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

Abstract

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.

The Human Mesenchymal Stem Cells and the Chick Embryo Chorioallantoic Membrane: The Key and the Lock in Revealing Vasculogenesis

“…It was previously demonstrated that SMA is present in the CAM capillary plexus, and its expression is intimately related to its endothelial layer at least after day 12 of incubation (16). Although the anti-SMA antibody was human specific, it was clearly positive in a population of cMSC and the chick pericytes.…”

“…What impresses in the SMA is its high expression in the angiogenic and vasculogenic areas, as it proves the close contact of the pericyte-like cells with the new capillaries, but also with CLS, even if they do not present a lumen yet. It is clear that SMA is needed for the stability of the pseudopode like projections present in the pericytes (16), offering a consecutive stability to the vessel wall, but the question is why is this SMA expressed so early in the cMSC? It was hypothesized that pericytes are recruited towards the capillary plexus early in CAM development (before day 10 of incubation) (16) and VEGF seems to stimulate this process (17).…”

“…It is clear that SMA is needed for the stability of the pseudopode like projections present in the pericytes (16), offering a consecutive stability to the vessel wall, but the question is why is this SMA expressed so early in the cMSC? It was hypothesized that pericytes are recruited towards the capillary plexus early in CAM development (before day 10 of incubation) (16) and VEGF seems to stimulate this process (17). Our results regarding the hMSC-stimulated vasculogenic process prove something else.…”

“…CAM is a frequently used tissue for studying vascular growth and remodeling, notably non-sprouting angiogenesis by formation of transluminal pillars (16), while VEGF seems to be involved in the augmentation of the intussusceptive microvascular growth (17). Ribatti et al suggests that sprouting angiogenesis may be identified in CAM in the early phase (days 5-7 of development), the later phases being characterized by the appearance of the intussusceptive angiogenesis and the absence of the sprouts (2).…”

State-of-the-art technologies, current opinions and developments, and novel findings: news from the field of histochemistry and cell biology

“…Intusseptive angiogenesis, nonsprouting angiogenesis by means of transluminal pillars and folds, has been extensively studied in the chicken chorionallantoic membrane (CAM; Kurz et al 2003), and pericytes have been shown to be important for this process. Kurz et al (2008a), using triple labelling with Xuorochrome-conjugated markers Sambucus nigra agglutinin, desmin (DES) or smooth muscle actin ( SMA), and DNA staining and confocal analysis, documented the presence of a delicate Wlamentous SMA-immunolabeling and a coarser, axially oriented DES-immunolabelling pattern in periendothelial cells of the mature CAM capillary plexus. Additionally, they developed a new automatic classiWcation method for DNA staining patterns to compare nuclei of DES or SMA-immunoreactive cells.…”