Notch1 promotes ordered revascularization through Semaphorin 3g modulation of downstream vascular patterning signalling factors.

Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain- or loss-of-function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well-ordered vascular regeneration. KEY POINTS: Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms. By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization. RNA-sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning. The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.

Comprehensive transcriptomic profiling reveals SOX7 as an early regulator of angiogenesis in hypoxic human endothelial cells.

Endothelial cells (ECs) lining the vasculature of vertebrates respond to low oxygen (hypoxia) by maintaining vascular homeostasis and initiating adaptive growth of new vasculature through angiogenesis. Previous studies have uncovered the molecular underpinnings of the hypoxic response in ECs; however, there is a need for comprehensive temporal analysis of the transcriptome during hypoxia. Here, we sought to investigate the early transcriptional programs of hypoxic ECs by using RNA-Seq of primary cultured human umbilical vein ECs exposed to progressively increasing severity and duration of hypoxia. We observed that hypoxia modulates the expression levels of approximately one-third of the EC transcriptome. Intriguingly, expression of the gene encoding the developmental transcription factor SOX7 (SRY-box transcription factor 7) rapidly and transiently increased during hypoxia. Transcriptomic and functional analyses of ECs following SOX7 depletion established its critical role in regulating hypoxia-induced angiogenesis. We also observed that depletion of the hypoxia-inducible factor (HIF) genes, HIF1A (encoding HIF-1α) and endothelial PAS domain protein 1 (EPAS1 encoding HIF-2α), inhibited both distinct and overlapping transcriptional programs. Our results indicated a role for HIF-1α in down-regulating mitochondrial metabolism while concomitantly up-regulating glycolytic genes, whereas HIF-2α primarily up-regulated the angiogenesis transcriptional program. These results identify the concentration and time dependence of the endothelial transcriptomic response to hypoxia and an early key role for SOX7 in mediating angiogenesis.

A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.

Embryonic Stem Cell Differentiation to Functional Arterial Endothelial Cells through Sequential Activation of ETV2 and NOTCH1 Signaling by HIF1α.

The generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue.

Effects of the psychotomimetic benzomorphan N-allylnormetazocine (SKF 10,047) on prepulse inhibition of startle in mice.

N-allylnormetazocine (NANM; SKF 10,047) is a benzomorphan opioid that produces psychotomimetic effects. (+)-NANM is the prototypical agonist for the sigma-1 (σ1) receptor, and there is a widespread belief that the hallucinogenic effects of NANM and other benzomorphan derivatives are mediated by interactions with σ1 sites. However, NANM is also an agonist at the κ opioid receptor (KOR) and binds to the PCP site located within the channel pore of the NMDA receptor, interactions that could potentially contribute to the effects of NANM. NMDA receptor antagonists such as phencyclidine (PCP) and ketamine are known to disrupt prepulse inhibition (PPI) of acoustic startle, a measure of sensorimotor gating, in rodents. We recently found that racemic NANM disrupts PPI in rats, but it is not clear whether the effect is mediated by blockade of the NMDA receptor, or alternatively whether interactions with KOR and σ1 receptors are involved. The present studies examined whether NANM and its stereoisomers alter PPI in C57BL/6J mice, and tested whether the effects on PPI are mediated by KOR or σ1 receptors. Racemic NANM produced a dose-dependent disruption of PPI (3-30mg/kg SC). (+)-NANM also disrupted PPI, whereas (-)-NANM was ineffective. Pretreatment with the selective KOR antagonist nor-binaltorphimine (10mg/kg SC) or the selective σ1 antagonist NE-100 (1mg/kg IP) failed to attenuate the reduction in PPI produced by racemic NANM. We also found that the selective KOR agonist (-)-U-50,488H (10-40mg/kg SC) had no effect on PPI. These findings confirm that NANM reduces sensorimotor gating in rodents, and indicate that the effect is mediated by interactions with the PCP receptor and not by activation of KOR or σ1 receptors. This observation is consistent with evidence indicating that the σ1 receptor is not linked to hallucinogenic or psychotomimetic effects.

The novel ketamine analog methoxetamine produces dissociative-like behavioral effects in rodents.

RATIONALE: Methoxetamine (MXE) is a ketamine analog sold online that has been subject to widespread abuse for its dissociative and hallucinogenic effects. Previous studies have shown that MXE has high affinity for the phencyclidine (PCP) binding site located within the channel pore of the NMDA receptor (NMDAR), but little is known about its behavioral effects. Dissociative anesthetics such as ketamine and PCP produce a characteristic behavioral profile in rats that includes locomotor hyperactivity and disruption of prepulse inhibition (PPI) of acoustic startle. METHODS: The goal of the present investigation was to determine whether MXE produces PCP-like effects in Sprague-Dawley rats using the PPI paradigm and the behavioral pattern monitor (BPM), which enables analyses of patterns of locomotor activity and investigatory behavior. PPI studies were conducted with several other uncompetitive NMDAR antagonists that produce dissociative effects in humans, including PCP, the S-(+) and R-(-) isomers of ketamine, and N-allylnormetazocine (NANM; SKF-10,047). RESULTS: MXE disrupted PPI when administered at 3 and 10 mg/kg SC. The rank order of potency of MXE and the other test compounds in the PPI paradigm (PCP > MXE > S-(+)-ketamine > NANM > R-(-)-ketamine) parallels their affinities for the PCP binding site reported in the literature. When tested in the BPM, 10 mg/kg MXE induced locomotor hyperactivity, reduced the number of rearings, increased the roughness of locomotor paths, and produced perseverative patterns of locomotion. Administration of PCP (2.25 and 6.75 mg/kg, SC) produced a similar profile of effects in the BPM. CONCLUSIONS: These results indicate that MXE produces a behavioral profile similar to that of other psychotomimetic uncompetitive NMDAR antagonists. Our findings support the classification of MXE as a dissociative drug and suggest that it likely has effects and abuse potential similar to that of PCP and ketamine.

Production of recombinant proteins in microalgae at pilot greenhouse scale.

Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae.