Stemness-Associated Markers Are Expressed in Extracranial Arteriovenous Malformation

Objectives: Arteriovenous malformation (AVM) consists of a nidus with poorly formed low-resistance vessels in place of a functional capillary network. The role of somatic mutations in embryonic stem cells (ESCs) and vascular anomalies and the presence of primitive populations in vascular anomalies led us to investigate the presence of a primitive population in extracranial AVM. Methods: Extracranial AVM tissue samples from 12 patients were stained for stemness-associated markers OCT4, SOX2, NANOG, KLF4, and c-MYC using immunohistochemical staining. In situ hybridization (ISH) was performed on six tissue samples to determine transcript expression. Western blotting and RT-qPCR were performed on two AVM-derived primary cell lines to determine protein and transcript expression of these markers, respectively. Immunofluorescence staining was performed on two tissue samples to investigate marker co-localization. Results: Immunohistochemical staining demonstrated the expression of OCT4, SOX2, KLF4, and c-MYC on the endothelium and media of lesional vessels and cells within the stroma of the nidus in all 12 AVM tissue samples. ISH and RT-qPCR confirmed transcript expression of all five markers. Western blotting showed protein expression of all markers except NANOG. Immunofluorescence staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ population within the endothelium and media of the lesional vessels and cells within the stroma of the AVM nidus. Conclusions: Our findings may suggest the presence of a primitive population within the AVM nidus. Further investigation may lead to novel therapeutic targeting of this population.


INTRODUCTION
Extracranial arteriovenous malformation (AVM) is a rare (1), non-heritable condition (2) with no sex or racial predilection (3). This high-flow vascular malformation (1,4) is present at birth and progresses during adolescence or adulthood (1,3). AVM is characterized by a central nidus, consisting of poorly formed vessels with direct connections between the high-flow arteries and low-flow veins, in place of normal capillaries (5) causing arteriovenous shunting (6). Histologically, AVM has a fibrous background underlying tortuous small vessels intermingled with thick-walled larger vessels with luminal dilation (5,7,8). Although predominantly affecting the head and neck, AVM can occur anywhere in the body. It is diagnosed by careful history and examination and confirmed by ultrasonography to identify arteriovenous shunting (9), magnetic resonance, and/or superselective angiography (10), demonstrating arterial feeders and draining vein(s) (1).
Standard treatment for AVM is surgical excision of the nidus and reconstruction following preoperative embolization (7), as required. Yakes (11) reports the efficacy of direct ethanol injection into the nidus, with ethanol embolization of arterial feeders and outflow vein(s) (12). Ethanol destroys the endothelium and induces thrombosis in the channels, preventing neovascular recruitment, or recanalization and minimizing risk of recurrence (11). Management of AVM remains challenging with a high incidence of recurrence (3).
The pathogenesis of AVM remains unclear and may result from an embryonic vascular developmental error (1). A somatic activating mutation in the MAP2K1 gene causing endothelial dysfunction via increased MEK1 activity has been proposed (2,13). Extracellular diffusion of von Willebrand factor (vWF) and staining on the pericellular rim in AVM has demonstrated endothelial cell dysfunction and leakage (14). Endothelial localization of the mutation correlates with severe phenotypic alterations in blood vessels of mutated mice (2,15).
Takahashi et al. have demonstrated induction of pluripotent stem cells from adult mouse (17) and human (20) fibroblasts by introducing the transcription factors OCT4, SOX2, c-MYC, and KLF4. Yu et al. (21) also achieve this with NANOG and LIN28 in place of c-MYC and KLF4, underscoring the sufficiency of these four transcription factors (17) to generate induced pluripotent stem cells (iPSCs).
OCT4, SOX2, and NANOG confer ESC identity through specification of ESC fate and self-renewal properties (22). OCT4 and SOX2 collaboratively induce nuclear transcription of target genes such as NANOG (23). Interestingly, NANOG-deficient iPSCs are transcriptionally similar to wild-type iPSCs (24). KLF4 additionally maintains vascular integrity during embryogenesis and reprograms somatic cells into iPSCs (25). c-MYC maintains cellular proliferation, growth, and survival by controlling cell cycle regulator expression (26). In this manuscript, these markers are referred to as stemness-associated markers.
The presence and, in particular, the patterns of expression of stemness-associated markers in vascular anomalies suggest a role for stem cells in their pathogenesis. Endothelial expression of stem cell marker CD133 has been demonstrated in vessels and stromal cells of lymphatic malformation, and NANOG and OCT4 genes are present within CD133+ patient-derived cells (27).
Negative controls for immunofluorescence staining were performed on AVM 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 Leica BOND TM RX auto-stainer using a BOND Detection system (cat#DS9455, Leica).

