Facial cutaneo-mucosal venous malformations can develop independently of mutation of TEK gene but may be associated with excessive expression of Src and p-Src
© The Author(s). 2017
Received: 7 September 2016
Accepted: 16 February 2017
Published: 20 March 2017
We aimed to search for mutations in the germline and somatic DNA of the TEK gene and to analyze the expression level of Src and phospho-Src (p-Src) in tumor and healthy tissues from patients with facial cutaneo-mucosal venous malformations (VMCM). Eligible patients from twelve families and thirty healthy controls were recruited respectively at the Departments of Stomatology and Oral Surgery, and Transfusion Medicine of Tlemcen University Medical Centre. Immunoblot analyses of Src and p-Src were performed after direct DNA sequencing. No somatic or germline mutations were found in all the 23 exons and their 5’ and 3’ intronic flanking regions, except for one case in which a c.3025+20-3025+22 del mutation was highlighted at the intron 15, both in the germline and somatic DNA. Additionally, elevated expression levels of Src and p-Src were observed only in the patient with such mutation. However, when normalized to β-actin, the overall relative expression levels of both Src and p-Src were significantly increased in VMCM tissues when compared to healthy tissues (for both comparisons, p <0.001). In conclusion, we confirm the outcomes of our previous work suggesting that VMCM can develop independently of mutation of the TEK gene. Additionally, the results for Src activity are of particular interest in the context of specific targeted therapies and biological diagnosis. Nevertheless, such a conclusion should be confirmed through a mechanistic study and/or in a satisfactory number of patients.
KeywordsCutaneo-mucosal venous malformations Direct sequencing Germline and somatic DNA p-Src Src TEK gene
Vascular malformations arise from an error of vascular morphogenesis and are named by their predominant vessel type: arterial, venous, capillary, lymphatic or different combinations of each of them . Venous malformations (VMs) are the most frequent vascular abnormalities but remain quite rare, with an incidence of approximately 1 in 10,000 [2, 3]. They are present at birth, and often become apparent afterward. Rapid growth may occur during puberty, pregnancy, or traumatic injury .
When venous lesions are located both at skin and mucous membranes, VMs are called cutaneo-mucosal venous malformations (VMCMs). Their pathogenesis is not yet fully understood. Nevertheless, it is assumed to be caused by abnormal development of the venous system . Further studies showed that somatic mutations in the gene of the receptor tyrosine kinase (TEK/TIE2, vascular endothelial cell specific receptor tyrosine kinase) was present in various single or multiple VMs and led to loss of TIE2 receptor function , and upregulated expression of other vascular endothelial growth factors, such as transforming growth factor (TGF)-β and fibroblast growth factor (FGF)-β, which exacerbated the severity of the lesion .
The TEK/TIE2 receptor tyrosine kinase plays a crucial role in angiogenesis and cardiovascular development . The main role of this receptor is triggering angiogenesis signals leading to the formation of blood vessels. This signaling process facilitates communication between two types of cells within the walls of blood vessels, endothelial cells and smooth muscle cells . Communication between these two cell types is necessary to direct angiogenesis and ensure the structure and integrity of blood vessels .
Angiogenesis, i.e. the formation of new blood vessels from preexisting ones, is a key event in tumor progression, which is controlled by a balance between positive and negative regulators [10, 11]. Among the several growth factors that can promote angiogenesis, vascular endothelial growth factor (VEGF) is the most widely studied and potent inducer of angiogenesis . One group of signaling molecules that may be involved in the VEGF signaling cascade is the proto-oncogene tyrosine-protein kinase Src.
It has been reported that Src kinases play an important role in cell cycle control and cell adhesion and movement, as well as in cell proliferation and differentiation in a numerous cells and tissues . They also play an important role in lymphokine-mediated cell survival and VEGF-induced angiogenesis . Of note, Src protein is one of the best characterized non-receptor protein tyrosine kinases that are involved in receptor signaling and cell communication. Multiple cellular functions are attributed to the activity of Src as a molecular switch allowing the external signal transduction across the plasma membrane, and then its conversion into internal message upon activation of the target molecules inside a cell. High expression of Src has been reported to be associated with increased VEGF expression , cellular proliferation and angiogenesis .
On the basis of these reports, we extend previously published research on germline DNA of the TEK gene  by including new eligible patients with VMCMs and additional controls for the examination of both germline and somatic mutation, as well as the evaluation of Src and p-Src expression levels.
