Molecular regulation of arteriovenous endothelial cell specification

Hedgehog

Binding of the morphogen Sonic Hedgehog (Shh) to its cell surface receptor Patched-1 (PTCH1) alleviates repression of the central downstream Shh effector, Smoothened (Smo). In turn, Smo induces the expression of numerous gene targets essential for embryonic development²⁸. In endothelial cells, Shh activates endothelial cell survival and alters cytoskeletal arrangement in culture²⁹, and studies in zebrafish show that Shh signaling is necessary for arteriovenous specification. Specifically, in zebrafish mutants lacking Shh, endothelial cells of the dorsal aorta fail to acquire expression of the arterial-enriched gene ephrinB2. This is thought to be the result of loss of VEGF expression in Shh-deficient somites, leading to reduced VEGF signaling and reduced downstream Notch³⁰. However, Shh also regulates endothelial cell identity independent of its stimulation of VEGF/Notch signaling. Specifically, Hedgehog represses venous identity³¹ and promotes arterial specification via calcitonin receptor-like receptor (Crlr) signaling³² as well as by directly upregulating expression of Notch signaling effectors³³.

Vascular endothelial growth factor

VEGF functions at multiple levels during vasculogenesis and vessel remodeling, including during arteriovenous specification. Although loss of even a single allele of VEGF-A is sufficient to disrupt vessel formation resulting in embryonic lethality in mice³⁴, VEGF-A knockdown in zebrafish morphants preserves embryonic survival albeit with arteriovenous specification defects³⁰. Recent studies indicate that whereas early VEGF signaling governs endothelial cell development from angioblasts, mid-somitogenic VEGF signaling primarily influences arterial specification by activating Etv2, a member of the Ets family of transcription factors³⁵, to regulate downstream Notch signaling effector expression^36,37.

Recent findings indicate that, in addition to its effects on Etv2 and downstream Notch, VEGF/VEGFR2 activation directly regulates the balance of signaling through either phosphatidylinositol-3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) pathways to determine arterial and venous cell fates. In a small-molecule screen, Hong et al.³⁸ report that inhibition of PI3K signaling induces ERK1/2 (MAPK signaling) activation—a signaling pathway that regulates endothelial cell proliferation (among other functions)³⁹—to promote arterial specification. This effect is capable of rescuing arteriovenous defects of the gridlock zebrafish mutant, where the Notch-targeted transcription factor Hey2 is affected, indicating that MAPK signaling influences arterial identity downstream of Notch signaling³⁸. In contrast, small-molecule inhibitors of MAPK, or constitutive activation of PI3K signaling via induction of protein kinase B (Akt), prevents arterial specification and instead induces venous identity³⁸, indicating that the antagonistic relationship between MAPK and PI3K signaling pathways strongly influences endothelial cell fate.

Notch

Members of the Notch family of transmembrane receptors, as well as their membrane-bound ligands, are expressed in multiple cell types of the developing and mature vasculature⁴⁰. In response to VEGF-activated expression of members of the Sox family of transcription factors (for example, Sox7, Sox17, and Sox18)⁴¹ or stimulation of the Wingless/Integrated (Wnt) signaling pathway⁴², primordial endothelial cells are induced to express the transmembrane receptor Notch1 and its ligand Dll4⁴³. Notch1 and related endothelial-expressed Notch receptors are activated by membrane-bound Notch ligands of adjacent endothelial cells (homocellular signaling) as well as those expressed by other stromal cell types (heterocellular signaling). Indeed, the developing vasculature expresses multiple Notch ligand and receptor types, and some are restricted to specific regions of the expanding and remodeling vascular tree⁴⁰. Binding of ligand to the Notch receptor results in proteolytic cleavage of the Notch intracellular domain, which translocates to the nucleus and binds to and activates DNA-binding protein RBPJκ, resulting in transcription of genes that influence endothelial cell cycle status¹⁷ and function, leading to induced expression of arterial identity genes⁴⁴.

In animals treated with Notch inhibitors or in transgenic animals lacking either Notch ligands or receptors, sprouting angiogenesis and arteriovenous specification fail to occur normally^{17,22,45–47}. Instead, vascular endothelial cells hyperproliferate and do not properly remodel into arteriovenous networks^17,45. In gridlock zebrafish mutants, ephrinB2 expression is lost and formation of the dorsal aorta is compromised but the cardinal vein is enlarged¹².

One possible explanation for the central role for Notch in arteriovenous specification is as a mechanism to couple mechanosensory receptor signaling to downstream endothelial cell specification pathways. Fluid shear stress activates Notch signaling in endothelial cells in a dose-dependent fashion with Notch activation peaking at¹⁷ or slightly above⁴⁸ physiologically arterial levels of shear. Ablation of Notch1 signaling compromises classic flow-sensitive endothelial cell responses, including quiescence¹⁷ and cell alignment⁴⁸, whereas constitutive activation of Notch4 induces focal vessel enlargement by disrupting normal hemodynamic signaling⁴⁹. Although the exact mechanosensory signaling complex or complexes that render Notch signaling flow-sensitive have yet to be identified, ligand-dependent Notch activation is force-dependent^50,51, which suggests that the Notch receptor itself may participate in an as-yet-undescribed mechanosensory complex.