Image Capture and Analysis
Immunohistochemical-stained slides were viewed and imaged on the Olympus BX53 light microscope with an Olympus SC100 camera (Olympus, Tokyo, Japan) and processed with cellSens 2.0 software (Olympus). Immunofluorescence-stained slides were viewed and imaged with an Olympus FV1200 biological confocal laser-scanning microscope and processed with cellSens Dimension 1.17 software (Olympus).

RT-qPCR
Total RNA was isolated from two AVM-derived primary cell lines. From frozen cell pellets of 5 × 10 5 viable cells, total RNA was extracted using the RNeasy Micro Kit (cat#74004, Qiagen, Hilden, Germany). An on-column DNAse digest (cat#79254, Qiagen) step was included to remove potentially contaminating genomic DNA. RNA quantity was determined using a NanoDrop2000 Spectrophotometer (ThermoFisher). 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. Primer probes used were OCT4 (Hs03005111_g1), SOX2 (Hs01053049_s1), NANOG (Hs02387400_g1), KLF4 (Hs00358836_m1), and c-MYC (Hs00153408_m1) (cat#4331182, ThermoFisher). Gene expression was normalized to the reference genes GAPDH (Hs99999905_m1), PUM1 (Hs00206469_m1), and PSMB4 (Hs00160598_m1) (cat#4331182, ThermoFisher). Universal human reference RNA (UHR; cat#CLT636690, Takara, Shiga, Japan), total RNA extracted from a range of healthy adult human tissues, was used as the calibrator for the 2 − Ct analysis. Nuclease-free water was added for the no template and no reverse transcriptase controls, and RNA from NTERA2 cells was used as the positive control. The presence of the correctly sized bands from the endpoint amplification products was confirmed using 2% agarose gel electrophoresis (cat#G402002, ThermoFisher) and imaged using the ChemiDoc MP (Bio-Rad, Hercules, CA, USA). Graphs were generated using GraphPad Prism (v8.0.2, Windows, San Diego, CA, USA) 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.

AVM Tissue Samples Showed the Characteristic Nidus
H&E staining demonstrated the characteristic nidus consisting of tortuous small vessels surrounded by arterioles and venules, intermingled with thickened arteries and dilated veins on a fibrous background (8) in 12 AVM tissue samples (Figure 1).

AVM Tissue Samples Expressed OCT4, SOX2, KLF4, and c-MYC Proteins
Immunohistochemical staining demonstrated nuclear and cytoplasmic expression of OCT4 on the endothelium and  media of lesional vessels, with nuclear staining of cells within the stroma of the nidus (Figure 2A), in all tissue samples. Nuclear and cytoplasmic expression of SOX2 and KLF4 and weaker nuclear expression of c-MYC were demonstrated on the endothelium and media of lesional vessels, with some staining on cells within the stroma of the nidus (Figures 2B,D) Figure 2F).

