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Background: We have recently demonstrated the presence of a NANOG + /pSTAT3 + /OCT4 + /SOX2 + /SALL4 + /CD44 + embryonic stem cell (ESC)-like subpopulation localized to the endothelium and a NANOG + /pSTAT3 + /SOX2 + /CD44 + subpopulation outside of the endothelium, within subcutaneous VM (SCVM) and intramuscular VM (IMVM). We have also shown the expression of components of the renin-angiotensin system (RAS): (pro)renin receptor (PRR); angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), in both SCVM and IMVM. This study investigated whether the ESC-like subpopulations within SCVM and IMVM expressed the RAS., Methods: Formalin-fixed paraffin-embedded sections of two representative samples from each of the seven SCVM and seven IMVM patients included in our previous studies underwent dual immunofluorescence (IF) immunohistochemical (IHC) staining for ESC marker OCT4 or SOX2 with PRR, ACE, ATIIR1, and ATIIR2., Results: IF IHC staining demonstrated the expression PRR by the OCT4 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ACE was expressed by the SOX2 + cells, predominantly in the endothelium in SCVM and IMVM. ATIIR1 was expressed by the SOX2 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ATIIR2 was expressed by the OCT4 + endothelium and outside the endothelium in SCVM and IMVM., Conclusions: Components of the RAS are expressed by ESC-like subpopulations within both SCVM and IMVM. These primitive subpopulations may be a novel therapeutic target by manipulation of the RAS using existing medications.

We have recently demonstrated the expression of embryonic stem cell markers on the endothelium of infantile hemangioma, a functional hemogenic endothelium with the capacity for primitive erythropoiesis in vitro . Despite recent work characterizing stem cells within proliferating infantile hemangioma, the expression of STAT proteins, well documented for their roles in stem cell signaling, has not been investigated. 3,3-Diaminobenzidine and immunofluorescence immunohistochemical staining revealed expression of pSTAT1, pSTAT3 and pSTAT5 in proliferating infantile hemangioma samples with the strongest expression of pSTAT3. There was reduced expression of these pSTAT proteins in the involuted infantile hemangioma samples. Western blotting confirmed the identification of all these three proteins in proliferating infantile hemangioma. It is therefore not surprising that the phosphorylated/activated forms of these proteins are relatively abundantly expressed in proliferating, in comparison to involuted infantile hemangioma samples. We speculate that the reduced STAT activation, as infantile hemangioma involutes, is a reflection of the depletion of the abundant stem cells within proliferating infantile hemangioma, as the lesion involutes.

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.