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Scientific Research (Molecular, Genetic, Histologic)

Expression of Cathepsins B, D, and G in Hypertrophic Port-wine Stain

Koh, Sabrina P.a; Brasch, Helen D.a; Patel, Josiea; Bockett, Nicholasa; Paterson, Erina; Davis, Paul F.a; Tan, Swee T.a,b,c

Author Information
Journal of Vascular Anomalies: September 2021 - Volume 2 - Issue 3 - p e022
doi: 10.1097/JOVA.0000000000000022

Abstract

Introduction

Port-wine stain (PWS), the most common form of capillary malformation, affects 0.3% of newborns.1–3 Present at birth as a flat pink macule, PWS grows with the child and progressively darkens with time, and the affected skin thickens, often forming nodules.2,4–6 PWS occurs most commonly in the head and neck region, typically along the distribution of the trigeminal nerve.3,6 It causes disfigurement and psychological stress and/or functional impairments such as visual obstruction.3

Histologically, PWS is characterized by the presence of ectatic capillary-venular channels within the papillary and upper reticular dermis, without endothelial proliferation.1 The mainstay treatment for PWS is selective photothermolysis of the dilated vessels using pulsed dye laser,2,3,5 with complete clearance achieved in less than 10% of the patients.6–8 Surgical debulking is used for hypertrophic PWS (hPWS) with modest results.1

PWS can be associated with syndromes such as Sturge-Weber syndrome—a neurocutaneous condition characterized by the presence of PWS along the distribution of the ophthalmic division of the trigeminal nerve and leptomeningeal and/or ocular involvement,3 and Klippel-Trenaunay syndrome characterized by capillary-lymphatico-venous malformation often with soft tissue and/or bony hypertrophy of the affected extremity.9

The biology of PWS is poorly understood. One theory proposes a deficiency of innervation of the affected capillary-venular channels, resulting in reduced sympathetic tone10 with subsequent dilation of the affected vessels.11,12 The role of somatic mutations in the pathogenesis of PWS is an area of emerging interest. A single amino acid substitution mutation (arginine to glutamine, p.Arg183GIn), encoding a somatic activating mutation of the GNAQ gene on chromosome 9q21, has been identified in syndromic13 and nonsyndromic13,14 PWS. Not all PWS express this GNAQ gene mutation, and mutations of the GNA11 gene15,16 and the PIK3CA gene17,18 have also been observed in PWS. The precise role of these mutations in the pathogenesis of PWS is yet to be elucidated.

A cell population that expresses stemness-associated markers has been demonstrated in vascular anomalies19 including many vascular tumors such as infantile hemangioma20,21 and pyogenic granuloma,22 and vascular malformations such as arterio-venous malformation (AVM),23 venous malformation,24 verrucous venous malformation,25 and lymphatic malformation.26 We have recently demonstrated the presence of an OCT4+/SOX2+/NANOG–/KLF4+/c-MYC+ embryonic stem cell (ESC)-like population on the endothelium and media of lesional vessels and cells within the stroma in hPWS.27 The expression of cathepsins B and D by the primitive population and cathepsin G by phenotypic mast cells has been demonstrated in infantile hemangioma28 and lymphatic malformation,29 in the microvessels of fibroproliferative conditions such as Dupuytren’s disease30 and keloid disorder,31 and many cancer types.32–35 Expression of cathepsins B and D has also been observed in abdominal aortic aneurysm,36 and cathepsin B in varicose veins37 and murine models of cerebral aneurysm.38,39

Cathepsins B, D, and G are proteolytic enzymes with a proposed role in regulating the aberrant growth of the primitive population within vascular anomalies, fibrotic conditions and cancer. The functional activity of various cathepsins is dependent on the surrounding environmental niche.40 Cathepsin B, a cysteine protease, cathepsin D, an aspartyl protease, and cathepsin G, a serine protease, are involved in extracellular matrix (ECM) remodeling.40 The homeostatic composition of the ECM is fundamental for maintaining tissue integrity, regulating gene expression, and other functions.40 Cathepsins B, D, and G also constitute bypass loops of the renin-angiotensin system (RAS), which is involved in many pathological states, by driving local aberrant processes including inhibition of apoptosis, promoting cellular proliferation and angiogenesis.41,42

This study investigated the expression of cathepsins B, D, and G in hPWS and their localization to the ESC-like population we have recently demonstrated in hPWS.27

Materials and methods

hPWS tissue samples

hPWS tissue samples from 8 male and 7 female patients aged 12–71 years (mean 39.13 years) (Supplementary Table 1, https://links.lww.com/JV9/A26), included in our previous study,27 were sourced from the Gillies McIndoe Research Institute Tissue Bank for this study. Ethics approval was approved by the Central Regional Health and Disability Ethics Committee (Ref. 13/CEN/130). Written informed consent was obtained from all patients.

hPWS-derived primary cell lines

Primary cell lines were derived from 3 fresh hPWS tissue samples from the original cohort of 15 patients. Tissues were cultured with Matrigel explant (Catalog No. 354234, Corning, Tewksbury, MA), in a 24-well plate, with a media containing Dulbecco’s Modified Eagle Medium with Glutamax (Catalog No. 10569010, Gibco, Rockford, IL), supplemented with 2% penicillin-streptomycin (Catalog No. 15140122, Gibco), and 0.2% gentamicin/amphotericin (Catalog No. R01510, Gibco). Cells were extracted using Dispase (Catalog No. 354235, Corning) and cultured and passaged in adherent culture flasks containing Dulbecco’s Modified Eagle Medium with Glutamax, supplemented with 10% fetal bovine serum (Catalog No. 10091148, Gibco), 5% mTeSR 1 Complete Medium (Catalog No. 85850, StemCell Technologies, Vancouver, BC, Canada), 1% penicillin-streptomycin (Catalog No. 15140122, Gibco), and 0.2% gentamicin/amphotericin B (Catalog No. R01510, Gibco). All cultures were maintained in a humidified incubator at 37°C with 5% CO2. All hPWS-derived primary cell lines used for the experiments were between passages 5 and 6.

Histochemical and immunohistochemical staining

Four μm-thick formalin-fixed paraffin-embedded sections of 15 hPWS tissue samples were subjected to hematoxylin and eosin staining to confirm diagnosis of hPWS by an anatomical pathologist. Immunohistochemical staining was performed on sections of the same 15 hPWS samples, with primary antibodies to cathepsin B (1:200; Catalog No. nBPI-19797, Novus. Biologicals, Denver, Littleton, CO), cathepsin D (1:2000; Catalog No. ab75852, Abcam, Cambridge, MA), and cathepsin G (1:100; Catalog No. nBP2-33498, Novus. Biologicals), using the Leica BOND RX auto-stainer (Leica, Nussloch, Germany), with 3,3′-diaminobenzidine as the chromagen. Antibodies were diluted with BOND primary antibody diluent (Catalog No. AR9352, Leica). Immunohistochemical-stained slides were mounted in Dako Mounting Medium (Catalog No. CS703, Dako, Glostrup, Denmark).

Positive human control tissues used were placenta for cathepsin B, breast carcinoma for cathepsin D, and tonsil for cathepsin G. Negative controls were performed on hPWS samples using rabbit (ready-to-use; Catalog No. IR600, Dako), or mouse (ready-to-use; Catalog No. IR750, Dako), matched isotype controls.

To confirm co-expression of proteins, immunofluorescence staining was performed on 2 representative hPWS tissue samples included in immunohistochemical staining, with the same primary antibodies with same concentrations. An additional cathepsin B antibody, raised in mouse (1:200; Catalog No. ab58802, Abcam), was used to facilitate multiplexing. Dual staining of the cathepsins was performed with CD31 as the endothelial marker (ready-to-use; Catalog No. PA0414, Leica); ESC markers OCT4 (1:30; Catalog No. 309-M-16, Cell Marque, Rocklin, CA) and SOX2 (1:500; Catalog No. PA1-094, Thermo Fisher Scientific, Waltham, MA), as surrogate markers of the primitive population, we have previously demonstrated within hPWS27; and mast cell markers tryptase (1:300; Catalog No. nCL-MCTRYP-428, Leica) and chymase (1:2000; Catalog No. ab2377, Abcam). An appropriate fluorescence secondary antibody of either Alexa Fluor anti-mouse 488 (1:500; Catalog No. A-21202, Life Technologies, Carlsbad, CA), Alexa Fluor anti-rabbit 594 (1:500; Catalog No. A-21207, Life Technologies), VectaFluor Excel anti-mouse 488 (ready-to-use; Catalog No. DK-2488, Vector Laboratories, Burlingame, CA), or VectaFluor Excel anti-rabbit 594 (ready-to-use; Catalog No. DK-1594, Vector Laboratories), was used to detect the primary antibodies. All antibodies were diluted using BOND primary antibody diluents (Catalog No. AR9352, Leica).

Immunofluorescence-stained slides were mounted in Vectashield HardSet mounting medium and counter-stained with 4′,6-diamino-2-phenylindole (Catalog No. H-1500, Vector Laboratories). To confirm specificity of the amplification cascade used in immunofluorescence staining, negative controls were performed on sections of hPWS using a matched isotype control for both mouse (ready-to-use; Catalog No. IR750, Dako) and rabbit (ready-to-use; Catalog No. IR600, Dako) primary antibodies.

Image analysis

Immunohistochemical-stained slides were visualized and imaged 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). An Olympus FV1200 biological confocal laser-scanning microscope was used to view and image immunofluorescence-stained slides, and the images were processed with cellSens Dimension 1.11 software (Olympus).

Western blotting

Six hPWS tissue samples and the 3 hPWS-derived primary cell lines from the original cohort of 15 patients were subject to western blotting. Total protein for hPWS tissue samples was extracted and subjected to pestle homogenization (Catalog No. PES-15-B-SI, Corning) in ice-cold Radioimmunoprecipitation Assay Buffer (Catalog No. 89900, Pierce Biotechnology, Rockford, IL, USA), supplemented with a protease and phosphatase inhibitor cocktail (Catalog No. 78440, Pierce Biotechnology). Protein from cell lines was extracted as above, without the homogenization step. Protein was quantified using a Pierce BCA Protein Assay Kit (Catalog. No. 23227, Thermo Fisher Scientific) and diluted in an equal volume of 2x LDS (Catalog No. B0007, Invitrogen, Carlsbad, CA). Gel electrophoresis was performed using 20 µg of total protein, separated using SDS page as it is a product name page on 4%–12% Bis-Tris gels (Catalog No. NW04122BOX, Invitrogen) in MES SDS running buffer as it is a product name running buffer (Catalog No. B0002, Invitrogen), and transferred to a polyvinylidene difluoride membrane (Catalog No. IB24001, Invitrogen) using the iBlot 2 (Catalog No. IB21001, Thermo Fisher Scientific). Protein was detected on the iBind Flex (Catalog No. SLF1010, Thermo Fisher Scientific), using primary antibodies for cathepsin B (1:1000; Catalog No. ab58802, Abcam, Cambridge, United Kingdom), cathepsin D (1:1000; Catalog No. ab75852, Abcam), and α-tubulin (1:2000; Catalog No. 62204, Thermo Fisher Scientific). Appropriate secondary antibodies of anti-rabbit horseradish peroxidase (1:1000; Catalog No. ab6721, Abcam) and anti-mouse (1:1000; Catalog No. 6789, Abcam) were used to detect primary antibodies. HepG2 cell line was used as a positive control for both cathepsins B and D, and human tonsil as a positive control for cathepsin B. Band detection and analysis was performed using Clarity Western ECL substrate (Catalog No. 1705061, Bio-Rad, Hercules, CA), the ChemiDoc MP Imaging System (Bio-Rad), and Image Lab 6.0 software (Bio-Rad). Western blot analysis was not performed for cathepsin G, as it was expressed only by a few cells scattered throughout hPWS tissues.

Reverse-transcription quantitative polymerase chain reaction

Total RNA was extracted from 6 snap-frozen hPWS tissue samples and 3 hPWS-derived primary cell lines from the original cohort of 15 patients. Tissue samples were homogenized using the Omni tissue homogenizer (Omni TH, Omni International, Kennesaw, GA), then subjected to the RNeasy Mini Kit protocol (Catalog No. 74104, Qiagen, Hilden, Germany). RNA was extracted from frozen cell pellets of 5 × 105 viable cells, using the RNeasy Micro kit protocol (Catalog No. 74004, Qiagen). An on-column DNase digest (Catalog No. 79254, Qiagen) step was included to remove potential contaminating genomic DNA. RNA yield of each sample was quantified using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Transcriptional expression was analyzed in triplicate using the Rotor-Gene Q (Qiagen), Rotor-Gene Multiplex RT-PCR Kit (Catalog No. 204974, Qiagen), and TaqMan Gene Expression Assay primer probes (Catalog No. 4331182, Thermo Fisher Scientific) on 40 ng of RNA. The TaqMan Assays used were cathepsin B (Hs00157194_m1), cathepsin D (Hs00157205_m1), and cathepsin G (Hs01113415_g1), with gene expression normalized to the reference genes glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1) and pumilio homolog 1 (Hs00206469_m1). Universal human reference RNA (UHR; Catalog No. CLT636690, Takara, Shiga, Japan) was used as the calibrator for the 2ΔΔCt analysis. Nuclease-free water was used as the No Template Control, and human tonsil tissue for the positive control. The specificity of the probes was confirmed using 2% agarose gel electrophoresis (Catalog No. G402002, Thermo Fisher Scientific), loaded with PCR amplification products and imaged using the ChemiDoc MP (Bio-rad). GraphPad Prism (v8.0.2, San Diego, CA) was used to generate graphs with results expressed as fold-change relative to UHR. A fold-change cutoff was set at greater than 2.0 for upregulated genes and less than 0.5 for downregulated genes.

Enzymatic activity assays

Enzymatic activity assays for cathepsin B (Catalog No. ab65300) and cathepsin D (Catalog No. ab65302) were performed on 6 snap-frozen hPWS tissue samples from the original cohort of 15 patients. These assays contained cathepsins B and D substrate, labeled with a fluorescent marker of amino-4-trifluoromethyl coumarin or 7-methoxycoumarin-4-acetic acid, respectively. Cell substrates when exposed to enzymatically active cathepsins, were cleaved to release the fluorescent marker, which was read in a fluorometer. Tonsil tissue was used as a positive control. To confirm the specificity of the reaction, a negative control was made by boiling an aliquot of the extracted sample for 10 minutes to denature the enzyme. An assay was not performed on cathepsin G, given that it was expressed only by scattered cells in hPWS tissues.

Results

Cathepsins B, D, and G were expressed in hPWS tissues

The diagnosis of hPWS was confirmed on hematoxylin and eosin staining on all 15 tissue samples (Figure 1) by an anatomical pathologist. Immunohistochemical staining demonstrated expression of cathepsin B (Figure 2A) and cathepsin D (Figure 2B) on the endothelium and weak staining in the media of the lesional vessels and cells scattered throughout the stroma. Cathepsin G (Figure 2C) was expressed by a small number of cells within the stroma. The insets show enlarged views of the corresponding images. The staining patterns of these cathepsins on all 15 hPWS samples are presented in Supplementary Table 2 (https://links.lww.com/JV9/A27).

F1
Figure 1.:
Representative hematoxylin and eosin-stained slide of hypertrophic port-wine stain tissue sample demonstrating presence of dilated capillary-venular channels, surrounded by stroma. Original magnification: ×100. The box shows a magnified view of the dilated capillary-venular channels.
F2
Figure 2.:
Representative immunohistochemical-stained sections of hypertrophic port-wine stain demonstrating expression of cathepsins B (A, brown) and cathepsin D (B, brown) on the endothelium (long arrows) and media (short arrows) of the lesional vessels and cells within the stroma (arrowheads). Cathepsin G (C, brown) is expressed heterogeneously throughout the stroma (arrowheads). Nuclei are counter-stained with hematoxylin (A–C, blue). Original magnification: ×200. The insets show enlarged views of the corresponding images.

Positive human control tissues demonstrated the expected staining patterns of cathepsin B on placenta (Supplementary Figure 1A, https://links.lww.com/JV9/A23), cathepsin D on breast carcinoma (Supplementary Figure 1B, https://links.lww.com/JV9/A23), and cathepsin G on tonsil (Supplementary Figure 1C, https://links.lww.com/JV9/A23). A negative matched anti-mouse and anti-rabbit isotype control confirmed specificities of the primary antibodies (Supplementary Figure 1D, https://links.lww.com/JV9/A23).

Cathepsins B and D were localized to the OCT4+/SOX2+ endothelium and media of lesional vessels and cells within the stroma, and cathepsin G was expressed by tryptase+/chymase+/OCT4+ mast cells within the stroma of hPWS tissues

Immunofluorescence dual staining was performed to localize the expression of the cathepsins in relation to the CD31+ endothelium. Immunofluorescence staining demonstrated expression of cathepsin B (Figure 3A, red) and cathepsin D (Figure 3B, red), but not cathepsin G (Figure 3C, red), on the CD31+ endothelium (Figure 3A–C, green) and media of the lesional vessels.

F3
Figure 3.:
Representative immunofluorescence-stained sections of hPWS demonstrating expression of cathepsin B (A, red), cathepsin D (B, red), but not cathepsin G (C, red), on the CD31+ endothelium of lesional vessels (A–C, green). Cathepsin B (D, red; E, green) is expressed by the OCT4+ (D, green) and SOX2+ (E, red) cells on the endothelium (long arrows) and media (short arrows) of the lesional vessels, and cells within the stroma (arrowheads). Expression of cathepsin D (F, red) is demonstrated on the OCT4+ (F, green) endothelium (long arrows) and media (short arrows) of the lesional vessels and within the stroma (arrowheads) of hPWS. Cathepsin B (G, green) is expressed by the cathepsin D+ (G, red) cells. Expression of cathepsin G (H, red) is not observed on the OCT4+ (H, green) endothelium and media of the lesional vessels but is observed in OCT4+ (H, green, arrowheads) cells within the stroma. Cathepsin G (I–J, red) is expressed by the tryptase+ (I, green) and chymase+ (J, green) mast cells within the stroma (arrowheads). All slides are counter-stained with 4′,6-diamino-2-phenylindole. Original magnification: ×400. Scale bars: 20 μm. The insets show enlarged views of the corresponding images. hPWS indicates hypertrophic port-wine stain.

To investigate if these cathepsins were expressed by the primitive population we previously identified in hPWS,27 dual staining of these cathepsins was performed with ESC markers OCT4 and SOX2 that were used as surrogate markers of the ESC-like population recently identified.27 Cathepsin B (Figure 3D, red; Figure 3E, green) was expressed by the OCT4+ (Figure 3D, green) and SOX2+ (Figure 3E, red) cells on the endothelium and media of the lesional vessels and cells within the stroma. Cathepsin D (Figure 3F, red) was expressed by the OCT4+ (Figure 3F, green) population on the endothelium and media of the lesional vessels and by cells within the stroma of hPWS tissue samples.

Immunofluorescence dual staining of cathepsin B and cathepsin D was performed to investigate if these cathepsins were expressed by the same population of cells. This showed that the cathepsin B+ (Figure 3G, green) cells on the endothelium and media of the lesional vessels, and cells within the stroma, also expressed cathepsin D (Figure 3G, red).

Cathepsin G (Figure 3H, red) was not expressed by the OCT4+ (Figure 3H, green) cells on the endothelium and media of the lesional vessels but was expressed by a small number of OCT4+ cells within the stroma. Immunofluorescence dual staining of cathepsin G with mast cells markers tryptase and chymase showed that cathepsin G (Figure 3I, J, red) was expressed by the tryptase+ (Figure 3I, green) and chymase+ (Figure 3J, green) mast cells within the stroma.

Magnified figure insets have been provided to show enlarged views of the corresponding images. Split images of individual markers presented in Figure 3 are shown in Supplementary Figure 2A–T (https://links.lww.com/JV9/A24). Specificity of the amplification cascade was confirmed using matched mouse and rabbit isotype controls (Supplementary Figure 2U, https://links.lww.com/JV9/A24).

Transcripts of cathepsins B, D, and G were expressed in hPWS tissues, and transcripts of cathepsins B and D but not cathepsin G were expressed in hPWS-derived primary cell lines

Reverse-transcription quantitative polymerase chain reaction detected transcripts of cathepsins B, D, and G in all 6 hPWS tissue samples (Figure 4A) normalized to the reference genes glyceraldehyde-3-phosphate dehydrogenase and pumilio homolog 1, with expression fold-change calculated, relative to UHR. Transcript expression of cathepsins B and D was not biologically significantly different to that of the UHR. There was a wide range in the levels of expression of cathepsin G across all 6 tissue samples, but the overall mean expression showed an 8-fold upregulation, relative to the UHR.

F4
Figure 4.:
Graph showing average 2ΔΔCt values of triplicate RT-qPCR runs for cathepsins B, D, and G in 6 hypertrophic port-wine stain (hPWS) tissue samples (A), and 3 hPWS-derived primary cell lines (B). Gene expression is normalized to reference genes GAPDH and PUM1, and expression expressed relative to that of healthy UHR. A fold-change cutoff is set at greater than 2.0 for upregulated genes and less than 0.5 for downregulated genes. CTSB indicates cathepsin B; CTSD, cathepsin D; CTSG, cathepsin G; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hPWS, hypertrophic port-wine stain; PUM1, pumilio homolog 1; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; UHR, universal human RNA.

Transcript expression of cathepsins B and D, but not cathepsin G, was present in the 3 hPWS-derived primary cell lines (Figure 4B). Transcript expression of cathepsin B was upregulated while expression of cathepsin D was downregulated in the cell lines, relative to the UHR.

Specific amplification of the PCR products was demonstrated using gel electrophoresis, which confirmed detection of cathepsins B, D, and G amplicons at the expected molecular weights for the hPWS tissue samples (Supplementary Figure 3A–E, https://links.lww.com/JV9/A25) and hPWS-derived cell lines (Supplementary Figure 3F–J, https://links.lww.com/JV9/A25).

