Lymphangiogenesis by blind‐ended vessel sprouting is concurrent with hemangiogenesis by vascular splitting

Abstract

Development of effective vascular therapies requires the understanding of all modes of vessel formation involved in angiogenesis (here termed "hemangiogenesis") and lymphangiogenesis. Two major modes of vessel morphogenesis include sprouting of a new vessel from a preexisting vessel and splitting of a preexisting parent vessel into two offspring vessels. In the quail chorioallantoic membrane (CAM) during mid-development (embryonic days E6 -E9), lymphangiogenesis progressed primarily via blind-ended vessel sprouting. Isolated lymphatic endothelial progenitor cells were recruited to the tips of growing vessels. During concurrent hemangiogenesis, parent blood vessels expanded from the capillary network and split into offspring vessels, accompanied by transient capillary expression of alpha smooth muscle actin (␣SMA) and recruitment of polarized mural progenitor cells. Lymphatics and blood vessels were identified by confocal/fluorescence microscopy of vascular endothelial growth factor (VEGF) receptor VEGFR-2, ␣SMA (specific to CAM blood vessels), homeobox transcription factor Prox1 (specific to lymphatics), and the quail hematopoetic marker, QH-1. VEGFR-2 was expressed intensely in isolated cells and lymphatics, and moderately in blood vessels. Prox1 was absent from isolated progenitor cells prior to lymphatic recruitment. Exogenous vascular endothelial growth factor-165 (VEGF 165 ) increased blood vessel density and anastomotic frequency without changing endogenous modes of vascular/lymphatic vessel formation or marker expression. Although VEGF 165 is a key cellular regulator of hemangiogenesis and vasculogenesis, the role of VEGF 165 in lymphangiogenesis is less clear. Interestingly, VEGF 165 increased lymphatic vessel diameter and density as measured by novel Euclidean distance mapping, and the antimaturational dissociation of lymphatics from blood vessels, accompanied by lymphatic reassociation into homogeneous networks. Published 2006 Wiley-Liss, Inc. †

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

“…In contrast to own and other earlier studies on CAM pericytes Hagedorn et al 2004;Nico et al 2004;Parsons-Wingerter et al 2006;Hlushchuk et al 2007), we could show here by combination with SNA ( Fig. 2a, b) that DES and SMA are present in the CAM capillary plexus, and are intimately related to its endothelial layer at least after E12 (even as early as E10, not shown).…”

“…Moreover, yet other reports are in conXict with respect to SMA expression, which was described either as not before E12 (Hlushchuk et al 2007) or transiently early: E8 (Parsons-Wingerter et al 2006). Taken together, despite the description of "endothelial-like cells" and their extensions during IA (Patan et al 1993(Patan et al , 1996, and their more recent interpretation as pericytes or myoWbroblasts (Burri et al 2004), cytoskeletal properties and arrangement of periendothelial cells and their processes remained enigmatic.…”

“…The avian chorioallantoic membrane (CAM) is an established system for studying vascular growth and remodeling (Patan et al 1993;Kurz et al 1995Kurz et al , 2003Schlatter et al 1997;Djonov et al 2000bDjonov et al , 2002, for evaluating growth factors or biocompatible materials (Wilting et al 1993(Wilting et al , 1996Kurz et al 1998;Ribatti et al 2001;Zwadlo-Klarwasser et al 2001;Hagedorn et al 2004;Parsons-Wingerter et al 2006;Hlushchuk et al 2007;Oates et al 2007), or angiogenesis-related processes like cell or tumor growth (Papoutsi et al 2000;Kurz et al 2002Kurz et al , 2004Hagedorn et al 2005;Lugassy et al 2007). When the role of vascular mural cells during embryonic angiogenesis received increasing attention about 10 years ago, we were among the Wrst to identify the cytoskeletal proteins desmin (DES) and -smooth muscle actin ( SMA) in cells of the CAM microvasculature and connective tissue; we showed that PDGF-B may stimulate myoWbroblast formation and formation of non-capillary microvessels (Oh et al 1998).…”

Cardiac Vasculature: Development and Pathology

“…Another equally important issue is the remodeling that eliminates nascent vessels during the transformation of the primitive vascular network into a mature branching vasculature. To complicate this picture of coronary vessel development, Qh-1 labels not just blood vessel precursors but distinctly labels lymphatic precursors (Parsons-Wingerter et al, 2006) in the chorioallantoic membrane (extraembryonic tissue) and we have recently found that the same is true in the developing epicardium (Karunamuni et al, 2010). Therefore, some of the structures labeled with Qh-1 and identified as nascent blood vessels may be nascent lymphatic vessels.…”

Lymphangiogenesis:in vitroandin vivomodels

“…Other lymphatic-gene mouse models are available, such as mice deficient for Net (93), podoplanin (94), neuropillin-2 (NRP-2) (95), angiopoietin-2 (Ang2) (96), integrin-␣ 9 (97), p85 subunit of PI3K (98) (102). The avian chorioallantoic membrane (CAM) was used to support this concept of progenitor cell recruitment and to demonstrate its dependence to VEGF-C (103,104).…”