Mouse Proepicardium Exhibits a Sprouting Response to Exogenous Proangiogenic Growth Factors in vitro

Abstract

Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A₁₂₀ and VEGF-A₁₆₄. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.

Proepicardium: Current Understanding of its Structure, Induction, and Fate

“…This was further confirmed with transmission electron microscopic studies, which showed the endothelium of proepicardial vessels to be covered by a discontinuous basement membrane; it also showed that cytoplasmic vesicles, which are typical for a mature and metabolically active endothelium, were scarce (Niderla‐Bielinska et al, ). PE explants, isolated from mice and cultured on collagen or Matrigel under the influence of angiogenic factors (VEGF, bFGF) can give rise to vascular sprouts, which are continuous with proepicardial explant vessels (Niderla‐Bielinska et al, , ). Avian PE explants are also capable of forming vascular sprouts, both spontaneously and under the influence of proangiogenic factors (VEGF, bFGF) (Guadix et al, ).…”

“…In vitro experiments showed that PE explants give rise to vascular sprouts, but since the PE contains its own EC population, the mechanism involves angiogenesis rather than de novo differentiation (Niderla‐Bielinska et al, , ).…”

Sulodexide inhibits angiogenesis via decreasing Dll4 and Notch1 expression in mouse proepicardial explant cultures

“…Proepicardium contains not only ECs, that are not fully differentiated and immature, but also various populations of mesenchymal cells, expressing different surface markers . Mouse PE is a new, distinctive model for angiogenesis studies that lacks immunocompetent cells . SDX strongly affects immunocompetent cells modulating cytokine, growth factor, and MMP expression and activity; thus, model devoid of such component would allow assessing the possible direct action of SDX on ECs and angiogenesis.…”

“…Since there is no single perfect model, researchers usually employ multimodel approach . Mouse PE is not widely used as an in vitro model for studying angiogenesis, but our previous observations lead us to the conclusion that PE could be a good tool for angiogenesis studies because: (i) it contains its own population of ECs that are not committed and undergo rapid changes, (ii) proepicardial ECs are embedded in the microenvironment, but they are devoid of immune system, and (iii) cultured explants of mouse or avian PE under the influence of various isoforms of VEGF‐A and bFGF produce vascular sprouts of different morphology, number, and phenotype .…”

Cardiac Development: A Glimpse on Its Translational Contributions

“…The embryonic epicardium originates from the proepicardium, an epithelial protrusion with a cauli-flower like structure emanating at the junction between the cardiac and the hepatic primordia [275,276] ( Figure 2B). The formation of the proepicardium is triggered by distinct factors [277][278][279][280], among which it is important to highly the antagonist roles of Bmp [282][283][284][285] and Fgf [288][289][290] family members. After covering the embryonic naked myocardium, the embryonic epicardium is instructed to an epithelial-to-mesenchymal transformation (EMT) leading to the epicardial-derived cells (EPDC) that will subsequently migrate into the embryonic myocardium and will progressively differentiate into distinct cell derivatives such as cardiac fibroblasts as coronary vasculature components such endothelial, smooth muscle cells, and adventitial fibroblasts [291][292][293][294][295][296][297][298][299] ( Figure 2D).…”