Port-wine stain (PWS) is the most common type of capillary malformation that affects the skin and/or mucosa of 0.3%–0.5% of the population,1–3 with no genetic predisposition.4 PWS is characterized by an increased number of dilated dermal capillary-venular channels with a flat endothelium2 that presents as a reddish to purplish “stain” that darkens over time.1,5 The involved skin often becomes hypertrophic and develops nodules, sometimes with overgrowth of the underlying soft tissue and bone,1,5 causing both cosmetic and functional problems.6
The standard treatment for PWS is photocoagulation with a pulsed dye laser5 and complete removal can be achieved in 10% of patients.6 Surgical debulking is used for hypertrophic PWS (HPWS) with modest results.5
The cause of PWS is likely to be multifactorial with unclear interactions between several biological components.6 One such component involves somatic mutations of GNAQ,7 which are associated with Sturge-Weber syndrome,7 a neurocutaneous syndrome characterized by facial PWS, and ocular and/or neurological anomalies.7,8 The somatic mutation in GNAQ demonstrated in PWS blood vessels of patients with Sturge-Weber syndrome9,10 activates extracellular signal-regulated kinase (ERK) (Figure 1) and may contribute to the development of PWS.11 Somatic mutations in the PIK3CA gene have also been demonstrated in PWS.6 Human umbilical vein endothelial cells that express PIK3CA have faster proliferation rates than normal human umbilical vein endothelial cells.6 Consequently, PIK3CA is proposed to cause excessive endothelial cell proliferation with hypertrophy of affected skin with nodular formation commonly seen in HPWS.6 The GNA11 gene that affects cellular function similar to GNAQ, and somatic mutations of GNA11 have been found in a variety of capillary malformations including PWS.12 Mutation of other genes has also been implicated in the development of PWS. For example, c-Jun N-terminal kinases (JNKs) that are associated with the progression of PWS are activated in PWS blood vessels of infants and adults.10 Overexpression of vascular endothelial growth factor (VEGF) on the endothelium of the lesional vessels causes vessel proliferation and dilation, in PWS.13
These gene mutations are implicated in the development of PWS although the exact mechanism remains unclear as some of these gene mutations are not always present in individuals with PWS. The missing key that could provide the link between these gene mutations and the development of PWS may lie within the early development of endothelial cells.
It has been hypothesized that the cause of PWS lies within the impairment of differentiation in endothelial progenitor cells (EPCs).14 EPCs are thought to develop into venule-like vasculatures commonly associated with PWS, and through the disruption of endothelial cell interactions by Eph receptor B1 (EphB1) and ephrin B2 (EfnB2), these vasculatures can dilate over time.14 This hypothesis is supported by the observation that: (1) when EphB1 and EfnB2 are expressed in normal microvascular endothelial cells, they are able to transform into PWS-like vessels14 and (2) that EphB1 and EfnB2 are expressed in infant, pediatric, and adult PWS endothelial cells.14 It has also been shown that the EPCs in nonnodular lesions of PWS show stemness properties as they express the EPC markers CD133 and CD166.14 What causes the upregulated signaling of EphB1 and EfnB2 in PWS remains unknown, although the stemness nature of the endothelial cells in PWS may provide a clue.
Stemness refers to cells that behave in a similar fashion to embryonic stem cells (ESCs) that are characterized by pluripotency and the capacity for self-renewal.15 OCT4, SOX2, NANOG, KLF4, and c-MYC are transcription factors involved in the generation of induced pluripotent stem cells (iPSCs) and have been used to characterize ESCs.16 Certain genes that are implicated in the progression of PWS are also implicated in the expression of ESC markers. For example, when JNK is activated through the Rac pathway, it induces the expression of Wnt and BMP-signaling proteins that are crucial in maintaining self-renewal of ESCs.17 Wnt has been shown to enhance the function of several stem cell markers, including OCT4, SOX2, and NANOG.18 BMP in turn limits the activity of NANOG, leading to differentiation to form ESCs.19 VEGF is a strong activator of Ras and its signaling pathway,20 which strongly activates ERK, amplifying the expression of OCT4, SOX2, and NANOG. In addition, activation of ERK via Ras signaling has been shown to induce phosphorylation of c-MYC21 and KLF422 in melanoma cells.
