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Expressions of stemness markers in keloid tissue

Authors:
Sri Suciati Ningsih at Universitas Muhammadiyah Prof. Dr. Hamka
Sri Suciati Ningsih
Dewi H. Sari
Dewi H. Sari
Dewi H. Sari
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Radiana Dhewayani Antarianto at University of Indonesia
Radiana Dhewayani Antarianto
Novi Silvia Hardiany at University of Indonesia
Novi Silvia Hardiany
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Abstract

strong>Background: Keloid is an abnormal wound healing process that extends beyond the site of injury. Keloid and tumor’s shared similarity of recurrence suggesting a shared underlying mechanism that involves stemness. Octamer-binding transcription factor-4 (Oct-4) and aldehyde dehydrogenase-1 (ALDH1) are stem cell stemness markers. This study aimed to analyze Oct-4 and ALDH1 expressions in keloid tissues. Methods: Samples were obtained from keloid tissue excisions from three keloid patients and post-circumcision preputial skin from three healthy donors (normal control) in accordance with the local ethical committee regulation. Total RNA was isolated using TriPure Isolation kit (Ameritech), and expressions of Oct4 and ALDH1 mRNA in keloid and preputial skin were determined by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) using Livak method. Results: The qRT-PCR analysis revealed the expressions of Oct4 and ALDH1 in keloid and preputial skin tissues. Keloid tissues exhibited lower expression levels of Oct-4 and ALDH1 than the preputial skin. The difference was statistically insignificant. Conclusion: Keloid tissues express Oct-4 and ALDH1 as stemness markers, and the stemness characteristics of keloid might be similar to a normal skin.
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Ningsih, et al.
Stemness markers in keloid 145
Expressions of stemness markers in keloid tissue
145–9
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     
            

1133
33
1 
 
3                

ABSTRACT
Background:       
            


Methods:
    


Results: 
               
Conclusion:

Medical Journal of Indonesia
Basic Medical Research
Sri W.A. Jusman

Keywords:
Med J Indones
Vol. 27, No. 3, September 2018
146
     
     
          
     
     
    
    
    

     
     
      
    
  
    3  
  

      
      

   

    
       
 
      

     

      

4   
  
      

   5   

       

  
      



      

      
    
       
      

METHODS
    
     
    
     
    

         


         
       
     
     


RNA isolation from keloid tissue and preputial
skin
    
      
      
 

       

     









mRNA expression analysis of Oct4 and ALDH1
by qRT-PCR (Livak method)
    
      

     
    
       
       
 

     



Ningsih, et al.
Stemness markers in keloid 147
 
       
    
    

RESULTS
Relative expression of Oct-4 mRNA
     
     

Relative expression of ALDH1 mRNA
     
 
      

DISCUSSION
      
 

      
     

     
6  
7
      
        
      13
 

14
15
    16 

Table 1.
       

Figure 1.   
      


 
         

 
      
       


      

Figure 2.   
      

 
18S  
  
  
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Preputial skin Keloid
mRNA relative expression of Oct -4
(fold)
1.26
0.60
2.06
0.44
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Preputium Keloid
mRNA relative expression of
ALDH1 (fold)
Medical Journal of Indonesia
Med J Indones
Vol. 27, No. 3, September 2018
148
     
      
17
 
  
     
1819 


    
     
      
    
       

     
         


       
     
      
   
     
       

       
      
     


 


    



      
      

      
  



     
      
  


       
       

Acknowledgment
   
     
     


REFERENCES
         
       
   

              
    
       
        

 

     

   


 
   

 
      


 
   
      

  


 

    

         
      
      
   

          
     
    


        

Ningsih, et al.
Stemness markers in keloid 149
  
         

 



  
       
     

              
   
      

16          
     
      

 

      

    
      
      

 
         

