Abstract
Venous malformation (VM) consists of a network of ectatic anomalous thin-walled venous channels. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs), led us to investigate the expression of ESC markers in subcutaneous VM (SCVM) and intramuscular VM (IMVM). Formalin-fixed paraffin-embedded sections of SCVM from seven patients and IMVM samples from seven patients were analyzed for the expression of Nanog, pSTAT3, OCT4, SOX2, SALL4, and CD44, using 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining. All these samples did not express lymphatic marker D2-40. NanoString mRNA analysis and RT-PCR were performed on snap-frozen samples of SCVM (n = 3) and IMVM (n = 3) from the respective original cohorts of patients included in DAB IHC staining. To confirm co-expression of two proteins, immunofluorescent (IF) IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed. DAB IHC staining demonstrated expression of all of the above ESC markers in both SCVM and IMVM samples. IF IHC staining showed that these markers were localized to the endothelium within these lesions and that Nanog, pSTAT3, SOX2, and CD44 were also expressed by cells outside of the endothelium. NanoString mRNA analysis confirmed transcription activation of pSTAT3, OCT4, and CD44. RT-qPCR confirmed transcription activation of Nanog, SOX2, and SALL4. Our findings support the presence of two ESC-like subpopulations, one within and one outside of the endothelium, of both SCVM and IMVM. Given that the endothelial ESC-like subpopulation expresses the more primitive marker, OCT4, it is exciting to speculate that they give rise to the non-endothelial subpopulation.
Highlights
Vascular anomalies are classified by the International Society for the Study of Vascular Anomalies classification system into vascular tumors of which infantile hemangioma is the most common, and vascular malformations of which venous malformation (VM) is the most common [1, 2]
We have recently demonstrated the expression of components of the renin–angiotensin system (RAS): prorenin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and potentially angiotensin II receptor 2 (ATIIR2), in Subcutaneous VM (SCVM) and intramuscular VM (IMVM), which suggests a role for the RAS in the biology of VM [20]
To confirm co-expression of two proteins, immunofluorescent (IF) IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed with the same primary antibodies at the same concentrations was performed with CD34 and ERG (1:200; cat# EP111, Cell Marque), as appropriate endothelial markers
Summary
Vascular anomalies are classified by the International Society for the Study of Vascular Anomalies classification system into vascular tumors of which infantile hemangioma is the most common, and vascular malformations of which venous malformation (VM) is the most common [1, 2]. Venous malformation, which affects 1% of the population [3], is composed of ectatic anomalous venous channels, lined by flat endothelial cells (ECs) [3,4,5] with absent or deficient smooth muscle cells (SMCs) within the thin vessel walls [3, 5]. VM is present at birth, it may not be noticed until later in life [3,4,5]. VM affects different topographic regions and tissues, commonly involving the subcutaneous tissues and less commonly muscle [6]. Subcutaneous VM (SCVM) usually presents as a compressible bluish swelling, whereas intramuscular VM (IMVM) often presents later in life with swelling, pain, or loss of function [5]. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs), led us to investigate the expression of ESC markers in subcutaneous VM (SCVM) and intramuscular VM (IMVM)
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