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Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery
Cryobiology xxx (2017) 1e3
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery Sara M. Awad, MD a, *, 1, Sahar A. Ismail, MD a, 1, Doaa S. Sayed, MD a, Abeer E. Refaiy, MD b, Rania Makboul, MD b a b
Department of Dermatology, Faculty of Medicine, Assiut University, Assiut, Egypt Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2016 Received in revised form 2 March 2017 Accepted 9 March 2017 Available online xxx
The biological mechanism underlying cryosurgical treatment of keloids remains unclear. Transforming growth factor-beta1 (TGF-b1) has been implicated in the pathogenesis of keloids and was reported to be the target of several therapeutic modalities. However, the effect of cryosurgery on its expression in keloid tissue has not been yet investigated. In this study, 26 consecutive keloid patients were treated with cryosurgery for 2e6 sessions. Keloids were biopsied before starting cryosurgery and after two treatment sessions for the immunohistochemical evaluation of TGF-b1 expression. The average volume reduction, after two treatment sessions (in 22 patients completing the study) was 68.77 ± 15.82%. Dermal overexpression of TGF-b1 was demonstrated in all keloid specimens before treatment. Following therapy, significant reduction of that expression was detected in all keloid specimens (P ¼ 0.016). In addition to attesting the clinical efficacy of cryosurgery, our findings indicate that cryosurgery effectively suppressed TGF-b1 expression, possibly contributing to keloid regression. © 2017 Elsevier Inc. All rights reserved.
Keywords: Cryosurgery Keloid Transforming growth factor-beta1
Keloid formation is a result of aberrant dermal fibroblast activity, exacerbated by several exogenous inflammatory and woundhealing signals leading to abnormal deposition of extracellular matrix components, particularly collagen. Transforming growth factor beta-1 (TGF-b1) has been implicated in the pathogenesis of keloids based on its over-expression in keloid fibroblasts (KFs) compared to normal fibroblasts, which is believed to stimulate collagen and fibronectin synthesis by fibroblasts [6,7]. Therefore, suppressing TGF-b1 in keloid tissue may be a potential mechanism for its involution. Cryosurgery, both contact and intralesional, has been proven to yield significant improvement of keloids [1,5,10]. In the skin, melanocytes die at - 4 to 7 C, while fibroblasts are rather resistant to cold being destroyed at - 30 to - 35 C. Previous studies have demonstrated uniform freezing during cryosurgical treatment at an end temperature, which was lethal for the fibroblasts i.e. - 30 C [5].
Abbreviations: TGF-b1, Transforming growth factor-beta1; KFs, keloid fibroblasts. * Corresponding author. Department of Dermatology, Assiut university hospital, Assiut University, 71526 Assiut, Egypt. E-mail address: [email protected] (S.M. Awad). 1 The first two authors contributed equally to the work.
