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Sudarshan Anand et al . show that endothelial cell expression of the microRNA miR-132 targets a negative regulator of Ras pathway signaling and thereby releases a brake to new blood vessel formation. miR-132 expression is upregulated in the endothelium of human hemangioma and tumor samples, and an antagonist of miR-132, delivered specifically to tumor endothelium using an integrin-targeted nanoparticle, was able to inhibit tumor angiogenesis and growth in mice. Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti–miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti–miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery 1 of anti–miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti–miR-132 inhibits neovascularization by maintaining vessels in the resting state.

Angoiogenesis and tumorigenesis: mixed messages from VEGF VEGF (vascular endothelial growth factor) is an important angiogenic factor that has been implicated in tumorigenesis. Two papers now show that the function of VEGF is far more complex, as VEFG can negatively regulate angiogenesis and limit tumorigenesis. In one study, Greenberg et al . found that VEGF can inhibit angiogenesis, by impeding the function of the PDGF (platelet-derived growth factor) receptor on pericytes, leading to a loss of pericyte coverage of blood vessels. This involves the formation of heterodimers between the receptors for VEGF and PDGF. In another paper, Stockmann et al . deleted VEGF production in myeloid cells, but not other cell types. Unexpectedly, they found more rapid tumour development in these mice, at the same time as attenuated tumour vascularization and the formation of morphologically and functionally normalized blood vessels. In contrast, tumours lacking VEGF altogether grew more slowly. VEGF is an important angiogenic factor that has been implicated in tumourigenesis. Two papers now show that the function of VEGF is far more complex, as VEGF can negatively regulate angiogenesis and limit tumourigenesis. This study found that VEGF can inhibit angiogenesis by impeding the function of the PDGF receptor on pericytes, leading to a loss of pericyte coverage of blood vessels. This involves the formation of heterodimers between the receptors for VEGF and PDGF. Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization 1 , 2 . These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively 3 , 4 . PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators 5 , 6 , 7 . Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rβ signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rβ and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rβ/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization 8 , 9 and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.

Background/objectivesProlonged-release (PR) naltrexone 32 mg/bupropion 360 mg (NB) is approved for chronic weight management as an adjunct to reduced-calorie diet and increased physical activity. Central nervous system-active medications have the potential to affect mood; therefore, post hoc analysis of clinical trial data was conducted to evaluate psychiatric adverse events (PAEs) and effects on mood of NB therapy versus placebo.Subjects/methodsData were pooled from 5 prospective, double-blind, randomized, placebo-controlled clinical trials (duration range, 24–56 weeks) of NB in subjects with overweight or obesity. PAEs were collected via AE preferred terms, organized into major subtopics (e.g., anxiety, depression, sleep disorders), and divided into category terms (e.g., anxiety, potential anxiety symptoms). Additionally, the Inventory of Depressive Symptomatology Self Report (IDS-SR; score range 0–84) and the Columbia Classification Algorithm of Suicide Assessment (C-CASA) evaluated treatment-emergent depressive/anxiety symptoms and suicidal behavior/ideation, respectively.ResultsBaseline characteristics and comorbidities were comparable for placebo (n = 1515) and NB (n = 2545). Most common PAEs in the NB group (using category grouping; NB vs placebo) were sleep disorders (12.7 vs 7.9%, P < 0.001), anxiety (5.4 vs 3.3%, P = 0.029), and depression (1.8 vs 2.7%, P = 0.014); PAEs were more frequent during dose escalation and generally mild or moderate. Mean (SD) changes in IDS-SR total score from baseline to endpoint were small in both groups: 0.13 (5.83) for NB and −0.45 (5.65) for placebo. Retrospective AE categorization via C-CASA confirmed no completed suicides, suicide attempts, or preparatory acts toward imminent suicidal behavior.ConclusionsThis large pooled analysis of 5 clinical trials provides additional safety information about the NB PAE profile. Anxiety and sleep disorder-related PAEs were more frequent with NB versus placebo but were mostly mild to moderate and generally occurred early. Depression-related PAEs were less common with NB than placebo, and NB was not associated with suicidal ideation or behavior in this patient population.

