Evaluation of the Role of High-mobility Group Box 1 Protein in Patients With Keloids: A Case Control Study

Background. Keloids cause cosmetic problems, pain, and pruritus. Different modes of therapy are limited by their efficacy and side effects. High-mobility group box 1 protein (HMGB1) may play a role in keloid pathogenesis; therefore, the therapeutic potential of box A1, an antireceptor of advanced glycation end products antibody, and other inhibitors of HMGB1 may play a role in the treatment of keloids. Objective. This study evaluates the role of HMGB1 in patients with keloids by comparing their serum level with healthy controls. Materials and Methods. Forty patients with keloids and 40 controls were enrolled in this study. Detailed history and clinical evaluation were performed. A 3-mL sample of whole blood was obtained from both patient groups and centrifuged immediately. The resultant supernatant serum was frozen at -20°C for the detection and quantification of HMGB1 using enzyme-linked immunosorbent assay. Results. There was a statistically significant elevation in the mean value of HMGB1 in keloid cases (74.38 ± 40.16) compared with the mean value of the controls (52.00 ± 5.41; P = .001). Mean value of HMGB1 was positively correlated with keloid severity. Conclusions. High-mobility group box 1 was found to be elevated in patients with keloids compared with their controls, suggesting its role in excessive scarring and the role of its antagonists in therapy.

Keloids are abnormalities of wound healing characterized by excessive production of collagen in the skin. In addition to the cosmetic problems caused by a raised and red appearance, keloids can cause pain, pruritus, and contractures. Keloids are visualized as scars that grow beyond the boundaries of the original wound and rarely regress over time. Although seen in patients of all races, keloids have a greater occurrence in dark-skinned individuals, with an incidence of 6% to 16% in African populations.¹

Large keloids can arise from minor injuries to the skin, such as acne and piercings.² Keloids develop from an improper balance between deposition and degradation of extracellular matrix (ECM) components, notably collagen. The excess collagen is produced by malfunctioning fibroblasts due to increased density and activation of growth factor receptors.³

High-mobility group box 1 protein (HMGB1) has dual functions. As an intracellular transcription factor, HMGB1 binds to bent DNA to promote the assembly of nucleoprotein complexes, which is critical in the processes of transcription, recombination, replication, and repair. As an extracellular mediator, HMGB1 acts as a potent inflammatory cytokine.⁴ Release of HMGB1 occurs actively by stimulated monocytes and macrophages and passively by necrotic/damaged cells.⁵ The protein exerts its effects by binding to cell surface receptors, particularly the advanced glycation end product (RAGE) receptor and toll-like receptor (TLR) 2 and TLR4.⁶ It affects wound healing by binding primarily to RAGE then translocating to the nucleus where it presumably alters gene expression, resulting in increased viability, proliferation, and migration of keratinocytes and fibroblasts. Several studies^4,7 have elucidated a potential role of HMGB1 in wound healing in murine and human models. 

In diabetic murine skin with both reduced HMGB1 levels and altered wound healing, adding HMGB1 increased fibroblast migration and wound closure rates.7 Although these studies^4,7 support the role of HMGB1 as a promoter of wound closure, the role of HMGB1 in scars remains unclear. By increasing the viability, proliferation, and migration of fibroblasts, HMGB1 presumably could contribute as a profibrotic molecule to produce collagen.

Dardenne et al⁸ reported that HMGB1, when applied to early embryonic (before embryonic day 16) murine skin wounds that normally heal with an absence of scar tissue, induced wound healing with scarring and fibrosis. In addition, there was a dose-dependent increase in scar size, fibroblast number, and collagen deposition.⁸ This correlated with enhanced and prolonged release of HMGB1 and healed via scar formation in wounds generated beyond murine embryonic day 16.⁸ However, Zhang et al⁹ concluded that HMGB1 decreases in vitro rat fibroblast collagen synthesis.

