Microvascular engineering in perfusion culture: immunohistochemistry and CLSM findings

BACKGROUND: One of the most challenging problems in tissue engineering is the establishment of vascular supply. A possible approach might be the engineering of microvasculature in vitro and the supply by engineered feeder vessels. METHODS: An in vitro model for a small-diameter vessel was developed and made from adipose tissue stromal cells and human umbilical vein endothelial cells in a tube-like gelatine scaffold. The number of "branches" emerging from the central lumen and the morphology of the central lumen of the vessel equivalent were assessed after 16 days of either pulsatile perfusion culture or culture in rotating containers by evaluation of immunohistochemically stained sections (n = 6 pairs of cultures). Intramural capillary network formation was demonstrated in five experiments with confocal laser scanning microscopy. RESULTS: Perfused specimens showed a round or oval lumen lined by a single layer of endothelial cells, whereas following rotation culture the lumen tended to collapse. Confocal laser scanning microscopy showed more extended network formation in perfused specimens as compared to specimens after rotation culture. Partially highly interconected capillary-like networks were imaged which showed orientation around the central lumen. Perfused specimens exhibited significantly more branches emerging from the central lumen. There were, however, hardly any capillary branches crossing the whole vessel wall. CONCLUSION: Pulsatile perfusion supports the development of vascular networks with physiological appearance. Advances in reactor development, acquisition of functional data and imaging procedures are however necessary in order to attain the ultimate goal of a fully functional engineered supplying vessel

Thymidine phosphorylase expression in normal, hyperplastic and neoplastic prostates: correlation with tumour associated macrophages, infiltrating lymphocytes, and angiogenesis

Thymidine phosphorylase is an angiogenic factor primarily expressed by cancer cells, stromal cells and tumour-associated macrophages in many human malignancies. These different types of thymidine phosphorylase-expressing cells, however, may have a distinct place in the angiogenic process, and this question was addressed in the present study. A series of 20 normal/hyperplastic prostate glands and 60 prostate carcinomas was investigated by immunohistochemistry, using specific antibodies for thymidine phosphorylase (P-GF.44C), tumour-associated macrophages (CD68), endothelium (CD31) and prostate specific antigen (ER-PR8). Thymidine phosphorylase expression by normal and hyperplastic epithelial or stromal cells occurred almost exclusively in the context of an intense lymphocytic infiltrate. High thymidine phosphorylase cancer cells and thymidine phosphorylase stromal cells expression was associated with high angiogenesis in prostate carcinomas, and this significant association was extended to include both tumour-associated macrophages and tumour-infiltrating lymphocytes. Thymidine phosphorylase expression and tumour-infiltrating lymphocytes were related inversely with prostate specific antigen reactivity. In conclusion, thymidine phosphorylase is a major angiogenic factor in prostate carcinomas and its up-regulation is likely to occur in the context of a host immune response

Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model

Diet and lifestyle produce major effects on tumour incidence, prevalence, and natural history. Moderate dietary restriction has long been recognised as a natural therapy that improves health, promotes longevity, and reduces both the incidence and growth of many tumour types. Dietary restriction differs from fasting or starvation by reducing total food and caloric intake without causing nutritional deficiencies. No prior studies have evaluated the responsiveness of malignant brain cancer to dietary restriction. We found that a moderate dietary restriction of 30–40% significantly inhibited the intracerebral growth of the CT-2A syngeneic malignant mouse astrocytoma by almost 80%. The total dietary intake for the ad libitum control group (n=9) and the dietary restriction experimental group (n=10) was about 20 and 13 Kcal day−1, respectively. Overall health and vitality was better in the dietary restriction-fed mice than in the ad libitum-fed mice. Tumour microvessel density (Factor VIII immunostaining) was two-fold less in the dietary restriction mice than in the ad libitum mice, whereas the tumour apoptotic index (TUNEL assay) was three-fold greater in the dietary restriction mice than in the ad libitum mice. CT-2A tumour cell-induced vascularity was also less in the dietary restriction mice than in the ad libitum mice in the in vivo Matrigel plug assay. These findings indicate that dietary restriction inhibited CT-2A growth by reducing angiogenesis and by enhancing apoptosis. Dietary restriction may shift the tumour microenvironment from a proangiogenic to an antiangiogenic state through multiple effects on the tumour cells and the tumour-associated host cells. Our data suggest that moderate dietary restriction may be an effective antiangiogenic therapy for recurrent malignant brain cancers

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference