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Cancer stem cell (CSC) subpopulations within moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNcSCC) express the components of the renin-angiotensin system (RAS). This study investigated the expression of cathepsins B, D, and G, which constitute bypass loops of the RAS, by CSCs in MDHNcSCC., Methods: Immunohistochemical staining was performed on MDHNcSCC tissue samples from 15 patients to determine the expression of cathepsins B, D, and G. Co-localization of these cathepsins with the embryonic stem cell markers Octamer-binding transcription factor 4 (OCT4) and c-MYC was investigated with immunofluorescence staining. Reverse transcription quantitative polymerase chain reaction was performed on 5 MDHNcSCC tissue samples to investigate transcript expression of cathepsins B, D and G. Western blotting and enzymatic activity assays were performed on 5 MDHNcSCC tissue samples and 6 MDHNcSCC-derived primary cell lines to confirm protein expression, transcript expression, and functional activity of these cathepsins, respectively., Results: Immunohistochemical staining demonstrated the expression of cathepsins B, D, and G in all MDHNcSCC tissue samples. Immunofluorescence staining showed localization of cathepsins B and D to the c-MYC + CSC subpopulations and the OCT4 + CSC subpopulations within the tumor nests and the peritumoral stroma. Cathepsin G was expressed on the tryptase + /c-MYC + cells within the peritumoral stroma. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of cathepsins B, D and G in the MDHNcSCC tissue samples. Western blotting and enzymatic activity assays confirmed protein expression and functional activity of cathepsins B and D in the MDHNcSCC tissue samples and MDHNcSCC-derived primary cell lines, respectively., Conclusions: Cathepsins B, D, and G are expressed in MDHNcSCC with functionally active cathepsins B and D localizing to the CSC subpopulations, and cathepsin G is expressed by mast cells, suggesting the potential use of cathepsin inhibitors in addition to RAS blockade to target CSCs in MDHNcSCC., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Background: We have previously shown that the endothelium of the microvessels of infantile hemangioma (IH) exhibits a hemogenic endothelium phenotype and proposed its potential to give rise to mesenchymal stem cells, similar to the development of hematopoietic cells. This endothelial-to-mesenchymal transition (Endo-MT) process involves the acquisition of a migratory phenotype by the endothelial cells, similar to epithelial-to-mesenchymal transition that occurs during neural crest development. We hypothesized that proliferating IH expresses Endo-MT-associated proteins and investigated their expression at the mRNA, protein, and functional levels., Methods: Immunohistochemical staining of paraffin-embedded sections of proliferating IH samples from 10 patients was undertaken to investigate the expression of the Endo-MT proteins Twist1, Twist2, Snail1, and Slug. Transcriptional analysis was performed for the same markers on proliferating IH tissues and CD34 + and CD34 - cells from proliferating IH-derived primary cell lines. Adipogenic and osteogenic differentiation plasticity was determined on the CD34-sorted fractions., Results: The endothelium of the microvessels and the cells within the interstitium of proliferating IH tissues expressed Twist1, Twist2, and Slug proteins. Twist1 was also expressed on the pericyte layer of the microvessels, whereas Snail1 was not expressed. Both CD34 + and CD34 - populations from the IH-derived primary cell lines underwent adipogenic and osteogenic differentiation., Conclusions: The expression of Endo-MT-associated proteins Twist1, Twist2, and Slug by both the endothelium of the microvessels and cells within the interstitium, and Twist1 on the pericyte layer of the microvessels of proliferating IH, suggest the presence of a process similar to Endo-MT. This may enable a tightly controlled primitive endothelium of proliferating IH to acquire a migratory mesenchymal phenotype with the ability to migrate away, providing a plausible explanation for the development of a fibrofatty residuum observed during involution of IH., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Keloid disorder (KD) is a fibroproliferative condition caused by dysregulated wound healing following wounding of the skin. The pathogenesis of KD has not been fully elucidated and current treatment is unsatisfactory. There is increasing evidence of the role of stem cells in KD. This review discusses the role of embryonic stem (ESC)-like cells and mesenchymal stem cells in the pathogenesis of KD. It is proposed that dysfunction of the ESC-like population localized to the endothelium of the microvessels and perivascular cells within the keloid-associated lymphoid tissues may give rise to the aberrant fibroblasts and myofibroblasts via a mesenchymal stem cell intermediate in keloid lesions, by undergoing an endothelial-to-mesenchymal transition. We also discuss the role of the renin-angiotensin system (RAS), the immune system, and the inflammatory response, on stem cell proliferation and differentiation. The understanding of the precise roles of these stem cells and interplay of the associated regulatory pathways could lead to the development of targeted therapy for this enigmatic and challenging condition. The demonstration of the expression of components of the RAS and cathepsins B, D, and G that constitute bypass loops of the RAS, by the ESC-like population, suggests that the primitive population may be a therapeutic target by modulation of the RAS, using existing medications.