The excess production of collagen seen in keloids could result from excess HMGB1 or increased responsiveness of fibroblasts to HMGB1, suggesting similar effects of HMGB1 and transforming growth factor beta (TGF-β) on the induction of keloids. If HMGB1 is elevated in these conditions, the therapeutic potential of mitogen-activated protein kinase kinase inhibitor, box A, glycyrrhizin, the anti-RAGE antibody, and other inhibitors of HMGB1 should be investigated.^4,7

This case control study evaluates the role of HMGB1 in patients with keloids by comparing the serum level in these patients with healthy controls and correlating the level of HMGB1 with scar severity and duration.

Case group
Forty patients of both sexes with keloids of any size were recruited from the outpatient clinic of dermatology at Kasr Al Ainy Hospital (Cairo, Egypt). Patient skin type, according to the Fitzpatrick scale,¹⁰ ranged from 2 to 4; 1 patient had albinism. A written informed consent form was obtained from each patient prior to study inclusion.

Patients with the following criteria were excluded: pregnancy, immunosuppression, long-term systemic corticosteroid therapy, diabetes, renal fibrosis, and history of the use of any treatment for keloids in the 4 weeks prior to the initiation of the study.

A full patient evaluation, including medical history, clinical scar history (duration and etiology), and previous therapy, was completed. Scar grade was determined as per the modified Vancouver scar scale.¹¹ 

Control group
Forty controls of both sexes with a skin type¹⁰ ranging from 2 to 4 were recruited. Exclusion criteria consisted of no keloid scarring and no concurrent therapy trial. Before study inclusion, written informed consent was obtained from each patient. A full patient evaluation, including medical history, was recorded.

Methodology
A 3-mL sample of whole blood was obtained from 40 patients with keloids and 40 controls. The samples were immediately centrifuged; the resultant supernatant serum was kept frozen at -20°C for detection and quantification of HMGB1 using enzyme-linked immunosorbent assay (ELISA).

The Sandwich ELISA method was employed using ELISA kit (SinoGeneClon Biotech Co, Ltd, Hang Zhou, Zhejiang, China). The micro-ELISA plate provided in this kit was precoated with an antibody specific to HMGB1. Samples were added to the appropriate micro-ELISA plate wells and combined with the specific antibody. Then, a biotinylated detection antibody specific for HMGB1 and avidin-horseradish peroxidase (A-HRP) conjugate was successively added to each microplate well and incubated. Free components were washed away. The substrate solution was added to each well. Only those wells that contain HMGB1, biotinylated detection antibody, and A-HRP conjugate appeared blue in color. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution and the color turned yellow. The optical density (OD) was spectrophotometrically measured at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of HMGB1. The concentration of HMGB1 was calculated in the samples by comparing the OD of the samples with the standard curve.

The stability of HMGB1 during ELISA practical work was ensured with the following procedures: incubation in dark at 37°C, wrapping the ELISA plate in aluminum foil, following all ELISA manual instructions, and duplication of each sample to avoid data disparity.

Ethics
Approval for the study was obtained from the Dermatology Research Ethical Committee Post conduction (Kasr Al Ainy Hospital).

Statistical analysis
Results are expressed as mean ± standard deviation or as number (percentage). Comparison between categorical data was performed using chi-square test. According to normality test, comparison between values of different parameters in the 2 patient groups was performed using either unpaired t test or Mann-Whitney U test whenever appropriate. Receiver operating curve (ROC) was used to determine the diagnostic indices of HMGB1. Correlation between HMGB1 and both scar duration and scar severity in the case group was performed using Spearman’s rank correlation coefficient test. Binary logistic regression test was used to study the predictive effect of HMGB1. For data analysis, SPSS for Windows, Version 16 (SPSS Inc, Chicago, IL), was used. A P value ≤ .05 was considered statistically significant.

Forty cases of keloid and 40 controls were enrolled in this study. The mean age of the control group was 32.43 ± 11.93 years (range, 15–66 years) and the case group was 26.81 ± 15.08 years (range, 5–62 years). There was no statistically significant difference in the mean value of age between the control group and case group (P = .069). Eight of the 40 controls were men (20.0%), and 20 patients in the case group were men (50.0%).

There was no statistically significant difference between the 2 groups regarding skin types. For skin type 2, there were 7 (17.5%) and 3 (7.5%) patients in the control and case groups, respectively. There were 19 (47.5%) control group patients and 22 (55.0%) case group patients with skin type 3 and 14 (35.0%) controls and 14 (35.0%) case patients with skin type 4. One (2.5%) patient in the case group had albinism.

There was a statistically significant difference in the mean value of HMGB1 in case patients (74.38 ± 40.16) compared with the mean value in controls (52.00 ± 5.41; P = .001) (Table 1).

There was no statistically significant difference in the mean HMGB1 value of gender between men (51.88 ± 5.79) and women (52.50 ± 3.78) of the control group (P = .774) and between men (79.25 ± 53.69) and women (69.50 ± 19.46) of the case group (P = .720).

The ROC analysis, applied to the HMGB1 level, showed the best diagnostic profile with an area under the curve of 0.898. The best cutoff point was 55 ng/mL. The sensitivity, specificity, and accuracy were 82.5%, 87.5%, 85.0%, respectively (Table 2, Figure 1).

A cutoff of 55 ng/mL had a sensitivity and specificity in detecting HMGB1 elevation of 82.50% and 87.50%, respectively (Table 3).

High-mobility group box 1 protein was found to be a predictor for scar severity (odds ratio [OR] = 1.557; 95% confidence interval [CI], 1.073–2.258; P = .020) but not a predictor for scar duration (OR = 0.980; 95% CI, 0.794–1.209; P = .848) (Table 4, Figure 2).

Keloids have remained a challenge for physicians and a significant quality of life issue for many patients. Keloids cause cosmetic problems as well as pain, pruritis, and contractures.¹² Despite advances over the past century, many patients still experience the negative effects of excessive scarring and presently are without effective treatment.

There are numerous modalities for treating keloids, but all have unsatisfying results for patients and physicians. Intralesional corticosteroid may improve scar pliability, diminish its volume and height, and reduce scar-related itching and pain.¹³ The main problem of this treatment is the high frequency of side effects, up to 63%,¹⁴ such as hypopigmentation, skin atrophy, telangiectasia, ineffectiveness, injection site pain, and osteoporosis secondary to a large injection area.¹⁵ Cryotherapy is another common therapeutic modality with side effects that include permanent hypo- and/or hyperpigmentation, moderate skin atrophy, blistering, and postoperative pain.¹³ Wound ulceration, hyperpigmentation, and pain are potential complications of treatment with fluorouracil.¹⁶ Bleomycin is an uncommon modality for keloids,¹⁷ and imiquimod adverse effects include irritation and hyperpigmentation.¹⁸ The neodymium-doped yttrium aluminum garnet laser (1064 nm), flashlamp-pumped pulsed dye laser (585 nm-595 nm), and carbon dioxide laser have been used frequently in the treatment of keloids¹⁹; however, none of the available laser modalities produced a satisfactory response until 2011.²⁰ Despite the numerous modalities for the treatment of keloids, there is no satisfactory therapeutic regimen because the exact pathogenesis is still unclear. 

High-mobility group box 1 protein has dual functions. As an intracellular transcription factor, HMGB1 binds to bent DNA to promote the assembly of nucleoprotein complexes, which is critical in the process of transcription, recombination, replication, and repair. As an extracellular mediator, HMGB1 acts as a potent inflammatory cytokine.⁴ The release of HMGB1 occurs actively by stimulated monocytes and macrophages and passively by necrotic/damaged cells.⁵ It exerts its effects by binding to cell surface receptors, particularly the receptors for RAGE, TLR2, and TLR4.²¹

Several studies have discussed the potential role of HMGB1 in wound healing, such as increasing the viability, proliferation, and migration of keratinocytes and fibroblasts.^4,7 In addition, adding HMGB1 to diabetic murine skin with reduced HMGB1 levels and altered wound healing increased fibroblast migration and wound closure rates.⁷

The role of HMGB1 in keloids was investigated in the present study. Because this protein is known to have a role in wound healing and is found in low levels in diabetic skin, it was hypothesized that HMGB1 elevation might play a role in keloid pathogenesis. Perhaps this could be a target for developing novel HMGB1 inhibitors for the prevention and/or reversal of keloid formation.