A proposed model of regulation of the iPSC markers OCT4, SOX2, NANOG, KFL4, and c-MYC, by the Ras pathway and Rac pathway, is shown in Figure 1. ESC markers OCT4, SOX2, and NANOG are able to bind to the promoter region of the EPHA1 gene, which encodes EphB1, and enhance transcription of that gene.19 Because the expression of OCT4, SOX2, and NANOG can be amplified through the Ras pathway,20 the transcription of EPHA1 can be even more enhanced.19 As EfnB2 is a ligand of EphB1,19 the enhanced transcription of EphB1 can cause upregulation of signaling, leading to aberration of EPCs.14 This suggests a possible oncogenic role of the ESC markers in PWS. Certain oncogenes have been implicated in pathogenesis of congenital diseases, especially those that are caused by somatic mutations23,24 and SOX2 has been described as working as an oncogene in certain cancers.25 If ESC markers are present in PWS, it is possible that these markers are behaving in an oncogenic-like manner, causing aberration of EPCs and other leading to the development of PWS.
iPSC markers OCT4, SOX2, NANOG, KLF4, and c-MYC have been used to identify and characterize ESC-like populations in venous malformation,26 lymphatic malformation,27 pyogenic granuloma,28 and arteriovenous malformation (Luke Krishnan et al, unpublished data, 2020). The findings of the presence of ESC-like cells in different types of vascular anomalies and previous research suggesting the existence of stem cells in PWS14 led us to investigate the presence of an ESC-like population within HPWS using these iPSC markers by immunohistochemical and immunofluorescence staining, Western blotting (WB), in situ hybridization (ISH), and reverse transcription quantitative polymerase chain reaction (RT-qPCR).
HPWS tissue samples from 8 male and 7 female patients, aged 14–71 (mean, 39) years (Supplemental Digital Content Table 1, http://links.lww.com/JV9/A8), were sourced from the Gillies McIndoe Research Institute Tissue Bank. This study was approved by the Central Health and Disability Ethics Committee (Reference 13/CEN/130), with written informed consent from all participants.
HPWS-derived primary cell lines
Primary cell lines were derived from 3 fresh HPWS tissue samples from the original cohort of 15 patients. Samples were cut into small pieces and incubated between layers of Matrigel (cat#354234, Corning, Tewksbury, MA) in a 24-well plate with a media containing Dulbecco’s Modified Eagle Medium with Glutamax (cat#10569010, Gibco, Rockford, IL), and supplemented with 2% penicillin-streptomycin (cat#15140122, Gibco) and 0.2% gentamycin-amphotericin (cat#R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving the Matrigel with Dispase (cat#354235, Corning) and transferred to an adherent culture flask with media containing Dulbecco’s Modified Eagle Medium with Glutamax supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSR Complete Medium (cat#85850, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin, and 0.2% gentamycin-amphotericin in a humidified incubator at 37°C and 5% CO2. Cells were expanded in culture and harvested at passages 5 or 6.
Histology and immunohistochemical staining
Hematoxylin and eosin staining was performed on 4-μm-thick consecutive formalin-fixed paraffin-embedded sections of 15 HPWS tissue samples to confirm the presence of HPWS by an anatomical pathologist. Immunohistochemical staining of tissue sections with primary antibodies for makers OCT4 (1:30; cat#309-M-16, Cell Marque, Rocklin, CA), SOX2 (1:500; cat#PA1-094, Thermo Fisher Scientific, Rockford, IL), NANOG (1:200; cat#443R, Cell Marque, Rocklin, CA), KLF4 (1:100; cat#NBP2-24749, Novus Biologicals, Centennial, CO), and c-MYC (1:1000; cat#ab32072, Abcam, Cambridge, UK) was performed on the BOND Rx autostainer (Leica, Nussloch, Germany) using the BOND Polymer Refine Detection (cat#9800, Leica). Immunohistochemical-stained slides were mounted in Dako Mounting Medium (cat#CS703, Dako, Glostrup, Denmark). Normal skin was also used for comparison with the staining pattern of the HPWS samples. Positive human control tissues used were seminoma for OCT4 and NANOG, normal skin epidermis for SOX2, breast carcinoma for KLF4, and normal colon mucosa for c-MYC. Negative controls were HPWS sections run with rabbit (ready-to-use; cat#IR600, Dako) or mouse (ready-to-use; cat#IR750, Dako) isotype controls.