Medical Journal of Indonesia
... Keloid therapy is both complicated and problematic. Until recently, surgical procedure, intralesional steroid (2), compressing silicone gel pads, and radiotherapy (3) have been used in treatment modalities, as having extra experimental treatments like bleomycin, interferons, and 5-fluorouracil (5-FU) (4,5). ...
Assessment of various intralesional injections in keloid: Comparative analysis
Article
Full-text available
Aim: Intralesional triamcinolone is a gold standard in treating the keloids to comparation its effectiveness versus intralesional 5-fluorouracil intralesional verapamil and intralesional platelet-rich plasma. Patients and methods: Several 160-cases were categorized into four groups of each Group-Containing 40 cases. Group-A (control) treated with intralesional triamcinolone and Group-B intralesional verapamil, Group-C intralesional 5-fluorouracil, and Group-D intralesional platelet-rich plasma. Patients were assessed for clinical response based on a decrease in the patient and observer scar assessment scale (POSAS) at baseline and the end of treatment. Results: The mean base-line POSAS score was 91 ± 10.98 SD check-in Group-A, 90 ± 10.85 in Group-B, 89 ± 10.06 in Group-C, and 92 ± 10.84 in Group-D.POSAS score after 24 weeks 36 ± 12.74 in Group-A, 29 ± 10.91 in Group-B, 39 ± 13.74 in Group-C, 36 ± 12.74 in Group-D. Statistically, a significant difference was observed between groups. Conclusion: Intralesional verapamil reported to be the most effective therapy and platelet-rich plasma was effective as intralesional triamcinolone acetonide with no serious side effects and 5-fluorouracil was less effective in treating the keloids.
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... Skin trauma is inevitable, and while some wounds may heal normally, some undergo abnormal wound healing which result in hypertrophic scars or keloids. 1 Although both are due to excessive deposition of collagen in the skin, however there are several distinctive characteristics. 2 Hypertrophic scars do not grow beyond the boundary of wound site while keloids grow and extend into the surrounding normal skin without any spontaneous regression. 1,3 A definitive diagnosis is made through histopathological examination where keloids present with dermal nodules and multiple thick eosinophilic collagen bundles. 4 This is in contrary to associated with keloid aggravation. ...
Evaluating the efficacy of intralesional verapamil over intralesional triamcinolone in management of keloid: an evidence based case report
Article
Full-text available
  • Dec 2019
Author present a case of 22-year-old female with keloid due to previous trauma three years prior. Keloids are excessive fibroblast growth present in pathological scars. Therapy for keloids still remain a challenge requiring an effective intervention. While the first line has always been the use of intralesional triamcinolone, recently intralesional verapamil has also been known to reduce growth of keloids. Aim of the study was to evaluate the efficacy of both of these drug options. Literature searching was performed from three databases namely PubMed, Cochrane library and Science Direct. Findings were systematically narrowed down through inclusion and exclusion criteria into four relevant randomized controlled trials. Selected studies were critically appraised for its validity, importance, and applicability using tools from Oxford Center of Evidence-Based Medicine. Both intralesional triamcinolone and verapamil show their own benefit and risk. Triamcinolone is more effective in reducing keloid with faster improvement as seen in scar height reduction, vascularity, pigmentation and pliability. However, verapamil has fewer side effects which serve as a safer treatment option. More clinical trials in the future may be needed to obtain more conclusive result.
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Site-specific gene expression profiling as a novel strategy for unravelling keloid disease pathobiology
Article
Full-text available
Keloid disease (KD) is a fibroproliferative cutaneous tumour characterised by heterogeneity, excess collagen deposition and aggressive local invasion. Lack of a validated animal model and resistance to a multitude of current therapies has resulted in unsatisfactory clinical outcomes of KD management. In order to address KD from a new perspective, we applied for the first time a site-specific in situ microdissection and gene expression profiling approach, through combined laser capture microdissection and transcriptomic array. The aim here was to analyse the utility of this approach compared with established methods of investigation, including whole tissue biopsy and monolayer cell culture techniques. This study was designed to approach KD from a hypothesis-free and compartment-specific angle, using state-of-the-art microdissection and gene expression profiling technology. We sought to characterise expression differences between specific keloid lesional sites and elucidate potential contributions of significantly dysregulated genes to mechanisms underlying keloid pathobiology, thus informing future explorative research into KD. Here, we highlight the advantages of our in situ microdissection strategy in generating expression data with improved sensitivity and accuracy over traditional methods. This methodological approach supports an active role for the epidermis