Blood samples were taken from 40 keloid cases and 40 controls to compare the level of HMGB1 in both groups and correlate the level with the severity and duration of keloids. A statistically significant elevation of HMGB1 in the case group compared with the control was found (P = .001), and the level of HMGB1 was found to positively correlate with the severity but not with skin type or duration. To the best of the authors’ knowledge, this is the first study comparing the levels of HMGB1 in patients with keloids and healthy controls.

Considering that HMGB1 plays a role in wound healing and excessive scarring, it was found to be elevated in other fibrotic diseases such as scleroderma, cystic fibrosis, and pulmonary fibrosis.²² Entezari et al²³ reported that inhibiting HMGB1 activity is beneficial to treat fibrotic diseases. Moreover, Lee et al²⁴ suggested HMGB1 promotes wound healing by inducing the proliferation and migration of fibroblasts.

Trauma and tissue damage trigger an inflammatory response, which is required for post injury tissue repair. Inflammation following tissue damage is a dynamic process driven by numerous inflammatory mediators.²⁵ The role of HMGB1 in inflammation as a chemoattractant for inflammatory cells has been defined, but the specific receptor (eg, for RAGE, TLR2, or TLR4) involved in this phenomenon is not clear.⁴

In addition, HMGB1 directly contributes to vessel formation through promoting endothelial cell (EC) proliferation, migration, and sprouting. Besides, HMGB1 may promote the release of proangiogenic cytokines from ECs and macrophages.²⁶

Emerging evidence indicates that HMGB1, a pluripotent mediator, contributes to many fibrotic diseases, and may be a promising therapeutic target for such diseases.²³ Based on these facts, it was suggested that HMGB1 also might play a role in the excessive scarring such as keloids. The excess production of collagen seen in keloids could result from excess HMGB1 or increased responsiveness of fibroblast receptors to HMGB1, suggesting similar effects of HMGB1 and TGF-β on the induction of keloids. This is why the present authors recommend examining the level of these receptors and their sensitivity in future studies. The elevated level of HMGB1, along with the positive correlation in keloid severity, may lead to the possibility of using HMGB1-blocking agents in the treatment of patients in whom other forms of therapy would be impractical. In addition, the HMGB1 protein can be targeted using anti-HMGB1 antibody.²⁷

There is no single mode of delivery for treatment, and further studies are needed to choose the best way to prevent and treat keloids without affecting the role of HMGB1 in transcription. It is possible that intralesional injection of HMGB1-targeted therapies in already formed keloids could help treatment or that intralesional injection in a postoperative wound could prevent further keloid appearance in a patient with a known history of keloid formation. The possibility of systemic delivery of a HMGB1-blocking agent to help individuals with spontaneous keloid formation still needs to be explored. However, future studies should aim to help treatment of excessive scarring with HMGB1 while considering its 2 dramatically opposed functions in the body as a biologically intrinsic requisite factor and as a proinflammatory cytokine. Though inhibiting the proinflammatory effects of HMGB1 is a therapy goal, research should recognize its essential nuclear function.

Limitations of this study include the small sample size and not measuring the receptors of the HMGB1. This should be examined in future studies.

This study hypothesizes that there is a role for HMGB1 in excessive scarring and thus blocking it may prevent and treat severe cases of keloids. Future research should focus on detecting the exact role of HMGB1 in scarring.

Authors: Omar Ahmad Azzam, MD; Marwa Salah El-Mesidy, MD; Moataz Maher Kamel, MD; and Amira Basyouny Nouh, MBBch

Affiliation: Kasr Al Ainy Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence: Marwa Salah El-Mesidy, MD, Cairo University Kasr Alainy Faculty of Medicine, Dermatology, 6 Faculty of Agriculture Street, Cairo, Giza 12211 Egypt; marwa.elmesidy@kasralainy.edu.eg 

Disclosure: The authors disclose no financial or other conflicts of interest.