Patients and subjects
The demographic data of patients with cutaneo-mucosal venous malformations
Patients with VMCM
Age at diagnosis (year)
13 ± 2
Total number of lesion (n)
1 ± 0
Lip VMCM (%)
Genio-cervical VMCM (%)
Blood samples were collected into ethylenediaminetetraacetic acid-containing Vacutainer tubes (BD Vacutainer EDTA, USA). VMCM and normal tissues were taken from patients after surgery, immediately placed into a sterile collection tube in liquid nitrogen and, then, stored at – 80 °C in dry ice. Extracted DNA from blood samples and tissues were used for polymerase chain reaction (PCR) and direct DNA sequencing for all exons and their flanking regions of the TEK gene. Immunoblot analysis of Src, p-Src and β-actin expression were performed on tissues.
DNA extraction and purification was carried out as we described . The search for mutation was performed by PCR amplification followed by direct sequencing of amplified DNA segments. Such analyses were performed in the Laboratory of Cell and Hormonal Biology, Arnaud de Villeneuve Hospital, Montpellier (France).
Sequences of the sense and antisense primers used for direct sequencing of all the exons of the TEK gene
Sense and anti-sense primers 5’-3’
5’ UTR region
3’ UTR region
The DNA was amplified in a thermocycler for PCR (Applied Biosystems, Foster, CA), using the primers described in Table 2. The medium of the DNA amplification reaction was composed of 50 ng of DNA, 25 \( \mu \)M of each primer, and 2X Promega PCR Master Mix (Promega). The PCR conditions were as follows: 5 min at 95 °C followed by 35 cycles of 30 s of denaturation at 95 °C, primer annealing at 60 °C for 30 s, and elongation at 72 °C followed by one cycle at 72 °C for 10 min.
After checking the quality and size of the PCR products by agarose gel (1.5%) electrophoresis, a bidirectional sequencing was performed by the use of Mix BigDye Terminator kit version 3.1 (ABI). The sequences of the 23 exons and their flanking regions were compared with the TEK gene reference sequence published in Ensembl using the SeqScape v2.5 software (ABI).
Src, p-Src and β-actin immunoblot assays
Venous malformation and healthy control tissues were homogenised for 10 min each in lysis buffer (20 mM HEPES, pH 7.3; 1 mM EDTA; 1 mM EGTA; 0.15 mM NaCl; 1% Triton X-100; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride; 2 mM sodium orthovanadate and 2 μl/ml anti-protease cocktail) and centrifuged (13000 g x 10 min). Protein concentrations in the supernatants were determined by bicinchoninic acid method (Pierce). Denatured proteins (40 μg) were separated by SDS-PAGE (10%) and transferred to PVDF membranes. Immunodetection was performed by using p-Src (cell signaling tech, OZYME, FRANCE), Src (cell signaling tech, OZYME, FRANCE) and β-actin (Sigma Aldrich, FRANCE) antibodies. β-actin was used as a loading control. Optimal dilutions of primary antibodies, including a monoclonal anti-β-actin, were 1:1000 (v/v). The horseradish peroxidase conjugated secondary antibodies were used at 1:5000 (v/v) dilution and the Enhanced Chemiluminescence (ECL) system (NEL121001EA, Perkin Elmer) was used for detection. Signal detection was done by ChemiDoc XRS System (Bio-Rad). Densitometry and protein band analysis were performed using ImageJ software (NIH, USA) as reported . Such analyses were performed at the UMR U866 INSERM/Université de Bourgogne/AgroSup (France). Additional verification analyses and experiments were carried out at the Laboratory of Applied Molecular Biology and Immunology (University of Tlemcen, Algeria).
Results and discussion
Facial VMCMs are often responsible for aesthetic and functional discomfort, but also cause detrimental changes in personal relationships, especially during childhood and adolescence. They are due to localized defects of angiogenesis that are caused by genetic modifications and anomalies in signaling pathways, including that of Src family kinases. From a genetic point of view, studies of rare familial cases have helped to suggest that these defects could be the result of mutations in the TEK gene (also referred to as TIE2), which is located on the band 21 of the short arm of chromosome 9 (9p21).
It has been reported that TEK is the only gene which mutations that can cause the development of VMCMs . As a matter of fact, the TEK gene was originally identified as a factor responsible for these defects thanks to a linkage analysis conducted in some families with autosomal dominant transmission [4, 22]. Mutated gene isolated by positional cloning experiment and the use of proteins expressed in insect cells have demonstrated that the mutation results in increased activity of the receptor tyrosine kinase TIE2, i.e. the angiopoietin receptor which is known to be specific for vascular endothelial cells. This mutation corresponds to a missense mutation resulting in an arginine-to-tryptophan substitution at position 849 (R849W) in the kinase domain of TIE2 .
Angiogenesis and blood vessel formation involves many signaling pathways that may interact with each other via Src [31, 32]. Src is considered as the focus of a variety of signaling pathways. It can be activated in multiple ways to become p-Src, which can in turn activate specific signaling pathways through phosphorylation of target proteins [33, 34]. In our study, the increased expression of Src and p-Src would be associated with the inducible effects of some angiogenic growth factors, including VEGF, but also the basic fibroblast growth factor (bFGF). Indeed, it has previously been reported that these two factors initiate the Src kinases signaling pathways, leading to the increased expression of Src in angiogenic tissues .
Although both VEGF and FGF stimulate Src activation in avian endothelial cells, only VEGF-induced angiogenesis is inhibited by treatment with a retrovirus that encodes for Src-251, which suppresses both angiogenesis and tumor growth. Moreover, overexpression of Src-251 in avian blood vessels induces apoptotic death, indicating that VEGF-induced activation of Src is essential for endothelial cell survival and angiogenesis. Similar results have been obtained in mice using a retrovirus encoding for the C-terminal Src kinase (CSK) a tyrosine kinase protein that blocks the action of Src through phosphorylation of the inhibitory site on Tyr527 .
The extended Src family includes at least ten proteins (Src, Frk, Lck, Lyn, Blk, Hck, Fyn, Yrk, Fgr, and Yes)  that engage jointly in the intracellular signal transduction [34, 36–38]. Numerous studies have shown an increase in Src and p-Src expression levels in tissues of different tumors, such as breast cancer, osteosarcoma and squamous cell carcinoma of the tongue [39–41]. Additionally, it has recently been shown that increased expression of Src is positively correlated with metastasis [42, 43].
A relationship between the TEK gene and Src signaling pathway can be suspected in the context of VEGF costimulation. Indeed, angiopoietin 1 (Ang1) activates TEK receptor, which triggers the activation of Rous sarcoma virus (Ras) homologous A (RhoA), which, in turn, inhibits Src proteins . It has recently been reported that intact TIE2 may be necessary to blunt Src activation . In our study, the dinucleotide deletion at intron 15 of the TEK gene may affect the function of this protein and consequently lead to an increased expression of Src and p-Src in VMCM tissue.
Here we confirm that VMCMs, especially non-family VMCMs, are not necessarily linked to mutations in the TEK gene. Although increased relative expression of the Src protein appears to be associated with VMCMs, such outcomes deserve to be verified in various populations. Indeed, this is a novel report on relative issues and an alternative reference for biological diagnosis and specific targeted treatment of angiogenesis, using monoclonal antibodies or pharmacological inhibitors. In order to confirm the efficacy of this approach, further investigations should be conducted, and among others, it would be wise to conduct a mechanistic study researching the link with the Src pathway.
C-terminal Src kinase
Fibroblast growth factor
Magnetic resonance imaging
Polymerase chain reaction
Arginine-to-tryptophan substitution at position 849
Rous sarcoma virus
Ras homologous A
Proto-oncogene tyrosine-protein kinase
- TEK :
Vascular endothelial cell specific receptor tyrosine kinase (also referred to as TIE2)
Transforming growth factor
- TIE2 :
Endothelial tyrosine kinase receptor
Vascular endothelial growth factor
Cutaneo-mucosal venous malformation
Vascular permeability factor
We are grateful to all patients and family members for their participation in this study. We also thank the entire teams of the Laboratory of Cell and Hormonal Biology (Arnaud de Villeneuve Hospital, Montpellier). Special thanks are addressed to Professor Naim Akhtar Khan (UMR U866 INSERM, Dijon, France) for his esteemed help. We are also grateful to Doctor Faïza Ghernaout for technical assistance and histological analysis.
This research was supported by the PHC TASSILI (Code 10MDU794) and grants from the Ministry of Foreign and European Affairs (Ministère des Affaires Étrangères et Européennes, MAEE).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
NB performed the DNA analyses and Src experiments, and participated in drafting the manuscript. SS performed experiments on Src. MSD substantially helped with the drafting of the manuscript. CE contributed to DNA analysis. POH was in charge of sequencing and analysis of DNA. BES was in charge of recruiting patients and controls, and surgical tumor resection. MA and GL designed the study concept, participated in data acquisition, and were involved in drafting the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The publication of the results of this study, including individual details, images and pictures from all participants, was approved as part of informed consent.
Ethics approval and consent to participate
This study was reviewed and approved by the Institutional Ethics Committee of Tlemcen University, the Hubert Curien Partnership (PHC Tassili, EGIDE, CAMPUS FRANCE) and the Ministry of Higher Education and Research of Algeria (Ministère Algérien de l’Enseignement Supérieur et de la Recherche Scientifique, MESRS). All participants or their parents or guardians provided signed informed consent in accordance with the principles of the Helsinki Declaration.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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