However, the biological mechanism by which cryosurgery induces keloid regression is not well-established. Cryosurgery has been claimed to modify collagen synthesis and fibroblast differentiation toward a normal phenotype [4]. A tendency toward normalization of the keloid collagen structure as well as the presence of young normal collagen fibers was observed after cryosurgery [5]. Recently, it was also reported to inhibit fibroblast proliferation, suppress vascular endothelial growth factor production and reduce tenascin C synthesis in keloids [1]. However, the effect of cryosurgery on TGF-b1 has not been investigated. In the present study, we investigated whether the aberrant expression of TGF-b1 found in keloids can be modulated by cryosurgery. Twenty-six consecutive keloid patients, attending the outpatient clinic of Dermatology Department at Assiut University Hospital during the period from May 2014 to August 2015, were enrolled in the study. They were 2 females and 24 males with their ages ranging from 18 to 31 years old. The study protocol was approved by the ethics committee of Assiut Faculty of Medicine and the study has been conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients before the start of treatment. Hypertrophic scars as well as scars in pregnant females and in
http://dx.doi.org/10.1016/j.cryobiol.2017.03.004 0011-2240/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004
2
S.M. Awad et al. / Cryobiology xxx (2017) 1e3
patients less than 12 years of age were excluded. In patients having more than one keloid, one lesion per patient was randomly chosen for treatment and evaluation. A period of at least 1 month was allowed between previous therapy/therapies and cryosurgery. Cryosurgery was performed using the handheld device (Brymill cryogenic system, CRYeAC, Ellington, CT, USA). Contact cryosurgery was performed for small lesions (19 patients) using a flat cryoprobe, 1 cm in diameter with a single freezing cycle (30e40 s). Intralesional cryosurgery was done for larger keloids (7 patients) using sterile 16egauge curved needles through which liquid nitrogen was sprayed until complete freezing of the keloid. Only 22 patients (84.6%) completed the study and 4 patients failed to show up after the second session. Baseline evaluation included site and duration of keloid, preceding injury, and associated symptoms. Keloid volume was assessed at baseline and at every session using skin fold caliper to determine the greatest length, width and height. Clinical improvement was defined as the percentage of volume reduction and graded into: Excellent improvement (>75%), Good improvement (50e75%) and Poor improvement (<50%). Photographs were taken at baseline and at each visit. Keloids were biopsied using 3 mm-punch from the central part of the lesion at baseline and 3 weeks after the second treatment session. Twenty normal skin specimens were examined as control (the pathological interpretation was performed blindly by 2 independent pathologists). Four mm sections of formalin-fixed, paraffin-embedded specimens were cut, deparaffinized and rehydrated. After blocking of endogenous peroxidase, antigen retrieval was done by citrate puffer for 8 min. The slides were then incubated for 1 h with TGFb1 (Clone 7F6, mouse monoclonal antibody, Novus Biologicals, USA) at a dilution of 1:100 and then incubated for 15 min with the one step poly HRP (Genemed Biotechnologies, South San Francisco, CA, USA). Diaminibenzidine was applied for 5 min, and then slides were counterstained with Mayer's hematoxylin. Sections from benign prostatic hyperplasia were stained as a positive control. Stained slides were examined and H score was generated for each slide by multiplying the extent expression (percentage of positive cells) by the intensity (0, 1, 2, and 3) and used for statistical analyses. SPSS system for Windows version 16.0 (SPSS Inc., Chicago, IL, U.S.A.) was used for statistical analysis. Data were expressed as mean ± SD for continuous variables and as number and percentage for categorical variables. Wilcoxon-signed rank test was used to compare differences in continuous variables at baseline and after treatment. Correlations among various parameters were tested by calculating Spearman's correlation coefficient. A p-value <0.05 was considered significant. Of the 26 patients enrolled, only 22 completed the study. Before treatment, biopsies were carried out for all 26 patients while posttreatment biopsies were performed in only 22 patients who continued treatment. All 22 treated keloids responded favorably to treatment in terms of reduction of keloid volume. After two sessions of treatment, an average volume reduction of 68.77 ± 15.82% was achieved. At the end of treatment, volume reduction was 89.29 ± 13.5%. Excellent clinical response was observed in 18 patients (81.8%); of these 12 showed complete resolution of their keloids (Table 1). After 2 treatment sessions, the mean keloid volume was reduced to 0.22 ± 0.34 cm3 compared to an initial volume of 0.73 ± 0.95 cm3 before treatment (P ¼ 0.001) and reached 0.06 ± 0.12 cm3 after completion of treatment sessions (P ¼ 0.001). The number of sessions ranged from 2 to 6. Moreover, improvement of subjective symptoms (pain and itching) was obtained in most of the patients (17/22).
Table 1 Clinical response to cryosurgery. Clinical responsea
n (%)
Excellent (>75e100%) Complete (100%) Good (50e75%) Poor (<50%)
18 (81.8) 12 (54.6) 4 (18.2) 0
a
Defined as percentage of volume reduction.