Background: We have recently demonstrated the presence of a NANOG + /pSTAT3 + /OCT4 + /SOX2 + /SALL4 + /CD44 + embryonic stem cell (ESC)-like subpopulation localized to the endothelium and a NANOG + /pSTAT3 + /SOX2 + /CD44 + subpopulation outside of the endothelium, within subcutaneous VM (SCVM) and intramuscular VM (IMVM). We have also shown the expression of components of the renin-angiotensin system (RAS): (pro)renin receptor (PRR); angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), in both SCVM and IMVM. This study investigated whether the ESC-like subpopulations within SCVM and IMVM expressed the RAS., Methods: Formalin-fixed paraffin-embedded sections of two representative samples from each of the seven SCVM and seven IMVM patients included in our previous studies underwent dual immunofluorescence (IF) immunohistochemical (IHC) staining for ESC marker OCT4 or SOX2 with PRR, ACE, ATIIR1, and ATIIR2., Results: IF IHC staining demonstrated the expression PRR by the OCT4 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ACE was expressed by the SOX2 + cells, predominantly in the endothelium in SCVM and IMVM. ATIIR1 was expressed by the SOX2 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ATIIR2 was expressed by the OCT4 + endothelium and outside the endothelium in SCVM and IMVM., Conclusions: Components of the RAS are expressed by ESC-like subpopulations within both SCVM and IMVM. These primitive subpopulations may be a novel therapeutic target by manipulation of the RAS using existing medications.

The pathogenesis of Dupuytren's disease (DD) remains unclear although there is increasing evidence supporting the role of stem cells in this and other fibrotic conditions. This review examines the role of DD tissue-associated embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), and circulating fibrocytes and circulating MSCs, in the biology of DD. It is exciting to infer that dysfunction of an upstream ESC-like population within the affected tissue leads to the downstream development and proliferation of aberrant myofibroblasts through a putative MSC intermediate. This ESC-like population may be a potential novel therapeutic target through modulation of the renin-angiotensin system. Furthermore, circulating CD34 + fibrocytes and MSCs either derived from the bone marrow, peripheral blood cells, or DD-associated ESC-like population, may serve as potential additional extra-palmar reservoirs that undergo endothelial-to-mesenchymal transition, eventually giving rise to the aberrant myofibroblasts. Further studies examining the relative roles of these stem cells and the precise regulatory pathways that govern them may lead to novel therapy that targets these populations.

