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Expression of Components of the Renin-angiotensin System by Embryonic Stem Cell-like Population within Keloid Lesions

Humphries, Hugo1; Brasch, Helen D MBChB FRCPA1; van Schaijik, Bede BTech (Hons)1,2,*; Tan, Swee T MBBS FRACS PhD1,*; Itinteang, Tinte MBBS PhD

Plastic and Reconstructive Surgery: May 7, 2019 - Volume PRS Online First - Issue - p
doi: 10.1097/PRS.0000000000005867
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Background This study investigated the expression of pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor (ATIIR2) by the embryonic stem cell-like population on the endothelium of the microvessels and the perivascular cells within keloid-associated lymphoid tissues (KALTs).

Methods 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for PRR, ACE, ATIIR1 and ATIIR2 was performed on 11 formalin-fixed paraffin-embedded sections of keloid tissue samples. Immunofluorescence (IF) IHC staining was performed on three keloid tissue samples by co-staining with OCT4, CD34, ERG and tryptase. RT-qPCR was performed on five keloid tissue samples and four keloid-derived primary cell lines. Western blotting (WB) was performed on the four keloid-derived primary cell lines, to investigate mRNA and protein expression of these proteins, respectively.

Results DAB and IF IHC staining showed expression of PRR, ACE, ATIIR1 and ATIIR2 in all 11 keloid tissue samples. PRR and ATIIR1 were expressed on the endothelium and the outer pericyte layer of the microvessels and the perivascular cells, ATIIR2 was localized to the endothelium of the microvessels and the tryptase+ perivascular cells, and ACE was localized to the endothelium of the microvessel, within the KALTs. RT-qPCR showed transcriptional activation of PRR, ACE and ATIIR1 in the keloid tissue samples and keloid-derived primary cell lines, while ATIIR2 was detected in keloid tissue samples only. WB confirmed the presence of PRR, ACE and ATIIR1 in the keloid-derived primary cell lines.

Conclusions PRR, ACE, ATIIR1 and ATIIR2 were expressed by the ESC-like population within the KALTs suggesting this primitive population may be a potential therapeutic target by modulation of the RAS.

1Gillies McIndoe Research Institute, Wellington, New Zealand

2Wellington Regional Plastic, Maxillofacial & Burns Unit, Hutt Hospital, Wellington, New Zealand.

* Equal senior authors

AUTHOR CONTRIBUTIONSHIP: TI and ST formulated the study hypothesis and designed the study. HH, HB, ST and TI interpreted the DAB and IF IHC data. ST and TI interpreted the RT-qPCR data. BvS conducted the WB analysis and interpreted the results. All authors drafted and approved the manuscript.

FINANCIAL DISCLOSURES: TI and ST are inventors of a PCT application Treatment of Fibrotic Conditions (PCT/NZ2016/050187). The authors are otherwise not aware of any commercial associations or financial relationships that might pose or create a conflict of interest with information presented in any submitted manuscript.

ETHICS STATEMENT: This study was carried out with the approval of the Central Health and Disability Ethics Committee (ref. no. 13/NTB/155) with written informed consent from all subjects in accordance with the Declaration of Helsinki.

ACKNOWLEDGEMENTS: We thank Mrs Liz Jones and Ms Erin Paterson at the Gillies and McIndoe Research Institute for performing the immunohistochemical staining, and cell culture, respectively. We also thank Ms Jennifer de Jongh for her assistance in performing the RT-qPCR experiments and interpreting the data.

Aspects of this work was presented at the Royal Australasian College of Surgeons’ 87th Annual Scientific Congress with the American College of Surgeons and the Australian and New Zealand College of Anaesthetists’ Annual Scientific Meeting, Sydney, Australia, May 7–11, 2018 and the 2nd International Keloid Symposium, Rome, Italy, June 7–8, 2018.

Correspondence author: Swee T Tan ONZM MBBS PhD FRACS, Gillies McIndoe Research Institute, PO Box 7184, Newtown 6242, Wellington, New Zealand. Ph: +64 4 2820366 Em: swee.tan@gmri.org.nz

©2019American Society of Plastic Surgeons