Initially, TGF-b1 was expressed in the dermis of all 26 cases. The dermal expression was strong in 7 patients (26.9%) and moderate in 19 patients (73.1%). The pattern of TGF-b1 expression was cytoplasmic and the expression was detected mainly in dermal fibroblasts and extracellular matrix. No expression was detected in endothelial cells (Fig. 1a). No statistically significant correlation was found between baseline TGF-b1 expression and keloids' duration or keloids' volume (r ¼ 0.217, P ¼ 0.287 and r ¼ 0.121, P ¼ 0.557 respectively). Almost all samples of normal skin (19/20), showed complete absence of TGF-b1 expression (Fig. 1b). Following therapy, statistically significant reduction in the extent of TGF-b1 dermal expression (P ¼ 0.013) was noted in all specimens of the 22 patients (Fig. 1c). The mean H-score of TGF-b1 expression was 180.40 ± 66.17 before treatment and decreased to 118.67 ± 70.19 after two treatment sessions (P ¼ 0.016) (Table 2). No statistically significant correlation was found between the magnitude of reduction of TGF-b1 expression following therapy and keloids' duration, volume or volume reduction percentage (r ¼ 0.097, P ¼ 0.637; r ¼ 0.020, P ¼ 0.946 and r ¼ 0.199, P ¼ 0.476 respectively). The over expression of TGF-b1 in keloid tissue, reported in this study is supported by several previous studies that confirmed its key role in the pathogenesis of the keloids as fibrosis-inducing cytokine [6,7]. Cryosurgery with liquid nitrogen has been proven to yield significant improvement up to total regression of keloids [1,5,10]. This was supported by our findings; where favorable results were achieved both in terms of volume reduction and alleviation of associated symptoms of pain and pruritus. In addition, complete keloid eradication was shown in 12/22 patients. Along with clinical improvement, our current novel data revealed the significant reduction of dermal TGF-b1 expression after cryosurgery which was detected in all keloid specimens of the 22 patients who completed the study. These results suggest that cryosurgery exhibits a suppressive effect on TGF-b1 expression in keloids. This could be the reason behind the reduced fibroblast proliferation and the decreased deposition of collagen observed after cryosurgery in keloids. Some studies have investigated the modulation of TGF-b1 production by other keloid therapies. Kontochristopoulos et al. [8] demonstrated a slight insignificant reduction of TGF-b1 expression after treatment with intralesional 5- fluorouracil. While some in vitro studies have demonstrated that corticosteroids decrease the production of TGF-b1 by human dermal fibroblasts [2], other studies reported that dexamethasone did not significantly reduce the expression of TGF-b1 in cultured-KFs [9]. It is possible that the therapeutic effect of these modalities may not be primarily mediated through the TGF-b1 pathway. On the other hand, Chen et al. [3] reported that captopril is able to inhibit TGF-b1 expression in keloids, and thereby inhibit fibroblast proliferation and collagen synthesis. These findings suggest that TGF-b1 inhibition may represent a therapeutic potential strategy for keloids. In conclusion, our results suggest that cryosurgery was able to reduce TGF-b1 expression in keloid tissue. Modulation of TGF-b1 production might be at least in part responsible for the
Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004
S.M. Awad et al. / Cryobiology xxx (2017) 1e3
3
Fig. 1. Immunohistochemical expression of Transforming Growth Factor-beta1. (a): extensive dermal expression of TGF-b1 in dermal fibroblasts and extracellular matrix with negative endothelial cells expression in keloid before treatment (X400). (b): no expression in skin control biopsy and (c): marked decrease in dermal TGF-b1 expression in keloid after cryosurgery (X400).
