Heart valve development: endothelial cell signaling and differentiation

Abstract

During the past decade, single gene disruption in mice and large-scale mutagenesis screens in zebrafish have elucidated many fundamental genetic pathways that govern early heart patterning and differentiation. Specifically, a number of genes have been revealed serendipitously to play important and selective roles in cardiac valve development. These initially surprising results have now converged on a finite number of signaling pathways that regulate endothelial proliferation and differentiation in developing and postnatal heart valves. This review highlights the roles of the most well-established ligands and signaling pathways, including VEGF, NFATc1, Notch, Wnt/beta-catenin, BMP/TGF-beta, ErbB, and NF1/Ras. Based on the interactions among and relative timing of these pathways, a signaling network model for heart valve development is proposed.

Figure 1

Anatomic overview of heart valve development. The developing heart tube contains an outer layer of myocardium and an inner lining of endothelial cells separated by an ECM referred to as the cardiac jelly. During heart valve formation, a subset of endothelial cells overlying the future valve site are specified to delaminate, differentiate, and migrate into the cardiac jelly, a process referred to as endothelial-mesenchymal transformation or transdifferentiation (EMT). Locally expanded swellings of cardiac jelly and mesenchymal cells are referred to as cardiac cushions. In a poorly understood process, cardiac cushions undergo extensive remodeling from bulbous swellings to eventual thinly tapered heart valves.

Figure 2

Model for VEGF in control of heart valve endothelial cell proliferation. VEGF expression must be tightly controlled during valve development. In situations of decreased VEGF, endothelial cells overlying the nascent cardiac cushion do not proliferate sufficiently, resulting in depletion of endothelial cells. In the presence of endothelial cell depletion, the cardiac cushions do not possess enough endothelial cells to undergo EMT. As a result, the cardiac cushions become hypoplastic, secondary to a decreased number of mesenchymal cells. In situations of increased VEGF, endothelial cells overlying the nascent cardiac cushion maintain their endothelial phenotype, thus preventing cells from undergoing EMT. If a subpopulation of endothelial cells does not undergo EMT, the cardiac cushions will not form. In the “physiologic window,” VEGF establishes an equilibrium between proliferation and differentiation to facilitate valve formation.

Figure 3

Model for NFATc1 as a transcriptional regulator of endothelial cell fate. VEGF signaling through NFATc1 increases the proliferation of pulmonary valve endothelial cells. In the developing cardiac cushion, Ca²⁺ may enter the endothelial cell through connexin-45 hexameric gap junctions and activate calcineurin. Calcineurin, in turn, dephosphorylates NFAT family isoforms, including NFATc1. NFATc1 is then transported into the nucleus, where it interacts with transcriptional regulators, including AP-1, to affect gene transcription. The endogenous calcineurin inhibitor DSCR1/MCIP1, a target of NFATc1, may establish a negative feedback loop by inhibiting calcineurin.

Figure 4

Model for Notch in specification for endothelial-mesenchymal transdifferentiation. In the developing cardiac cushion, Notch signaling increases the level of TGF-β₂, which is known to increase the activity of the transcription factor Snail. Snail activity may lead to downregulation of VE-cadherin, an adhesion molecule needed for homotypic cell-cell interactions. The increased cell-cell separation resulting from downregulation of VE-cadherin may be the initial event in the activation of EMT. Experiments also suggest that Notch signaling may activate Snail independent of TGF-β signaling (dashed line).

Figure 5

Model for TGF-β/BMP signaling in the initiation of EMT. Type III TGF-β receptor (TGF β-RIII) presents TGF-β₂ to TGFβ-RII in the developing heart valve. TGF-β signaling acts through snail/slug transcription factors, and may decrease the expression of VE-cadherin. Receptors ALK3 and BMP-RII have both been shown to be crucial for valve development; BMPs in the developing valve may therefore signal through these receptors. Smad6 antagonizes the interaction of Smad1 with Smad4, thereby decreasing BMP signaling. Synergy between TGF-β and BMP signaling in cardiac cushion explants has been shown to facilitate EMT.

Figure 6

Model for ErbB signaling in integration of extracellular matrix signals. Transmembrane precursor pro-HB-EGF is cleaved by TACE to HB-EGF, a ligand for erbB1 and erbB4. Binding of HB-EGF to erbB1, and possibly formation of a heterodimer with erbB2, may limit the extent of EMT and also decrease BMP expression. In contrast, activation of erbB2/3 heterodimers by the ECM polysaccharide hyaluronic acid (HA) increases EMT and migration, an effect that appears to be mediated by Ras signaling. Synthesis of HA from glucuronic acid and N-acetylglucosamine (GlcNAc) is dependent on the enzymes UDP-glucose dehydrogenase (UGDH) and hyaluronic acid synthase-2 (HAS-2).

Figure 7

Model for NF1 in feedback control of EMT. Neurofibromin is a Ras-specific GTPase activating protein (GAP) that cycles Ras from an active, GTP-bound state to an inactive, GDP-bound, state. Ras signaling is activated by receptor tyrosine kinases (RTKs) and usually proceeds through activation of downstream targets to increase mesenchymal proliferation. Evidence also suggests that the downstream signaling targets of Ras interact with NFATc1 to alter gene transcription. Neurofibromin may therefore decrease endothelial and/or mesenchymal cell proliferation by modulating Ras signaling.

Figure 8

Signaling network model for heart valve development and remodeling. In the signaling network model for cardiac cushion development, numerous signaling pathways and transcriptional regulators act to coordinately regulate the process of heart valve formation. Each signaling pathway is a simplified schema of the signaling events that occur; see Figures 2 through 7 for the details of these signaling pathways. Red arrows denote positive/synergistic interactions between pathways. Blunt red arrows denote inhibitory effects between pathways.