Western blot analysis confirmed protein expression of cathepsins B and D in hPWS tissues and in hPWS-derived primary cell lines

Western blot analysis confirmed protein expression of cathepsin B (Figure 5A) in all 6 hPWS tissue samples and 3 hPWS-derived primary cell lines, at the expected molecular weight of 22 kDa. A band at 28 kDa was also observed in the 3 cell lines (Figure 5A). Both 22 kDa and 28 kDa bands are likely to represent varying weights of mature cathepsin B, when cleaved from its precursor pro-cathepsin B. Cathepsin D (Figure 5B) was expressed in all 6 hPWS tissue samples and 3 primary cell lines, at the expected molecular weight of 28 kDa. A larger band at 46 kDa was detected in all samples, which could either represent an intermediate form of cathepsin D or alternatively, pro-cathepsin D. α-Tubulin (Figure 5C) was used to demonstrate equal total protein loading of all tissue samples and cell lines. Positive controls were demonstrated using HepG2 for both cathepsins B and D, and also tonsil for cathepsin B (Figure 5A, B).

F5
Figure 5.:
Representative images of western blot analysis of 6 hPWS tissue samples and 3 hPWS-derived primary cell lines demonstrating expression of cathepsin B at the appropriate molecular weights of 28 kDa and 22 kDa (A), and cathepsin D at the appropriate molecular weights of 28 kDa (B). Western blot of α-tubulin (C, ~50 kDa) demonstrates equal protein loading of the samples, and protein expression at the expected molecular weight. Lanes 1–6 indicate the 6 hPWS tissue samples used, lanes 7–10 indicate the 3 hPWS-derived cell lines. HepG2 cells were used as positive controls. hPWS indicates hypertrophic port-wine stain.

Enzymatic activity assays showed that cathepsins B and D were active

Enzymatic activity assays confirmed activity of cathepsin B (Figure 6A) and cathepsin D (Figure 6B) in all 6 hPWS tissue samples. Human tonsil was used as the positive control, and denatured protein as the negative control to confirm specificity of the reaction.

F6
Figure 6.:
Enzymatic activity assays demonstrating activity of cathepsin B (A) and cathepsin D (B), in relative florescent units per 50 µg of total protein, on 6 snap-frozen hPWS tissue samples. Lanes 1–6 indicate the 6 hPWS tissue samples used, lane 7 indicates the positive control of human tonsillar tissue, and lane 8 is that of the negative control of denatured tonsil, which demonstrates no activity. hPWS indicates hypertrophic port-wine stain; PWS, port-wine stain; RFU, relative fluorescence unit.

Discussion

PWS affects 0.3% of the population.1–3 The mainstay treatment of PWS is pulsed dye laser therapy that achieves complete clearance in less than 10% of patients following multiple treatment sessions.6–8 Surgical debulking is used for patients with hPWS with modest results.1

We here demonstrate the novel finding of the expression of cathepsins B, D, and G in hPWS at the transcriptional and translational levels. Immunohistochemical staining showed expression of cathepsins B and D most prominently on the endothelium, and to a lesser extent, on the media of the lesional vessels and cells within the stroma, of hPWS. Western blotting confirmed protein expression of cathepsins B and D, and enzymatic activity assays showed that these cathepsins were active. Western blot analysis and enzymatic activity assays were not performed for cathepsin G, as expression was heterogeneously scattered throughout hPWS tissues. Reverse-transcription quantitative polymerase chain reaction demonstrated transcript expression of cathepsins B and D in all 6 hPWS tissue samples at fairly consistent levels, and in all 3 hPWS-derived primary cell lines, while cathepsin G transcripts were present in all 6 tissue samples at varying levels of expression, but absent in all hPWS-derived primary cell lines. This is consistent with the results of immunofluorescence staining that demonstrated cathepsin G expression by phenotypic mast cells, which may not have survived the culture process.

We have recently demonstrated the presence of an OCT4+/SOX2+/NANOG–/KLF4+/c-MYC+ ESC-like population within hPWS, localized to the endothelium and media of the lesional vessels and cells within the stroma of hPWS.21 In this study, OCT4 and SOX2 were used as surrogate markers of this primitive population. Immunofluorescence staining demonstrated expression of both cathepsins B and D on the OCT4+/SOX2+ population in all 3 compartments, while cathepsin G was expressed by the chymase+/tryptase+ mast cells within the stroma. The expression of OCT4 by these cathepsin G+ cells suggests a primitive phenotype. A primitive population has also been demonstrated in other vascular anomalies19 including IH,20 pyogenic granuloma,22 AVM,23 venous malformation,24 verrucous venous malformation,25 and lymphatic malformation.26

While the presence of cathepsins B, D, and G in hPWS is a novel finding, these cathepsins have been demonstrated in other vascular anomalies including IH,28 AVM,43 and LM,29 with cathepsins B and D similarly expressed by the primitive population on the endothelium and media of lesional vessels, and also cells within the stroma, while cathepsin G is expressed by phenotypic mast cells.28,29,43 Cathepsins B and D have also been observed in other vascular pathologies, with cathepsins B and D expressed in abdominal aortic aneurysm,36 and cathepsin B in varicose veins37 and in murine models of cerebral aneurysms.38,39 Similar to the findings from our study, these studies show that cathepsins B and D are localized to the endothelium and media of the affected vessels.36–38

While the role of cathepsins B, D, and G in vascular anomalies and other vascular diseases is an area of emerging research, these cathepsins have a recognized role in oncogenesis.44–50 Synthesized as an inactive pro-enzyme, pro-cathepsin B is activated to its active form either through autocatalysis or by other proteases including mature cathepsin B and cathepsin D.46 Cathepsin D, an aspartic protease, is synthesized as inactive pre-pro-cathepsin D, which is cleaved to generate pro-cathepsin D, which is subject to further proteolytic cleavage to form an active intermediate form, and mature active cathepsin D.51,52 Both cathepsins B and D contribute to ECM remodeling and proangiogenic processes.45,51–53 Secreted by mast cells and monocytes, cathepsin G, a serine protease, is associated with ECM remodeling40 and promoting cell aggregation.49 In tumorigenesis, the expression of cathepsin B is associated with angiogenesis, tumor invasion, and metastasis.46 Overexpression of cathepsin B has been observed in high-grade human gliomas.44,45 Cathepsin B knockdown models of human glioblastoma cell lines, using siRNA, are associated with reduced tumor-induced angiogenesis in in vitro conditions, and in in vivo environments using murine xenografts.44,45 Inhibition and up to 50% regression in tumor growth is also observed in murine glioblastoma models treated with cathepsin B siRNA vectors compared with controls.44 Less is known about the functional role of cathepsin B in vascular pathologies, although its expression is observed in murine models of cerebral aneurysms.38,39 Cathepsin B expression, which is either weak or absent in nonaneurysmal vascular walls, significantly increases following aneurysm induction.38,39 Furthermore, rats treated with the cathepsin inhibitor NC-2300, have a significant reduction in incidence of advanced cerebral aneurysms, compared with the noncathepsin inhibitor-treated control rats (50% versus 90%, respectively, P = 0.022),38 and murine models treated with cathepsin B siRNA demonstrate a reduction in rate of apoptosis of vascular smooth muscle cells.39 Overexpression of cathepsin D is a recognized marker of poor prognosis in human breast cancer and contributes to tumor angiogenesis, apoptosis, and proliferation.47,48 In an in vivo murine xenograft tumor model of 3Y1-Ad12 cells transfected with either human active cathepsin D, catalytically inactive pro-cathepsin D, or an empty vector, cathepsin D transfected models are associated with an increase in tumor surface area, vascular density and reduction in rate of apoptosis compared with the control group.47 In a murine model of breast tumor-related bone metastasis, cathepsin G inhibited models demonstrate reduced tumor microvessel density and reduced expression of proangiogenic factors including vascular endothelial growth factor, compared with controls.50

Cathepsins B, D, and G may also contribute to disease processes through their role as bypass loops of the RAS.41 Commonly recognized as a endocrine system with a prominent role in cardiovascular homeostasis, recent literature highlights the presence of a local (paracrine) RAS that may contribute to disease states.41 Components of the RAS have been identified on the primitive population in many types of vascular anomalies including IH,54 VM,55 and LM.56 In the classical RAS, angiotensinogen is cleaved by renin to form angiotensin I (ATI), which is further activated by angiotensin-converting enzyme, to the active peptide angiotensin II (ATII). ATII exerts its actions by binding with ATII receptor 1 and ATII receptor 2.41,57 The RAS is implicated in numerous pathological states, through actions mediated by the interaction of ATII with ATII receptor 2, by inhibiting apoptosis and promoting cellular proliferation and angiogenesis.41,57 Cathepsins B and D contribute to the activation pro-renin to renin. Cathepsin D has a further role in mediating the conversion of angiotensinogen to ATI, while cathepsin G increases ATII yield from angiotensinogen and ATI.41

The role of somatic mutations13,14,17,18,58 in both syndromic and nonsyndromic PWS is an emerging area of interest. It is interesting to note that these somatic mutations of the GNAQ and PIK3CA genes are most prominently identified on the endothelium, and to a lesser extent, in the media and stroma.17,58 Investigation into the relationship between somatic mutations and the primitive population that expresses cathepsins B and D, is worthwhile.

To the best of our knowledge, this is the first study demonstrating the expression of cathepsins B, D, and G in hPWS, with cathepsins B and D localizing to the primitive population and cathepsin G localizing to the phenotypic mast cells. While the functional role of these cathepsins in hPWS has yet to be fully elucidated, the role of these cathepsins in the pathogenesis of other vascular pathologies and malignancies has been demonstrated. Further studies with a larger sample size, and including functional investigations, are needed to fully elucidate the functional role of cathepsins B, D and G in hPWS.

Conclusions

Cathepsins B, D, and G are expressed in hPWS. Cathepsins B and D are localized to the ESC-like population within hPWS on the endothelium and media of the lesional vessels and cells in the stroma. Cathepsin G is expressed by phenotypic chymase+/tryptase+/OCT4+ mast cells within the stroma of hPWS. Further studies including a larger sample size and functional investigations, are needed to fully elucidate the functional role of cathepsins B, D and G in hPWS.

Acknowledgments

The authors would like to thank Ms Liz Jones of the Gillies McIndoe Research Institute for her assistance with the hematoxylin and eosin, immunohistochemical, and immunofluorescence staining.

Author contributions

Drs Davis and Tan conceived the idea and designed the study. Drs Koh, Brasch, and Tan interpreted the immunohistochemical data. Mr Bockett carried out confocal microscopy. Dr Koh, Mr Bockett, and Dr Tan interpreted the immunofluorescence data. Mr Bockett performed the western blot analysis. Dr Koh, Mr Bockett, and Dr Tan interpreted the western blot data. Ms Patel performed the reverse-transcription quantitative polymerase chain reaction experiments and interpreted the data. Ms Paterson performed cell culture experiments. Mr Bockett performed the enzymatic activity assays and interpreted the data. Dr Koh drafted the article. Dr Tan critically revised the article. All authors commented on and approved the article.

Previous presentations

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; and the American Society of Plastic Surgeons (virtual) Annual Meeting: Plastic Surgery—The Meeting, October 16–18, 2020.