Table 2 Comparison of Transforming growth factor-beta1 expression before and after treatment. Parameter (mean ± SD)
Before treatment
After treatment
P -Value
TGF-b1 extent (%) TGF-b1 H-score
77.87 ± 16.05 180.40 ± 66.17
55.33 ± 21.67 118.67 ± 70.19
P ¼ 0.013* P ¼ 0.016*
Wilcoxon Signed Ranks Test * Statistical significant difference (P < 0.05). Data expressed as mean ± standard deviation.
normalization of collagen structure and reduced fibroblast proliferation after cryosurgery, which might in turn contribute to the superior efficacy of cryosurgery in inducing keloid scar regression compared to other modalities. This preliminary immunohistochemical study, although only from twenty-two patients (relatively small number of patients), has provided a scientific basis for the destructive effect that cryosurgery has on keloid tissue, and thus yields significant clinical improvement of keloids. Acknowledgements The authors are grateful to all paramedical personnel and technicians who aided in this research. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cryobiol.2017.03.004. Conflicts of interest None declared.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1] A.M. Abdel-Meguid, A.H. Weshahy, D.S. Sayed, A.E. Refaiy, S.M. Awad, Intralesional vs. contact cryosurgery in treatment of keloids: a clinical and immunohistochemical study, Int. J. Dermatol 54 (2015) 468e475. [2] L.A. Carroll, M.M. Hanasono, A.A. Mikulec, M. Kita, R.J. Koch, Triamcinolone stimulates bFGF production and inhibits TGF-beta1 production by human dermal fibroblasts, Dermatol. Surg. 28 (2002) 704e709. [3] J. Chen, S. Zhao, Y. Liu, Y. Cen, C. Nicolas, Effect of captopril on collagen metabolisms in keloid fibroblast cells, Aust. N. Z. J. Surg. (2014), http://dx.doi.org/ 10.1111/ans.12670. [4] A. Dalkowski, S. Fimmel, C. Beutler, C.C. Zouboulis, Cryotherapy modifies synthetic activity and differentiation of keloidal fibroblasts in vitro, Exp. Dermatol 12 (2003) 673e681. [5] Y. Har-Shai, E. Sabo, E. Rohde, M. Hyams, C. Assaf, C.C. Zouboulis, Intralesional cryosurgery enhances the involution of recalcitrant auricular keloids: a new clinical approach supported by experimental studies, Wound Repair Regen. 14 (2006) 18e27. [6] S. He, X. Liu, Y. Yang, W. Huang, S. Xu, S. Yang, X. Zhang, M.S. Roberts, Mechanisms of transforming growth factor beta (1)/Smad signaling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts, Br. J. Dermatol 162 (2010) 538e546. [7] J. Jagadeesan, A. Bayat, Transforming growth factor beta (TGF beta) and keloid disease, Int. J. Surg. 5 (2007) 278e285. [8] G. Kontochristopoulos, C. Stefanaki, A. Panagiotopoulos, K. Stefanaki, T. Argyrakos, A. Petridis, A. Katsambas, Intralesional 5-fluorouracil in the treatment of keloids: an open clinical and histopathologic study, J. Am. Acad. Dermatol 52 (2005) 474e479. [9] W.S. Wu, F.S. Wang, K.D. Yang, C.C. Huang, Y.R. Kuo, Dexamethasone induction of keloid regression through effective suppression of VEGF expression and keloid fibroblast proliferation, J. Invest. Dermatol 126 (2006) 1264e1271. [10] C.C. Zouboulis, E. Zouridaki, A. Rosenberger, A. Dalkowski, Current developments and uses of cryosurgery in the treatment of keloids and hypertrophic scars, Wound Repair Regen. 10 (2002) 98e102.
Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery Sara M. Awad, MD a, *, 1, Sahar A. Ismail, MD a, 1, Doaa S. Sayed, MD a, Abeer E. Refaiy, MD b, Rania Makboul, MD b a b
Department of Dermatology, Faculty of Medicine, Assiut University, Assiut, Egypt Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2016 Received in revised form 2 March 2017 Accepted 9 March 2017 Available online xxx
The biological mechanism underlying cryosurgical treatment of keloids remains unclear. Transforming growth factor-beta1 (TGF-b1) has been implicated in the pathogenesis of keloids and was reported to be the target of several therapeutic modalities. However, the effect of cryosurgery on its expression in keloid tissue has not been yet investigated. In this study, 26 consecutive keloid patients were treated with cryosurgery for 2e6 sessions. Keloids were biopsied before starting cryosurgery and after two treatment sessions for the immunohistochemical evaluation of TGF-b1 expression. The average volume reduction, after two treatment sessions (in 22 patients completing the study) was 68.77 ± 15.82%. Dermal overexpression of TGF-b1 was demonstrated in all keloid specimens before treatment. Following therapy, significant reduction of that expression was detected in all keloid specimens (P ¼ 0.016). In addition to attesting the clinical efficacy of cryosurgery, our findings indicate that cryosurgery effectively suppressed TGF-b1 expression, possibly contributing to keloid regression. © 2017 Elsevier Inc. All rights reserved.
Keywords: Cryosurgery Keloid Transforming growth factor-beta1
Keloid formation is a result of aberrant dermal fibroblast activity, exacerbated by several exogenous inflammatory and woundhealing signals leading to abnormal deposition of extracellular matrix components, particularly collagen. Transforming growth factor beta-1 (TGF-b1) has been implicated in the pathogenesis of keloids based on its over-expression in keloid fibroblasts (KFs) compared to normal fibroblasts, which is believed to stimulate collagen and fibronectin synthesis by fibroblasts [6,7]. Therefore, suppressing TGF-b1 in keloid tissue may be a potential mechanism for its involution. Cryosurgery, both contact and intralesional, has been proven to yield significant improvement of keloids [1,5,10]. In the skin, melanocytes die at - 4 to 7 C, while fibroblasts are rather resistant to cold being destroyed at - 30 to - 35 C. Previous studies have demonstrated uniform freezing during cryosurgical treatment at an end temperature, which was lethal for the fibroblasts i.e. - 30 C [5].
Abbreviations: TGF-b1, Transforming growth factor-beta1; KFs, keloid fibroblasts. * Corresponding author. Department of Dermatology, Assiut university hospital, Assiut University, 71526 Assiut, Egypt. E-mail address: [email protected] (S.M. Awad). 1 The first two authors contributed equally to the work.
However, the biological mechanism by which cryosurgery induces keloid regression is not well-established. Cryosurgery has been claimed to modify collagen synthesis and fibroblast differentiation toward a normal phenotype [4]. A tendency toward normalization of the keloid collagen structure as well as the presence of young normal collagen fibers was observed after cryosurgery [5]. Recently, it was also reported to inhibit fibroblast proliferation, suppress vascular endothelial growth factor production and reduce tenascin C synthesis in keloids [1]. However, the effect of cryosurgery on TGF-b1 has not been investigated. In the present study, we investigated whether the aberrant expression of TGF-b1 found in keloids can be modulated by cryosurgery. Twenty-six consecutive keloid patients, attending the outpatient clinic of Dermatology Department at Assiut University Hospital during the period from May 2014 to August 2015, were enrolled in the study. They were 2 females and 24 males with their ages ranging from 18 to 31 years old. The study protocol was approved by the ethics committee of Assiut Faculty of Medicine and the study has been conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients before the start of treatment. Hypertrophic scars as well as scars in pregnant females and in
http://dx.doi.org/10.1016/j.cryobiol.2017.03.004 0011-2240/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004
2
S.M. Awad et al. / Cryobiology xxx (2017) 1e3
patients less than 12 years of age were excluded. In patients having more than one keloid, one lesion per patient was randomly chosen for treatment and evaluation. A period of at least 1 month was allowed between previous therapy/therapies and cryosurgery. Cryosurgery was performed using the handheld device (Brymill cryogenic system, CRYeAC, Ellington, CT, USA). Contact cryosurgery was performed for small lesions (19 patients) using a flat cryoprobe, 1 cm in diameter with a single freezing cycle (30e40 s). Intralesional cryosurgery was done for larger keloids (7 patients) using sterile 16egauge curved