control skin. These findings were in agreement with Cho et al.16 who observed that hypertrophic scars were associated with decreased epidermal expression and serum levels of galectin-7. Previous studies 17–20 reported that epidermal keratinocytes intercommunicate with underlying fibroblasts, and this intercommunication plays an important role in the pathogenesis and development of hypertrophic scars allowing us to suggest that reduced galectin-7 expression in the epidermis of hypertrophic scars may participate in its pathogenesis through epidermal-mesenchymal interactions with subsequent activation of dermal fibroblasts.

To our knowledge, no previous study had evaluated galectin-7 expression in keloids. The current study detected that galectin-7 expression in the epidermal keratinocytes of keloids was significantly increased compared with hypertrophic scars. This finding may refer to the presence of different pathways for the emergence of both types of cutaneous scars, which already differed in morphology either clinically or histologically and in their molecular pathogenesis 1,2,7.

In the current study, galectin-7 was significantly overexpressed in dermal fibroblasts in both hypertrophic scars and keloids compared with the normal control skin. It is well known that galectin-7 is a proapoptotic protein that is produced by p53 tumor suppressor gene 10,12. The open question is how apoptosis can be triggered in hypertrophic scars and keloids, which are characterized by excessive deposition of extracellular matrix mainly collagen.

Excessive extracellular matrix deposition in hypertrophic scars and keloids is caused by accumulation of dermal fibroblasts. This is the result of imbalances between fibroblast proliferation and apoptosis. Regarding proliferation, fibroblasts proliferate with higher densities in hypertrophic scars and keloids in comparison with normal fibroblasts. Regarding fibroblast apoptosis, hypertrophic scars and keloids-derived fibroblasts are significantly resistant to apoptosis, in contrast to normal dermal fibroblasts 5. From our point of view, resistance of hypertrophic scars and keloids-derived fibroblasts to apoptosis may be responsible for overexpression of proapoptotic molecules including galectin-7 as a feedback mechanism suggesting a potential function for galectin-7 in regulating and controlling fibroblasts proliferation during wound healing. Subsequently, it could be suggested that galectin-7 overexpression is not likely to be a primary event, but mostly a secondary consequence to excessive fibroblasts proliferation during wound healing.

The current work detected significant galectin-7 overexpression in the dermal fibroblasts of hypertrophic scars in comparison with keloids. It could be suggested that the relative overexpression of galectin-7 in hypertrophic scars fibroblasts could lead to a time-dependent apoptosis that explained the better evolution and spontaneous regression reported in hypertrophic scars and not in keloids. On the contrary, relatively decreased galectin-7 expression in dermal fibroblasts of keloids than hypertrophic scars is an important mechanism for the maintenance of their progressive nature.

One of the possible mechanisms of galectin-7-induced apoptosis was suggested by Park et al.21 who found that galectin-7 can activate matrix metalloproteinase (MMP)-9 transcription. In addition, Guo and Li 22 detected that galectin-7 promotes the expression of both MMP-2 and MMP-9. As previous studies 7,23–25 provided evidence of the role of MMPs in keloids and hypertrophic scars, it is possible to suspect that galectin-7 may induce apoptosis through activation of MMPs in these scars.

In conclusion, the results of the current study point out to interesting different pathways in hypertrophic scars and keloids apoptosis. Galectin-7 may have a potential role in the pathogenesis of hypertrophic scars and keloids through controlling both epidermal keratinocytes and dermal fibroblasts. Galectin-7 overexpression in hypertrophic scars fibroblasts in comparison with keloids could lead to a time-dependent apoptosis that explained the better evolution and spontaneous regression reported in hypertrophic scars and not in keloids. Furthermore, evaluation of galectin-7 expression could be taken into consideration in the future as a useful tool to differentiate hypertrophic scars from keloids for successive therapeutic intervention.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG. Hypertrophic scarring and keloids: pathomechanisms and current and emerging treatment strategies. Mol Med 2011; 17:113–125.
2. Wolfram D, Tzankov A, Pülzl P, Piza-Katzer H. Hypertrophic scars and keloids – a review of their pathophysiology, risk factors, and therapeutic management. Dermatol Surg 2009; 35:171–181.
3. Seo BF, Jung SN. The immunomodulatory effects of mesenchymal stem cells in pevention or treatment of excessive scars. Stem Cells Int 2016; 2016:1–8.
4. Rees PA, Greaves NS, Baguneid M, Bayat A. Chemokines in wound healing and as potential therapeutic targets for reducing cutaneous scarring. Adv Wound Care 2015; 4:687–703.
5. Huang C, Murphy GF, Akaishi S, Ogawa R. Keloids and hypertrophic scars: update and future directions. Plast Reconstr Surgery Glob Open 2013; 1:e25.
6. Atiyeh BS, Costagliola M, Hayek SN. Keloid or hypertrophic scar: the controversy: review of the literature. Ann Plast Surg 2005; 54:676–680.
7. Lee DE, Trowbridge RM, Ayoub NT, Agrawal DK. High-mobility group box protein-1, matrix metalloproteinases, and vitamin D in keloids and hypertrophic scars. Plast Reconstr Surgery Glob Open 2015; 3:e425.
8. Chen H-Y, Lo C-H, Li1 C-S, Hsu DK, Liu F-T. Galectins and cutaneous immunity. Dermatol Sinica 2012; 30:121–127.
9. Biron-Pain K, Grosset AA, Poirier F, Gaboury L, St-Pierre Y. Expression and functions of galectin-7 in human and murine melanomas. PLoS One 2013; 8:e63307.
10. Kim S-J, Hwang J-A, Ro JY, Lee YS, Chun K-H. Galectin-7 is epigenetically-regulated tumor suppressor in gastric cancer. Oncotarget 2013; 4:1461–1471.
11. Barkan B, Cox AD, Kloog Y. Ras inhibition boosts galectin-7 at the expense of galectin-1 to sensitize cells to apoptosis. Oncotarget 2013; 4:256–268.
12. Saussez S, Kiss R. Galectin-7. Cell Mol Life Sci 2006; 63:686–697.
13. Gendronneau G, Sidhu SS, Delacour D, Dang T, Calonne C, Houzelstein D, et al. Galectin-7 in the control of epidermal homeostasis after injury. Mol Biol Cell 2008; 19:5541–5549.
14. Panjwani N. Role of galectins in re-epithelialization of wounds. Ann Transl Med 2014; 2:89.
15. Labrie M, Vladoiu MC, Grosset AA, Gaboury L, St-Pierre Y. Expression and functions of galectin-7 in ovarian cancer. Oncotarget 2014; 5:7705–7721.
16. Cho S, Kim J, Zheng Z, Choi M, Choi I, Oh H, et al. Decreased tissue and serum expression of galectin-7 in patients with hypertrophic scars. Acta Derm Venereol 2013; 93:669–673.
17. Edriss AS, Mešták J. Epidermal keratinocytes may have an important role in hypertrophic scarring pathogenesis: an immunohistochemical study (using P63 and Ki-67 staining). Ann Burns Fire Disasters 2005; 18:133–139.
18. Ghahary A, Ghaffari A. Role of keratinocyte-fibroblast cross-talk in development of hypertrophic scar. Wound Repair Regen 2007; 15:S46–S53.
19. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol 2007; 127:998–1008.
20. Funayama E, Chodon T, Oyama A, Sugihara T. Keratinocytes promote proliferation and inhibit apoptosis of the underlying fibroblasts: an important role in the pathogenesis of keloid. J Invest Dermato 2003; 121:1326–1331.
21. Park JE, Chang WY, Cho M. Induction of matrix metalloproteinase-9 by galectin-7 through p38 MAPK signaling in HeLa human cervical epithelial adenocarcinoma cells. Oncol Rep 2009; 22:1373–1379.
22. Guo P, Li XG. Galectin-7 promotes the invasiveness of human oral squamous cell carcinoma cells via activation of ERK and JNK signaling JIA. Oncol Lett 2017; 13:1919–1924.
23. Imaizumi R, Akasaka Y, Inomata N, Okada E, Ito K, Ishikawa Y, et al. Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression. Histopathology 2009; 54:722–730.
24. Tanriverdi-Akhisaroglu S, Menderes A, Oktay G. Matrix metalloproteinase-2 and -9 activities in human keloids, hypertrophic and atrophic scars: a pilot study. Cell Biochem Funct 2009; 27:81–87.
25. Ulrich D, Ulrich F, Unglaub F, Piatkowski A, Pallua N. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with different types of scars and keloids. J Plast Reconstr Aesthet Surg 2010; 63:1015–1021.
Keywords:

cutaneous scars; galectin-7; hypertrophic scars; keloids

© 2018 Egyptian Women's Dermatologic Society