Embryonic Stem Cell-like Population within Venous Malformation Expresses the Renin-Angiotensin System.

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.

Phosphorylated Forms of STAT1, STAT3 and STAT5 Are Expressed in Proliferating but Not Involuted Infantile Hemangioma.

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.

Embryonic Stem Cell-Like Subpopulations in Venous Malformation.

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.

Neuropeptide Y receptor 1 is expressed by B and T lymphocytes and mast cells in infantile haemangiomas.

AIM: We investigated the expression of neuropeptide Y (NPY), NPY receptor 1 (NPYR1) and NPY receptor 2 (NPYR2) in infantile haemangiomas (IHs). METHODS: Immunohistochemical (IHC) staining was performed on proliferating IHs from six patients aged 4-13 (mean 8.7) months and involuted IHs from six patients aged 5-59 (mean 18.7) years, for the expression of NPY, NPYR1 and NPYR2. Protein and messenger ribonucleic acid expression corresponding to these proteins was investigated by Western blotting and NanoString analysis, respectively. RESULTS: IHC staining, Western blotting and NanoString analysis demonstrated the presence of NPYR1, but not NPYR2, within proliferating and involuted IHs. IHC staining showed NPYR1 was expressed by B and T lymphocytes expressing CD45 and mast cells expressing tryptase. IHC staining demonstrated the presence of NPY on the NPYR1+ cells, but it was not detected by Western blotting or NanoString analysis. CONCLUSION: NPYR1, but not NPYR2, was present in IHs. The localisation of NPYR1 to B and T lymphocytes and mast cells suggests its role in the biology of IHs. The demonstration of NPY on the NPYR1+ cells, without active transcription, suggests that NPY was not being produced within IHs.

Characterisation of lymphocyte subpopulations in infantile haemangioma.

AIMS: Interstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and the expression of Thy-1, a marker of thymocyte progenitors, which have the ability to give rise to both B and T cells. METHODS: Immunohistochemical (IHC) staining was performed on proliferating and involuted IHs for the expression of CD45, CD3, CD20, CD79a, Thy-1 and CD34. The presence of mRNA corresponding to CD45, CD3G, CD20 and Thy-1 was confirmed by reverse transcriptase-polymerase chain reaction in snap-frozen IH tissues. Cell counting of 3,3-diaminobenzidine IHC-stained slides was performed on CD45+ only cells and dually stained CD45+/CD3+ cells or CD45+/CD20+ cells and analysed statistically. In situ hybridisation and mass spectrometry were also performed to confirm the presence and abundance of Thy-1, respectively. RESULTS: IHC staining showed a subpopulation of CD45+ interstitial cells that expressed the T lymphocyte marker, CD3, and another subpopulation that expressed the B lymphocyte marker, CD20, in proliferating and diminished in involuted IHs. The abundant expression of Thy-1 on the endothelium of proliferating, but not involuted IH, was demonstrated by IHC staining and confirmed by in situ hybridisation and mass spectrometry. CONCLUSIONS: Both B and T lymphocytes are present within the interstitium of proliferating and involuted IH. The expression of Thy-1 by the endothelium suggests that B and T cells in IH may have originated from within the lesion, rather than migrating from the peripheral circulation.

Characterisation of subpopulations of myeloid cells in infantile haemangioma.

AIMS: Cells expressing markers of mast cells, macrophages and dendritic cells have previously been demonstrated within the interstitium of infantile haemangioma (IH). This study characterised these myeloid cellular subpopulations within IH. METHODS: Immunohistochemical staining was performed on proliferating and involuted IHs for the expression of Nanog, tryptase, CD163, DC-SIGN and CD45. The presence of mRNA corresponding to Nanog, tryptase α/ß-1, tryptase ß-2, CD163 and DC-SIGN was confirmed by NanoString and RT-PCR in snap-frozen IH tissues. RESULTS: Immunohistochemical staining showed expression of Nanog by interstitial phenotypical mast cells within proliferating IH, which were separate from the interstitial M2-polarised macrophages that also expressed DC-SIGN, a dendritic cell marker. These two myeloid cellular subpopulations in IH did not express the pan-haematopoietic marker, CD45. CONCLUSIONS: There are two interstitial subpopulations of myeloid cells within IH: phenotypical mast cells which also express Nanog, indicating a primitive phenotype; and M2-polarised macrophages which also express DC-SIGN.