Quantitative and morphometric evaluation of the angiogenic effects of leptin

Abstract

Angiogenesis is a dynamic process that requires an interaction of pro-and antiangiogenic factors. It is known that the cytokine leptin stimulates endothelial cell growth and angiogenesis, but further quantitative analysis is necessary to understand leptin angiogenic effects. The quail chorioallantoic membrane ͑CAM͒ assay has been used to study angiogenesis in vivo by focusing on morphometric parameters that quantify vascular complexity and density. We quantify the angiogenic activity of leptin using the CAM assay by digital morphometry and a computer-assisted image analysis to evaluate more precisely vessel length, diameter, branching, and tortuousity. CAM images are obtained from ex ovo cultures of E8-E9 quail embryos. MATLAB® and custom software are used for our analysis. The effects of leptin, vascular endothelial growth factor-165 ͑VEGF 165 ͒, and their corresponding neutralizing antibodies are compared. Our results show that CAM treated with leptin and VEGF 165 has a significant increase in vascular complexity and density. A corresponding decrease is observed using neutralizing antibodies. Notably, leptin induced more significant changes than VEGF in vessel length and tortuousity. Conversely, VEGF induced a greater increase in vessel branching than leptin. These results underscore the importance of using multiparametric quantitative methods to assess several aspects of angiogenesis and enable us to understand the proangiogenic effects of leptin.

Angiogenic Effect of Leptin in the Quail Chorioallantoic Membrane

“…However, the administration of leptin to CAM together with anti FGF-2 antibodies reduced the angiogenic effect of leptin by approximately 40%, indicating that the activation of endogenous FGF-2 mediated at least in part leptin action (Ribatti et al 2001). Recent data obtained with quail CAM demonstrate that leptin acts also in cooperation with the vascular endothelial growth factor (VEGF) inducing more significant changes than VEGF in vessel length and tortuousity (Talavera-Adame et al 2008). An important finding supporting the idea of leptin effects on angiogenesis was the identification of leptin receptors in endothelial cells and mouse adipose tissue while its expression was not proved in adipose tissue and endothelial cells in leptin receptor-deficient (db/db) mice (Bornstein et al 2000).…”

“…However, the mechanisms of how leptin can accelerate embryonic growth are not understood. Since leptin has been shown to stimulate angiogenesis under in vitro and in vivo conditions (Sierra-Honigmann et al 1998;Talavera-Adame et al 2008) we expect that activation of vessel development can contribute to better exchange of nutrients and gasses between the developing embryo and the yolk and gas environment. Therefore, the aim of our study was to determine the effect of leptin on the development of CAM in Japanese quail under in vivo conditions using two experimental approaches (ex ovo and in ovo).…”

Leptin Neuroprotection in the Central Nervous System

“…Leptin also has a potent angiogenic effect [47,48]. Administration of leptin has induced neovascularization in corneas of normal rats but not in corneas from fa/fa Zucker rats that do not have LEPRs capable of signal transduction [49].…”

Endothelium-derived essential signals involved in pancreas organogenesis

“…For instance, high signaling of the canonical WNT/β-catenin pathway promotes endoderm posterior pattern with foregut-derived structures such as pancreas and liver [68] . Interestingly it has been found that some of these factors are produced by ECs [13,42,53,[69][70][71] .…”

“…Additionally, at later embryo stage, flk-1 -/-mice showed dorsal mesenchyme that does not express Isl-1 in the absence of aortic ECs suggesting that endothelial-cell signaling promotes Isl-1 cell expression from dorsal mesenchymal cells [64] . In addition, it has been reported that mesenchymal cells also express bone morphogenetic proteins (BMPs) and that these proteins have a pivotal role in pancreas development [71,72] .…”