An OCT4+/SOX2+/KLF4+/c-MYC+ Population Was Present on the Endothelium and Media of the Lesional Vessels and Stroma of the Nidus of AVM
Dual immunofluorescence staining of vWF and SMA demonstrated vessel architecture with a vWF+ inner endothelium (Figure 3A, red) and SMA+ outer media (Figure 3A, green). The CD34+ endothelium (Figure 3B, green) and media of lesional vessels and cells within the stroma of the nidus expressed KLF4 (Figure 3B, red). NANOG (Figure 3C, red) was minimally expressed on the CD34+ endothelium (Figure 3C, green) and media and cells within the stroma of the nidus. SOX2 (Figure 3D, red) was expressed on the CD34+ endothelium ( Figure 3D, green) with nuclear staining in the media and in cells within the stroma of the nidus. Dual staining of KLF4 (Figures 3E,F, red) with OCT4 ( Figure 3E, green) and c-MYC (Figure 3F, green) confirmed the co-expression of KLF4 with both markers. NANOG (Figure 3G, red) was expressed on some OCT4+ (Figure 3G, green) endothelium of the lesional vessels. SOX2 (Figure 3H, red) and c-MYC (Figure 3H, green) were co-expressed on the endothelium and media and in cells within the stroma of the nidus.   (Figure 5D, red) and c-MYC was detected at 55 kDa (Figure 5E, red). Specificity was confirmed on the positive control (NTERA2 cell line). α-Tubulin loading control confirmed equal and consistent protein loading (Figure 5F).

AVM-Derived Primary Cell Lines Expressed OCT4, SOX2, NANOG, KLF4, and c-MYC Transcripts
RT-qPCR performed on two AVM-derived primary cell lines confirmed mRNA expression of OCT4, SOX2, NANOG, KLF4, and c-MYC (Figure 6). The results compared the expression of the stemness-associated markers in AVM-derived cell lines, relative to the expression of the same markers within healthy UHR. c-MYC was the only marker with a higher expression in AVM-derived primary cell lines, of approximately 1.5-and 3fold, relative to healthy UHR. KLF4 transcript expression was similar to healthy UHR. OCT4, followed by NANOG and SOX2, showed the least amounts of transcripts. NANOG and SOX2 showed minimal expression, relative to healthy UHR. Electrophoresis of qPCR products on 2% agarose gels (Supplementary Figure 5) demonstrated specific amplification of the products. The expected size amplicons were observed, and no products were observed in the no template or no reverse transcriptase control reactions (Supplementary Figure 5).

