Rationale: Defects in the morphogenesis of the 4th 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 interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery (AAA) and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5β1 and fibronectin (Fn1) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5β1 and Fn1 regulate AAA 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 uniform 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 α5β1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates AAA morphogenesis at multiple steps: 1) accumulation of SHF-derived ECs in the pharyngeal arches, 2) remodeling of the uniform EC plexus in the 4th 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 multi-step 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 AAA and its major branches. Cell-ECM interactions regulated by integrin α5β1 and Fn1 play essential roles at each of these developmental stages.

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Rationale: Defects in the morphogenesis of the aortic arch arteries (AAAs) are among the most severe congenital birth defects. Understanding genes and mechanisms regulating AAA formation and remodeling will provide important insights into the etiology and potential treatments of congenital heart disease.Objective: Cell-ECM interactions play essential roles in the AAA morphogenesis; however, their specific functions are not well-understood. Previously, we demonstrated that integrin a5b1 and fibronectin (Fn1) expressed in the Isl1 lineage and its derivatives regulate the formation of the pharyngeal arch arteries (PAAs), the vessels giving rise to the AAAs. The objective of these studies was to investigate the mechanisms by which integrin a5b1 and Fn1 regulate AAA morphogenesis. Methods and Results:Using temporal lineage tracing, we found that endothelial progenitors of the AAA endothelium arise early during the development of the second heart field (SHF) and that the 4 th PAAs contain the highest percentage of the SHFderived ECs (ECs). To understand the role of cell-extracellular matrix (ECM) interactions in AAA development, we deleted either integrin a5 or its major extracellular ligand Fn1 in the Isl1 lineage. We used whole-mount confocal imaging to define the complex spatial and temporal EC dynamics during PAA formation at the quantitative level and assessed how cell-ECM interactions modulated these dynamics. Our analyses demonstrated that integrin a5b1 and Fn1 mediate AAA morphogenesis by regulating the accrual of SHF-derived endothelium into the 4 th pharyngeal arches and the remodeling of the 4 th pharyngeal arch EC plexus into the PAAs. Following PAA formation, integrin a5b1 is essential for the activation of Notch in the neural crestderived cells surrounding the 4 th PAAs and for the differentiation of the neural crest cells into vascular smooth muscle cells. Conclusions:Our data demonstrate that cell-ECM interactions regulated by integrin a5b1 and Fn1 function reiteratively during AAA development to mediate the multi-step process of AAA morphogenesis.

Venous malformations (VMs) are slow-flow malformations of the venous vasculature and are the most common type of vascular malformation with a prevalence of 1%. Germline and somatic mutations have been shown to contribute to VM pathogenesis, but how these mutations affect VM pathobiology is not well understood. The goal of this study was to characterize VM endothelial and mural cell expression by performing a comprehensive expression analysis of VM vasculature. VM specimens (n = 16) were stained for pan-endothelial, arterial, venous, and endothelial progenitor cell proteins; proliferation was assessed with KI67. Endothelial cells in the VM vessels were abnormally orientated and improperly specified, as seen by the misexpression of both arterial and endothelial cell progenitor proteins not observed in control vessels. Consistent with arterialization of the endothelial cells, VM vessels were often surrounded by multiple layers of disorganized mural cells. VM endothelium also had a significant increase in proliferative endothelial cells, which may contribute to the dilated channels seen in VMs. Together the expression analysis indicates that the VM endothelium is misspecified and hyperproliferative, suggesting that VMs are biologically active lesions, consistent with clinical observations of VM progression over time.

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