To confirm coexpression of 2 proteins, immunofluorescence staining was performed on 2 HPWS tissue samples of the 15 patients, using the same primary antibodies against the iPSC markers at the same concentrations (as described in the immunohistochemical staining methods above) and costaining with either endothelial marker CD3429,30 (ready-to-use, cat#PA0212, Leica, Newcastle-upon-Tyne, United Kingdom), smooth muscle actin (ready-to-use; cat#PAO943, Leica), or von Willebrand factor (1:200, cat#AO052, Dako). Appropriate secondary antibodies and amplification kits were used for immunofluorescence detection; Alexa Fluor antimouse 488 (1:500; cat#A-21202, Life Technologies, Carlsbad, CA), Alexa Fluor antirabbit 594 (1:500; cat#A-21207, Life Technologies), VectaFluor Excel antimouse 488 (ready-to-use; cat#DK-2488, Vector Laboratories, Burlingame, CA), and VectaFluor Excel antirabbit 594 (ready-to-use; cat#DK-1594, Vector Laboratories). All antibodies were diluted with BOND primary antibody diluents (cat#AR9352, Leica). Slides were mounted using Vectashield hardset medium with 4’,6-diamidino-2-phenylindole (cat#H-1500, Vector Laboratories). Negative controls for immunofluorescence staining were performed on HPWS sections using primary isotype mouse (ready-to-use; cat#IR750, Dako) and rabbit (ready-to-use; cat#IR600, Dako) isotype controls. All immunofluorescence staining was performed on the BOND Rx autostainer (Leica) using a BOND detection system (cat#DS9455, Leica).
Image capture and analysis
Immunohistochemical-stained slides were viewed and the images were captured using an Olympus BX53 light microscope fitted with an Olympus SC100 digital camera (Olympus, Tokyo, Japan) and processed with the cellSens 2.0 software (Olympus). Immunofluorescence-stained slides were viewed and imaged with an Olympus FV1200 biological confocal laser-scanning microscope and processed with the cellSens Dimension 1.17 software (Olympus).
In situ hybridization
Four-micrometer-thick formalin-fixed paraffin-embedded sections of HPWS tissue samples from 6 of the 15 patients were subjected to ISH mRNA analysis. Staining was performed using the Leica BOND Rx autostainer (Leica) with probes for OCT4 (cat#592868), SOX2 (cat#477658), KLF4 (cat# 457468), and c-MYC (cat#311768). The probes were detected using the RNAscope 2.5 LSx Reagent Brown Kit (cat# 322700; Advanced Cell Diagnostics, Newark, CA). Positive human control tissues were seminoma for OCT4, melanoma for SOX2, breast carcinoma for KLF4, and normal colon mucosa for c-MYC. RNAscope 2.5LS Positive Control Probe—PPIB Probe targeting common housekeeping gene was demonstrated on HPWS tissue samples. Negative controls were demonstrated on HPWS tissue samples using a probe for the bacterial gene DapB (cat#312038). All probes were sourced from Advanced Cell Diagnostics.
