Giving Voice to the Voiceless in Environmental Gene Editing.

Participatory deliberation, whereby diverse experts and publics collectively engage in decision-making, can ensure a more informed and just decision by centering historically marginalized perspectives and engaging a spectrum of value systems. Broad and diverse participation is crucial for the equitable distribution of risks and benefits resulting from complex and uncertain decisions such as environmental gene editing. From an ethical position that gives intrinsic value to the nonhuman and recognizes the interconnectedness of species across generations, we argue that deliberation over environmental gene editing must include the voice of nature and the voice of future generations. Inclusion of these key participant groups can encourage reflection on the human relationship with nature and help safeguard intergenerational equity of decisions reached. By drawing from the legal rights of nature movement, the Boardman River Dams Project, and methods for representative participation, we offer strategies for inclusion of nonhuman nature and future generations in deliberative processes about environmental gene editing and other crucial decisions about our shared environments.

FRS2α-dependent cell fate transition during endocardial cushion morphogenesis.

Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFß signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2αiECKO). Analysis of the FRS2αiECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis.

Inhibition of Jagged-Specific Notch Activation Reduces Luteal Angiogenesis and Causes Luteal Hemorrhaging of Hormonally Stimulated Ovaries.

Robust angiogenesis in the corpus luteum is critical for maintenance of pregnancy and thus mammalian female fertility. During angiogenesis, blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Pericytes are associated with capillaries and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial induction of Notch signaling in adjacent pericytes helps recruit and maintain pericyte coverage in some but not all tissue types. We have employed a Notch decoy, N110-24, which blocks Notch signaling in a ligand-specific manner, and determined that pharmacological inhibition of Notch ligand Jagged blocks luteal angiogenesis after normal ovulation, resulting in reduced luteal vasculature. Conversely, after ovarian hyperstimulation, a condition which occurs during fertility treatments, Jagged inhibition causes vascular dilation and hemorrhage. These results indicate that Jagged inhibition has effects in different ovarian angiogenic conditions, promoting vascular growth in the corpus luteum and vascular stability in hyperstimulated ovaries.

Treatment of heritable diseases using CRISPR: Hopes, fears, and reality.

CRISPR gene editing is poised to transform the therapeutic landscape for diseases of genetic origin. The ease and agility by which CRISPR can make specific changes to DNA holds great promise not only for the treatment of heritable diseases, but also their prevention through germline editing. CRISPR-based therapeutic strategies are currently under development for numerous monogenic diseases. These strategies range from proof of concept studies demonstrating pre-fertilization gamete editing to recently initiated clinical trials for postnatal ex vivo therapies. The promise of CRISPR's human genome editing potential has captivated the public's attention. It is of paramount importance that medical professionals who work with patients who may have or carry a monogenic heritable disease understand CRISPR technology in order to have informed and compassionate discussions with their patients. Understanding CRISPR means understanding its evolving therapeutic applications' nuances, limitations, and barriers to access as well as the regulatory landscape they inhabit. In this piece we provide a review of the promises and pitfalls of CRISPR germline gene editing and their implications for patient decision-making throughout various stages of the reproductive process.

The Rab-effector protein RABEP2 regulates endosomal trafficking to mediate vascular endothelial growth factor receptor-2 (VEGFR2)-dependent signaling.

As a master regulator of endothelial cell function, vascular endothelial growth factor receptor-2 (VEGFR2) activates multiple downstream signaling pathways that are critical for vascular development and normal vessel function. VEGFR2 trafficking through various endosomal compartments modulates its signaling output. Accordingly, proteins that regulate the speed and direction by which VEGFR2 traffics through endosomes have been demonstrated to be particularly important for arteriogenesis. However, little is known about how these proteins control VEGFR2 trafficking and about the implications of this control for endothelial cell function. Here, we show that Rab GTPase-binding effector protein 2 (RABEP2), a Rab-effector protein implicated in arteriogenesis, modulates VEGFR2 trafficking. By employing high-resolution microscopy and biochemical assays, we demonstrate that RABEP2 interacts with the small GTPase Rab4 and regulates VEGFR2 endosomal trafficking to maintain cell-surface expression of VEGFR2 and VEGF signaling. Lack of RABEP2 also led to prolonged retention of VEGFR2 in Rab5-positive sorting endosomes, which increased VEGFR2's exposure to phosphotyrosine phosphatase 1b (PTP1b), causing diminished VEGFR2 signaling. Finally, the loss of RABEP2 increased VEGFR2 degradation by diverting VEGFR2 to Rab7-positive endosomes destined for the lysosome. These results implicate RABEP2 as a key modulator of VEGFR2 endosomal trafficking, and demonstrate the importance of RABEP2 and Rab4 for VEGFR2 signaling in endothelial cells.

