J Clin Aesthet Dermatol. 2018;11(10):36–39
by Feizollah Niazi, MD; Seyed Hassan Hooshyar, MD; Keshvad Hedayatyanfard, PhD; Seyed Ali Ziai, PhD; Farideh Doroodgar, MD; Sana Niazi, MS; Behnam Habibi, PhD; and Ali Asadirad, PhD
Drs. Niazi, Hooshyar, Doroodgar, and Niazi are with the Department of Plastic and Reconstructive Surgery at Modarres Hospital School of Medicine at Shahid Beheshti University of Medical Sciences in Tehran, Iran. Drs. Hedayatyanfard, Ziai, and Habibi are with the Department of Pharmacology at the School of Medicine, Shahid Beheshti University of Medical Sciences in Tehran, Iran. Dr. Asadirad is with the Department of Immunology at the School of Medicine, Shahid Beheshti University of Medical Sciences in Tehran, Iran.
FUNDING: The study was supported by a grant from the Shahid Beheshti University of Medical Sciences.
DISCLOSURES: The authors have no conflicts of interest relevant to the content of this article.
ABSTRACT: Background.Keloid and hypertrophic scars (HTS) are formed by excessive collagen formation. Angiotensin II, through the AT1 receptor, plays an important role in extracellular matrix production. However, less is known about angiotensin II and AT1 receptor concentrations in HTS and keloid tissues.
Objective. The purpose of this study was to determine the angiotensin II and AT1 receptor concentrations in keloid, HTS, and normal skin tissues.
Methods.Skin biopsy samples from patients with HTS (n=26), keloid (n=20), and normal (n=30) skin tissues were evaluated for angiotensin II and AT1 receptor concentrations by use of the enzyme-linked immunosorbent assay technique.
Results.The angiotensin II concentration in patients with HTS was higher than that in the normal (P<0.0067) and keloid (P>0.9553) groups, while the AT1 receptor concentration in patients with keloid was higher than that in the HTS (P<0.0001) and normal (P<0.0048) groups.
Conclusion. Angiotensin II and AT1 receptor concentrations could stimulate the formation of HTS and keloid. Angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors may be suitable compounds for the treatment of scar tissue.
KEYWORDS: Angiotensin II, AT1, ELISA, hypertrophic scar, keloid
The wound healing process occurs in three phases: inflammation, proliferation, and remodeling.1 Hypertrophic scars (HTS) and keloids are formed by abnormal healing2 and imbalance in the production and destruction of collagen.1 The current treatments for HTS and keloid scar are excision, cryotherapy, intralesional corticosteroid injections, laser therapy, topical applications, and emerging therapies such as interferon and fluorouracil.3 Researchers have suggested that chronic inflammation is a key factor in HTS and keloid formation.2,4 Keloid and HTS differ in terms of histology, predilection site, incidence, appearance, and time course.3 Human skin could completely express the rennin–angiotensin system (RAS) and operate independently.5 Angiotensin II is a peptide that has two major receptors: AT1 and AT2. These receptors act through G-protein-coupled receptor signaling, and their effects in many tissues are opposite to each other.6 During the wound healing process, the expression of angiotensin II is increased.7 Angiotensin II, through the AT1 receptor, induced the expression of proinflammatory chemokines and also increased inflammation responses, vasoconstriction, and oxidative stress.8 Furthermore, AT1 receptor signaling stimulated the migration of keratinocytes and fibroblasts and increased the expression and production of collagen content.9–12 AT1 receptor antagonists additionally inhibits the level of cytokines, the inflammatory pathway gene expression, and collagen deposition.10,11,13–16 In a previous unpublished study conducted by the authors of this paper, it was found that losartan ointment relieved some signs of HTS and keloid (data unpublished). The purpose of the present study was to determine the angiotensin II and AT1 receptor concentrations in keloid, HTS, and normal skin tissues.
Materials and Methods
Ethical statement. This study was approved by the ethics committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran. In addition, written informed consent was obtained from all the participants prior to surgery.
