Sirolimus and propranolol inhibit endothelial proliferation while detergent sclerosants induce endothelial activation,microparticle release and apoptosis in vitro

Abstract

Objectives

To investigate the effects of detergent sclerosants, sodium tetradecyl sulphate and polidocanol, on endothelial cell activation and microparticle release and the effects of detergent sclerosants, sirolimus and propranolol, on apoptosis in vitro.

Methods

Cultured human umbilical vein endothelial cells and murine haemangioendothelioma (EOMA) cell lines were incubated with different concentrations of sodium tetradecyl sulphate and polidocanol, as well as sirolimus and propranolol. Endothelial activation was assessed using flow cytometry for CD62e (E-Selectin), CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-Cadherin), CD146 (MCAM) and the release of endothelial microparticles. Cell proliferation was assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and carboxyfluorescein succinimidyl ester assays. Apoptosis was assessed using flow cytometry for lactadherin/propidium iodide staining and for Caspase-3 expression.

Results

Sublytic concentrations of sodium tetradecyl sulphate and polidocanol (0.075%–0.3%) increased the expression of the activation markers CD62e and CD54. The expression of CD105 decreased in sclerosant treated cultured human umbilical vein endothelial cells. Both sodium tetradecyl sulphate and polidocanol induced the release of endothelial microparticles. All agents inhibited cell proliferation. Sodium tetradecyl sulphate and polidocanol-induced apoptosis as evidenced by increased phosphatidylserine exposure and caspase-3 expression, whereas sirolimus and propranolol increased caspase-3 expression only.

Conclusion

Sublytic concentrations of detergent sclerosants induce endothelial activation and the release of endothelial microparticles. All agents were anti-proliferative in EOMA cell lines, with sodium tetradecyl sulphate and polidocanol inducing cellular apoptosis.

Keywords

Endothelial cells haemangioma sclerosants sirolimus propranolol

Introduction

Sclerotherapy involves the administration of detergent sclerosing agents with the aim of inducing endothelial lysis, vessel closure and the eventual absorption of the vein. We have previously demonstrated that sublytic concentrations of detergent sclerosants induce platelet¹ and leukocyte² activation, as well as the release of microparticles from platelets and erythrocytes.^3–5 In this study, we postulated that the agents can also induce endothelial activation at sublytic concentrations.

Upon activation, endothelial cells (EC) express markers such as intracellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62e). In addition, endothelial cells also release endothelial microparticles (EMPs) in response to cellular activation or apoptosis. These microparticles harbour the procoagulant phospholipid phosphatidylserine that acts as the catalytic site for activated Factor X and the subsequent formation of thrombin from prothrombin.⁶ Other studies have shown that the infusion of foam sclerosants induces the release of endothelin-1 into the circulation, with increased endothelin-1 concentrations found minutes following infusion.^7,8

We have also demonstrated that detergent sclerosants induce apoptosis in cultured endothelial cells at sublytic concentrations through a caspase-3, -8 and -9-dependent pathways.⁹ Given this novel finding, we hypothesised that detergent sclerosants could be used for the treatment of haemangioma, or as an adjunct to current treatment options such as sirolimus (rapamycin) and propranolol. Sirolimus was the first FDA approved mammalian target of rapamycin (mTOR) inhibitor indicated for use in haemoangioma. Propranolol is a beta-blocker and has been shown to inhibit the proliferation of cultured haemangioma cell lines and induces apoptosis through the activation of caspase 3 and 9.¹⁰

In this study, we aimed to compare the in vitro apoptotic and anti-proliferative effects of detergent sclerosants with those of propranolol and sirolimus. We also aimed to investigate whether these agents cause EC activation at sublytic concentrations. We utilised human umbilical vein endothelial cells (HUVECs) to model endothelial cells for studies of activation (increases in activation markers and endothelial microparticles) and murine EOMA cells as a model for haemangioma cells for studies of proliferation (viability, apoptosis, morphology, proliferation markers).

Materials and methods

Materials

HUVECs were obtained from Life Technologies (Carlsbad, CA, USA). The EOMA cell line (CRL-2586), originally derived in 1980 from a mixed haemangioendothelioma arising in an adult mouse, was obtained from the ATCC (Manassas, VA, USA).

