Haemangioma: A Study of the Biology

Infantile haemangioma (IH), considered a primary tumour of the microvasculature, is the most common tumour of infancy affecting about 10% of Caucasian infants. IH predominantly affects white, female and premature infants. IH typically undergoes an initial rapid proliferation during infancy (proliferative phase) characterised by aggressive angiogenesis, followed by spontaneous involution over the next 1-5 years (involuting phase) and continued improvement up to 10 years (involuted phase), often with a fibro-fatty residuum. IH consists of cells of various lineages, with the presence of mesenchymal stem cells, endothelial progenitor cells, endothelial cells, myeloid haematopoietic cells, and pericytes. This thesis demonstrates the expression of primitive (stem/progenitor cell) markers on the endothelium of IH. The expression of the transcription factors brachyury, Tal-1 and GATA-2, along with the demonstration of erythropoiesis in IH explants in vitro supports the hypothesis that IH consists of a primitive endothelium similar to an embryonic haemogenic endothelium. The expression of the erythropoietin receptor and haemoglobin zeta chain by the endothelium of IH further strengthens the notion that IH is a haemogenic endothelium. Consistent with the primitive embryonic origin, the expression of the placental markers human chorionic gonadotrophin (hCG) and human placenta lactogen (hPL), but not cytokeratin 7 (CK7) or human leucocyte antigen- G (HLA-G) by the endothelium in IH, supports a placental chorionic villous mesenchymal core cell, and not a trophoblast, origin for IH. IH thus has an extraembryonically derived primitive mesodermal origin. This primitive mesoderm is able to account for the haemogenic endothelium phenotype of the endothelium of proliferating IH microvessels with its capacity for both erythropoietic and mesenchymal differentiation. Additionally, data are presented to show that IH expresses key components of the renin-angiotensin system (RAS), angiotensin converting enzyme (ACE), angiotensin II (ATII), angiotensin receptor 2 (ATR2). Cultured IH-derived stem cells can be induced to proliferate and form blast colonies in response to ATII treatment. The crucial regulatory role of RAS in the proliferation and differentiation of the stem/progenitor cell population within IH accounts for the natural progression of IH. A model is proposed to provide a rational explanation for the serendipiditous discovery of the dramatic effect that the β-blocker, Propranolol has in accelerating involution of IH. The hypothesis that Propranolol exerts its action on IH through modulation of the RAS by blocking renin activity and preventing the conversion of angiotensinogen to angiotensin I, thereby reducing ATII levels, has led to a clinical trial using Captopril, an ACE inhibitor in the treatment of problematic proliferating IH. The observed accelerated involution of IH by Captopril which blocks the conversion of angiotensin I to ATII confirms a key regulatory role for RAS in the biology of IH This discovery underpins the development of potentially safer and novel treatment modalities for this enigmatic condition

Cancer Stem Cells in Oral Cavity Squamous Cell Carcinoma: A Review

Cancer stem cells (CSCs) have been identified in oral cavity squamous cell carcinoma (OCSCC). CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells and cancer cells that drive tumor growth. Studies of many cancer types including OCSCC have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple CSC subtypes within OCSCC, making investigation reliant on the use of multiple markers. This review examines the current knowledge in CSC markers OCT4, SOX2, NANOG, ALDH1, phosphorylated STAT3, CD44, CD24, CD133, and Musashi-1, specifically focusing on their use and validity in OCSCC CSC research and how they may be organized into the CSC hierarchy. OCSCC CSCs also express components of the renin–angiotensin system (RAS), which suggests CSCs may be novel therapeutic targets by modulation of the RAS using existing medications

Expression of Embryonic Stem Cell Markers on the Microvessels of WHO Grade I Meningioma

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined.Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively.Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers.Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG

Expression of Components of the Renin-Angiotensin System by the Putative Stem Cell Population Within WHO Grade I Meningioma

Aim: We have recently demonstrated a putative stem cell population within WHO grade I meningioma (MG) that expressed embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC, localized to the endothelial and pericyte layers of the microvessels. There is increasing recognition that the renin-angiotensin system (RAS) plays a critical role in stem cell biology and tumorigenesis. This study investigated the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on the putative stem cell population on the microvessels of WHO grade I MG.Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on WHO grade I MG tissue samples from 11 patients for PRR, ACE, ATIIR1, and ATIIR2. Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these components of the RAS in using combinations of CD34 and ESC marker SOX2 or OCT4. NanoString mRNA expression analysis and Western blotting (WB), were performed on six snap-frozen MG tissue samples to confirm mRNA and protein expression of these proteins, respectively.Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 within all 11 MG tissue samples. WB and NanoString mRNA analyses, confirmed protein and mRNA expression of these proteins, respectively. IF IHC staining showed PRR, ATIIR1 and ATIIR2 were localized to the OCT4+ and SOX2+ endothelium and the pericyte layer of MG while ACE was localized to the OCT4+ endothelium of the microvesels.Conclusion: The novel finding of the expression of PRR, ACE, ATIIR1, and ATIIR2 on the putative stem cell population on the microvessels of WHO grade I MG, suggests that these stem cells may be a potential therapeutic target by manipulation of the RAS

Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma

Background: There is a growing body of research demonstrating expression of the renin-angiotensin system (RAS) by a putative embryonic stem cell (ESC)-like population within vascular anomalies. This study investigated the expression of components of the RAS in relation to the putative ESC-like population within pyogenic granuloma (PG) that we have recently reported.Methods: PG samples from 14 patients were analyzed for the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), using 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining. Immunofluorescence (IF) IHC staining was performed to localize these proteins on four of the PG samples. RT-qPCR was performed on two snap-frozen PG samples. Western blotting (WB) was performed on one snap-frozen PG sample and two PG-derived primary cell lines.Results: DAB IHC staining demonstrated the expression of ACE, PRR, ATIIR1, and ATIIR2 in all 14 PG tissue samples. RT-qPCR analysis confirmed abundant mRNA transcripts for PRR, ACE, AIITR1 and ATIIR2, relative to the housekeeping gene. WB confirmed the presence of PRR, ATIIR1, and ACE in the PG tissue sample, and PRR and ATIIR1, in the PG-derived primary cell lines. IF IHC staining demonstrated the expression of PRR, ACE, ATIIR1 on the primitive population that expressed NANOG and SOX2 on the ERG+ endothelium of the microvessels within PG.Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma

Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated.Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression (n = 6) and Western blotting (WB; n = 5) analyses, and enzyme activity assays (EAAs; n = 3), were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively.Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA+ pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels.Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG

Haemangioma: A Study of the Biology

Infantile haemangioma (IH), considered a primary tumour of the microvasculature, is the most common tumour of infancy affecting about 10% of Caucasian infants. IH predominantly affects white, female and premature infants. IH typically undergoes an initial rapid proliferation during infancy (proliferative phase) characterised by aggressive angiogenesis, followed by spontaneous involution over the next 1-5 years (involuting phase) and continued improvement up to 10 years (involuted phase), often with a fibro-fatty residuum. IH consists of cells of various lineages, with the presence of mesenchymal stem cells, endothelial progenitor cells, endothelial cells, myeloid haematopoietic cells, and pericytes. This thesis demonstrates the expression of primitive (stem/progenitor cell) markers on the endothelium of IH. The expression of the transcription factors brachyury, Tal-1 and GATA-2, along with the demonstration of erythropoiesis in IH explants in vitro supports the hypothesis that IH consists of a primitive endothelium similar to an embryonic haemogenic endothelium. The expression of the erythropoietin receptor and haemoglobin zeta chain by the endothelium of IH further strengthens the notion that IH is a haemogenic endothelium. Consistent with the primitive embryonic origin, the expression of the placental markers human chorionic gonadotrophin (hCG) and human placenta lactogen (hPL), but not cytokeratin 7 (CK7) or human leucocyte antigen- G (HLA-G) by the endothelium in IH, supports a placental chorionic villous mesenchymal core cell, and not a trophoblast, origin for IH. IH thus has an extraembryonically derived primitive mesodermal origin. This primitive mesoderm is able to account for the haemogenic endothelium phenotype of the endothelium of proliferating IH microvessels with its capacity for both erythropoietic and mesenchymal differentiation. Additionally, data are presented to show that IH expresses key components of the renin-angiotensin system (RAS), angiotensin converting enzyme (ACE), angiotensin II (ATII), angiotensin receptor 2 (ATR2). Cultured IH-derived stem cells can be induced to proliferate and form blast colonies in response to ATII treatment. The crucial regulatory role of RAS in the proliferation and differentiation of the stem/progenitor cell population within IH accounts for the natural progression of IH. A model is proposed to provide a rational explanation for the serendipiditous discovery of the dramatic effect that the β-blocker, Propranolol has in accelerating involution of IH. The hypothesis that Propranolol exerts its action on IH through modulation of the RAS by blocking renin activity and preventing the conversion of angiotensinogen to angiotensin I, thereby reducing ATII levels, has led to a clinical trial using Captopril, an ACE inhibitor in the treatment of problematic proliferating IH. The observed accelerated involution of IH by Captopril which blocks the conversion of angiotensin I to ATII confirms a key regulatory role for RAS in the biology of IH This discovery underpins the development of potentially safer and novel treatment modalities for this enigmatic condition