Nogo isoforms (Nogo-A and -B) have been implicated in regulating neural and cardiovascular functions, such as cell spreading and chemotaxis. Unlike the loop domain (Nogo-66) found in all Nogo isoforms that can interact with a neural-specific Nogo-66 receptor, the receptor for the amino terminus of Nogo-B that mediates vascular function is unknown. Here, we identify a previously uncharacterized Nogo-B receptor specific for the amino terminus of Nogo-B and show that Nogo-B receptor localizes with the ligand Nogo-B during VEGF and wound healing angiogenesis in vivo, mediates chemotaxis in a heterologous expression system and chemotaxis, and 3D tube formation in native endothelial cells. Thus, identification of this receptor may lead to the discovery of agonists or antagonists of this pathway to regulate vascular remodeling and angiogenesis.

Background Pathogenesis in non-alcoholic steatohepatitis (NASH) involves abnormal cholesterol metabolism and hepatic accumulation of toxic free cholesterol. Apical sodium-dependent bile acid transporter (ASBT) inhibition in the terminal ileum may facilitate removal of free cholesterol from the liver by reducing recirculation of bile acids (BAs) to the liver, thereby stimulating new BA synthesis from cholesterol. The aim of this phase 1 study in adult healthy volunteers (HVs) and patients with type 2 diabetes mellitus (T2DM) was to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of ASBT inhibition with volixibat (SHP626; formerly LUM002). Methods Participants were randomised 3:1 to receive once-daily oral volixibat (0.5 mg, 1 mg, 5 mg or 10 mg) or placebo for 28 days in two cohorts (HV and T2DM). Assessments included safety, faecal BA and serum 7α-hydroxy-4-cholesten-3-one (C4; BA synthesis biomarker). Results Sixty-one individuals were randomised (HVs: placebo, n  = 12; volixibat, n  = 38; T2DM: placebo, n  = 3; volixibat, n  = 8). No deaths or treatment-related serious adverse events were reported. Mild or moderate gastrointestinal adverse events were those most frequently reported with volixibat. With volixibat, mean total faecal BA excretion on day 28 was ~1.6–3.2 times higher in HVs (643.73–1239.3 μmol/24 h) and ~8 times higher in T2DM (1786.0 μmol/24 h) than with placebo (HVs: 386.93 μmol/24 h; T2DM: 220.00 μmol/24 h). With volixibat, mean C4 concentrations increased by ~1.3–5.3-fold from baseline to day 28 in HVs and by twofold in T2DM. Conclusions Volixibat was generally well tolerated. Increased faecal BA excretion and serum C4 levels support the mechanistic rationale for exploring ASBT inhibition in NASH. The study was registered with the Dutch clinical trial authority (Centrale Commissie Mensgebonden Onderzoek; trial registration number NL44732.056.13; registered 24 May 2013).

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRβ and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRβ and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRβ and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its “scaffolding domain” to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo , we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82–101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.

Although Nogo-A has been identified in the central nervous system as an inhibitor of axonal regeneration, the peripheral roles of Nogo isoforms remain virtually unknown. Here, using a proteomic analysis to identify proteins enriched in caveolae and/or lipid rafts (CEM/LR), we show that Nogo-B is highly expressed in cultured endothelial and smooth muscle cells, as well as in intact blood vessels. The N terminus of Nogo-B promotes the migration of endothelial cells but inhibits the migration of vascular smooth muscle (VSM) cells, processes necessary for vascular remodeling. Vascular injury in Nogo-A/B-deficient mice promotes exaggerated neointimal proliferation, and adenoviral-mediated gene transfer of Nogo-B rescues the abnormal vascular expansion in those knockout mice. Our discovery that Nogo-B is a regulator of vascular homeostasis and remodeling broadens the functional scope of this family of proteins.

Robo4 expression in emerging blood vessels can neutralize signaling through the angiogenic factor vascular endothelial growth factor (VEGF) and maintain vessel integrity. The findings could lead to new therapeutic targets for angiogenesis and vascular leakage ( pages 448–453 ).