Sublethal Photic Stress and the Motility of RPE Phagosomes and Melanosomes

PURPOSE. To determine whether sublethal oxidative stress to the retinal pigment epithelium by visible light treatment affects the translocation of organelles, notably phagosomes and melanosomes. METHODS. Isolated porcine melanosomes were phagocytized by ARPE-19 cells, then cultures were treated with blue light to generate reactive oxygen intermediates (ROIs) by endogenous retinal pigment epithelial (RPE) chromophores throughout the cytoplasm. Other melanosomes were preloaded with a photosensitizer before phagocytosis, and cells were light treated to generate ROIs specifically at the granule surface. Phagosome movement was analyzed by live cell imaging. Also analyzed were phagocytized black latex beads, phagocytized melanosomes pretreated to simulate age-related melanin photobleaching, and endogenous RPE melanosomes in primary cultures of porcine retinal pigment epithelium. RESULTS. Sublethal blue light treatment slowed the movement of some, but not all, phagocytized melanosomes. All phagosomes slowed when ROIs were generated near the organelles through a photosensitized reaction. Melanosome photobleaching, which makes granules more photoreactive, increased the effects of blue light. Blue light treatment also slowed the motility of phagosomes containing latex beads and endogenous pigment granules. CONCLUSIONS. Blue light-induced stress impairs phagosome motility in RPE cells but affects individual organelles differently, suggesting that the effects of mild oxidative injury vary with subcellular location. The mechanisms underlying slowed motility are at least partially local because slowing can be induced by a photosensitized reaction in the subdomain of the organelle and the magnitude of the slowing is greater when the phagosome contents are photoreactive. Photic stress may impair the movement and positioning of RPE organelles, which would have widespread consequences for maintaining a functionally efficient subcellular organization. (Invest Ophthalmol Vis Sci

Photoreactivity of aged human RPE melanosomes: A comparison with lipofuscin

purpose. To determine whether aging is accompanied by changes in aerobic photoreactivity of retinal pigment epithelial (RPE) melanosomes isolated from human donors of different ages, and to compare the photoreactivity of aged melanosomes with that of RPE lipofuscin. methods. Human RPE pigment granules were isolated from RPE cells pooled into groups according to the age of the donors. Photoreactivity was determined by blue-light-induced oxygen uptake and photogeneration of reactive oxygen species. Short-lived radical intermediates were detected by spin-trapping, hydrogen peroxide by an oxidase electrode, singlet oxygen by cholesterol assay, and lipid hydroperoxides by iodometric assay. results. Blue-light photoexcitation of melanosomes resulted in age-related increases in both oxygen uptake and the accumulation of superoxide anion spin adducts. The efficiencies of these processes, however, were still significantly lower than that induced by photoexcited lipofuscin. During irradiation of melanosomes, a substantial amount of oxygen was converted into hydrogen peroxide, whereas for lipofuscin, hydrogen peroxide accounted for not more than 3% of oxygen consumed. In contrast to lipofuscin, photoexcited melanosomes did not substantially increase the rate of oxidative reactions in the presence of polyunsaturated lipids or albumin. However, oxygen uptake was significantly elevated in the presence of ascorbate. Thus, the rate of photo-induced oxygen uptake in samples containing both ascorbate and melanosomes approached that observed in lipofuscin samples. conclusions. Blue-light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction

The Occurrence of Rocky Habitable-zone Planets around Solar-like Stars from Kepler Data

We present the occurrence rates for rocky planets in the habitable zones (HZs) of main-sequence dwarf stars based on the Kepler DR25 planet candidate catalog and Gaia-based stellar properties. We provide the first analysis in terms of star-dependent instellation flux, which allows us to track HZ planets. We define η⊕ as the HZ occurrence of planets with radii between 0.5 and 1.5 R⊕ orbiting stars with effective temperatures between 4800 and 6300 K. We find that η⊕ for the conservative HZ is between 0.37^(+0.48)_(−0.21) (errors reflect 68% credible intervals) and 0.60^(+0.90)_(−0.36) planets per star, while the optimistic HZ occurrence is between 0.58^(+0.73)_(−0.33) and 0.88^(+1.28)_(−0.51) planets per star. These bounds reflect two extreme assumptions about the extrapolation of completeness beyond orbital periods where DR25 completeness data are available. The large uncertainties are due to the small number of detected small HZ planets. We find similar occurrence rates between using Poisson likelihood Bayesian analysis and using Approximate Bayesian Computation. Our results are corrected for catalog completeness and reliability. Both completeness and the planet occurrence rate are dependent on stellar effective temperature. We also present occurrence rates for various stellar populations and planet size ranges. We estimate with 95% confidence that, on average, the nearest HZ planet around G and K dwarfs is ~6 pc away and there are ~4 HZ rocky planets around G and K dwarfs within 10 pc of the Sun