Background: The pathogenesis of Dupuytren's disease (DD) remains unclear. An embryonic stem cell (ESC)-like population in the endothelium of the microvessels around tissues that expresses components of the renin-angiotensin system (RAS) has been reported. This study investigated if this primitive population expresses cathepsins B, D, and G, that contribute to RAS bypass loops., Methods: 3,3-Diaminobenzidine immunohistochemical (IHC) staining for cathepsins B, D, and G was performed on sections of formalin-fixed paraffin-embedded DD cords (n = 10) and nodules (n = 10). Immunofluorescence IHC staining was utilized to demonstrate co-expression of these cathepsins with ESC markers. Protein and gene expression of these cathepsins was investigated in snap-frozen DD cords (n = 3) and nodules (n = 3) by Western blotting and NanoString analysis, respectively. Enzymatic activity of these cathepsins was investigated by enzymatic activity assays., Results: 3,3-Diaminobenzidine IHC staining demonstrated expression of cathepsins B, D, and G in DD cords and nodules. Gene expression of cathepsins B, D, and G was confirmed by NanoString analysis. Western blotting confirmed expression of cathepsins B and D, but not cathepsin G. Immunofluorescent IHC staining demonstrated high abundance of cathepsins B and D on the OCT4 + /angiotensin converting enzyme + endothelium and the smooth muscle layer of the microvessels. Cathepsin G was localized to trypase + cells within the stroma in DD cords and nodules with limited expression on the microvessels. Enzyme activity assays demonstrated functional activity of cathepsins B and D., Conclusions: Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

The renin-angiotensin system (RAS) mediates cardiac and renal fibrosis. Dupuytren's disease (DD) is a proliferative fibromatosis affecting the hands. This study investigated the expression of the RAS in DD., Methods: 3,3-Diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) was performed on 4-μm thick formalin-fixed paraffin-embedded sections of DD cords and nodules from 6 patients. Western blotting (WB) and NanoString mRNA analysis were performed to confirm RAS protein expression and transcriptional activation, respectively., Results: IHC staining demonstrated the expression of PRR, ACE, ATIIR1, and ATIIR2 on the ERG + and CD34 + endothelium of the micro vessels surrounding the DD cords and nodules. PRR was also expressed on the pericyte layer of these microvessels. WB confirmed protein expression of PRR, ACE, and ATIIR2 but not ATIIR1. NanoString analysis confirmed transcriptional activation of PRR, ACE, ATIIR1, but ATIIR2 was below detectable levels., Conclusions: We demonstrated expression of PRR, ATIIR1, ATIIR2, and ACE on the embryonic stem cell-like cell population on the microvessels surrounding DD nodules and cords by IHC staining, although the expression of ATIIR1 was not confirmed by WB and that of ATIIR2 was below detectable levels on NanoString analysis. These findings suggest the embryonic stem cell-like cell population as a potential therapeutic target for DD, by using RAS modulators., Competing Interests: Disclosure: Drs. Tan and Itinteang are inventors of a provisional patent application (no. 62/260,953) Treatment of Fibrotic Conditions. 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 article. The Article Processing Charge was paid for by the Gillies McIndoe Research Institute general fund.

Background: Recent research has identified mesenchymal stem cells (MSCs) within Dupuytren's disease (DD) tissue and they have been proposed to give rise to the myofibroblasts, implicated in the progression of this condition. The aim of this study was to identify and characterize the primitive population that might be upstream of the MSC population, within DD., Methods: Formalin-fixed paraffin-embedded 4-µm-thick sections of DD cords and nodules obtained from 6 patients underwent 3,3-diaminobenzidine and immunofluorescent immunohistochemical staining for embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, pSTAT3, and SALL4 and endothelial markers CD34 and ERG. NanoString gene expression analysis was performed to determine the transcriptional activation of these markers., Results: Immunohistochemical staining demonstrated the expression of ESC markers OCT4, NANOG, SOX2, pSTAT3, and SALL4 on the endothelium of the microvessels expressing CD34 and ERG, particularly those surrounding the DD nodules. NanoString analysis confirmed the transcriptional activation of OCT4, NANOG, STAT3, and SALL4, but not SOX2., Conclusion: This article demonstrates the novel finding of an ESC-like population expressing ESC markers OCT4, NANOG, SOX2, pSTAT3, and SALL4, localized to the endothelium of the microvessels within DD tissue, suggesting a potential therapeutic target for this condition.