The expanding role of neuropilin: regulation of transforming growth factor-ß and platelet-derived growth factor signaling in the vasculature.

PURPOSE OF REVIEW: Long recognized for its role in regulation of vascular endothelial growth factor signaling, neuropilin (Nrp)1 has emerged as a modulator of additional signaling pathways critical for vascular development and function. Here we review two novel functions of Nrp1 in blood vessels: regulation of transforming growth factor-ß (TGFß) signaling in endothelial cells and regulation of platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells. RECENT FINDINGS: Novel mouse models demonstrate that Nrp1 fulfills vascular functions independent of vascular endothelial growth factor signaling. These include modulation of TGFß-dependent inhibition of endothelial sprouting during developmental angiogenesis and PDGF signaling in vascular smooth muscle cells during development and disease. SUMMARY: Broadening our understanding of how and where Nrp1 functions in the vasculature is critical for the development of targeted therapeutics for cancer and vascular diseases such as atherosclerosis and retinopathies.

Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations.

Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.

Pericytes are progenitors for coronary artery smooth muscle.

Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.

Angiogenesis versus arteriogenesis: neuropilin 1 modulation of VEGF signaling.

In development and disease, vascular endothelial growth factor (VEGF) regulates the expansion of the vascular tree. In response to hypoxia, VEGF promotes new capillary formation through the process of angiogenesis by inducing endothelial cell sprouting, proliferation, and migration. Wound healing, tissue regeneration, and tumor growth depend on angiogenesis for adequate nutrient and oxygen delivery. Under different conditions, VEGF promotes arterial growth, modulates lumen expansion, and induces collateral vessel formation, events collectively referred to as arteriogenesis. Induction of arteriogenesis after cardiac or cerebral arterial occlusion can reduce ischemia and improve disease outcome. Endothelial VEGF receptor 2 (VEGFR2) signaling governs both processes. However, modulation of downstream VEGF signaling effectors, such as extracellular-signal-regulated kinase (ERK) activation, differs in order to achieve angiogenic versus arteriogenic outcomes. Recent reports show that neuropilin 1 (NRP1), a VEGF receptor, can instill VEGF signaling outcomes that specifically regulate either angiogenesis or arteriogenesis. Here, we discuss how NRP1 functions as a VEGFR2 co-receptor in angiogenesis and a modulator of VEGFR2 trafficking in arteriogenesis. The unique role played by neuropilin in different endothelial processes makes it an exciting therapeutic target to specifically enhance angiogenesis or arteriogenesis in disease settings.

NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.

UNLABELLED: A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.

Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis.

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.

Dissociation of the glucose and lipid regulatory functions of FoxO1 by targeted knockin of acetylation-defective alleles in mice.

FoxO1 integrates multiple metabolic pathways. Nutrient levels modulate FoxO1 acetylation, but the functional consequences of this posttranslational modification are unclear. To answer this question, we generated mice bearing alleles that encode constitutively acetylated and acetylation-defective FoxO1 proteins. Homozygosity for an allele mimicking constitutive acetylation (Foxo1(KQ/KQ)) results in embryonic lethality due to cardiac and angiogenesis defects. In contrast, mice homozygous for a constitutively deacetylated Foxo1 allele (Foxo1(KR/KR)) display a unique metabolic phenotype of impaired insulin action on hepatic glucose metabolism but decreased plasma lipid levels and low respiratory quotient that are consistent with a state of preferential lipid usage. Moreover, Foxo1(KR/KR) mice show a dissociation between weight gain and insulin resistance in predisposing conditions (high fat diet, diabetes, and insulin receptor mutations), possibly due to decreased cytokine production in adipose tissue. Thus, acetylation inactivates FoxO1 during nutrient excess whereas deacetylation selectively potentiates FoxO1 activity, protecting against excessive catabolism during nutrient deprivation.