The Occurrence of Rocky Habitable Zone Planets Around Solar-Like Stars from Kepler Data

We present occurrence rates for rocky planets in the habitable zones (HZ) of main-sequence dwarf stars based on the Kepler DR25 planet candidate catalog and Gaia-based stellar properties. We provide the first analysis in terms of star-dependent instellation flux, which allows us to track HZ planets. We define $\eta_\oplus$ as the HZ occurrence of planets with radius between 0.5 and 1.5 $R_\oplus$ orbiting stars with effective temperatures between 4800 K and 6300 K. We find that $\eta_\oplus$ for the conservative HZ is between $0.37^{+0.48}_{-0.21}$ (errors reflect 68\% credible intervals) and $0.60^{+0.90}_{-0.36}$ planets per star, while the optimistic HZ occurrence is between $0.58^{+0.73}_{-0.33}$ and $0.88^{+1.28}_{-0.51}$ planets per star. These bounds reflect two extreme assumptions about the extrapolation of completeness beyond orbital periods where DR25 completeness data are available. The large uncertainties are due to the small number of detected small HZ planets. We find similar occurrence rates using both a Poisson likelihood Bayesian analysis and Approximate Bayesian Computation. Our results are corrected for catalog completeness and reliability. Both completeness and the planet occurrence rate are dependent on stellar effective temperature. We also present occurrence rates for various stellar populations and planet size ranges. We estimate with $95\%$ confidence that, on average, the nearest HZ planet around G and K dwarfs is about 6 pc away, and there are about 4 HZ rocky planets around G and K dwarfs within 10 pc of the Sun.Comment: To appear in The Astronomical Journa

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

High levels of E-/P-cadherin: Correlation with decreased apical polarity of Na-K ATPase

PURPOSE. The adherens junction protein E-cadherin induces a basolateral polarity of Na/K ATPase in most epithelial cells that express it, whereas in retinal pigment epithelium (RPE) cells, Na/K ATPase is largely apical. The purpose of this study was to determine whether the distribution of Na/K ATPase differs in RPE cells in situ, that differ in levels of junctional E-cadherin. METHODS. Bovine RPE cells in situ were immunostained with an E-cadherin antibody (which has some cross-reactivity with the closely related epithelial cadherin P-cadherin), and RPE cells with different levels of junctional stain were identified. RPE cells with low and high E-/P-cadherin were costained in various combinations with Na/K ATPase and interacting proteins of the membrane cytoskeleton (ankyrin, fodrin, and actin) and analyzed by confocal imaging. RESULTS. Individual RPE cells within the same monolayer differed in amount of Na/K ATPase, with a lower frequency of high expressing cells in the area centralis. High expressing Na/K ATPase cells were found among cells with both low and high E-/P-cadherin levels. In cells with low E/P-cadherin, Na/K ATPase localized to apical microvilli, whereas in high E-/P-cadherin cells, Na/K ATPase was on basolateral surfaces in addition to microvilli. Actin staining showed that microvillar domains were smaller and that lateral membrane domains were taller in high E-/P-cadherin cells. In high but not low E-/P-cadherin cells, ankyrin and fodrin levels varied among cells, with a subset of cells showing distinctly higher expression. Both ankyrin and fodrin had complex subcellular distribution patterns, although they tended to be enriched basal to rather than apical to the adherens junction. Cells with high Na/K ATPase did not necessarily have commensurately higher levels of ankyrin or fodrin. Where both Na/K ATPase and ankyrin were high, they codistributed weakly in apical microvilli but more prominently on the basal cell surface. CONCLUSIONS. Within the same RPE monolayer, the polarity of Na/K ATPase differs among cells, with a more basal polarity found in cells with high levels of junctional E-/P-cadherin. The increased basal Na/K ATPase was due to a combination of a smaller microvillar domain, a taller lateral domain, and more basolateral staining for Na/K ATPase, perhaps because of an enrichment of a basal ankyrinfodrin membrane cytoskeleton with which Na/K ATPase is known to associate. (Invest Ophthalmol Vis Sci. 2000;41:1945-1952 T he calcium-dependent adhesion protein E-cadherin forms cell-cell attachments at the zonula adherens junction of most monolayer epithelial cells. Retinal pigment epithelial (RPE) cells were believed to be an exception and to express N-cadherin rather than E-cadherin

Expression of Ecadherin by human retinal pigment epithelium: Delayed expression

PURPOSE. To determine whether retinal pigment epithelial (RPE) cells, which reportedly express N-cadherin as their major cadherin cell adhesion protein, also express the more common epithelial cadherin, E-cadherin. METHODS. Cadherins expressed by human RPE cells in situ were examined by western blot analysis of extracts prepared from the RPE of human adult eyes. Cadherins expressed in vitro were examined by analysis of confluent and postconfluent human RPE cultures, using the methods of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Protein distribution was examined by conventional fluorescence microscopy, confocal imaging, or both. Proteins whose expression, distribution, or both correlated with E-cadherin expression in other epithelial cells were examined by similar methods in cultured RPE cells. RESULTS. In addition to N-cadherin, E-cadherin (and P-cadherin) was found in adult human RPE in situ. In cultured human RPE cells, N-cadherin was ubiquitous, but E-cadherin was limited to patches of cells and was not expressed until several weeks after confluence, a time when several phenotypic variants become prominent. E-cadherin was absent from RPE cells of fusiform shape but was found in only a subset of epithelioid RPE cells. Unlike epithelial cell lines expressing E-cadherin, cultured RPE cells with E-cadherin did not show diminished coexpression of N-cadherin, increased expression of desmosomal proteins, or a preferential expression of the ␣E-(rather than ␣-N) isoform of the cadherin linker protein ␣-catenin. Na/K ATPase distributed to both apical and basolateral membranes in RPE cells with junctional E-cadherin and not preferentially to the basolateral domain as in most epithelial cells with E-cadherin. CONCLUSIONS. RPE cells express E-cadherin, a cadherin found in most other epithelial cells, but which was believed to be absent from RPE. In RPE in vitro, E-cadherin expression is a late developmental event, occurring in late confluence in cells that already express N-cadherin. Ecadherin is an established epithelial morphoregulatory protein, but it does not induce the same properties in RPE cells as in other epithelial cells, suggesting tissue-specific differences in the potential of E-cadherin to determine an epithelial phenotype. (Invest Ophthalmol Vis Sci. 1999;40: 2963-2970 I n the process of mediating cell-cell attachment, the adhesion molecule E-cadherin also appears to confer phenotype on cultured epithelial cells. E-cadherin localizes to junctional sites shortly after confluence where it triggers the timedependent development of polarized plasma membrane domains 1-5 and the acquisition of a grossly epithelial cell shape. In addition to E-cadherin, epithelial cells also often express P-cadherin, although little is known about the morphoregulatory properties of this member of the cadherin family. In contrast to most monolayer epithelial cells, cells of the retinal pigment epithelium (RPE) have been reported to lack E-cadherin 6 -8 and to express N-cadherin, 7,9 -12 which is typically found in nonepithelial cells. In embryonic chick development, RPE cells have also been shown to express B-cadherin, 13 which is likely the avian homologue of mammalian P-cadherin. 14 Aside from expressing N-cadherin, RPE cells have several other unusual properties that distinguish them from most epithelia. The RPE monolayer is located between two tissues rather than facing a lumen, the sodium pump of RPE cells is reportedly polarized to the apical rather than basolateral membrane domain, 22 Because E-cadherin plays a role in directing Na/K ATPase polarity When propagated in vitro, human RPE cells display another feature that differs from epithelial cell lines. Rather than producing cultures consisting of cells with a fairly unifor