Reverse transcriptase quantitative polymerase chain reaction
Total RNA was isolated from 6 snap-frozen HPWS tissue samples and from 3 HPWS-derived primary cell lines from the original cohort of 15 patients. Approximately 20 mg of each tissue sample was homogenized using the Omni Tissue Homogenizer (Omni TH, Omni International, Kennesaw, GA) before following the RNeasy Mini Kit protocol (cat#74104, Qiagen, Hilden, Germany). RNA was extracted from frozen-cell pellets of 5 × 105 viable cells, using the RNeasy Micro kit protocol (cat#74004, Qiagen). An on-column DNase digest (cat#79254, Qiagen) step was included to remove potentially contaminating genomic DNA. RNA quantity was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Transcriptional expression was analyzed in triplicate using the Rotor-Gene Q (Qiagen), Rotor Gene Multiplex RT-PCR Kit (cat#204974, Qiagen), and TaqMan Gene Expression Assay primer probes on 40 ng of RNA. The primer probes were: OCT4 (Hs03005111_g1), SOX2 (Hs00602736_s1), NANOG (Hs02387400_g1), KLF4 (Hs00358836_m1), and c-MYC (Hs00153408_m1) (cat#4331182, Thermo Fisher Scientific). Gene expression was normalized to the reference genes GAPDH (Hs99999905_m1) and PUM1 (Hs00206469_m1; cat#4331182, Thermo Fisher Scientific). Universal human reference (UHR) RNA (cat#CLT636690, Takara, Shiga, Japan), total RNA extracted from a range of healthy adult human tissues, was used as the calibrator for the 2ΔΔCt analysis. Nuclease-free water was added for the no template control and RNA from NTERA2 cells was used as a positive control. The presence of the correctly sized bands from the endpoint amplification products was confirmed using 2% agarose gel electrophoresis (cat#G402002, Thermo Fisher Scientific) and imaged using the ChemiDoc MP (Bio-Rad, Hercules, CA). Graphs were generated using GraphPad Prism (v8.0.2, San Diego, CA) and results expressed as fold-change relative to UHR. A fold-change cutoff was set at 2.0 for upregulated and 0.5 for downregulated genes.
Total protein was extracted from 3 snap-frozen HPWS-derived primary cell lines used for RT-qPCR. Cell pellets were lysed in ice-cold radioimmunoprecipitation assay buffer (cat#89900, Pierce Biotechnology, Rockford, IL) supplemented with a protease and phosphatase inhibitor cocktail (cat#78440, Pierce Biotechnology), protein was quantified using
a BCA assay (cat#23227, Pierce Biotechnology), and subsequently diluted in an equal volume of 2× LDS (cat#B0007, Invitrogen, Carlsbad, CA). About 20 μg of total protein was separated by SDS-PAGE on 4%–12% Bis-Tris gels (cat#NW04122BOX, Invitrogen) in an MES SDS running buffer (cat#B0002, Invitrogen) and transferred to a PVDF membrane (cat#IB24001, Invitrogen) using an iBlot 2 (cat#IB21001, Thermo Fisher Scientific). Protein was detected on the iBind Flex (cat#SLF2000, Thermo Fisher Scientific), using primary antibodies for OCT4 (1:1000; cat#ab109183, Abcam), SOX2 (1:500; cat# 48-1400, Thermo Fisher Scientific), NANOG (1:500; cat#ab109250, Abcam), KLF4 (1:1000; cat# NBP2-24749, Novus Biologicals), c-MYC (1:1000; cat#ab32072, Abcam), and α-tubulin (1:2000; cat# 62204, Thermo Fisher Scientific). Secondary antibodies were Alexa 488 goat antirabbit for α-tubulin (1:1000; cat#a21202, Abcam) and goat antirabbit HRP (1:1000; cat#ab6721, Abcam) for all other markers. Positive control was NTERA-2 cells (cat#CRL-1973, ATCC, Gaithersburg, Maryland). Human dermal fibroblast cell line (Detroit 551, cat#CCL-110, ATCC, Gaitherberg, MD) was used as negative controls.
To visualize HRP protein bands, Clarity Western ECL substrate (cat#1705061, Bio-Rad) was used with the ChemiDoc MP Imaging System (Bio-Rad) and the Image Lab 6.0 software (Bio-Rad) to analyze protein bands.
HPWS tissue samples demonstrated characteristic lesional vessels
Hematoxylin and eosin staining demonstrated the presence of HPWS consisting of dilated capillary-venular channels with irregular shapes filled with red blood cells in all 15 tissue samples (Figure 2).