Medium 199 (M199), l-glutamine, penicillin/streptomycin, carboxyfluorescein succinimidyl ester (CFSE), HEPES and Trypsin/EDTA were obtained from Invitrogen (Carlsbad, CA, USA). Foetal bovine serum (FBS) was obtained from JRH Biosciences (Lenexa, KS, USA). All cultureware was obtained from Corning (New York, USA) or Becton Dickinson (Franklin Lakes, NJ, USA). All flow cytometry antibodies, active-caspase-3 apoptosis kits, TRU-Count tubes and flow cytometry tubes were obtained from Becton Dickinson. Paraformaldehyde was obtained from ProSciTech (Queensland, Australia). Lactadherin-FITC was obtained from Haematologic Technologies (Essex Junction, VT, USA). [3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) was obtained from Promega (Madison, WI, USA). Phenazine methosulfate (PMS), 1.1-µm latex beads, propidium iodide (PI), bovine serum albumin (BSA) and endothelial cell growth supplement (ECGS) were obtained from Sigma-Aldrich (Maryland, WI, USA). Heparin 100 µg/ml was obtained from Pharmacia and Upjohn (Kalamazoo, USA).

Agents

Sodium tetradecyl sulphate (STS) was obtained as FIBRO-VEIN 3% (w/v), 47.4 mM (STD Pharmaceuticals, Hereford, UK); polidocanol (POL) as AETHOXYSKLEROL 3% (w/v), 51.5 mM (Kreussler, Wiesbaden, Germany). Rapamycin (2.5mg/mL) was obtained in ready-made solution of DMSO (Sigma–Aldrich); propranolol as Hemangiol® 3.75 mg/ml (Pierre Fabre Australia Pty Limited, NSW, Australia).

Cell culture

HUVECs were cultured in M199 medium containing, 20% FBS, 10-mM l-glutamine, 10 µg/ml penicillin and streptomycin and 25 µg/ml HEPES 25 µg/ml. EOMA cell lines were maintained in DMEM supplemented with 10% FBS in T25 and T75 culture flasks. The cells seeded at a concentration of 1.25 × 10⁴ cells/ml and grown to confluency. EOMA cells were culture in DMEM medium supplemented with 10% FBS in T25 and T75 culture flasks. The cells were seeded at a concentration of 2.5 × 10⁴ cells/ml and grown to confluency. Culture medium was changed every 2–3 days. Upon reaching confluence, cells were detached by trypsinisation for subculture and experimentation was done using 0.05% trypsin/EDTA solution.

Measurement of endothelial activation and microparticle formation

Confluent HUVECs were trypsinised for 5 min before washing with 10% BSA in PBS. About 180 µl of cells (containing 10⁶ cells) were then added to 120 µl of varying concentrations of detergent sclerosants (final concentrations 0–0.3% in 10% BSA) and incubated for 15 min.

For endothelial activation following incubation with detergent sclerosants, 100 µl of cells was added to flow cytometry tubes containing 5 µl of the following antibodies: CD54-FITC, CD62e-PeCy5, CD144-PE, CD105-V450, CD31-AF647 and CD146-PE-Cy7. Samples were incubated for 20 min before the addition of 2 ml of PBS. The cells were then washed by centrifugation at 300 g for 10 min, following which the supernatant was discarded and cells resuspended in 0.5 mL of 0.5% paraformaldehyde in PBS. Samples were analysed on an LSR-II Flow Cytometer. Endothelial cells identified using forward/side scatter and defined at CD31+ events and assessed for their expression of each endothelial marker.

For microparticle counting following incubation with detergent sclerosants, 67.5 µl of sample was added to flow cytometry tubes containing 5-µl CD31-PE, 2.5-µl Lactadherin-FITC, and 27.5-µl of PBS. After 30 min room temperature incubation, 1 ml of PBS was added. Counting of EMPs was performed using TRU-Count tubes and analysed on an LSR-II flow cytometer. EMPs were defined as CD31+/Lactadherin+ events, with forward scatter profile of less than that of 1.1-µm latex beads. EMPs were then expressed as relative increase or decrease when compared to the 0% control.

Incubation of EOMA cells with sclerosants, sirolimus and propranolol

EOMA cells were trypsinised and seeded onto culture plates in complete medium at concentrations of 5000 cells/ml. The cells were allowed to attach over 24 h, each respective agent was added. For sclerosant incubations, varying concentrations of STS or POL in 10% BSA were added to wells and incubated for 15 min. Following this, the cells were analysed by flow cytometry, or the media was replaced with fresh DMEM. For the remaining experiments, DMEM culture media was supplemented with varying concentrations of sirolimus or propranolol (all at concentrations specified in the figures). Culture media was replaced after three days, with cells cultured for a total of six days.

MTS cell proliferation assay

After six days, viable cell counts were estimated using the MTS assay. The MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay is a colorimetric method for estimating the number of viable cells following cell culture. Metabolically active cells produce dehydrogenase enzymes that convert MTS tetrazolium to formazan, which can be quantified by absorbance at 490 nm.

The culture media in each well was replaced with fresh DMEM, to which 20 µl of an MTS and phenazine methosulfate (PMS) solution was added to each well. This was incubated at 37°C/5% CO₂ for 2 h. Absorbance was read at 490 nm using a 96-well microplate reader (Rayto, RT-6100).

Measurement of apoptosis by lactadherin/propidium iodide staining

About 100 µl of treated cells were added to an antibody mixture containing 5 µl of propidium iodide (PI) and 2.5 µl of lactadherin-FITC in flow cytometry tubes. We elected to use lactadherin as a measure of phosphatidylserine exposure as opposed to the traditional agent Annexin V, as our previous studies demonstrated decrease Annexin V binding on STS-treated cells, presumably due to interference of the negatively charged detergent interfering with the positively charged calcium ions required for Annexin V binding.¹ This was incubated for 15 min before the reaction was terminated by the addition of 400 µl of PBS. This was then analysed on an LSR Fortessa-X20 with apoptotic cells defined as cells that were both lactadherin and PI positive.

Active caspase 3 expression

Active caspase 3 expression was assessed using an FITC active caspase-3 apoptosis kit (Becton Dickinson) according to the manufacturer’s instructions. Analysis was performed on an LSR Fortessa X-20 flow cytometer.

Light microscopy

Cell morphology was assessed in treated cells using an Olympus CKX41 light microscope at 40× magnification.

Cell proliferation analysis by CFSE staining

Cell proliferation was assessed by CFSE staining using a Cell TraceTM CFSE Cell proliferation kit (Invitrogen) according to the manufacturer’s instructions, with cells incubated for six days in culture medium. CFSE staining was then assessed using an LSR-Fortessa X20 flow cytometer, with cell cycle analysis performed for 11 generations using ModFit software (version 4.0.5 for Windows).

Statistical analysis

All data are presented as means ± standard error of the mean (SEM), where n equals the number of experiments performed. Statistical analyses were performed using a paired t-test using GraphPad Prism software (v7) Statistical significance was defined as P-value < 0.05 (*).

Results

Endothelial cell activation

Incubation of HUVEC cells with varying concentration STS- and POL-induced endothelial cell activations, with significantly increased expression of the endothelial activation markers CD54 and CD62e (Figure 1). There was also a significant decrease in CD105 expression on HUVECs incubated in the presence of 0.3% of either STS or POL. There was no significant difference in the expression of CD144 or CD146.

Endothelial cell microparticles

Incubation of HUVECS with varying concentrations of STS and POL induced the release of endothelial microparticles at low concentrations (Figure 2). Similar to our previous publications,^1,4 there was no detectable release of microparticles at high sclerosant concentrations (>0.3%), presumably due to the high concentration of detergent resulting in the lysis of both endothelial cells and microparticles.

The effect of sclerosants, sirolimus and propranolol on EOMA viability

Viable EOMA cell counts after six days of cell culture following a 15-min exposure of cells to varying concentrations of detergent sclerosants was estimated using an MTS assay (Figure 3(a)). All concentrations of STS and POL resulted in a significant decrease in viable cell counts as assessed by decreased MTS absorbance when compared to the control.

Figure 1.

Flow cytometric analysis of endothelial surface marker expression following sclerosant incubation. Human umbilical vein endothelial cells were incubated with varying concentrations of sodium tetradecyl sulphate (STS, red) or polidocanol (POL, blue) for 15 min before analysis using flow cytometry. Compared to unstimulated endothelial cells, there was a significant increase in the expression of endothelial activation markers (a) ICAM-1 (CD54) and (b) E-Selectin (CD62e), a significant decrease in (c) endoglin (CD105) expression and no change in the expression of (d) VE-Cadherin (CD144) and (e) MCAM (CD146); n = 6, *P < 0.05, **P < 0.01

Figure 2.

The effect of sclerosants on the release of endothelial microparticles. Human umbilical vein endothelial cells were incubated with varying concentrations of sodium tetradecyl sulphate (STS, red) or polidocanol (POL, blue) for 15 min before analysis using flow cytometry for the release of CD31+/lactadherin + endothelial microparticles. n = 7, *P < 0.05, **P < 0.01 versus the zero concentration.

Figure 3.

Endothelial cell growth following incubation with sclerosants, sirolimus or propranolol. EOMA cells were cultured for (a) 15 min in the presence varying concentrations of sodium tetradecyl sulphate (STS, squares) or polidocanol (POL, circles) or varying concentrations of (b) sirolimus or (c) propranolol. The cells were then cultured for six days before in DMEM culture media alone or DMEM supplemented with sirolimus or propranolol. An MTS assay was then performed where absorbance was used as an estimation of the viable cell count. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 versus the zero concentration.

EOMA cells incubated for six days with culture media supplemented with varying concentrations of sirolimus or propranolol exhibited significantly decreased viable cell counts when compared with control (Figure 3(b) and (c)). From these experiments, concentrations of 20-nM sirolimus and 100-µM propranolol were used for all subsequent experiments.

The effect of sclerosants, sirolimus and propranolol on EOMA apoptosis

Apoptosis was quantified as the percentage of EOMA cells stained as lactadherin + propidium iodide as assessed by flow cytometry. The percentage of EOMA cells undergoing apoptosis was significantly increased following 15 min exposure of cells to >0.075% STS and >0.0375% POL (Figure 4(a)).

Figure 4.

EOMA cell apoptosis. Apoptosis in EOMA cells was quantified using flow cytometry using lactadherin/propidium iodide staining following incubation with (a) varying concentrations of either sodium tetradecyl sulphate (STS) or polidocanol (POL) or (b) sirolimus (20 nM) or (c) propranolol (100 µM). n = 3, *P < 0.05, **P < 0.01 versus the control sample.

Figure 5.

Expression of active caspase-3. Active caspase expression was assessed in EOMA cells cultured in the presence of sodium tetradecyl sulphate (STS), polidocanol (POL), sirolimus, propranolol or with a serum deprived control. The cells were then assessed using flow cytometry for active caspase-3 expression. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Following six-day incubation of EOMA cells in culture media supplemented with in 20-nM sirolimus and 100-µM propranolol, there was no significant difference in the percentage of cells undergoing apoptosis when compared to control (Figure 4(b)).

Active capase-3 expression was then assessed using flow cytometry for cells exposed to STS, POL, sirolimus and propranolol and subsequent culture for six days (Figure 5). For these experiments, a serum-deprived sample of cells cultured in media without serum was used as a positive control for apoptosis. Active caspase-3 expression was significantly increased for all agents.

Effects of sclerosants, sirolimus and propranolol on cell morphology

Light microscopy was used to assess cellular morphology following exposure of EOMA cells to STS, POL, sirolimus and propranolol and subsequent culture for six days. When compared to control samples, all agents displayed fewer cell numbers after 6 days in culture (Figure 6). Cells treated with STS, POL or propranolol all demonstrated an altered rounded cell morphology. In contrast, cells treated with sirolimus demonstrated a morphology consistent with the control samples, but with fewer cell numbers.

Effects of sclerosants, sirolimus and propranolol on cell proliferation

EOMA cells stained with CFSE and exposed to STS, POL, sirolimus and propranolol and subsequent culture for six days were assessed for remaining CFSE staining using flow cytometry to determine the passage of cells into different generations (Figure 7). This was then converted into a proliferation index. All agents, including the serum deprived positive control demonstrated a significant decrease in proliferation compared to the control sample.

Figure 6.