needles through which liquid nitrogen was sprayed until complete freezing of the keloid. Only 22 patients (84.6%) completed the study and 4 patients failed to show up after the second session. Baseline evaluation included site and duration of keloid, preceding injury, and associated symptoms. Keloid volume was assessed at baseline and at every session using skin fold caliper to determine the greatest length, width and height. Clinical improvement was defined as the percentage of volume reduction and graded into: Excellent improvement (>75%), Good improvement (50e75%) and Poor improvement (<50%). Photographs were taken at baseline and at each visit. Keloids were biopsied using 3 mm-punch from the central part of the lesion at baseline and 3 weeks after the second treatment session. Twenty normal skin specimens were examined as control (the pathological interpretation was performed blindly by 2 independent pathologists). Four mm sections of formalin-fixed, paraffin-embedded specimens were cut, deparaffinized and rehydrated. After blocking of endogenous peroxidase, antigen retrieval was done by citrate puffer for 8 min. The slides were then incubated for 1 h with TGFb1 (Clone 7F6, mouse monoclonal antibody, Novus Biologicals, USA) at a dilution of 1:100 and then incubated for 15 min with the one step poly HRP (Genemed Biotechnologies, South San Francisco, CA, USA). Diaminibenzidine was applied for 5 min, and then slides were counterstained with Mayer's hematoxylin. Sections from benign prostatic hyperplasia were stained as a positive control. Stained slides were examined and H score was generated for each slide by multiplying the extent expression (percentage of positive cells) by the intensity (0, 1, 2, and 3) and used for statistical analyses. SPSS system for Windows version 16.0 (SPSS Inc., Chicago, IL, U.S.A.) was used for statistical analysis. Data were expressed as mean ± SD for continuous variables and as number and percentage for categorical variables. Wilcoxon-signed rank test was used to compare differences in continuous variables at baseline and after treatment. Correlations among various parameters were tested by calculating Spearman's correlation coefficient. A p-value <0.05 was considered significant. Of the 26 patients enrolled, only 22 completed the study. Before treatment, biopsies were carried out for all 26 patients while posttreatment biopsies were performed in only 22 patients who continued treatment. All 22 treated keloids responded favorably to treatment in terms of reduction of keloid volume. After two sessions of treatment, an average volume reduction of 68.77 ± 15.82% was achieved. At the end of treatment, volume reduction was 89.29 ± 13.5%. Excellent clinical response was observed in 18 patients (81.8%); of these 12 showed complete resolution of their keloids (Table 1). After 2 treatment sessions, the mean keloid volume was reduced to 0.22 ± 0.34 cm3 compared to an initial volume of 0.73 ± 0.95 cm3 before treatment (P ¼ 0.001) and reached 0.06 ± 0.12 cm3 after completion of treatment sessions (P ¼ 0.001). The number of sessions ranged from 2 to 6. Moreover, improvement of subjective symptoms (pain and itching) was obtained in most of the patients (17/22).
Table 1 Clinical response to cryosurgery. Clinical responsea
n (%)
Excellent (>75e100%) Complete (100%) Good (50e75%) Poor (<50%)
18 (81.8) 12 (54.6) 4 (18.2) 0
a
Defined as percentage of volume reduction.
