ABSTRACT
During embryogenesis, the initial vascular network forms by the process of vasculogenesis, or the specification of vascular progenitors de novo. In contrast, the majority of later-forming vessels arise by angiogenesis from the already established vasculature. Here, we show that new vascular progenitors in zebrafish embryos emerge from a distinct site along the yolk extension, or secondary vascular field (SVF), incorporate into the posterior cardinal vein, and contribute to subintestinal vasculature even after blood circulation has been initiated. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of the function of vascular progenitor marker etv2/etsrp prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. Similar late-forming etv2+ progenitors were also observed in mouse embryos, suggesting that SVF cells are evolutionarily conserved. Our results characterize a distinct mechanism by which new vascular progenitors incorporate into established vasculature.
Subject(s)
Zebrafish Proteins , Zebrafish , Animals , Blood Vessels/metabolism , Embryo, Nonmammalian/metabolism , Endothelial Cells/metabolism , Gene Expression Regulation, Developmental , Mice , Neovascularization, Physiologic , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
RATIONALE: Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. OBJECTIVE: Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5ß1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5ß1 and Fn1 regulate aortic arch artery morphogenesis. METHODS AND RESULTS: Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5ß1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells. CONCLUSIONS: PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5ß1 and Fn1 play essential roles at each of these developmental stages.
Subject(s)
Aorta, Thoracic/metabolism , Cell-Matrix Junctions/metabolism , Endothelial Progenitor Cells/metabolism , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Animals , Aorta, Thoracic/embryology , Cell Lineage , Cell-Matrix Junctions/genetics , Extracellular Matrix/genetics , Extracellular Matrix Proteins/genetics , Fibronectins/genetics , Fibronectins/metabolism , Gene Expression Regulation, Developmental , Gestational Age , Integrin alpha5beta1/genetics , Integrin alpha5beta1/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Morphogenesis , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
PURPOSE: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need.Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. RESULTS: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. CONCLUSIONS: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC.