DISCUSSION
In this study, immunohistochemical staining demonstrated the expression of the stemness-associated markers OCT4, SOX2, KLF4, and c-MYC but not NANOG on the endothelium and media of lesional vessels and cells within the stroma of the nidus in all 12 AVM tissue samples. This was confirmed by the transcript expression of OCT4, SOX2, KLF4, and c-MYC on ISH analysis of AVM tissue samples and by the protein expression on WB analysis of AVM-derived primary cell lines. RT-qPCR performed on the same cell lines detected all five stemnessassociated markers, although NANOG and SOX2 showed minimal expression. Different combinations of the markers of interest were used during immunofluorescence staining, allowing us to efficiently investigate the co-expression within the tissues. Based on the co-expression of two markers on the same cells, we then assumed that in subsequent combinations where cells visibly expressed one of those markers, the other would also be expressed, as it was not feasible to co-stain for all five markers in one sample. This allowed us to deduce that all markers except NANOG were present and localized to the same areas within the vessels, demonstrating an OCT4+/SOX2+/KLF4+/c-MYC+ ESC-like population on the endothelium and media of lesional vessels and on cells within the stroma of the AVM nidus. RT-qPCR showed the lowest expression of SOX2, contrasting with its distinct immunohistochemical and immunofluorescence staining and transcript expression on ISH analysis of AVM tissues. However, it has been suggested that transcript levels are not entirely predictive of protein expression (32). Discrepancies between transcript and protein expression levels, as seen with SOX2 in this study, have been previously observed (32)(33)(34). It has been attributed to several factors, such as the role of posttranslational processing (32,33) in the variability of protein levels relative to mRNA transcripts. The discrepancy has also been explained by the variable lengths of mRNA and protein half-lives (32,34), the rate of translation, and its modulation by several factors (32) and by technical error which generates background noise (33,34). c-MYC expression on immunohistochemical ( Figure 2E) and immunofluorescence (Figures 3G,H) staining and WB ( Figure 5E) aligned with abundant transcript expression on ISH ( Figure 4D) and RT-qPCR analysis on AVM-derived primary cell lines (Figure 6). Interestingly, the oncoprotein c-MYC showed higher expression levels in AVM tissue than in healthy UHR. This supports the proliferative tendencies of AVM, suggesting a similar mechanism in cancer characterized by uncontrolled proliferation and dysregulated cellular processes, e.g., apoptosis and cell division (35). As KLF4 is associated with maintaining vascular integrity (25), its relatively high transcript and protein expression is consistent with the thickened vessels in AVM (8). Absent NANOG transcript expression aligns with the lack of NANOG protein expression on immunohistochemical ( Figure 2C) and WB (Figure 5C) analysis.
Variable transcript expression is characteristic of iPSCs and ESCs, which display heterogeneous expression and temporal fluctuation of pluripotency genes including KLF4 and NANOG (36). OCT4, SOX2, and NANOG are the most primitive ESC markers (35), regulated by feedback loops (36) which allow downregulation and compensatory upregulation of other markers. This could explain the low SOX2 expression on RT-qPCR, alongside the relatively higher expression of NANOG and OCT4. NANOG was detected in the cell line samples using RT-qPCR, but not by WB. This may suggest transcriptional but not translational activation of NANOG. Although it is possible that this is due to the cell line constituents failing to accurately represent the composition of the tissues, the absence of NANOG on WB is unsurprising, as it has been proposed that NANOG is dispensable in induced pluripotency and the formation of iPSC under optimal cell culture conditions (24).
iPSCs are generated from somatic cells by overexpression of transcription factors OCT4, SOX2, NANOG, KLF4, and c-MYC, which are markers of iPSCs and facilitate the reprogramming of somatic cells back to a pluripotent state (37). The presence of the stemness-associated markers within lymphatic malformation (31) and AVM suggests the presence of an iPSC phenomenon which may be initiated and perpetuated by certain somatic mutations. A causative link between the genetic instability of iPSCs during reprogramming and an increased risk of disease has been proposed (37), with pre-existing mutations in somatic cells identified as the primary cause (15).
Somatic mutations have been identified in many types of vascular anomalies, including extracranial AVM (2,15). It has been proposed that AVM results from a vascular developmental error during embryogenesis (3,7,38,39), namely a somatic activating mutation in the MAP2K1 gene causing endothelial dysfunction through increased MEK1 activity (2).
Vaskova et al. (37) suggest that iPSCs often acquire genetic and epigenetic aberrations such as mutations during the reprogramming, when they display genomic instability and DNA damage (40). We speculate that somatic activating mutations may perpetuate a similar population underscoring the progression of AVM. The demonstration of cells in AVM that express stemness-associated markers may suggest the presence of ESC-like cells, and this warrants further investigation. The presence of such a primitive population would be consistent with the finding of ESC-like cells in other vascular anomalies such as infantile hemangioma (28), pyogenic granuloma (29), venous malformation (22), verrucous venous malformation (30), and lymphatic malformation (31).
This study demonstrated the presence of an OCT4+/SOX2+/KLF4+/c-MYC+ population on the endothelium and media of lesional vessels and on cells within the stroma of the AVM nidus. Further studies with a larger sample size including functional experiments are required to confirm our findings. Components of the renin-angiotensin system (RAS) have been shown to be expressed by similar populations in infantile hemangioma (41), pyogenic granuloma (42), and venous malformation (43,44). Investigation into the expression of RAS on a primitive population in extracranial AVM may lead to novel targeting of the population using available medications.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Central Health and Disability Ethics Committee (Ref. 13/CEN/130). The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
ST formulated the study hypothesis and design. CLK, PD, HB, and ST interpreted the immunohistochemical data. NB performed the confocal microscopy. CLK, PD, NB, and ST interpreted the immunofluorescence data. NB performed the WB analysis. NB, CLK, PD, and ST interpreted the WB data. JP performed the RT-qPCR experiments. JP, CLK, and ST interpreted the RT-qPCR data. EP performed the cell culture.
CLK and ST drafted the manuscript. CLK, PD, and ST revised the manuscript. All authors commented on and approved the manuscript.