Other studies suggest that Notch may also enable arteriovenous specification by determining the cell cycle state of remodeling endothelial cells. In response to flow, Notch signaling activation alters the expression of cell cycle regulators¹⁷. In addition, Notch-mediated G₁ arrest is required for acquisition of arterial^17,45 as well as hemogenic²⁷ cell fates. In contrast, suppression of Notch signaling by COUP-TFII drives venous specification⁵², and transgenic ablation of Notch signaling components enhances lymphatic endothelial cell specification⁵³. Taken together, these data suggest that, in the developing vasculature, Notch signaling may play a central role in precisely coupling endothelial cell cycle state to hemodynamic flow sensing to achieve proper fate specification. However, it is still unclear whether venous and lymphatic endothelial cell fates are similarly specified in distinct cell cycle states, which requires further intensive investigation. Nonetheless, in support of this hypothesis, dysregulated Notch signaling leads to focal appearance of AVMs at sites of high flow that are associated with failure to acquire (or maintain) specialized endothelial cell identities^49,54. Whether in animals lacking Notch (or Alk, see below) signaling AVMs are a direct result of disrupted arteriovenous specification or whether EC fail to undergo proper arteriovenous specification as a by-product of enlarged, malformed vessels that result from aberrant responses to shear (such as failure to migrate against the direction of flow^49,55) remains unclear.

Lastly, Notch is an important regulator of hemogenic endothelial cell development in the yolk sac and embryonic aorta–gonad–mesonephros region^27,56, and circulating yolk sac-endothelium–derived hematopoietic progenitors have recently been shown to reintegrate into the developing vasculature^9,10. Thus, it is interesting to speculate that Notch may contribute to arteriovenous network formation, in part, by regulating the relative abundance of these two endothelial cell sources, which may have different propensities for arterial versus venous identity. However, much more work is needed to address these possibilities.

Ephrin/Eph

The ephrin family of transmembrane ligands and their cognate Eph receptors mediate cell–cell signaling between adjacent cells and often involve the repulsion of Eph receptor–expressing cells from ephrin-expressing neighbors. In a landmark study of the developing vasculature, Wang et al.¹¹ found that ephrinB2 expression is highly enriched in arteries and EphB4 expression is enriched in veins. Expression of both genes is observed prior to the onset of blood flow, suggesting that they participate in a genetic arteriovenous program. Furthermore, EphB4 venous expression depends upon arterial expression of ephrinB2¹¹, suggesting that during development arterial specification may drive venous specification via ephrin-Eph signaling. Mutations that affect the EphB4 gene or downstream Ras signaling are associated with the autosomal-dominant congenital vascular disease, capillary malformation-arteriovenous malformation (CM-AVM), wherein patients present with numerous cutaneous capillary malformations as well as AVMs⁵⁷.

Transforming growth factor-beta

The TGF-β superfamily of soluble ligands and their cognate membrane-bound receptors play a variety of key roles during vessel development. This pathway includes signaling through the TGF-β1-TGFβR2-Alk5 ligand-receptor complex, which predominantly activates Smads2/3 signaling, as well as Bone morphogenetic protein (BMP) 9/10-Alk1-Eng ligand-receptor signaling, which predominantly activates Smads1/5/8⁵⁸. Specifically, signaling through TGF-β1-TGFβR2 typically mediates mural cell recruitment and differentiation⁵⁹, whereas Alk1/Eng signaling regulates endothelial cell quiescence, limits vessel caliber, and enables arteriovenous specification^54,55,60. However, there is also evidence of significant cross-talk between distinct TGF-β signaling pathways^61,62 as well as between TGF-β superfamily pathways and other cell signaling pathways (for example, Notch) and that it is the balance of Smad signaling activation via these distinct pathways that establishes proper vessel formation^63,64. Indeed, patients bearing heterozygous mutations affecting either Alk1 or Eng, or downstream Smad4, exhibit the congenital disease hereditary hemorrhagic telangiectasia (HHT), which is characterized by microvascular overgrowth and the focal appearance of large-caliber AVMs that lack clear arterial or venous identity^60,65.

Several recent studies have focused on the cross-talk between BMP and Notch signaling pathways to modulate endothelial cell behavior during vessel development. BMP-activated sprouting angiogenesis is negatively regulated by Notch upregulation of Smad6, an inhibitor of BMP signaling⁶⁶. Meanwhile, nuclear translocation of BMP-activated phospho-Smads not only upregulates BMP target genes but also participates in the Notch/RBPJ-κ gene regulatory complex to regulate Notch-activated transcriptional responses⁶⁷. Consequently, inhibited expression of endothelial-expressed BMP regulatory proteins, BMPER and TWSG1, disrupts Notch signaling and expression of arterial identity genes, resulting in increased venous specification in zebrafish embryos⁶⁸. Alk1 inhibition also depresses Notch signaling and produces AVMs in mice⁵⁴. In separate studies, BMPER is reported to activate ERK1/2 signaling⁶⁹ (which promotes arterial specification³⁸) while the Alk1 co-receptor Endoglin suppresses PI3K/Akt signaling (which promotes venous identity³⁸) to support endothelial cell migration against the direction of blood flow, a process hypothesized to support arteriogenesis and prevent AVMs⁷⁰. Thus, currently available evidence suggests that BMP9/10-Alk1 signaling may regulate arteriovenous specification, at least in part, by modulating endothelial cell responsiveness to VEGF- and Notch-activated signaling. However, other studies suggest that other endothelial cell behaviors, such as responsiveness to shear or migration (or both), are also affected^49,55.