Initially, TGF-b1 was expressed in the dermis of all 26 cases. The dermal expression was strong in 7 patients (26.9%) and moderate in 19 patients (73.1%). The pattern of TGF-b1 expression was cytoplasmic and the expression was detected mainly in dermal fibroblasts and extracellular matrix. No expression was detected in endothelial cells (Fig. 1a). No statistically significant correlation was found between baseline TGF-b1 expression and keloids' duration or keloids' volume (r ¼ 0.217, P ¼ 0.287 and r ¼ 0.121, P ¼ 0.557 respectively). Almost all samples of normal skin (19/20), showed complete absence of TGF-b1 expression (Fig. 1b). Following therapy, statistically significant reduction in the extent of TGF-b1 dermal expression (P ¼ 0.013) was noted in all specimens of the 22 patients (Fig. 1c). The mean H-score of TGF-b1 expression was 180.40 ± 66.17 before treatment and decreased to 118.67 ± 70.19 after two treatment sessions (P ¼ 0.016) (Table 2). No statistically significant correlation was found between the magnitude of reduction of TGF-b1 expression following therapy and keloids' duration, volume or volume reduction percentage (r ¼ 0.097, P ¼ 0.637; r ¼ 0.020, P ¼ 0.946 and r ¼ 0.199, P ¼ 0.476 respectively). The over expression of TGF-b1 in keloid tissue, reported in this study is supported by several previous studies that confirmed its key role in the pathogenesis of the keloids as fibrosis-inducing cytokine [6,7]. Cryosurgery with liquid nitrogen has been proven to yield significant improvement up to total regression of keloids [1,5,10]. This was supported by our findings; where favorable results were achieved both in terms of volume reduction and alleviation of associated symptoms of pain and pruritus. In addition, complete keloid eradication was shown in 12/22 patients. Along with clinical improvement, our current novel data revealed the significant reduction of dermal TGF-b1 expression after cryosurgery which was detected in all keloid specimens of the 22 patients who completed the study. These results suggest that cryosurgery exhibits a suppressive effect on TGF-b1 expression in keloids. This could be the reason behind the reduced fibroblast proliferation and the decreased deposition of collagen observed after cryosurgery in keloids. Some studies have investigated the modulation of TGF-b1 production by other keloid therapies. Kontochristopoulos et al. [8] demonstrated a slight insignificant reduction of TGF-b1 expression after treatment with intralesional 5- fluorouracil. While some in vitro studies have demonstrated that corticosteroids decrease the production of TGF-b1 by human dermal fibroblasts [2], other studies reported that dexamethasone did not significantly reduce the expression of TGF-b1 in cultured-KFs [9]. It is possible that the therapeutic effect of these modalities may not be primarily mediated through the TGF-b1 pathway. On the other hand, Chen et al. [3] reported that captopril is able to inhibit TGF-b1 expression in keloids, and thereby inhibit fibroblast proliferation and collagen synthesis. These findings suggest that TGF-b1 inhibition may represent a therapeutic potential strategy for keloids. In conclusion, our results suggest that cryosurgery was able to reduce TGF-b1 expression in keloid tissue. Modulation of TGF-b1 production might be at least in part responsible for the
Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004
S.M. Awad et al. / Cryobiology xxx (2017) 1e3
3
Fig. 1. Immunohistochemical expression of Transforming Growth Factor-beta1. (a): extensive dermal expression of TGF-b1 in dermal fibroblasts and extracellular matrix with negative endothelial cells expression in keloid before treatment (X400). (b): no expression in skin control biopsy and (c): marked decrease in dermal TGF-b1 expression in keloid after cryosurgery (X400).
Table 2 Comparison of Transforming growth factor-beta1 expression before and after treatment. Parameter (mean ± SD)
Before treatment
After treatment
P -Value
TGF-b1 extent (%) TGF-b1 H-score
77.87 ± 16.05 180.40 ± 66.17
55.33 ± 21.67 118.67 ± 70.19
P ¼ 0.013* P ¼ 0.016*
Wilcoxon Signed Ranks Test * Statistical significant difference (P < 0.05). Data expressed as mean ± standard deviation.
normalization of collagen structure and reduced fibroblast proliferation after cryosurgery, which might in turn contribute to the superior efficacy of cryosurgery in inducing keloid scar regression compared to other modalities. This preliminary immunohistochemical study, although only from twenty-two patients (relatively small number of patients), has provided a scientific basis for the destructive effect that cryosurgery has on keloid tissue, and thus yields significant clinical improvement of keloids. Acknowledgements The authors are grateful to all paramedical personnel and technicians who aided in this research. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cryobiol.2017.03.004. Conflicts of interest None declared.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Please cite this article in press as: S.M. Awad, et al., Suppression of transforming growth factor-beta1 expression in keloids after cryosurgery, Cryobiology (2017), http://dx.doi.org/10.1016/j.cryobiol.2017.03.004