Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites.

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M-1 cm-1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light-dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.

Hepatic retinaldehyde dehydrogenases are modulated by tocopherol supplementation in mice with hepatic steatosis.

OBJECTIVES: An altered retinol metabolism might play a role in the development of nonalcoholic fatty liver disease (NAFLD). Tocopherols (TF) modulate metabolic pathways and have been proposed as a complementary treatment of obesity-induced metabolic alterations. Moreover, there is evidence suggesting that TF may modulate retinol metabolism. The aim of this study was to evaluate whether the dietary supplementation of α- and γ-TF modulates the expression of hepatic retinaldehyde dehydrogenases, RALDH1, RALDH2, and RALDH3 (involved in retinol metabolism) and, lipogenic factors sterol regulatory element binding protein-1c (SREBP-1c) and cluster differentiation 36 (CD36) in an animal model of diet-induced NAFLD. METHODS: Male C57BL/6J mice were divided into four groups: a control diet (CD) group (10% fat, 20% protein, 70% carbohydrates); a CD + TF group (α-tocopherol: 0.7 mg·kg·d-1, γ-tocopherol: 3.5 mg·kg·d-1); a high-fat diet (HFD) group (60% fat, 20% protein, 20% carbohydrates); and a HFD + TF group (0.01 mL·g body weight·d-1), for 12 wk. General parameters (body-adipose tissue weight, glucose-triacylglyceride serum levels), liver steatosis (histology, liver triacylglycerides content), and hepatic RALDH1, RALDH2, RALDH3, SREBP-1c and CD36 (qPCR, quantitative polymerase chain reaction; IHQ, immunohistochemistry) were measured. RESULTS: TF supplementation in HFD-fed mice decreased the presence of lipid vesicles (90%) and total lipid content (75%) and downregulated the expression of RALDH1, RALDH3, SREBP-1c, and CD36. CONCLUSIONS: The present study demonstrated that α- and γ-TF (1:5 ratio) might play a role in modulating retinol metabolism in the prevention of NAFLD induced by a HFD.

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR).

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as "visual cycle" involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.

Horizontal cells expressing melanopsin x are novel photoreceptors in the avian inner retina.

In the vertebrate retina, three types of photoreceptors-visual photoreceptor cones and rods and the intrinsically photosensitive retinal ganglion cells (ipRGCs)-converged through evolution to detect light and regulate image- and nonimage-forming activities such as photic entrainment of circadian rhythms, pupillary light reflexes, etc. ipRGCs express the nonvisual photopigment melanopsin (OPN4), encoded by two genes: the Xenopus (Opn4x) and mammalian (Opn4m) orthologs. In the chicken retina, both OPN4 proteins are found in ipRGCs, and Opn4x is also present in retinal horizontal cells (HCs), which connect with visual photoreceptors. Here we investigate the intrinsic photosensitivity and functioning of HCs from primary cultures of embryonic retinas at day 15 by using calcium fluorescent fluo4 imaging, pharmacological inhibitory treatments, and Opn4x knockdown. Results show that HCs are avian photoreceptors with a retinal-based OPN4X photopigment conferring intrinsic photosensitivity. Light responses in HCs appear to be driven through an ancient type of phototransduction cascade similar to that in rhabdomeric photoreceptors involving a G-protein q, the activation of phospholipase C, calcium mobilization, and the release of the inhibitory neurotransmitter GABA. Based on their intrinsic photosensitivity, HCs may have a key dual function in the retina of vertebrates, potentially regulating nonvisual tasks together with their sister cells, ipRGCs, and with visual photoreceptors, modulating lateral interactions and retinal processing.

A novel cream formulation containing nicotinamide 4%, arbutin 3%, bisabolol 1%, and retinaldehyde 0.05% for treatment of epidermal melasma.