OCT4, SOX2, KLF4, and c-MYC were expressed in HPWS tissue samples
Immunohistochemical staining demonstrated cytoplasmic and nuclear expression of OCT4 on the endothelium and the media of the lesional vessels with nuclear staining of the cells within the stroma (Figure 3A). Cytoplasmic and nuclear expression of SOX2 was present on the endothelium of the lesional vessels and the cells within the stroma with strong expression in the media of the lesional vessels (Figure 3B). Weak cytoplasmic expression of KLF4 was observed on the endothelium of the lesional vessels (Figure 3C). Nuclear expression of c-MYC was demonstrated on the endothelium and the media of the lesional vessels and within the cells in the stroma (Figure 3D). NANOG was not expressed in any of the 15 samples (Figure 3E).
Positive staining was demonstrated on human control tissues: seminoma for OCT4 (Supplemental Digital Content Figure 1A, http://links.lww.com/JV9/A2), skin epidermis for SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), breast carcinoma for KLF4 (Supplemental Digital Content Figure 1C, http://links.lww.com/JV9/A2), normal colon mucosa for c-MYC (Supplemental Digital Content Figure 1D, http://links.lww.com/JV9/A2), and seminoma for NANOG (Supplemental Digital Content Figure 1E, http://links.lww.com/JV9/A2). Immunohistochemical staining of normal skin showed no expression of OCT4 (Supplemental Digital Content Figure 1F, http://links.lww.com/JV9/A2) and NANOG (Supplemental Digital Content Figure 1G, http://links.lww.com/JV9/A2). SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2), and c-MYC (Supplemental Digital Content Figure 1I, http://links.lww.com/JV9/A2) were present in the epidermis of the normal skin. In addition, SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2) and KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2) were expressed by some cells but not blood vessels within the stroma. A negative stain using combined Flex Negative Control Mouse and Flex Negative Control Rabbit on a section of HPWS (Supplemental Digital Content Figure 1J, http://links.lww.com/JV9/A2) showed no staining, confirming the specificity of the primary antibodies.
An OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population was present on the endothelium and media of lesional vessels and within the stroma of HPWS
Immunofluorescence staining demonstrated von Willebrand factor + inner endothelium (Figure 4A, red, long arrows) and SMA+ outer media (Figure 4A, green, short arrows) of the lesional vessels of HPWS surrounded by stroma (arrowheads). Strong nuclear staining of SOX2 (Figure 4B, red) was present on the CD34+ (Figure 4B, green) endothelium and the media of the lesional vessels and cells within the stroma. To further characterize the SOX2+ population, dual-staining with c-MYC (Figure 4C, green) and OCT4 (Figure 4D, green) was performed. The SOX2+ endothelium of the lesional vessels and cells within the stroma showed weak coexpression of c-MYC (Figure 4C, green). Weak cytoplasmic and nuclear staining of OCT4 (Figure 4D, green) was present on the SOX2+ (Figure 4D, red) endothelium with coexpression of the 2 markers in the media of the lesional vessels.
Very weak expression of NANOG (Figure 4E, red) was seen on the CD34+ (Figure 4E, green) endothelium and the media of the lesional vessels and cells within the stroma. There was low OCT4 (Figure 4F, green) expression on the endothelium and media of the lesional vessels and cells within the stroma. KLF4 (Figure 4G, red) was expressed on the CD34+ (Figure 4G, green) endothelium and media of the lesional vessels, and some cells within the stroma. There was strong KLF4 (Figure 4H, red) expression in the endothelium of the lesional vessels and low expression of OCT4 (Figure 4H, green) on cells within the stroma. KLF4 (Figure 4I, red) and c-MYC (Figure 4I, green) were expressed on the endothelium and media of the lesional vessels, with colocalization seen in the cells within the stroma (Figure 4I). Magnified figure insets have been provided to show enlarged views of the corresponding images.
Images of individual stains of the merged images presented in Figure 4 are provided in Supplemental Digital Content Figure 2A–R (http://links.lww.com/JV9/A3). Minimal to no staining was present on the negative controls (Supplemental Digital Content Figure 2S, http://links.lww.com/JV9/A3), confirming the specificity of the primary antibodies used.
OCT4, SOX2, KLF4, and c-MYC transcripts were present in HPWS tissue samples
ISH demonstrated the transcript expressions of OCT4 (Figure 5A), SOX2 (Figure 5B), KLF4 (Figure 5C), and c-MYC (Figure 5D) were present primarily in the endothelium of the lesional vessels, with expression OCT4 (Figure 5A), KLF4 (Figure 5C), and c-MYC (Figure 5D) also in cells outside the endothelium, in all 6 HPWS tissue samples.
Positive staining was demonstrated on human control tissues for OCT4 (Supplemental Digital Content Figure 3A, http://links.lww.com/JV9/A4) on seminoma, SOX2 (Supplemental Digital Content Figure 3B, http://links.lww.com/JV9/A4) on normal skin epidermis, KLF4 (Supplemental Digital Content Figure 3C, http://links.lww.com/JV9/A24) on malignant melanoma, and c-MYC (Supplemental Digital Content Figure 3D, http://links.lww.com/JV9/A4) on normal colonic mucosa. The negative control showed no staining, indicating no background activity (Supplemental Digital Content Figure 3E, http://links.lww.com/JV9/A4).
OCT4, SOX2, NANOG, KLF4, and c-MYC transcripts were present in HPWS tissue samples and HPWS-derived primary cell lines
RT-qPCR analysis of the 6 HPWS tissue samples (Figure 6A) and 3 HPWS-derived primary cell lines (Figure 6B) demonstrated mRNA expression of OCT4, SOX2, NANOG, KLF4, and c-MYC. In the tissue samples, c-MYC and KLF4 demonstrated the highest levels of transcript expression and were upregulated relative to UHR with an average fold-change above 2.0. Elevated levels of expression were also seen for OCT4 and NANOG but not above the 2-fold cutoff. For SOX2, only 1 tissue sample had an expression higher than the UHR with the remaining 4 tissue samples showing downregulated expression relative to UHR. In the HPWS-derived primary cell lines, c-MYC was the only marker that had a higher expression level relative to UHR for all 3 HPWS tissue samples. Higher expression of KLF4 relative to UHR was seen in only 1 cell line sample. The expression of NANOG, SOX2 and OCT4 was downregulated relative to UHR.
Specific amplification of the products was demonstrated by electrophoresis with only the expected size amplicons observed, and no products observed in the no template control or no RT reactions (Supplemental Digital Content Figure 4, http://links.lww.com/JV9/A5).
OCT4, SOX2, KLF4, and c-MYC proteins were present in HPWS-derived primary cell lines
WB was used to demonstrate protein expression of the iPSC markers in 3 HPWS-derived primary cell lines (Figure 7). OCT4 was detected in all 3 samples at approximately 36 kDa (Figure 7A). SOX2 was detected in all 3 samples at approximately 38 kDa (Figure 7B). NANOG was not detected in any of the 3 samples (Figure 7C). KLF4 was present in all 3 samples with a band at approximately 55 KDa. There was a further band at approximately 27 kDa (Figure 7D), which is likely to be a nonspecific band, possibly IgG light chain. c-MYC was detected in all 3 samples at approximately 53 kDa (Figure 7E). NTERA2 was used as a positive control for all 3 samples. Immunostaining of human dermal fibroblast cell line showed no expression of these iPSC markers (Supplemental Digital Content Figure 5A–E, http://links.lww.com/JV9/A6). α-Tubulin staining confirmed approximately equal total protein loading for the cell lines (Figure 7F, and Supplemental Digital Content Figure 5F, http://links.lww.com/JV9/A6; ~50 kDa).
The pathogenesis of PWS remains unknown and current treatment is empirical and unsatisfactory. The presence of genes commonly found in PWS, such as ERK and JNK, has been shown to enhance the function of ESC markers through the Ras pathway21,22 (Figure 1). Dysregulation of EPC differentiation by EphB1 and EfnB2 has been linked to the development of PWS, and this process can be amplified by the presence of the ESC markers OCT4, SOX2, and NANOG.14,19 Along with the identification of the EPC markers CD133 and CD1666 on the lesional vessels in PWS, the demonstration of the expression of the iPSC markers in this study suggests the existence of ESC-like cells within PWS. Figure 1 highlights the potential action of the Rac pathway and of VEGF acting directly and GNAQ mutation acting indirectly on the Ras pathway on the expression of iPSC markers that we have demonstrated in HPWS in this study.
Immunohistochemical staining demonstrated the expression of OCT4, SOX2, and c-MYC on the endothelium and media of the lesional vessels and also cells within the stroma, and KLF4 was expressed on the endothelium of the lesional vessels in all 15 HPWS tissue samples, whereas NANOG was not detected in any sample. This was supported by the presence of OCT4, SOX2, KLF4, and c-MYC transcripts in all 6 tissue samples by ISH. RT-qPCR analysis on the same 6 HPWS tissue samples confirmed the presence of transcripts of all 5 iPSC markers with low expression of SOX2, and all 5 iPSC markers in the 3 HPWS-derived primary cell lines, with low expression of NANOG, SOX2, and OCT4, relative to UHR. Variability in transcript expression of the iPSC markers for both the HPWS tissue samples and HPWS-derived primary cells lines was demonstrated, with the largest variation seen with c-MYC and KLF4 in the tissue samples and KLF4 in the primary cell lines. WB on the same 3 HPWS-derived primary cell lines confirmed protein expression of OCT4, SOX2, KLF4 and c-MYC at their appropriate molecular weights, whereas NANOG was not detected. Expression of iPSC markers in normal vessels has not been reported. Immunohistochemical staining showed no expression of OCT4 and NANOG, whereas SOX2, c-MYC, and KLF4 were present on the epidermis of the normal skin with SOX2 and KLF4 also expressed by some cells but not blood vessels within the stroma. Immunofluorescence staining showed the presence of an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population within the endothelium and media of the lesional vessels and cells within the stroma. Although some cells within the stroma of normal skin expressed SOX2 and KLF4, the colocalization of OCT4 and c-MYC to these SOX2+/KLF4+ cells within the stroma of HPWS tissues suggests a unique population.
The expression of OCT4 and NANOG is intricately connected.26 High expression of OCT4 has been shown to increase NANOG expression, so as to maintain pluripotency of a cell.27 The weak NANOG expression in the presence of a strong OCT4 expression on the endothelium and media of the lesional vessels and cells within the stroma, demonstrated by immunofluorescence dual-staining, is interesting. Repression of NANOG may be necessary for the formation of dilated capillary-venular channels. During embryogenesis, repression of NANOG is needed for cells to differentiate (Luke Krishnan et al, unpublished data, 2020).28 In conditions in which repression of NANOG activity is absent, such as in some ovarian cancers, NANOG is responsible for increased cell proliferation, invasion, and migration, along with creating a population of cells that are poorly differentiated.29 NANOG activity is also autorepressive and is independent of the expression and/or interaction it may have with OCT4 and other ESC markers such as SOX2.29 Unlike OCT4 and SOX2, NANOG is not required for the viability of ESCs, consistent with the fact that NANOG is not a regulator of OCT4/SOX2 expression.29 The limited function of NANOG supports the involvement of JNK in PWS, as JNK activates BMP, which is a suppressor of NANOG19 (Figure 1). The limited function of NANOG also suggests that although OCT4, SOX2, and NANOG have the capability of enhancing the transcription of EphB1, OCT4 and SOX2 may be the only markers that actually have a role in the function within PWS.
We have demonstrated the presence of an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population within HPWS. It is possible that the absence of NANOG leads to abnormal differentiation of the primitive cells,19 leading to the aberrant formation of capillaries in PWS. Further work including functional studies is needed to confirm the presence of such a primitive population and its relationship with somatic mutations of GNAQ9 and other genes observed in PWS vessels, through their action on the Ras pathway, initiate, and/or perpetuate the ESC-like cells in PWS.
The authors would like to thank Ms. Liz Jones of the Gillies McIndoe Research Institute for her assistance with the hematoxylin and eosin, immunohistochemical staining, and in situ hybridization staining. Aspects of this work were presented at the International Study for the Society of Vascular Anomalies On-line Workshop, May 14–15, 2020, Vancouver, BC, Canada.
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