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Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions.Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall.Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1β activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis.In silicoanalysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively.Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.Non-standard Abbreviations and Acronymscryo-EMcryogenic electron microscopyECendothelial cellEVextracellular vesicleGOgene ontologyHAEChuman aortic endothelial cellSMChuman aortic vascular smooth muscle cellIL-1βinterleukin 1 betaKEGGKyoto encyclopedia of genes and genomesLC-MSlabel-free liquid-chromatography mass spectrometryMVBmultivesicular bodymiRNAmicroRNARNAseqRNA sequencingTEMtransmission electron microscopyTIRFtotal interal reflection fluorescence microscopymiRNAmicroRNAGraphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

Atherosclerosis results from the deposition and oxidation of low-density lipoprotein (LDL) and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Numerous studies have now shown that transcytosis transports the circulating LDL across endothelial cells lining the blood vessel. LDL transcytosis is initiated by binding to either Scavenger Receptor B1 (SR-B1) or Activin A receptor-like kinase 1 (ALK1) on the apical side of endothelial cells leading to its transit to the basolateral side. Individuals with elevated levels of circulating high-density lipoprotein (HDL) are partly protected from atherosclerosis due to its ability to remove excess cholesterol and act as an antioxidant. Apolipoprotein A1 (ApoA1), an HDL constituent, can bind to SR-B1, suggesting that ApoA1/HDL may also compete with LDL for SR-B1 binding and thereby oppose its deposition in the sub-arterial space. To examine this possibility, we used in vitro approaches to quantify the internalization and transcytosis of fluorescently labelled LDL in coronary endothelial cells. Using microscale thermophoresis and affinity capture, we find that SR-B1 and ApoA1 directly interact and that binding is enhanced when using the cardioprotective variant of ApoA1 termed Milano (ApoA1-Milano). In vivo injection of either wild-type ApoA1 or ApoA1-Milano significantly reduced the acute accumulation of LDL in the atheroprone inner curvature of the murine aorta; inhibition was greater with Milano than the wild-type ApoA1. Together, the results suggest that an additional mechanism for HDL’s atheroprotective effects is attenuating LDL transcytosis through competition for the SR-B1 receptor.

Background Evaluating the patient with adrenal disease is challenging due to the lack of precise clinical and biochemical parameters for disease control. Quality of life (QOL) evaluation aims to measure the patient's subjective experience. Objective To describe how QOL is defined and measured in adrenal disease, critically appraise the use of QOL tools in published literature, discuss the implications of these findings and provide direction for further research in this field. Materials and methods We searched the Cochrane library, EMBASE, Google Scholar, PsycINFO, PubMed, Web of Science databases to identify only primary studies where self-reported QOL was measured as a parameter in adults with confirmed adrenal disease, and results presented in English. Key data were independently extracted from each study and adherence to reporting guidelines evaluated. Results A total of 117 studies involving 13 717 subjects were included. The vast majority of studies did not define QOL. The most common approach was to combine generic and domain-specific tools, although disease-specific tools are increasingly being used. Adherence to reporting guidelines was variable. A narrative synthesis of the findings was performed. Conclusion We present the first systematic review of QOL in adrenal disease. Quality of life is reduced in patients with adrenal disease, irrespective of adrenal hyperfunction or hypofunction. Quality of life improved with therapy but was not completely reversed despite biochemical remission. Authors should adhere to consistent reporting practices which are interpretable by clinicians. Further research is required to explain the mechanisms driving impaired QOL and value of QOL evaluations in the clinical context.

Background and Purpose Endocannabinoids are a family of lipid mediators involved in the regulation of gastrointestinal (GI) motility. The expression, localization and function of their biosynthetic enzymes in the GI tract are not well understood. Here, we examined the expression, localization and function of the enzyme diacylglycerol lipase-α (DAGLα), which is involved in biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Experimental Approach Cannabinoid CB1 receptor-deficient, wild-type control and C3H/HeJ mice, a genetically constipated strain, were used. The distribution of DAGLα in the enteric nervous system was examined by immunohistochemistry. Effects of the DAGL inhibitors, orlistat and OMDM-188 on pharmacologically induced GI hypomotility were assessed by measuring intestinal contractility in vitro and whole gut transit or faecal output in vivo. Endocannabinoid levels were measured by mass spectrometry. Key Results DAGLα was expressed throughout the GI tract. In the intestine, unlike DAGLβ, DAGLα immunoreactivity was prominently expressed in the enteric nervous system. In the myenteric plexus, it was colocalized with the vesicular acetylcholine transporter in cholinergic nerves. In normal mice, inhibiting DAGL reversed both pharmacologically reduced intestinal contractility and pharmacologically prolonged whole gut transit. Moreover, inhibiting DAGL normalized faecal output in constipated C3H/HeJ mice. In colons incubated with scopolamine, 2-AG was elevated while inhibiting DAGL normalized 2-AG levels. Conclusions and Implications DAGLα was expressed in the enteric nervous system of mice and its inhibition reversed slowed GI motility, intestinal contractility and constipation through 2-AG and CB1 receptor-mediated mechanisms. Our data suggest that DAGLα inhibitors may be promising candidates for the treatment of constipation.

Training student pharmacists to administer vaccinations requires a substantial investment in vaccines, supplies, and time. Few schools of pharmacy seek out or receive any reimbursement for the provision of vaccines, despite the fact it is a covered service. This study sought to implement, deliver, and demonstrate an innovative, financially sustainable curriculum-based immunization program by trained pharmacy students as part of their experiential learning. Thirty-nine community health clinics targeting Medicare beneficiaries were conducted throughout Northern/Central California during Medicare&rsquo, s fall open enrollment periods between 2014&ndash, 2016. American Pharmacists Association (APhA)-trained student pharmacists (under licensed pharmacist supervision) administered 1777 vaccinations. Vaccines were billed via a secure Health Insurance Portability and Accountability Act of 1996 (HIPAA)-compliant web-based portal. The total net income was $11,905 and $8032 for 2015 and 2016, respectively. Return on investment was greatest for the influenza vaccine &gt, Tdap &gt, pneumococcal. Pharmacy students are already being trained to provide immunizations and can utilize their skills to deliver financially viable public health programs.

Cannabinoid (CB) agonists suppress nausea in humans and animal models; yet, their underlying neural substrates remain largely unknown. Evidence suggests that the visceral insular cortex (VIC) plays a critical role in nausea. Given the expression of CB1 receptors and the presence of endocannabinoids in this brain region, we hypothesized that the VIC endocannabinoid system regulates nausea. In the present study, we assessed whether inhibiting the primary endocannabinoid hydrolytic enzymes in the VIC reduces acute lithium chloride (LiCl)-induced conditioned gaping, a rat model of nausea. We also quantified endocannabinoid levels during an episode of nausea, and assessed VIC neuronal activation using the marker, c-Fos. Local inhibition of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of 2-arachidonylglycerol (2-AG), reduced acute nausea through a CB1 receptor mechanism, whereas inhibition of fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of anandamide (AEA), was without effect. Levels of 2-AG were also selectively elevated in the VIC during an episode of nausea. Inhibition of MAGL robustly increased 2-AG in the VIC, while FAAH inhibition had no effect on AEA. Finally, we demonstrated that inhibition of MAGL reduced VIC Fos immunoreactivity in response to LiCl treatment. Taken together, these findings provide compelling evidence that acute nausea selectively increases 2-AG in the VIC, and suggests that 2-AG signaling within the VIC regulates nausea by reducing neuronal activity in this forebrain region.