Epidermal melasma is a common hyperpigmentation disorder that can be challenging to treat. Although current treatment options for melasma are limited, topical skin-lightening preparations have widely been used as alternatives to hydroquinone. In this prospective, single-arm, open-label study, treatment of epidermal melasma with a novel cream formulation containing nicotinamide 4%, arbutin 3%, bisabolol 1%, and retinaldehyde 0.05% was associated with reductions in Melasma Area and Severity Index (MASI) scores as well as total melasma surface area as measured by medical imaging software. Treatment outcomes including tolerance and safety profiles as well as patient satisfaction and product appreciation showed this novel cosmetic compound may be valuable in the treatment of epidermal melasma.

Atomic and molecular analysis highlights the biophysics of unprotonated and protonated retinal in UV and scotopic vision.

During the photoreaction of rhodopsin, retinal isomerizes, rotating the C11[double bond, length as m-dash]C12 π-bond from cis to an all-trans configuration. Unprotonated (UR) or protonated (PR) retinal in the Schiff's base (SB) is related to UV and light vision. Because the UR and PR have important differences in their physicochemical reactivities, we compared the atomic and molecular properties of these molecules using DFT calculations. The C10-C11[double bond, length as m-dash]C12-C13 dihedral angle was rotated from 0° to 180° in 45° steps, giving five conformers, and the following were calculated from them: atomic orbital (AO) contributions to the HOMO and LUMO, atomic charges, bond length, bond order, HOMO, LUMO, hardness, electronegativity, polarizability, electrostatic potential, UV-vis spectra and dipole moment (DM). Similarly, the following were analyzed: the energy profile, hybridization, pyramidalization and the hydrogen-out-of-plane (HOOP) wagging from the H11-C11[double bond, length as m-dash]C12-H12 dihedral angle. In addition, retinal with a water H-bond (HR) in the SB was included for comparison. Interestingly, in the PR, C11 and C12 are totally the LUMO and the HOMO, respectively, and have a large electronegativity difference, which predicts an electron jump in these atoms during photoexcitation. At the same time, the PR showed a longer bond length and lower bond order, with a larger DM, lower HOMO-LUMO gap, lower hardness and higher electronegativity. In addition, the AOs of -45° and -90° conformers changed significantly, from pz to py, during the rotation concomitantly with marked hybridization, smooth pyramidalization and lower HOOP activity. Clearly, the atomic and molecular differences between the UR and PR are overwhelming, including the rotational energy profile and light absorption spectra, which indicates that light absorption of UR and PR is already determined by the retinal characteristics of the SB protonation. The HR-model compared with UR shows a lower energy barrier and a discreet bathochromic effect in the UV region.

Electron density deformations provide new insights into the spectral shift of rhodopsins.

Spectral shifts of rhodopsin, which are related to variations of the electron distribution in 11-cis-retinal, are investigated here using the method of deformed atoms in molecules. We found that systems carrying the M207R and S186W mutations display large perturbations of the π-conjugated system with respect to wild-type rhodopsins. These changes agree with the predicted behavior of the bond length alternation (BLA) and the blue shifts of vertical excitation energies of these systems. The effect of the planarity of the central and Schiff-base regions of retinal chain on the electronic structure of the chromophore is also investigated. By establishing nonlinear polynomial relations between BLA, chain distortions, and vertical excitation energies, we are also able to provide a semiquantitative approach for the understanding of the mechanisms regulating spectral shifts in rhodopsin and its mutants.

Understanding rhodopsin mutations linked to the retinitis pigmentosa disease: a QM/MM and DFT/MRCI study.

