Embryonic Stem Cell-Like Subpopulations in Venous Malformation

Abstract

Background: 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).

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: embryonic; markers; pathogenesis; stem cells; venous malformation.

Figure 1

Representative hematoxylin and eosin stained subcutaneous venous malformation (SCVM) (A) and intramuscular venous malformation (IMVM) (B) sections demonstrating the characteristic ectatic venous channels. Representative sections of SCVM (C) and IMVM (D) showing minimal staining for D2-40 [(C,D), brown]. Nuclei were counterstained with hematoxylin [(A–D), blue]. Original magnifications: 400× (A,B) and 100× (C,D).

Figure 2

3,3′-Diaminobenzidine immunohistochemical-stained images demonstrating the expression of Nanog [(A,B), red], pSTAT3 [(C,D), brown], OCT4 [(E,F), brown], SOX2 [(G,H), brown], SALL4 [(I,J), brown], and CD44 [(K,L), brown] in subcutaneous venous malformation (SCVM) (A,C,E,G,I,K) and intramuscular venous malformation (IMVM) (B,D,F,H,J,L). Endothelial staining of all six embryonic stem cell markers was present on the endothelium within both SCVM and IMVM samples. Nanog (A,B), pSTAT3 (C,D), SOX2 (G,H), and CD44 (K,L) were also expressed on cells (arrowheads) away from the endothelium in both SCVM and IMVM samples. Nuclei were counterstained with hematoxylin (blue). Original magnification: 400×.

Figure 3

Representative immunofluorescent immunohistochemical-stained sections of subcutaneous venous malformation (SCVM) (A) and intramuscular venous malformation (IMVM) (B) samples, demonstrating the endothelium consisted of CD34⁺ (green)/ERG⁻ (red) (long arrows), ERG⁺(red)/CD34⁻ (green) endothelium (arrowheads), and CD34⁺ (red)/ERG⁺ (red) (short arrows) phenotypes. The CD34⁺ (green) endothelium expressed Nanog (red, arrows) in SCVM (C) and IMVM (D) lesions with cells away from the endothelium also expressing Nanog (red, arrowheads) within SCVM (C) and IMVM (D) lesions. The CD34⁺ (green) endothelium expressed pSTAT3 (red, arrows) in both SCVM (E) and IMVM (F) lesions. Cells away from the endothelium also expressed pSTAT3 (red, arrowheads) within SCVM (E) and IMVM (F) lesions. The ERG⁺ (red) endothelium also expressed OCT4 (green, arrows) in both SCVM (G) and IMVM (H) lesions. The CD34⁺ (green) endothelium expressed SOX2 (red, arrows) in SCVM (I) and IMVM (J) lesions. Cells away from the endothelium also expressed SOX2 (red, arrowheads) in SCVM (I) and IMVM (J) lesions. The ERG⁺ endothelium (red) expressed SALL4 (green, arrows) in SCVM (K) and IMVM (L) lesions. Dual staining of with SOX2 and SALL4 demonstrated the SALL4⁺ [(M,N), green] endothelial population expressed SOX2 [(M,N), red] in both SCVM (M) and IMVM (N). The ERG⁺ endothelium (red) expressed CD44 (green, arrows) in SCVM (O) and IMVM (P) lesions with cells away from the endothelium also expressing CD44 (green, arrowheads) in SCVM (O) and IMVM (P). Cells outside of the endothelium in both SCVM (Q) and IMVM (R) co-expressed Nanog [(Q,R), red] and CD44 [(Q,R), green]. Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole [(A–R), blue]. Scale bars: 20 µm.

Figure 4

Log₁₀ relative expression of OCT4, STAT3, and CD44 (A) and SOX2, Nanog, and SALL4 (B) mRNA transcripts in three subcutaneous venous malformation (SCVM) and three intramuscular venous malformation (IMVM) samples analyzed by NanoString (A) and RT-qPCR (B) analyses. Expression is depicted relative to the housekeeping gene GAPDH. OCT4 was detected in two SCVM and two IMVM samples (A). STAT3 and CD44 (A) and SOX2 and SALL4 (B) were detected in all three samples. Nanog was detected in all three SCVM samples and two out of three IMVM samples (B).