Connexins

Membrane-expressed connexin (also known as Cx) proteins form multimeric complexes (termed connexons) that dock with connexons of adjacent cells to form intercellular channels (termed gap junction channels) that mediate passage of ions and small signaling molecules to support electrochemical coupling and intercellular communication. At least four connexin proteins (Cx37, Cx40, Cx43, and Cx45) are commonly reported at endothelial cell–cell junctions of the blood vasculature^71,72, and some studies report endothelial expression of a fifth connexin (Cx32)^73,74. Furthermore, an additional connexin (Cx47) is expressed in lymphatic endothelial cells and contributes to lymphatic vessel development⁷⁵. Of the commonly studied vascular connexins, Cx40 (encoded by the gene Gja5) is well recognized as a potent marker of arterial endothelial cells owing to its high expression in arteries invested with smooth muscle^14,17. Deletion of this connexin inhibits flow-activated arterial specification in the chick¹⁴ and affects sprouting angiogenesis and mural cell recruitment in the neonatal mouse retina⁷⁶. Furthermore, loss of Cx40 potentiates the appearance of AVMs in Alk1-haploinsufficient animals⁷⁷, suggesting that it may suppress the formation of these vascular defects in wild-type animals, at least in part, by functioning downstream of BMP9/10-Alk1 signaling. Recently, Su et al.²⁶ employed a single-cell transcriptomic analysis of developing coronary vessels to identify a Cx40-enriched population of venous-originating “pre-artery” cells that express markers of mature arteries. The majority of these cells were later found to line the coronary arteries and were excluded from coronary veins, suggesting that expression of Cx40 is a critical intermediary step for arterial identity acquisition.

Endothelial-expressed Cx37 is also almost exclusively expressed in large arteries of the adult vasculature⁷⁸ as well as in developing arteries and arterioles of remodeling vessels¹⁷. However, unlike Cx40, Cx37 is additionally expressed in remodeling capillaries and in arteriolar vessels that have yet to be invested with mural cells¹⁷, suggesting that it may play an earlier role than Cx40 in arteriovenous specification during development. Deletion of Cx37 disrupts developmental and injury-induced vessel growth and remodeling^17,79, and transgenic ablation of both connexins in combination results in embryonic lethality due to failure of the vasculature to form⁸⁰, suggesting that Cx37 and Cx40 play essential and possibly distinct roles during vessel development. For example, many connexins regulate cell proliferation, and Cx37 is a particularly potent inhibitor of cell cycle progression⁸¹. Cx37 directly modulates endothelial cell cycle status downstream of flow-activated Notch signaling by upregulating p27, causing late G₁ arrest to enable expression of arterial genes Cx40 and ephrinB2¹⁷. It is possible that Cx37 plays a similar cell cycle arrest role to enable specification towards other endothelial cell fates. In support of this possibility, transgenic ablation of one or both copies of Cx37 is associated with endothelial cell hyperproliferation and defects in not only arterial development¹⁷ but also venous⁸² and lymphatic^83,84 development.

MicroRNAs

A growing body of evidence indicates that microRNA (miRNA) species likely play an important, if currently underappreciated, role in endothelial cell specification. Although several studies show that miRNAs are necessary for endothelial cell differentiation from angioblasts, less is known about their involvement in specifying endothelial cell phenotypes. Endothelial and mural cells express numerous miRNA species, and miRNA processing machinery, including Drosha and Dicer, appears to be crucial for vessel development^85,86. Mutants lacking expression of Dicer in Etv2-positive mesodermal progenitor cells exhibit defects in vessel remodeling and patterning due, at least in part, to loss of miR-130a expression⁸⁷. Meanwhile, miR-27b is required for venous formation in zebrafish⁸⁸, and miR-181 destabilizes expression of Prox1, a key regulator of lymphatic endothelial cell specification and maintenance⁸⁹. In addition, endothelial-specific mutation of Drosha in mice produces leaky, dilated microvessels and aberrant arteriovenous connections (but lack clear AVMs), and missense point mutations in the Drosha gene are more prevalent among patients with HHT compared with healthy populations, suggesting that miRNA processing defects may contribute to the pathogenesis of this disease or modulate its severity or do both⁸⁶. Taken together, these studies suggest that miRNAs likely play a broad role in endothelial cell specification and vascular remodeling. However, more work is needed to identify critical miRNA regulators of these processes and to fully elucidate their molecular roles.