Abstract
Angiogenesis is a key factor in normal development and in the development and progression of most malignancies, including oral cancer. It is also an integral component of some inflammatory hyperplastic lesions including those that occur in the oral regions such as pyogenic granulomas (PG). Physiological angiogenesis is controlled mostly by vascular endothelial growth factor A (VEGF)/VEGF receptor 2 (VEGFR2) signalling, which promotes endothelial cell migration and proliferation, and blood vessel growth and by vasohibin 1 (VASH-1), an intrinsic inhibitor of angiogenesis. VEGFR2 is also known as kinase domain receptor (KDR) and this term will be used in this thesis. During pathological processes, the loss of angiogenic regulation can contribute to inflammatory hyperplasias and tumour progression.
Aim:
To investigate angiogenic regulation in hyperplastic vascular oral lesions and oral squamous cell carcinoma (OSCC).
Methods:
Archival formalin-fixed paraffin embedded (FFPE) tissue from ten gingival PGs, ten cases of OSCC and five normal oral mucosal (NOM) specimens were analysed for angiogenic markers using standard immunohistochemistry techniques with the chromogen 3, 3’-diaminobenzidine (DAB). Anti-VEGF, anti-KDR and anti-VASH-1 antibodies identified angiogenic and anti-angiogenic activity in the tissues. Mean vessel density (MVD) was assessed using the endothelial cell marker anti-CD146 antibody. Blood vessels were counted using the anti-CD34 antibody as a marker of positive endothelial cells. Immuno-positivity was evaluated by light microscopy for qualitative analysis and five randomly chosen fields were photographed for semi-quantitative and quantitative analysis. VEGF was analysed semi-quantitatively at 200x magnification and the percentage of positive cells analysed using chi-squared statistics. VASH-1+, KDR+ and CD34+ were counted at 400x magnification while CD146+ and VEGF+ cells were counted at 200x magnification. These groups were analysed using a log transformation and oneway analysis of varience (ANOVA). Statistical significance for quantitative analyses was at the 5% level. Qualitative analysis of overall staining patterns was also undertaken. Gene expression analysis was performed on granulation tissue and cell lines from three OSCC and three normal epithelial cell lines. A quality control assay was run using a granulation tissue sample as a control for specificity and validity of the assays. Gene expression was analysed using quantitative real-time polymerase chain reaction (qRT-PCR), 7500 Fast System SDS software v.1.3.1, and SA Biosciences RT2 Profiler PCR Array Data Analysis v3.5 with a 5 % level of significance, significant fold change was >2.
Results:
All tissues were positive for endothelial cell and angiogenic and anti-angiogenic markers. Endothelial cells were positively identified in all tissues. VEGF was found in the stroma and the overlying epithelium, especially in the OSCC group. VASH-1 was positive in blood vessels in all tissues but also in some malignant epithelial cells. The MVD was significantly greater in PGs than in normal oral mucosa and in OSCC (p<0.001) and the number of CD34+ cells was also significant (p=0.01). In respect to expression in blood vessels, qualitative analysis of the tissues showed a greater epithelial intensity of staining for VEGF in OSCC. More VASH-1+endothelial cells were present in PGs than in the other tissues with a trend towards significance (p= 0.05). There was no significant difference in VEGF staining observed in OSSC by comparison with PG or NOM. The expression of staining for KDR showed no significant difference between the tissue types. Gene expression mRNA analysis of cell lines showed a statistically significant increase in expression of VEGF in OSCC cell lines compared with normal epithelial cells (p=0.01). The levels of VASH-1 and KDR could not be determined from the analysis, because of low levels of expression.
Discussion and Conclusion:
The presence of VEGF, KDR and VASH-1 in OSCC tissue demonstrates the presence of angiogenic signalling. OSCC showed greater epithelial VEGF expression in IHC and OSCC cell lines but VASH-1 expression showed a tendency towards fewer stromal cells staining positively compared with PG tissues, indicating that angiogenesis self-regulating mechanisms may be altered in OSCC. PG tissues had a greater MVD and showed expression of VASH-1 in many endothelial cells. From this, it appears that angiogenic factors may be expressed differently in hyperplastic lesions compared with OSCC. Expression of these angiogenic factors was seen in mRNA analysis of vascular granulation tissue and in OSCC and epithelial cell lines, with greater relative expression of VEGF in OSCC cells compared with normal epithelium. Further research may show the implications of this for treatment of OSCC and diseases related to angiogenesis, with potential for regulation with angiogenesis inhibitors, and development of prognostic biological markers.