cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior

The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/beta-catenin signaling

Blood vessel stability is essential for embryonic development; in the adult, many diseases are associated with loss of vascular integrity. The ETS transcription factor ERG drives expression of VE-cadherin and controls junctional integrity. We show that constitutive endothelial deletion of ERG (ErgcEC-KO) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (ErgiEC-KO) results in defective physiological and pathological angiogenesis in the postnatal retina and tumors, with decreased vascular stability. ERG controls the Wnt/β-catenin pathway by promoting β-catenin stability, through signals mediated by VE-cadherin and the Wnt receptor Frizzled-4. Wnt signaling is decreased in ERG-deficient endothelial cells; activation of Wnt signaling with lithium chloride, which stabilizes β-catenin levels, corrects vascular defects in ErgcEC-KO embryos. Finally, overexpression of ERG in vivo reduces permeability and increases stability of VEGF-induced blood vessels. These data demonstrate that ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Molecular regulation of tip cell competition during sprouting angiogenesis

Sprouting angiogenesis describes the formation of blood vessels from preexisting ones, a process guided by leading endothelial tip cells, followed by stalk cells. It has previously been established that the transient and dynamic specification of both phenotypes is mediated by DLL4/NOTCH signalling, which is primarily actuated by vascular endothelial growth factor (VEGF) and the corresponding vascular endothelial growth factor receptor 2 (VEGFR-2). The ability of a cell to outcompete its neighbour is determined by VEGFR signalling mediated production of DLL4 and subsequent activation of NOTCH in the adjacent cell. The stalk cell phenotype, induced by NOTCH activation, is reinforced by SMAD signalling through a crosstalk between both pathways. However, the downstream effectors of NOTCH and the molecular link to SMAD signalling in tip cell competition are unknown. During my PhD I have identified Neuropilin-1 (NRP1) as a critical NOTCHregulated determinant of tip/stalk specification. I have found that endothelial cells lacking one or both functional alleles of NRP1 are unable to form tip cells when competing with WT cells. Despite NRP1 having been previously described as a coreceptor of VEGFR-2, my data indicate that NRP1 functions independently of VEGFR-2 during tip/stalk selection. Furthermore, I have shown that inhibition of NOTCH is not sufficient to rescue the profound stalk cell phenotype of NRP1 deficient cells. Thus, I have identified NRP1 as the first bona fide downstream effector of NOTCH signalling in regulating angiogenesis. Furthermore, my data provides the missing link between NOTCH and SMAD signalling in stalk cell specification, as it shows NRP1 to be a negative regulator of the TGF- β/ALK5/SMAD2 pathway. Additionally, in contrast to NOTCH ablation, inhibition of ALK5 quantitatively restores the ability of NRP1 null cells to contribute to the tip position. I conclude that NRP1 is a key regulator of tip/stalk cell specification in sprouting angiogenesis; differential NRP1 levels act as key effecto

The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity

10.1016/j.celrep.2015.05.006Cell Reports11121919-192

Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation

FOXO1 couples metabolic activity and growth state in the vascular endothelium

Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements(1). Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation(2,3). Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function(1,4), yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth(5,6). MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation