Pericytes in the mature chorioallantoic membrane capillary plexus contain desmin and alpha-smooth muscle actin: relevance for non-sprouting angiogenesis.

The chicken chorioallantoic membrane (CAM) is a frequently used tissue for studying vascular growth and remodeling, notably non-sprouting angiogenesis by formation of transluminal pillars. Vascular pericytes have received increasing attention in the field of angiogenesis research and appear important for pillar growth. Our earlier observation that desmin (DES), but not alpha-smooth muscle actin (alphaSMA) was expressed in pericytes of the mature CAM capillary plexus after E12 was confirmed by others. However, in different species or vascular beds, either marker or both have been used to identify pericytes, raising the questions if (1) expression of these cytoskeletal proteins really was mutually exclusive; or (2) different types of pericytes existed in the same vascular bed. Using triple labeling with fluorochrome-conjugated markers Sambucus nigra agglutinin, DES or alphaSMA, and DNA-specific YoPro-1, we report here for the first time a delicate filamentous, circumferentially oriented alphaSMA pattern in periendothelial cells of the mature CAM capillary plexus, quite different from the coarser, axially oriented DES pattern. A new method for automatic classification of DNA-staining pattern was applied to compare nuclei of DES- or alphaSMA-positive cells. It predicted colocalisation of both proteins in most capillary pericytes, which was confirmed by double immunostaining for DES and alphaSMA. We conclude that (1) in contrast to published work, DES and alphaSMA are not mutually exclusive in most pericytes; (2) different types of pericytes may co-exist in the same vascular bed; (3) on average, one pericyte is associated with two transluminal pillars; (4) a novel imaging modality may be useful for cell identification in angiogenesis research and elsewhere.