Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models

Abstract

SUMMARYArteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Deltalike 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic lossand gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4 +/− mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4 +/− mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

“…Endothelial loss of CCN1 reduced Dll4 expression and produced striking morphological changes, similar to those reported for Dll4/Notch inhibition ( Cristofaro et al, 2013). Thus far, hypoxia-driven VEGF-VEGF-R2 signaling has been widely reported as a major inducer of the expression of Dll4 in tip cells (Sainson and Harris, 2006).…”

Molecular Controls of Arterial Morphogenesis

“…Numerous genes affecting native collateral density have been identified including CD44 56 , chloride intracellular channel-4 57 , gap junction proteins connexin-37 58 and connexin-40 28 , PECAM-1 59 , NFkB 60 , Delta-like-4 (Dll4) 61 , HIF2α 62 and RGS5 63 among others (Table 1). Broadly speaking, these fall into three distinct categories: genes affecting endothelial ERK activation and Delta-Notch signaling, genes affecting shear stress and SMC G-protein signaling and genes affecting monocyte/macrophage recruitment and “inflammatory” response.…”

“…Endothelial Notch activation induced by Dll1 is involved in regulation of arteriogenesis 132 while Dll4 binding is considered the principle mechanism controlling the extent of branching 133–135 . Loss of a single Dll4 allele, or inhibition of Notch signaling, significantly augments the extent of arterial branching and artery-to-artery connections 61, 135 . Yet, despite the overall increase in arterial density and the number of collateral connections, tissue perfusion is not improved at baseline and is distinctly reduced in adult mice following a major arterial trunk ligation 61 .…”

Fox(y) Regulators of VEGF Receptors

“…The reduction in VEGFR2 expression and the consequent decrease in VEGF-driven ERK activation as well as a profound decline in ephrin B2 and Sox17 levels are particularly interesting as these have been linked to the arterial fate specification program 22–24 . At the same time, a marked increase in Dll4 would be expected to result in increased Notch signaling leading to decreased vascular branching 25, 26 while elevated Ang2 levels may result in vascular destabilization 27, 28 . Some or all of these mechanisms may account for alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) observed in patients with FOXF1 mutations.…”