P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Feasibility and efficacy of a hospital-based violence intervention program on reducing repeat violent injury in youth: a randomized control trial.

OBJECTIVES: To determine feasibility and efficacy of an Emergency Department Violence Intervention Program (EDVIP) to reduce violence related injuries in youth. METHODS: One hundred and thirty youth aged 14-24 presenting to an emergency with violence related injury were randomized in parallel to receive EDVIP for 1 year (n = 65) or a waitlist control (n = 65). The primary outcome was to determine feasibility. Secondary outcomes are incidence, number/severity of repeat violence related injury, justice and education systems interactions, substance misuse and mental health presentations, and ED length of stay (LOS). RESULTS: This study established feasibility in recruitment, outcomes collection and safety. Fidelity and adherence measures required optimization during the study. Efficacy analysis of EDVIP vs. the control group demonstrates an absolute decrease of 10.4% in repeat violence related injury (13.7% vs. 24.1%) (p = 0.15), reduction in new interactions in the justice system (OR = 0.36 (0.07-1.77)), improved engagement in education (11.8% EDVIP vs. 7.6% control, p = 0.42) and no change in repeat visits for substance or mental health. LOS decreased by 59.5 min (p = 0.21). CONCLUSIONS: This program is feasible for ED implementation and for completion of a future RCT to measure effectiveness.

PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells.

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1-3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.

Development of an Emergency Department Violence Intervention Program for Youth: An Integrated Knowledge Translation Approach.

BACKGROUND: Violent interpersonal injury is a common presentation to emergency departments (EDs) and is increasingly being treated as a preventable condition. Given the complexity of the issue, it is key to ensure interventions are feasible and acceptable within the communities that are affected by violence. Our team consists of ED staff, community members who work with youth affected by violence, people who were affected by violence in their youth, and researchers. OBJECTIVES: We describe how an integrated knowledge translation (KT) process was used to develop an ED violence intervention program (EDVIP) for youth affected by violence. METHODS: We used the Canadian Institutes of Health Research Guidelines for integrated KT (iKT) to develop an EDVIP. Specifically, we report the Knowledge to Action process which involves both knowledge creation and an action cycle. RESULTS: Our team determined the research question, the research approach, assessed feasibility and determined outcomes for our study. Using the iKT approach facilitated initiation of a funded trial that is now active. CONCLUSIONS: This paper highlights the benefit of including community experts at the beginning of and throughout the research process.

Once-weekly albiglutide in the management of type 2 diabetes: patient considerations.

This review describes the pharmacologic, pharmacokinetic, and pharmacodynamic properties of albiglutide, as well as its clinical efficacy and safety. Albiglutide is a novel, once-weekly, injectable glucagon-like peptide-1 receptor agonist for the treatment of type 2 diabetes. The European Commission recently granted marketing authorization for the drug in the European Union and on April 15, 2014, the US Food and Drug Administration approved albiglutide (Tanzeum™ [GlaxoSmithKline LLC, Wilmington, DE, USA]) to improve glycemic control in adults with type 2 diabetes. Albiglutide has been studied in Phase I, II, and III clinical trials. In the Phase III clinical trials, known as the Harmony series, weekly dosing of albiglutide demonstrated reductions in fasting plasma glucose, postprandial plasma glucose, and glycated hemoglobin, and was associated with weight loss. In all phases of the clinical trials, albiglutide administered once weekly showed a safety and tolerability profile similar to that of placebo, with mild gastrointestinal-related complaints and injection site erythema being the most commonly encountered adverse effects. Compared with pioglitazone and liraglutide, albiglutide has been shown to be clinically less effective. However, it offers the benefit of weight loss that pioglitazone does not, with fewer gastrointestinal side effects than liraglutide. As guidelines continue to advocate for patient-centered treatment strategies, once-weekly albiglutide will be an important addition to the growing armamentarium of treatment options for adults with type 2 diabetes needing target glycemic control.

Extracellular ATP is a pro-angiogenic factor for pulmonary artery vasa vasorum endothelial cells.

Expansion of the vasa vasorum network has been observed in a variety of systemic and pulmonary vascular diseases. We recently reported that a marked expansion of the vasa vasorum network occurs in the pulmonary artery adventitia of chronically hypoxic calves. Since hypoxia has been shown to stimulate ATP release from both vascular resident as well as circulatory blood cells, these studies were undertaken to determine if extracellular ATP exerts angiogenic effects on isolated vasa vasorum endothelial cells (VVEC) and/or if it augments the effects of other angiogenic factors (VEGF and basic FGF) known to be present in the hypoxic microenvironment. We found that extracellular ATP dramatically increases DNA synthesis, migration, and rearrangement into tube-like networks on Matrigel in VVEC, but not in pulmonary artery (MPAEC) or aortic (AOEC) endothelial cells obtained from the same animals. Extracellular ATP potentiated the effects of both VEGF and bFGF to stimulate DNA synthesis in VVEC but not in MPAEC and AOEC. Analysis of purine and pyrimidine nucleotides revealed that ATP, ADP and MeSADP were the most potent in stimulating mitogenic responses in VVEC, indicating the involvement of the family of P2Y1-like purinergic receptors. Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC. However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel. Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that can be observed in hypoxic conditions.

A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion.

This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications.