Cynomolgus monkey's choroid reference database derived from hybrid deep learning optical coherence tomography segmentation.

Cynomolgus monkeys exhibit human-like features, such as a fovea, so they are often used in non-clinical research. Nevertheless, little is known about the natural variation of the choroidal thickness in relation to origin and sex. A combination of deep learning and a deterministic computer vision algorithm was applied for automatic segmentation of foveolar optical coherence tomography images in cynomolgus monkeys. The main evaluation parameters were choroidal thickness and surface area directed from the deepest point on OCT images within the fovea, marked as the nulla with regard to sex and origin. Reference choroid landmarks were set underneath the nulla and at 500 µm intervals laterally up to a distance of 2000 µm nasally and temporally, complemented by a sub-analysis of the central bouquet of cones. 203 animals contributed 374 eyes for a reference choroid database. The overall average central choroidal thickness was 193 µm with a coefficient of variation of 7.8%, and the overall mean surface area of the central bouquet temporally was 19,335 µm2 and nasally was 19,283 µm2. The choroidal thickness of the fovea appears relatively homogeneous between the sexes and the studied origins. However, considerable natural variation has been observed, which needs to be appreciated

Emergent temporal signaling in human trabecular meshwork cells: role of TRPV4-TRPM4 interactions

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway

TRPC4/TRPC5 channels mediate adverse reaction to the cancer cell cytotoxic agent (-)-Englerin A

(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg⁻¹ i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation

Drinking motives, personality traits and life stressors-identifying pathways to harmful alcohol use in adolescence using a panel network approach

BACKGROUND AND AIMS: Models of alcohol use risk suggest that drinking motives represent the most proximal risk factors on which more distal factors converge. However, little is known about how distinct risk factors influence each other and alcohol use on different temporal scales (within a given moment versus over time). We aimed to estimate the dynamic associations of distal (personality and life stressors) and proximal (drinking motives) risk factors, and their relationship to alcohol use in adolescence and early adulthood using a novel graphical vector autoregressive (GVAR) panel network approach.DESIGN, SETTING AND CASES: We estimated panel networks on data from the IMAGEN study, a longitudinal European cohort study following adolescents across three waves (aged 16, 19 and 22 years). Our sample consisted of 1829 adolescents (51% females) who reported alcohol use on at least one assessment wave.MEASUREMENTS: Risk factors included personality traits (NEO-FFI: neuroticism, extraversion, openness, agreeableness and conscientiousness; SURPS: impulsivity and sensation-seeking), stressful life events (LEQ: sum scores of stressful life events), and drinking motives [drinking motives questionnaire (DMQ): social, enhancement, conformity, coping anxiety and coping depression]. We assessed alcohol use [alcohol use disorders identification test (AUDIT): quantity and frequency] and alcohol-related problems (AUDIT: related problems).FINDINGS: Within a given moment, social [partial correlation (pcor) = 0.17] and enhancement motives (pcor = 0.15) co-occurred most strongly with drinking quantity and frequency, while coping depression motives (pcor = 0.13), openness (pcor = 0.05) and impulsivity (pcor = 0.09) were related to alcohol-related problems. The temporal network showed no predictive associations between distal risk factors and drinking motives. Social motives (beta = 0.21), previous alcohol use (beta = 0.11) and openness (beta = 0.10) predicted alcohol-related problems over time (all P  &lt; 0.01).CONCLUSIONS: Heavy and frequent alcohol use, along with social drinking motives, appear to be key targets for preventing the development of alcohol-related problems throughout late adolescence. We found no evidence for personality traits and life stressors predisposingtowards distinct drinking motives over time.</div

Drinking Motives, Personality Traits, Life Stressors - Identifying Pathways to Harmful Alcohol Use in Adolescence Using a Panel Network Approach

BACKGROUND AND AIMS: Models of alcohol use risk suggest that drinking motives represent the most proximal risk factors on which more distal factors converge. However, little is known about how distinct risk factors influence each other and alcohol use on different temporal scales (within a given moment vs. over time). We aimed to estimate the dynamic associations of distal (personality and life stressors) and proximal (drinking motives) risk factors, and their relationship to alcohol use in adolescence and early adulthood using a novel graphical vector autoregressive (GVAR) panel network approach.DESIGN, SETTING, AND CASES: We estimated panel networks on data from the IMAGEN study, a longitudinal European cohort study following adolescents across three waves (ages 16, 19, 22). Our sample consisted of 1829 adolescents (51% females) who reported alcohol use on at least one assessment wave.MEASUREMENTS: Risk factors included personality traits (NEO-FFI: neuroticism, extraversion, openness, agreeableness, and conscientiousness; SURPS: impulsivity and sensation seeking), stressful life events (LEQ: sum scores of stressful life events), and drinking motives (DMQ: social, enhancement, conformity, coping anxiety, coping depression). We assessed alcohol use (AUDIT: quantity and frequency) and alcohol-related problems (AUDIT: related problems).FINDINGS: Within a given moment, social (partial correlation (pcor) =0.17) and enhancement motives (pcor=0.15) co-occurred most strongly with drinking quantity and frequency, while coping depression motives (pcor=0.13), openness (pcor=0.05), and impulsivity (pcor=0.09) were related to alcohol-related problems. The temporal network showed no predictive associations between distal risk factors and drinking motives. Social motives (beta=0.21), previous alcohol use (beta=0.11), and openness (beta=0.10) predicted alcohol-related problems over time (all p&lt;0.01).CONCLUSIONS: Heavy and frequent alcohol use, along with social drinking motives, appear to be key targets for preventing the development of alcohol-related problems throughout late adolescence. We found no evidence for personality traits and life stressors predisposing towards distinct drinking motives over time.</p

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Background: Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV. Methods: We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619. Results: OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV. Conclusion: These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG

Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on β-adrenergic versus Angiotensin II (Ang II)-dependent (Gs vs. Gαq mediated) modulation of Ca2+i-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca2+ currents and Ca2+i transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca2+ currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca2+/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, β-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca2+-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca2+i-dependent hypertrophic growth response to Ang II, but not to β-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT1 signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for β-adrenergic Ca2+i-stimulation in adult myocytes

Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice

Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE