Tumourigenesis in experimental models is associated with the formation of new blood vessels (angiogenesis). Recent studies have suggested that tumour angiogenic activity may be inferred in histological sections by measuring the density of the vasculature. The purpose of this study was to determine whether the transition from normal to dysplastic and neoplastic tissue in the oral mucosa is accompanied by quantitative or qualitative changes in the vascularity of the tissue, and how the estimate of vascularity is influenced by the vessel marker and method of assessment. A total of 100 specimens of normal oral mucosa, dysplastic lesions, and squamous cell carcinomas were examined. Sections were immunostained with the pan‐endothelial antibodies to von Willebrand Factor (vWF) and CD31, or with an antibody to the αvβ3 integrin, previously reported to be a marker of angiogenic vessels. Vascularity was quantitated by two different methods: highest microvascular density (h‐MVD) and microvascular volume, as determined by point counting (MVV). The results showed that vascularity, measured by the MVV method using antibodies to either vWF or CD31, increased significantly (P<0·0001) with disease progression from normal oral mucosa, through mild, moderate, and severe dysplasia to early and late carcinoma (76 paraffin‐embedded tissues examined). In contrast, h‐MVD did not discriminate between dysplastic lesions and carcinoma. A similar percentage of the total vessel volume (MVV) and density (h‐MVD) were positive for αvβ3 in 24 frozen tissues examined, including normal oral mucosa. It is concluded that there is a close association between vascularity and tumour progression in the oral mucosa. Morphometric analysis reflecting microvascular volume is more informative than the currently popular analysis of microvascular density. The expression of αvβ3 in the vasculature of oral tissues does not necessarily reflect the presence of angiogenic vessels. © 1997 by John Wiley & Sons, Ltd.

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Experimental animal models have demonstrated that angiogenesis is essential for tumour progression, whilst sustained tumour growth requires a positive balance between tumour cell proliferation and cell death (apoptosis). The aim of this study was to determine the relative contribution of apoptosis, proliferation, and angiogenesis to disease progression in the oral mucosa. Histological sections of 47 archival specimens were examined; these included four groups of oral tissues: normal mucosa (n=12), moderate dysplasia (n=11) severe dysplasia (n=6), and squamous cell carcinoma (n=18). Apoptotic cells were visualized by in-situ end-labelling of DNA, proliferative cells by staining with Ki-67 antibody, and blood vessels with von Willebrand factor (vWF) antibody. One-way analysis of variance showed that indices of apoptosis (AI), proliferation (PI), and angiogenesis (vascularity) increased significantly with disease progression from normal oral mucosa, through dysplasia, to carcinoma (p<0.0001 for every index). The increase from normal mucosa to moderate dysplasia was significant for PI and vascularity, but not for AI. In contrast, the increase from dysplasia to carcinoma was significant for AI and vascularity, but not for PI. These data suggest that disease progression in the oral mucosa is accompanied by angiogenesis and increases in both epithelial proliferation and apoptosis. Net epithelial growth results from proliferation starting earlier and proceeding at a higher rate than apoptosis.

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Summary The aim of this study was to test the hypotheses that (a) microvascular density (MVD) measured in histological sections of resected non-small cell lung carcinomas is an index of angiogenesis and (b) the measurement of MVD in a single block is representative of the overall MVD of the tumour. MVD was quantitated in one block per specimen of 60 lung tumours and nine normal lung tissues, and in 47 blocks taken from different regions of four tumours. Blood vessels were stained with antibody to von Willebrand Factor and MVD was quantitated using two methods: average density throughout the section (a-MVD) and density in the most vascularized area or 'hot spot' (h-MVD). Similar h-MVD values were found in tumours and in normal bronchus, whereas a-MVD was greater in the latter (P < 0.01). When 47 blocks from four tumours were analysed, inter-tumour variation was significant (P < 0.001) in spite of significant intra-tumour variation. The highest MVD value was not necessarily found in the periphery of the tumour. The four tumours were ranked into either two or four tiers according to their overall MVD. In 50 random selections of one block per tumour, the correct ranking was achieved in 68-74% of cases with the two-tier ranking and in 6-16% of cases with the four-tier ranking (h-MVD and a-MVD values respectively). These results suggest that elevated MVD values do not necessarily represent angiogenesis in non-small cell lung carcinomas. When only one block per tumour is examined, the chance of obtaining an accurate estimate of the vascularity of that tumour may be lower than 68%.

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TSP-1 is a large extracellular matrix glycoprotein implicated in angiogenesis. Its specific role is not clear, as both stimulatory and inhibitory effects have been demonstrated in animal models ( I , 2). Angiogenesis cannot be measured directly in human tumours, however, the density of the microvasculature in tissue sections has been commonly used as an index of this process. Although angiogenesis is important for tumour growth and dissemination, the value of microvascular density as a prognostic indicator in breast carcinoma remains controversial (3)(4)(5).In order to assess the possible role of TSP-I in angiogenesis, we have quantitated vascularity and TSP-I expression in resected human breast carcinomas. Vascularity was assessed by four different methods following immunostaining of histological sections with antibodies to Von Willebrand factor (vWF). These methods were: (a) average microvascular density (a-MVD), (b) highest microvascular density (h-MVD), (c) microvascular volume ( M W ) and (d) image analysis of stained area (vWF Area). a-MVD and h-MVD were assessed by counting the vessels that were contained within a grid covering an area of 0.476 mm2. To measure a-MVD, vessels in 15-20 random fields across the section were counted. To measure h-MVD, the tumour sections were scanned for the area of highest vascular density; the number of vessels in 5 fields in this area were then counted and the highest value was taken. In both cases the counts were expressed as number of vessels per square millimetre. MVV was measured by point counting using an eyepiece graticule which contained 100 points; 15 random fields (1500 points) were counted across each section. TSP-I mRNA expression was determined by (a) northern blotting (nb-TSP) using a 32P-labelled cDNA probe to human endothelial TSP-I, and (b) in situ hybridisation (ish-TSP) using a digoxigenin-labelled RNA probe. nb-TSP was quantitated by densitometric scanning and ish-TSP was quantitated morphometrically by point counting of 15 random fields. We found good correlation between the four methods of assessing vascularity in the tumours (a-MVD v h-MVD, r = 0.92, p

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