Interaction between alk1 and blood flow in the development of arteriovenous malformations

Abstract

SUMMARYArteriovenous malformations (AVMs) are fragile direct connections between arteries and veins that arise during times of active angiogenesis. To understand the etiology of AVMs and the role of blood flow in their development, we analyzed AVM development in zebrafish embryos harboring a mutation in activin receptor-like kinase I (alk1), which encodes a TGFb family type I receptor implicated in the human vascular disorder hereditary hemorrhagic telangiectasia type 2 (HHT2). Our analyses demonstrate that increases in arterial caliber, which stem in part from increased cell number and in part from decreased cell density, precede AVM development, and that AVMs represent enlargement and stabilization of normally transient arteriovenous connections. Whereas initial increases in endothelial cell number are independent of blood flow, later increases, as well as AVMs, are dependent on flow. Furthermore, we demonstrate that alk1 expression requires blood flow, and despite normal levels of shear stress, some flow-responsive genes are dysregulated in alk1 mutant arterial endothelial cells. Taken together, our results suggest that Alk1 plays a role in transducing hemodynamic forces into a biochemical signal required to limit nascent vessel caliber, and support a novel two-step model for HHT-associated AVM development in which pathological arterial enlargement and consequent altered blood flow precipitate a flow-dependent adaptive response involving retention of normally transient arteriovenous connections, thereby generating AVMs.

Circulating Bmp10 acts through endothelial Alk1 to mediate flow-dependent arterial quiescence

“…Both of these genes are flow responsive in cultured endothelial cells (Wang et al, 1993;Melchionna et al, 2005), suggesting that Alk1 might lie upstream of cxcr4a and edn1 in a mechanosensitive signaling pathway. In support of this hypothesis, blood flowmediated repression of cxcr4a correlates with diminished endothelial cell protrusive activity in nascent zebrafish arteries (Bussmann et al, 2011), and zebrafish alk1 mutants, which exhibit abnormally high levels of arterial cxcr4a, develop enlarged arteries containing supernumerary endothelial cells, suggestive of failed flow-induced suppression of endothelial cell migration or proliferation (Roman et al, 2002;Corti et al, 2011). Evidence from mice further supports the idea that Alk1 functions in a flowresponsive pathway to promote quiescence of nascent arteries.…”

“…When appropriate, embryo medium was supplemented with 0.003% phenylthiourea (PTU) (Sigma, St Louis, MO, USA) at 24 hours post-fertilization (hpf) to prevent melanin synthesis. Mutant line alk1 y6 and the alk1 y6 genotyping assay have been described previously (Roman et al, 2002 have been described previously (Roman et al, 2002;Traver et al, 2003;Choi et al, 2007;Corti et al, 2011 (Villefranc et al, 2007), pME-alk1 and p3E-MTpA (Kwan et al, 2007). To drive ligand-and type II receptor-independent, constitutively active alk1 (Roman et al, 2002) in endothelial cells, we generated ptolfli1a.ebs:alk1 CA -mCherry, recombining pDESTtol2pA2, p5E fli1a.ebs (fli1a Ets-binding site; a kind gift from N. Lawson, University of Massachusetts Medical School, Worcester, MA, USA), pME alk1 CA and p3E mCherry-pA (Kwan et al, 2007).…”

“…We could not rescue bmp10 or bmp10-like morphant phenotypes by injecting morpholino-resistant mRNA because embryos ventralized (data not shown). The alk1, tnnt2a and control morpholinos have been described previously (Sehnert et al, 2002;Corti et al, 2011).…”

Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice

“…Increased Pai1 suggests increased TGFβ signaling following endothelial deletion of Rbpj. Mutations in TGFβ pathway members are associated with AVMs in humans, mice and zebrafish (Corti et al, 2011;Johnson et al, 1996;Kim et al, 2012;McAllister et al, 1994;Park et al, 2008Park et al, , 2009Urness et al, 2000). Recently, TGFβ and Notch were shown to synergistically regulate retinal vascular morphogenesis (Larrivee et al, 2012) and BAVM formation (Yao et al, 2013) in mice.…”

Computational hemodynamic optimization predicts dominant aortic arch selection is driven by embryonic outflow tract orientation in the chick embryo

“…Though the exact molecular mechanisms involved in the relationship between aortic arch hemodynamics, gene expression, and tissue remodeling are not yet fully understood, τ w is believed to be the primary mechanical stimulus for vascular remodeling (Culver and Dickinson 2010;Rodbard 1975), clinically referred to as the "flow-dependency principle" (Kamiya and Togawa 1980;Lu et al 2001;Rodbard 1975). The temporal and spatial events associated with flow sensitive altered aortic morphogenesis (at the first aortic arch) in the Akl-1 mutant zebrafish model can be more complex as illustrated by recent studies Corti et al 2011;Patrick et al 2011). …”