A Novel Ogremorphin Group of First-in-Class GPR68 Inhibitors Upregulate Endothelial Barrier Function and Rescue Bacterial Pathogens or Acidosis-Induced Inflammation in Lung Endothelium

Rationale: An increase in endothelial permeability resulting from the disruption of endothelial barrier and aggravated inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID-19. Drugs that enable the preservation and restoration of endothelial function represent attractive therapeutic targets to treat endothelial dysfunction-derived cardiopulmonary diseases. A role of G protein-coupled receptors (GPCRs), especially a sub-family of proton-sensing GPCRs including GPR4 and GPR68, has been suggested in modulation of endothelial function. In this study, we analyzed the barrier protective and anti-inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM-1.Methods: Transendothelial electrical resistance (TER) was monitored in human pulmonary arterial endothelial cells (HPAECs) to evaluate endothelial barrier function. Quantitative real time PCR and western blot analyses were performed to determine mRNA and protein expression of endothelial inflammation markers, respectively. Acidic pH (6.5) medium was used to induce acidosis, and luciferase-based Tango assay was employed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heatkilled Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples. Results: A robust dose-dependent increase in basal EC barrier function was observed with OGM8345 (1-5 μM) and OGM-1 (0.3-1.5 μM) evident by an 150-200% increase in TER values. Both inhibitors also effectively rescued LPS- and HKSA-induced EC hyperpermeability. RT-PCR analysis demonstrated that LPS or HKSA-induced upregulation of inflammatory cytokines/chemokines genes TNF-α, ICAM-1, VCAM-1, IL-6, IL-8, IL- 1β, and CXCL5 was significantly attenuated by OGMs. Consistently, both OGMs suppressed LPSand HKSA-induced protein expression of VCAM-1 and ICAM-1. In contrast, pharmacologic inhibition of GPR4 by NE 52-QQ57 failed to alleviate LPS or HKSA-induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression and GPR68 activity that was inhibited by OGMs. Intratracheal injection of LPS or HKSA in C57BL/6 mice caused vascular leak and lung inflammation that was attenuated by both OGMs as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of accumulation of inflammatory cells and protein content in bronchoalveolar lavage samples. Conclusion: These results establish a critical role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68-selective inhibitors in improving endothelial dysfunction caused by bacterial infections and acidosis associated with acute and chronic lung injury.

REassessment Campaign On VEterinary Resuscitation: Has the time come for horses?

9 Hoehne, S.N., Hopper, K. and Epstein, S.E. (2019) Prospective evaluation of cardiopulmonary resuscitation performed in dogs and cats according to the RECOVER guidelines. Keywords: horse;cardiopulmonary arrest;CPR;international guidelines;RECOVER EN horse cardiopulmonary arrest CPR international guidelines RECOVER 117 119 3 02/09/22 20220301 NES 220301 It is disconcerting that equine anaesthetic-related fatalities for elective procedures are nearly 10- to 20-fold more frequent than their small animal counterparts, and between 100- and 1000-fold more likely than human anaesthesia-associated fatalities (Brodbelt I et al i . 2008;Li I et al i . 2009;Dugdale I et al i . 2016;Laurenza I et al i . 2019). The RECOVER guidelines, published in the summer of 2012, aimed to systematically review the body of literature on CPR from a veterinary standpoint and to generate practical consensus guidelines to be used in dogs and cats. Horse, CPR, RECOVER, cardiopulmonary arrest, international guidelines. [Extracted from the article] Copyright of Equine Veterinary Education is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Development of experimental model of ARDS in SHR strain old rats

Introduction. ARDS (acute respiratory distress syndrome) is a direct cause of death due to lung lesions of various origins including SARS-CoV-2 infection. Most lung and respiratory diseases are characterized by inflammation, which in turn causes occlusions, mucous hypersecretion, shortness of breath, cough and other symptoms of airway inflammation. Understanding the pathological processes involved in the regulation of the immune response may lead to the discovery of new mechanisms that support or suppress inflammatory processes in the lungs and respiratory tract. Methods. In order to develop the experimental model of ARDS we used precision pulmonary hyperventilation and intratracheal administration of poly I:C, which reproduces the body's response to viral infection (mimic viral infection). The severity of ARDS was estimated by the following parameters: the ratio of wet lung weight to dry lung (wet/dry lung weight), extensibility, impedance, lung stiffness, protein level and the number of neutrophils in the bronchial lavage, the level of proinflammatory cytokines in lung tissue. Moreover, we provide a method of simultaneous registration of the lungs vagal nerve activation parameters due to ARDS induction. It is known that the lungs have a massive innervation of the peripheral nervous system and such innervation has a powerful effect on the cells of the immune system. Results. The biologically active substances of neurons affect the activity of immune cells, and the activity of the immune system affects the functioning of the nervous system. That is why the investigation of neuro-immune interaction has a great potential in studying ARDS. Evaluation of dynamic changes in respiratory function due to simulated ARDS showed a significant increase in such parameters as Newtonian resistance, tissue stiffness, static elongation, and a decrease in elasticity and tissue dumping, which is fully consistent with the pathogenesis of ARDS in patients. Analysis of the dry/wet lung ratio showed a two-fold increase in pulmonary edema, a severe, life-threatening condition that develops as a result of ARDS. Significant increase of the protein content and concentration of neutrophils in bronchopulmonary lavage indicates an increase in the permeability of the pulmonary capillaries due to ARDS. The real time PCR identified significant increase of proinflammatory chemokine Cxl2 concentration. Also we observed an increase of neutrophil-activating protein 3 cytokine Gro1, which stimulates the migration of neutrophils. Electrophysiological registration of the activity of the vagal nerve innervating the lungs showed a significant increase in the activity of nociceptive and mechanosensitive fibers, especially in the last stage of acute respiratory distress syndrome, which confirms the role of the nervous system in this pathology. Conclusion. As a result we obtained a model that reproduces ARDS most relevant to human pathological condition.