EOMA cell morphology. Cells were cultured DMEM culture media supplemented with either sodium tetradecyl sulphate (STS, 0.1%), polidocanol (POL, 0.1%), sirolimus (20nM) or propranolol (100 µM), with representative light microscopy images obtained 15 min from the commencement of cultures for day 0 and then imaged for cells continuing in culture for days 3 and 6.

Figure 7.

EOMA cell proliferation. Cell proliferation was quantified using a CFSE assay in EOMA cells following incubation with (a) culture medium alone, (b) sodium tetradecyl sulphate (STS, 0.1%), (c) polidocanol (POL, 0.1%), (d) sirolimus (20nM), (e) propranolol (100 µM) or (f) serum-deprived media used as a positive control. (g) From this data, a proliferation index was calculated. n = 3, *P < 0.05 versus the control sample.

Discussion

In this study, we have demonstrated that at sublytic concentrations <0.3%, the detergent sclerosants STS and POL induce endothelial cell activation, as demonstrated by the increased expression of surface markers of cellular activation and the release of endothelial cell microparticles. Similar to our previous publications, these concentrations of detergent sclerosants also induced cellular apoptosis in a mouse haemangioendothelioma cell line. When compared to the current treatments for haemangioma (sirolimus and propranolol), these agents also increased the expression of active caspase-3 and decreased cellular proliferation.

A number of previous studies have implicated the vasoconstrictor endothelin-1 in visual disturbances following sclerotherapy.^7,8 In these studies, both rats and humans injected with foam sclerosant demonstrated a significant increase in circulating endothelin-1 expression within minutes. These results suggested endothelial cell activation resulting from sclerotherapy. The results of our study have confirmed that endothelial cell activation occurs as a direct result of the exposure of sclerosants to endothelial cells. As our experiments were performed using liquid rather than foam sclerosants, this also confirms endothelial activation as a direct result of the detergent and not from the foam format of the agent.

This is the first report describing the release of endothelial microparticles from endothelial cells following exposure to detergent sclerosants. As these microparticles bind lactadherin to exposed phosphatidylserine, it is expected that they are procoagulant. Due to the prothrombotic nature of microparticles, the results of this study demonstrating endothelial activation and microparticle release may account for some of the neurological complications found following foam or liquid sclerotherapy that can occur within seconds of injection.¹¹

Prior to this study, we demonstrated that low concentrations of both STS and POL were capable of inducing cellular apoptosis in leukocytes¹² and in endothelial cells.⁹ In both cell types, we were able to demonstrate increased lactadherin/propidium iodide staining and that apoptosis occurred as a result of increased active caspase-8, -9 and -3 expression and following increased Bax expression in endothelial cells. The induction of apoptosis has also been observed by other investigators, with increased p53 and ICAM-1 expression found in harvested veins that were exposed to either STS or POL.¹³

In the current study, we were able to demonstrate increased lactadherin/propidium iodide in EOMA cells following exposure to detergent sclerosants but not following exposure to either sirolimus or propranolol. This was also accompanied by increased active caspase-3 expression for all agents. As the effect of sclerosants on the expression of other caspases (caspase-3, caspase-8, caspase-9 and Bax) had been characterised by our previous studies in endothelial cells,⁹ we elected to investigate only caspase-3 expression as this is the point where both the intrinsic and extrinsic pathways of apoptosis converge. It is likely that the mechanisms involved in the induction of apoptosis in HUVEC cells are similar for EOMA cells.

Interestingly, increased caspase-3 expression was found in sirolimus and propranolol treated cells. This occurred despite there being no further evidence of apoptosis, such as increased lactadherin/propridium iodide staining. There was evidence of increased lactadherin expression in propranolol-treated cells (data not shown), commonly referred to as early apoptosis; however, this did not translate into late apoptosis. The mechanism by which caspase-3 results in apoptosis following sclerosant incubation but not sirolimus or propranolol incubation warrants further investigation.

It should be noted that the morphology of cells treated with sirolimus are distinctly different to cells treated with STS, POL or propranolol following six days of culture. Although cell counts were decreased following all treatments, cells treated with sirolimus maintained their typical endothelial (flattened) morphology, whereas cells treated with STS, POL and propranolol developed a rounded morphology. This provides further evidence that the mechanisms behind the antiproliferative nature of sirolimus are distinct from the pro-apoptotic nature of STS and POL; however, this distinction for propranolol needs further investigation.

All these agents, however, inhibited cellular proliferation, including the serum-deprived positive control as demonstrated using a CFSE assay. In this assay, a fixed concentration of CFSE dye added to all cells that becomes distributed evenly throughout subsequent generations of cells following cell divisions and can be used to quantify cellular proliferation. In this study, we found all agents, irrespective of whether they induced apoptosis, were antiproliferative.

Given the morphological changes, phenotype antiproliferative nature of STS- and POL-incubated cells, these agents appear to be pro-apoptotic, whereas sirolimus appears to be antiproliferative only. Whether the antiproliferative nature of propranolol is due at least in part to apoptosis should be investigated in further studies.

Given that we observed evidence of increased endothelial activation but decreased cell proliferation following exposure to similar concentrations of detergent sclerosants, it is possible that these effects may be due to independent pathways within the endothelial cells. The use of the term “activation” in this article to describe the release of endothelial markers and the increase in CD54 and CD62e are distinct from the effects on proliferation, apoptosis and morphology.

This study introduces the potential for detergent sclerosant to be used in the treatment of haemangioma, due to both their lytic properties and their ability to induce cellular apoptosis. This could be used as an alternative or as an adjunct to traditional therapies such as with sirolimus or propranolol.

In terms of limitations for this study, it should be emphasised that these experiments were performed using a cultured monolayer of endothelial cells. This therefore does not take into account any potential effects of sclerosants on other components of the vessel wall, such as an increased potential for exposure of collagen or von Willebrand Factor from the basement membrane following endothelial cell lysis.

These experiments were also performed using liquid agents added to this cultured monolayer of cells; however in practice, sclerosants may be administered as either a liquid or a foam format, with foams generated as a mixture of the liquid agent with a gas such as room air, carbon dioxide or a mixture of carbon dioxide and oxygen. Furthermore, foams may be generated using manual techniques, employing the Tessari or a double-syringe methods¹⁴ or using automated systems such as using the polidocanol endovenous microfoam (Varithena®) delivery device,¹⁵ ultrasonic systems or by other laboratory-made devices.¹⁶ The foams produced by these devices may possess different basic physiochemical properties^17,18 and it is yet to be determined whether the results of this study would extend to these alternatives.

The cell proliferation experiments were also performed on a mouse haemangioendothelioma cell line, as there were few reproducible haemangioma cell lines available. This cell line was selected as it had been used in a number of other publications as a model for haemangioma, due to the ability of cells to transform into haemangiomas following injection into mice and due to their sensitivity to sirolimus.¹⁹ The results of this study should be confirmed using haemangioma cells derived from humans.

Given the effects of detergent sclerosants of cell morphology and microparticle release, it is possible that observed results of this study may simply be due to the removal of material from the cellular membrane. Given the increase in caspase-3 for all agents, with the maximal increase seen for STS and propranolosl, it is also possible that these effects could be a secondary effect on the cell, potentially unrelated to the detergent effect and similar to the anti-proliferative effects of sirolimus and propranolol. Further studies using other cell lines and more concentrations of agents would be helpful in elucidating the exact antiproliferative and proapoptotic mechanisms.

In conclusion, the detergent sclerosants STS and POL induce endothelial activation at sublytic concentrations. In addition they are pro-apoptotic and anti-proliferative, mediated by distinct pathways to the antiproliferative nature of both sirolimus and propranolol. The clinical utility of sclerosants in the treatment of haemangioma should be the subject of further studies.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by a grant from the American College of Phlebology (now the American Venous and Lymphatic Society) and a grant from the International Union of Phlebology (Kreussler Young Scientists’ Sclerotherapy Award).

Ethical approval

Not required.

Guarantor

DC.

Contributorship

DC was involved in study design, experimental work, data analysis and manuscript preparation. JG was involved in experimental work, data analysis and manuscript preparation. ANC was involved in experimental work, data analysis and manuscript preparation. OCA was involved in study design, experimental work and data analysis. DG was involved in experimental work and data analysis. KC was involved in experimental work and data analysis. KP was involved in study design and manuscript preparation.

ORCID iDs

David E. Connor

Kurosh Parsi

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