in the pathogenesis of KD through identification of genes and upstream regulators implicated in epithelial-mesenchymal transition, inflammation and immune modulation. We describe dermal expression patterns crucial to collagen deposition that are associated with TGFβ-mediated signalling, which have not previously been examined in KD. Additionally, this study supports the previously proposed presence of a cancer-like stem cell population in KD and explores the possible contribution of gene dysregulation to the resistance of KD to conventional therapy. Through this innovative in situ microdissection gene profiling approach, we provide better-defined gene signatures of distinct KD regions, thereby addressing KD heterogeneity, facilitating differential diagnosis with other cutaneous fibroses via transcriptional fingerprinting, and highlighting key areas for future KD research.
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MiR-21-5p Links Epithelial-Mesenchymal Transition Phenotype with Stem-Like Cell Signatures via AKT Signaling in Keloid Keratinocytes
Article
Full-text available
  • Sep 2016
Keloid is the abnormal wound healing puzzled by the aggressive growth and high recurrence rate due to its unrevealed key pathogenic mechanism. MicroRNAs contribute to a series of biological processes including epithelial-mesenchymal transition (EMT) and cells stemness involved in fibrotic disease. Here, using microRNAs microarray analysis we found mir-21-5p was significantly up-regulated in keloid epidermis. To investigate the role of miR-21-5p in keloid pathogenesis, we transfected miR-21-5p mimic or inhibitor in keloid keratinocytes and examined the abilities of cell proliferation, apoptosis, migration and invasion, the expressions of EMT-related markers vimentin and E-cadherin and stem-like cells-associated markers CD44 and ALDH1, and the involvement of PTEN and the signaling of AKT and ERK. Our results demonstrated that up-regulation or knockdown of miR-21-5p significantly increased or decreased the migration, invasion and sphere-forming abilities of keloid keratinocytes, and the phenotype of EMT and cells stemness were enhanced or reduced as well. Furthermore, PTEN and p-AKT were shown to participate in the regulation of miR-21-5p on EMT phenotypes and stemness signatures of keloid keratinocytes, which might account for the invasion and recurrence of keloids. This molecular mechanism of miR-21-5p on keloid keratinocytes linked EMT with cells stemness and implicated novel therapeutic targets for keloids.
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Expressions of Collagen I and III in Hypoxic Keloid Tissue
Article
Full-text available
  • Jul 2016
Background: Wound heals itself spontaneously as physiological process. However, in some individuals, small wounds such as parenteral injections or body piercings may cause increased expression of collagen synthesis. The condition is known as keloid. Histopathology of keloid demonstrates extensive tissue proliferation that extends beyond the margin of primary wound. As a result, it develops uncontrolled or excessive fibrogenesis and tremendous source of collagen that still causes clinical problems until now. A wound, no matter how small the size is, will be followed by increased expression of collagen synthesis. Procollagen I and III is one of markers indicating the development of fibrosis. In fibrosis, there is hypoxia, which is characterized by stabilization of HIF-1α. Therefore, our study was aimed to obtain information about expression of collagen I and III in hypoxic keloid tissue. Method: The study design was observational descriptive. Keloid specimens were obtained from biopsy and preputium skins as the control specimens were obtained from circumcision. There were 10 tissue specimens for each specimen group. The analysis performed were evaluation of mRNA expression on collagen I, collagen III and HIF-1α using RT-PCR, the evaluation of HIF-1α protein level using ELISA and the expression of collagen I and collagen III protein using immunohistochemistry. Statistically, data was analyzed by unpaired t-test. Results: In keloid with excessive cell proliferation, we found that the expression of procollagen I mRNA increased 35 times and the expression of procollagen III mRNA increased 27.1 times compared to preputium control group (p<0.05). The expression of procollagen I protein in the dermal layer of keloid was 61% and in the preputium was 37% (p<0.05). The expression of collagen III protein in the dermal layer of keloid was 39% and in the preputium was 16% (p<0.05). There was a 5-fold increase on expression of HIF-1α mRNA in keloid tissue compared to those in preputium (p<0.05). The levels of HIF-1α protein in keloid tissue was 0.201 ng/mg protein and the level in preputium was 0.122 ng/mg protein (p<0.05). There was a strong positive and extremely significant correlation between the expression of HIF-1α protein and procollagen III (R=0.744; p<0.05, Pearson), but HIF-1α with procollagen I are weak correlation (R=0.360; p>0.05, Pearson) Conclusion: Expression of collagen I and III have important role in hypoxic keloid tissue characterized by increased expressions. The expression of collagen I and III is associated with stable HIF-1α in keloid tissue.
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Cutaneous basal cell carcinoma arising within a keloid scar: A case report
Article
Full-text available
  • Aug 2016
Basal cell carcinomas (BCCs) are one of the most frequent cutaneous malignancies. The majority of BCCs are reported to occur on the auricular helix and periauricular region due to ultraviolet light exposure. Despite the frequency of BCCs, those that develop within scar tissue are rare, and the phenomenon of keloid BCCs has rarely been reported in the literature. Keloid collagen within BCCs is associated with morphoeiform characteristics, ulceration, or necrosis. Extensive keloid collagen is often seen in BCCs of the ear region, a site prone to keloid scarring. This article presents a rare case of a secondary tumor (BCC) which arose on top of a primary tumor (keloid scar) on the right auricle region in a healthy 23-year-old female after an ear piercing 2 years prior. To our knowledge, the tumor described in this case, in contrast to keloidal BCCs, has never been reported in the literature.
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Expression of embryonic stem cell markers in keloid-associated lymphoid tissue
Article
Full-text available
  • Mar 2016
Aims: To identify, characterise and localise the population of primitive cells in keloid scars (KS). Methods: 5-µm-thick formalin-fixed paraffin-embedded sections of KS samples from 10 patients underwent immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers OCT4, SOX2, pSTAT3 and NANOG, and keloid-associated lymphoid tissue (KALT) markers CD4 and CD20. NanoString gene expression analysis and in situ hybridisation (ISH) were used to determine the abundance and localisation of the mRNA for these ESC markers. Results: IHC staining revealed the expression of the ESC markers OCT4, SOX2, pSTAT3 and NANOG by a population of cells within KS tissue. These are localised to the endothelium of the microvessels within the KALTs. NanoString gene expression analysis confirmed the abundance of the transcriptional expression of the same ESC markers. ISH localised the expression of the ESC transcripts to the primitive endothelium in KS tissue. Conclusions: This report demonstrates the expression of ESC markers OCT4, SOX2, pSTAT3 and NANOG by the endothelium of the microvessels within the KALTs. These findings show a unique niche of primitive cells within KS, expressing ESC markers, revealing a potential therapeutic target in the treatment of KS.
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Aldehyde Dehydrogenase 1 (ALDH1) Is a Potential Marker for Cancer Stem Cells in Embryonal Rhabdomyosarcoma
Article
Full-text available
Cancer stem cells (CSCs) are defined as a small population of cancer cells with the properties of high self-renewal, differentiation, and tumor-initiating functions. Recent studies have demonstrated that aldehyde dehydrogenase 1 (ALDH1) is a marker for CSCs in adult cancers. Although CSCs have been identified in some different types of pediatric solid tumors, there have been no studies regarding the efficacy of ALDH1 as a marker for CSCs. Therefore, in order to elucidate whether ALDH1 can be used as a marker for CSCs of pediatric sarcoma, we examined the characteristics of a population of cells with a high ALDH1 activity (ALDH1high cells) in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. We used the human embryonal RMS (eRMS) cell lines RD and KYM-1, and sorted the cells into two subpopulations of ALDH1high cells and cells with a low ALDH1 activity (ALDH1low cells). Consequently, we found that the ALDH1high cells comprised 3.9% and 8.2% of the total cell population, respectively, and showed a higher capacity for self-renewal and tumor formation than the ALDH1 low cells. With regard to chemoresistance, the survival rate of the ALDH1high cells was found to be higher than that of the ALDH1low cells following treatment with chemotherapeutic agents for RMS. Furthermore, the ALDH1high cells exhibited a higher degree of pluripotency and gene expression of Sox2, which is one of the stem cell markers. Taken together, the ALDH1high cells possessed characteristics of CSCs, including colony formation, chemoresistance, differentiation and tumor initiation abilities. These results suggest that ALDH1 is a potentially useful marker of CSCs in eRMS.
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Keloid-derived keratinocytes acquire a fbroblast-like appearance and an enhanced invasive capacity in a hypoxic microenvironment in vitro
Article
Full-text available
  • Mar 2015
  • INT J MOL MED
A keloid scar is an overgrowth of dense fibrous tissue that develops around a wound. These scars are raised scars that spread beyong the margins of the orinigal wound to normal skin by invasion. Keloid tissue consists of both an epithelium and dermal fibroblasts. Recent studies have primarily focused on keloid fibroblasts; however, the precise role of keratinocytes in the invasion process of keloids remains to be identified. Hypoxia is a typical characteristic of keloid scars, as well as other solid tumors. The expression of the transcription factor, hypoxia‑inducible factor‑1α (HIF‑1α), is mainly induced by hypoxia and is known for its ability to induce proliferative and transformative changes in cells; its expression has been shown to correlate with tumor invasion and metastasis. In the present study, we used immunohistochemistry, fluorescence staining and western blot analysis and demonstrated that HIF‑1α was highly expressed in both the epithelial layer of keloid tissue specimens and in