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Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor. Int. J. Cancer 86:768–776, 2000. © 2000 Wiley-Liss, Inc.

Amyloid-? (A?) deposition in cerebral vessels (cerebral amyloid angiopathy, CAA) is accompanied by degeneration of vascular cells, including pericytes and smooth muscle cells. Previous studies indicated that specific A? protein isoforms are toxic for cultured human brain pericytes and smooth muscle cells. In particular, A?1-40carrying the E22Q mutation, as in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), is toxic. We investigated the effects of the A?-binding protein apolipoprotein E (ApoE) on the toxicity of A? for cultured human brain pericytes. We compared the toxicity of HCHWA-D A?1-40for pericyte cultures with different ApoE genotypes, studied the accumulation of A? and ApoE in these different cell cultures, and investigated the effects of exogenous ApoE. Pericyte cultures with an ApoE 23 genotype were more resistant to HCHWA-D A?1-40treatment than cultures with a 33 or 34 genotype. Cell death was highest in cultures homozygous for ApoE 4. The extent to which both A? and ApoE accumulated at the cell surface was parallel to the degree of toxicity. The addition of purified ApoE resulted in a decrease in cell death. These data suggest that ApoE4 may direct A? more efficiently than other ApoE isoforms into a pathological interaction with the HBP cell surface. The results of this study are in line with the observations that inheritance of the ApoE 4 allele increases the risk of developing Alzheimer's disease, and that the ApoE 2 allele has a relatively protective effect.

In a number of recent papers, the degree of tumour vascularization has been described as a promising new prognostic factor. Methods for the assessment of vascular density involve immunohistochemical staining of the vasculature, followed by counting the number of vessel profiles in the angiogenic hot spot. One of the problems of this procedure is the selection of the angiogenic hot spot, which has been described as being subject to inter-observer variation. In this study, the value of true colour image analysis in reducing inter-observer variation has been assessed. Highly (MV3) and poorly (M14) vascularized human melanoma xenografts were used to evaluate the image analysis procedure, and the image analysis results were compared with results from the conventional manual hot-spot procedure. Assessment by image analysis was performed on measurement fields covering the entire tumour tissue specimens rather than on a single hot-spot field. Also, by selecting the most densely vascularized area from all fields assessed by the semi-automatic procedure, it was possible to objectify the hot spot selection (automated hot-spot procedure). Manual assessment showed a good correlation between two independent observers for MV3 xenografts (r=0·74, P=0·014), but a poor correlation for M14 xenographs (r=0·4, P>0·05). Automated assessment by different operators showed good correlations for both MV3 xenografts (r=0·99, P<0·001) and M14 xenografts (r=0·80, P=0·006). It is concluded that although both manual vessel counting and semi-automated image analysis can differentiate between the level of vascularization in the two types of xenograft (P<0·001 for both methods), the automated method is favourable in that it showed no significant inter-observer effects. In M14 xenografts, the manual hot-spot vessel densities did not correlate well with the automated hot-spot densities (r=0·27, P>0·05), indicating that selection of angiogenic hot spots in this tumour type is indeed subject to observer bias. The automated hot-spot vessel densities were a reliable indicator of overall tumour vessel density in both tumour types. Image analysis allows analysis of vessel subclasses based on morphological criteria such as vessel profile area or diameter. In the model system used, the discrimination between MV3 and M14 xenografts was further enhanced by selectively examining vessels with diameters between 6 and 9 μm (P<0·0005). In conclusion, image analysis appears to offer an objective and more reproducible method to quantify tumour vascularity than manual counting of vessel profiles in the hot spot. Analysis of subclasses of vessels may further enhance the value of vessel density measurements in discriminating between tumour types differing in biological behaviour. © 1998 John Wiley & Sons, Ltd.