References

1. Richter GT, Friedman AB. Hemangiomas and vascular malformations: current theory and management. Int J Pediatr. 2012;2012:645678.
2. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains. Clin Cosmet Investig Dermatol. 2015;8:27–33.
3. Ortiz AE, Nelson JS. Port-wine stain laser treatments and novel approaches. Facial Plast Surg. 2012;28:611–620.
4. Ashinoff R, Geronemus RG. Flashlamp-pumped pulsed dye laser for port-wine stains in infancy: earlier versus later treatment. J Am Acad Dermatol. 1991;24:467–472.
5. Kelly KM, Choi B, McFarlane S, et al. Description and analysis of treatments for port-wine stain birthmarks. Arch Facial Plast Surg. 2005;7:287–294.
6. Nguyen V, Hochman M, Mihm MC Jr, Nelson JS, Tan W. The pathogenesis of port wine stain and sturge weber syndrome: complex interactions between genetic alterations and aberrant MAPK and PI3K activation. Int J Mol Sci. 2019;20:E2243.
7. van der Horst CM, Koster PH, de Borgie CA, Bossuyt PM, van Gemert MJ. Effect of the timing of treatment of port-wine stains with the flash-lamp-pumped pulsed-dye laser. N Engl J Med. 1998;338:1028–1033.
8. Yohn JJ, Huff JC, Aeling JL, Walsh P, Morelli JG. Lesion size is a factor for determining the rate of port-wine stain clearing following pulsed dye laser treatment in adults. Cutis. 1997;59:267–270.
9. Jacob AG, Driscoll DJ, Shaughnessy WJ, Stanson AW, Clay RP, Gloviczki P. Klippel-Trénaunay syndrome: spectrum and management. Mayo Clin Proc. 1998;73:28–36.
10. Rosen S, Smoller B. Pathogenesis of port wine stains. A new hypothesis. Med Hypotheses. 1987;22:365–368.
11. Rydh M, Malm M, Jernbeck J, Dalsgaard CJ. Ectatic blood vessels in port-wine stains lack innervation: possible role in pathogenesis. Plast Reconstr Surg. 1991;87:419–422.
12. Smoller BR, Rosen S. Port-wine stains. A disease of altered neural modulation of blood vessels? Arch Dermatol. 1986;122:177–179.
13. Shirley MD, Tang H, Gallione CJ, et al. Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ. N Engl J Med. 2013;368:1971–1979.
14. Frigerio A, Wright K, Wooderchak-Donahue W, et al. Genetic variants associated with port-wine stains. PLoS One. 2015;10:e0133158.
15. Couto JA, Ayturk UM, Konczyk DJ, et al. A somatic GNA11 mutation is associated with extremity capillary malformation and overgrowth. Angiogenesis. 2017;20:303–306.
16. Polubothu S, Al-Olabi L, Carmen Del Boente M, et al. GNA11 mutation as a cause of Sturge-Weber syndrome: expansion of the phenotypic spectrum of Gα/11 mosaicism and the associated clinical diagnoses. J Invest Dermatol. 2020;140:1110–1113.
17. Yin R, Gao L, Tan W, et al. Activation of PKCα and PI3K kinases in hypertrophic and nodular port wine stain lesions. Am J Dermatopathol. 2017;39:747–752.
18. Lian CG, Sholl LM, Zakka LR, et al. Novel genetic mutations in a sporadic port-wine stain. JAMA Dermatol. 2014;150:1336–1340.
19. Kilmister EJ, Hansen L, Davis PF, Hall SRR, Tan ST. Cell populations expressing stemness-associated markers in vascular anomalies. Front Surg. 2020;7:610758.
20. Itinteang T, Tan ST, Brasch HD, et al. Infantile haemangioma expresses embryonic stem cell markers. J Clin Pathol. 2012;65:394–398.
21. Khan ZA, Boscolo E, Picard A, et al. Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice. J Clin Invest. 2008;118:2592–2599.
22. Blackwell MG, Itinteang T, Chibnall AM, Davis PF, Tan ST. Expression of embryonic stem cell markers in pyogenic granuloma. J Cutan Pathol. 2016;43:1096–1101.
23. Luke Krishnan CS, Brasch HD, Patel J, et al. Stemness-associated markers are expressed in extracranial arteriovenous malformation. Front Surg. 2021;8:621089.
24. Tan EMS, Siljee SD, Brasch HD, Enriquez S, Tan ST, Itinteang T. Embryonic stem cell-like subpopulations in venous malformation. Front Med (Lausanne). 2017;4:162.
25. Laing EL, Brasch HD, Steel R, et al. Verrucous hemangioma expresses primitive markers. J Cutan Pathol. 2013;40:391–396.
26. Eady EK, Brasch HD, de Jongh J, Marsh RW, Tan ST, Itinteang T. Expression of embryonic stem cell markers in microcystic lymphatic malformation. Lymphat Res Biol. 2019;17:496–503.
27. Williams J, Brasch H, Bockett N, et al. Induced embryonic stem cell-like population in hypertrophic port-wine stain. J Vasc Anom. 2021;2:e006..
28. Itinteang T, Chudakova DA, Dunne JC, Davis PF, Tan ST. Expression of cathepsins B, D, and G in infantile hemangioma. Front Surg. 2015;2:26.
29. Mehrotra S, van Schaijik B, Boyes K, et al. Expression of cathepsins B, D, and G in microcystic lymphatic malformation [published online ahead of print December 18, 2020]. Lymph Biol Res. doi:10.1089/lrb.2020.0047.
30. Tan K, Brasch HD, van Schaijik B, et al. Expression and localization of cathepsins B, D, and G in Dupuytren’s disease. Plast Reconstr Surg Glob Open. 2018;6:e1686.
31. Paterson C, Lee VMY, Brasch HD, et al. Expression of cathepsins B, D, and G by the embryonic stem cell-like population within human keloid tissues and keloid-derived primary cell lines. Plast Reconstr Surg. 2019;144:1338–1349.
32. Koh SP, Wickremesekera AC, Brasch HD, Marsh R, Tan ST, Itinteang T. Expression of cathepsins B, D, and G in isocitrate dehydrogenase-wildtype glioblastoma. Front Surg. 2017;4:28.
33. Mehrotra S, Wickremesekera SK, Brasch HD, et al. Expression and localization of cathepsins B, D and G in cancer stem cells in liver metastasis from colon adenocarcinoma. Front Surg. 2018;5:40.
34. Rahman RMA, van Schaijik B, Brasch HD, et al. Expression of cathepsins B, D, and G in WHO grade I meningioma. Front Surg. 2019;6:6.
35. Featherston T, Marsh RW, van Schaijik B, Brasch HD, Tan ST, Itinteang T. Expression and localization of cathepsins B, D, and G in two cancer stem cell subpopulations in moderately differentiated oral tongue squamous cell carcinoma. Front Med (Lausanne). 2017;4:100.
36. Lohoefer F, Reeps C, Lipp C, et al. Histopathological analysis of cellular localization of cathepsins in abdominal aortic aneurysm wall. Int J Exp Pathol. 2012;93:252–258.
37. Xu N, Zhang YY, Lin Y, et al. Increased levels of lysosomal cysteinyl cathepsins in human varicose veins: a histology study. Thromb Haemost. 2014;111:333–344.
38. Aoki T, Kataoka H, Ishibashi R, Nozaki K, Hashimoto N. Cathepsin B, K, and S are expressed in cerebral aneurysms and promote the progression of cerebral aneurysms. Stroke. 2008;39:2603–2610.
39. Guo D, Wang YW, Ma J, et al. Study on the role of cathepsin B and JNK signaling pathway in the development of cerebral aneurysm. Asian Pac J Trop Med. 2016;9:499–502.
40. Patel S, Homaei A, El-Seedi HR, Akhtar N. Cathepsins: proteases that are vital for survival but can also be fatal. Biomed Pharmacother. 2018;105:526–532.
41. Munro M, Wickremesekera A, Davis P, Marsh R, Tan S, Itinteang T. Renin-angiotensin system and cancer: a review. Integr Cancer Sci Ther. 2017;4:1–6.
42. George AJ, Thomas WG, Hannan RD. The renin-angiotensin system and cancer: old dog, new tricks. Nat Rev Cancer. 2010;10:745–759.
43. Hansen L, Brasch HD, Paterson E, et al. Expression of cathepsins B, D and G by embryonic stem cell-like cells in extracranial arterio-venous malformation.
44. Lakka SS, Gondi CS, Yanamandra N, et al. Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene. 2004;23:4681–4689.
45. Malla RR, Gopinath S, Gondi CS, et al. Cathepsin B and uPAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma. Cancer Gene Ther. 2011;18:419–434.
46. Podgorski I, Sloane BF. Cathepsin B and its role(s) in cancer progression. Biochem Soc Symp. 2003;70:263–276.
47. Berchem G, Glondu M, Gleizes M, et al. Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene. 2002;21:5951–5955.
48. Garcia M, Platet N, Liaudet E, et al. Biological and clinical significance of cathepsin D in breast cancer metastasis. Stem Cells. 1996;14:642–650.
49. Morimoto-Kamata R, Mizoguchi S, Ichisugi T, Yui S. Cathepsin G induces cell aggregation of human breast cancer MCF-7 cells via a 2-step mechanism: catalytic site-independent binding to the cell surface and enzymatic activity-dependent induction of the cell aggregation. Mediators Inflamm. 2012;2012:456462.
50. Wilson TJ, Nannuru KC, Futakuchi M, Singh RK. Cathepsin G-mediated enhanced TGF-beta signaling promotes angiogenesis via upregulation of VEGF and MCP-1. Cancer Lett. 2010;288:162–169.
51. Liaudet-Coopman E, Beaujouin M, Derocq D, et al. Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis. Cancer Lett. 2006;237:167–179.
52. Benes P, Vetvicka V, Fusek M. Cathepsin D–many functions of one aspartic protease. Crit Rev Oncol Hematol. 2008;68:12–28.
53. Kostoulas G, Lang A, Nagase H, Baici A. Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases. FEBS Lett. 1999;455:286–290.
54. Itinteang T, Brasch HD, Tan ST, Day DJ. Expression of components of the renin-angiotensin system in proliferating infantile haemangioma may account for the propranolol-induced accelerated involution. J Plast Reconstr Aesthet Surg. 2011;64:759–765.
55. Siljee S, Keane E, Marsh R, Brasch HD, Tan ST, Itinteang T. Expression of the components of the renin-angiotensin system in venous malformation. Front Surg. 2016;3:24.
56. Papali’i-Curtin JC, Brasch HD, van Schaijik B, et al. Expression of components of the renin-angiotensin system in pyogenic granuloma. Front Surg. 2019;6:13.
57. Ager EI, Neo J, Christophi C. The renin-angiotensin system and malignancy. Carcinogenesis. 2008;29:1675–1684.
58. Tan W, Nadora DM, Gao L, Wang G, Mihm MC Jr, Nelson JS. The somatic GNAQ mutation (R183Q) is primarily located within the blood vessels of port wine stains. J Am Acad Dermatol. 2016;74:380–383.
Keywords:

Capillary malformation; Cathepsin B; Cathepsin D; Cathepsin G; Embryonic stem cells; Hypertrophic; Induced pluripotent stem cell; Port-wine stain; Stemness-associated markers; Transcription factors

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Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Society for the Study of Vascular Anomalies.