hypoxia‑exposed keratinocytes, which suggested that the keloid keratinocytes underwent epithelial‑to‑mesenchymal transition (EMT) in vitro. The high expression of mesenchymal markers, such as as vimentin and fibronectin was confirmed, as well as the reduced expression of E‑cadherin and zonula occludens-1 (ZO‑1) during this process by detection at the protein and mRNA level. Moreover, siRNA targeting HIF‑1α reversed the changes which had occurred in the morphology of the keratinocytes (cells had acquired a fibroblast‑like appearance) and suppressed the invasive ability of the keratinocytes. In conclusion, the present findings demonstrate that the hypoxia/HIF‑1α microenvironment provides a favorable environment for keloid‑derived keratinocytes to adopt a fibroblast‑like appearance through EMT. This transition may be responsible for the enhanced capacity of keloid keratinocytes to invade, allowing the keloids to extend beyond the wound margin.
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The Oct4 protein: More than a magic stemness marker
Article
Full-text available
  • Sep 2014
The Oct4 protein, encoded by the Pou5f1 gene was the very first master gene, discovered 25 years ago, to be absolutely required for the stemness properties of murine and primate embryonic stem cells. This transcription factor, which has also been shown to be essential for somatic cell reprogrammation, displays various functions depending upon its level of expression and has been quoted as a "rheostat" gene. Oct4 protein is in complexes with many different partners and its activity depends upon fine post-translational modifications. This review aims at revisiting some properties of this protein, which has not yet delivered all its potentialities.
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[The expressions of notch genes in human keloid-derived mesenchymal-like stem cells]
Article
  • May 2014
Objective: To study the expressions of Notch1-4 gene in human keloid-derived mesenchymal-like stem cells, and to explore the Notch signaling pathway's role in the formation of keloid. Methods: Keloid samples were collected to harvest human keloid-derived mesenchymal-like stem cells through two-step enzymatic dissociation method. By flow cytometry, cell phenotype of primary and P3 generation were analyzed. By immunocytochemistry, the expressions of Oct4, vimentin and CK19 were examined. Keloid-derived mesenchymal-like stem cells were induced into osteoblasts in vitro and calcium deposition was detected by Alizarin red S stain. Realtime polymerase chain reaction (RT-PCR) was used to detect the expressions of Notch1-4 mRNA in keloid-derived mesenchymal-like stem cells. Results: Flow cytometry showed that keloid-derived mesenchymal-like stem cells of primary and P3 generation highly expressed CD29, CD44, CD90 from the typical MSC phenotype marker, but they failed to express HSC phenotype markers, such as CD34 and CD45. The results of immunocytochemistry showed that Oct4 from pluripotent stem cell markers and vimentin from mesenchymal cell markers was positive and CK19 from epithelial cell markers was negative. After induced differentiation into osteoblasts in vitro after 21 day, calcium nodules could be seen clearly; Notch1-4 gene were expressed in keloid-derived mesenchymal-like stem cells through RT-PCR. The relative quantitative of Notch2, Notch3 gene were higher than Notch1, Notch4 gene (P < 0.05). Conclusions: The expression difference of different subtypes from Notch gene in human keloid-derived mesenchymal-like stem ceils may be related to self-renewal, proliferation, differentiation, and participate in the formation of keloid.
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Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology
Article
  • Jul 2013
  • WOUND REPAIR REGEN
Keloids are disfiguring scars that extend beyond the original wound borders and resist treatment. Keloids exhibit excessive extracellular matrix deposition, although the underlying mechanisms remain unclear. To better understand the molecular basis of keloid scarring, here we define the genomic profiles of keloid fibroblasts and keratinocytes. In both cell types, keloid-derived cells exhibit differential expression of genes encompassing a diverse set of functional categories. Strikingly, keloid keratinocytes exhibited decreased expression of a set of transcription factor, cell adhesion, and intermediate filament genes essential for normal epidermal morphology. Conversely, they exhibit elevated expression of genes associated with wound healing, cellular motility, and vascular development. A substantial number of genes involved in epithelial-mesenchymal transition were also up-regulated in keloid keratinocytes, implicating this process in keloid pathology. Furthermore, keloid keratinocytes displayed significantly higher migration rates than normal keratinocytes in vitro and reduced expression of desmosomal proteins in vivo. Previous studies suggested that keratinocytes contribute to keloid scarring by regulating extracellular matrix production in fibroblasts. Our current results show fundamental abnormalities in keloid keratinocytes, suggesting they have a profoundly more direct role in keloid scarring than previously appreciated. Therefore, development of novel therapies should target both fibroblast and keratinocyte populations for increased efficacy.
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