Patient selection. This was an experimental study of 70 volunteers, aged 18 to 60 years, with keloid (n=20; mean±standard deviation [SD]=23.55±8.69), HTS (n=26; mean±SD=25.4 ± 6.88), and normal (n=30; mean±SD=25.72±8.69) skin tissues who were referred to Modarres Hospital in Tehran, Iran between April 2016 and April 2017 (Tables 1–3). The Shahid Beheshti University Research Ethics Committee approved this research, and informed consent was obtained from every patient. The inclusion criteria were ages between 18 and 70 years and presence of a mature scar. Patients with hypertension, pregnancy, radiotherapy, diabetes, chemotherapy, cognitive disabilities, suicide attempt with drugs, addiction, malignancy condition, bleeding, local infection and/or discharge in scar tissue, scar treatment in the last two months, and/or antihypertensive medications were excluded. Biopsies were obtained from the center of the scar sites, and normal skin samples were obtained from healthy skin tissues. All samples were immediately frozen in liquid nitrogen and stored at -86°C for the immunological assay.
Enzyme-linked immunosorbent assay (ELISA). Levels of angiotensin II and AT1 receptor in tissue were measured by using the ELISA technique (angiotensin II and AT1 receptor; Cloud Clone Corp., Houston, Texas). According to the manufacturer’s instructions, 100mg of tissues were mechanically homogenized in 1mL of extraction buffer containing protease inhibitors (Roche Holding AG, Basel, Switzerland). The homogenate was then centrifuged at 13,000g for five minutes at 4°C. The resulting supernatant was used for the quantification of angiotensin II and AT1 receptor levels. The amount of angiotensin II and AT1 receptor per gram of tissue was calculated.
Statistical analysis. The data were summarized in the format of mean±SD and all analyses were performed using the GraphPad Prism software (GraphPad Software, La Jolla, California). P values of <0.05 were considered to be significant. Within-group comparisons were performed by use of Tukey’s multiple comparison test.
The ELISA technique was used to measure the concentration of angiotensin II and AT1 receptor in normal, HTS, and keloid tissues in human skin. One-way analysis of variance indicated there were significant differences between the three groups in terms of angiotensin II and AT1 receptor concentrations (P<0.05). The results indicated that the angiotensin II concentrations in the HTS and keloid groups were significantly higher than that in the normal group (P<0.0067 and P<0.0306; Figure 1). Conversely, although the concentration of angiotensin II in the HTS group was higher than that in the keloid group, this value was not significant (P>0.9553). As presented in Figure 2, the AT1 receptor concentration in the keloid group was significantly higher than that in the HTS (P<0.0001) and normal (P<0.0048) groups. In addition, the AT1 receptor concentration in the normal group was higher than that in the HTS group, but no significant difference between the two groups was seen (P>0.3428).
The purpose of this study was to measure the angiotensin II and AT1 receptor concentrations in keloid, HTS, and normal skin tissues by using the ELISA technique.
Steckelings et al7 found that angiotensin II and AT1 receptor are upregulated in human cutaneous wounds. The RAS is completely expressed in human skin and AT1 receptor signaling increased gene expression and collagen production, whereas AT1 antagonists inhibited these pathways.5,10–12,17 Furthermore, the angiotensin-converting enzyme (ACE) activity in wounded skin and scar tissue was higher than that in normal skin.18 Angiotensin II could play an important role in the inflammatory process.8 One of the most important cytokines in the inflammatory process and in fibrosis production is transforming growth factor beta (TGF-beta).16–19 The current literature has shown that angiotensin II increases the level of TGF-beta.11, 17 Some studies have also suggested that ACE inhibitors and AT1 receptor antagonists reduce TGF-beta levels.11–14,17,20,21–23 In addition, angiotensin II increased some factors involved in collagen production and extracellular matrix formation. A number of studies have demonstrated that angiotensin II increased the connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1.12–17 Colwell et al25 presented that CTGF inhibition could reduce scar formation.The level of vessels in keloid and HTS were higher than in normal skin tissue and this was a key factor in the development of scar, whereas the AT1 receptor antagonist could reduce the angiogenesis.6,26,27 Some studies revealed that using ACE inhibitors such as captopril and quinapril reduced the scar and keloid.28, 29 Ardekani et al28 reported that captopril cream was able to reduce the keloid in an 18-year-old girl. Khalil et al14 showed that using losartan could reduce renal cortical scarring in mice. Criss et al30 illustrated that the AT1 receptor antagonist could delay the rate of wound healing in a rat model. The previous unpublished study conducted by the authors showed that using losartan ointment on volunteers with keloid and HTS leads to a decrease in vascularity, pigmentation, pliability, and height (unpublished data).
The authors believe that angiotensin II and AT1 receptor could stimulate the formation of HTS and keloid. Angiotensin II receptor blockers and ACE inhibitors could potentially be used in the treatment of HTS and keloid.
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