Microvascular Density in Terms of Number and Length of Microvessel Segments per Unit Tissue Volume in Mammalian Angiogenesis

Abstract

The number and length of all the microvessel segments present per unit of tissue volume are the two determinants of microvascular density. In the present study, a microscopic interactive image-analysis method was introduced, using the length of individual microvessel segments extending between two successive points of bifurcation (Le. MS) and their number (No. MS) per unit of tissue volume in large populations of microvessel segments, which were measured using the nonsurgical rat mesenteric-window angiogenesis assay. In addition, microvessel tortuosity was quantitatively assessed. The method presented here was applied to the angiogenic response over 3 weeks to the directly acting angiogen VEGF165, which was administered i.p. at three dose levels, i.e., 4.8, 48, and 480 pM. Following the VEGF165-treatment, statistically significant changes were found not only in the No. MS but also in the distribution of Le. MS: shortening of vessels being an indicator of vessel branching and increased vessel length being an indicator of angiogenesis through elongation. The shape of the overall distribution of Le. MS was, however, basically the same on any observation occasion regardless of whether significant angiogenesis was present or not. VEGF165induced a roughly dose-dependent angiogenic response in terms of No. MS and the length of the shortest (0–10% percentile) and/or the longest (90–100% percentile) microvessel segments. Moreover, the onset of the early stimulating effect of VEGF165on microvessel branching was also dose-dependent. The variables which were introduced here were shown to display a very high degree of sensitivity and resolution and are apparently unrivalled when it comes to the study of density in populations of microvessels.