Retinitis pigmentosa (RP) is a pathological condition associated with blindness due to progressive retinal degeneration. RP-linked mutations lead to changes at the retinal binding pocket and in the absorption spectra. Here, we evaluate the geometries, electronic effects, and vertical excitation energies in the dark state of mutated human rhodopsins carrying the abnormal substitutions M207R or S186W at the retinal binding pocket. Two models are used, the solvated protein and the protein in a solvated POPC lipid bilayer. We apply homology modeling, classical molecular dynamics simulations, density functional theory (DFT), and quantum mechanical/molecular mechanical (QM/MM) methods. Our results for the wild type bovine and human rhodopsins, used as a reference, are in good agreement with experiment. For the mutants, we find less twisted QM/MM ground-state chromophore geometries around the C(11)-C(12) double bond and substantial blue shifts in the lowest vertical DFT excitation energies. An analysis of the QM energies shows that the chromophore-counterion region is less stable in the mutants compared to the wild type, consistent with recent protein folding studies. The influence of the mutations near the chromophore is discussed in detail to gain more insight into the properties of these mutants. The spectral tuning is mainly associated with counterion effects and structural features of the retinal chain in the case of the hM207R mutant, and with the presence of a neutral chromophore with deprotonated Lys296 in the case of the hS186W mutant.

Increased blood oxidative stress in experimental menopause rat model: the effects of vitamin A low-dose supplementation upon antioxidant status in bilateral ovariectomized rats.

Menopause has been reported to be associated with increased oxidative stress and metabolic disorders among women worldwide. Disarrangements in the redox state similar to those observed in women during the decline of ovarian hormonal activity can be obtained experimentally through rat bilateral ovariectomy. The search for alternative treatments to improve life quality in postmenopausal woman is really important. The aim of this study was to evaluate biochemical and oxidative stress parameters that distinguish sham-operated female rats from Wistar rats bilaterally ovariectomized (OVX). Additionally, we have also investigated the effects of retinol palmitate (a vitamin A supplement) low-dose supplementation (500 or 1500 IU/kg/day, during 30 days) upon blood and plasma antioxidant status in OVX rats. Ovariectomy caused an increase in body weight gain, pronounced uterine atrophy, decreased plasma triglycerides and increased total cholesterol levels, and reduced acid uric content. Moreover, we found increased blood peroxidase activities (catalase and glutathione peroxidase), decreased plasma non-enzymatic antioxidant defenses total reactive antioxidant potential and total antioxidant reactivity, decreased protein and non-protein SH levels, accompanied by increased protein oxidative damage (carbonyl). In addition, vitamin A low-dose supplementation was capable to ameliorate antioxidant status in OVX rats, restoring both enzymatic and non-enzymatic defenses, promoting reduction in plasma SH content, and decreasing protein oxidative damage levels. This is the first work in the literature showing that vitamin A at low dose may be beneficial in the treatment of menopause symptoms. Further studies will be made to better understand the effects of vitamin A supplementation in menopause rat model.

Retinaldehyde/hyaluronic acid fragments: a synergistic association for the management of skin aging.

BACKGROUND: Retinaldehyde (RAL) was proven effective in treating photodamaged skin. Topical treatments with specific intermediate-size hyaluronate fragments (HAFi, 50-400 kDa) have been shown to stimulate keratinocytes proliferation and epidermal hyperplasia. The aim of this open, multicentric, international study was to assess the efficacy of the combination RAL-HAFi in the correction of skin photoaging. PATIENTS/METHODS: Either RAL 0.05%-HAFi 0.5% (Eluage® cream; group 1) or RAL 0.05%-HAFi 1% (Eluage® antiwrinkle concentrate; group 2) or both products (group 3) were applied daily to the 1462 subjects during 90 days. Overall photoaging severity was evaluated in the three groups by the dermatologists at D0, D30, and D90 based on the Larnier's scale. Wrinkles and/or furrows and clinical signs of aging were evaluated using a 4-point scale. The skin microrelief of the crow's feet, evaluated by optical profilometry, was performed in subjects from group 3. RESULTS: The 3-month application significantly improved overall photoaging through decrease of the Larnier's score in the three groups (P<0.001). At D90, significant improvement of wrinkles was shown in groups 2 and 3 [forehead wrinkles (-19% and -10%, respectively, P<0.001), nasolabial folds (-20% and -16%, P<0.001), crow's feet (-27% in the two groups, P<0.001), and perioral wrinkles (-34% and -23%, P<0.001)]. Clinical signs of photoaging on the entire face improved significantly in groups 1 and 3 [elasticity (-32% and -33%, respectively, P<0.001), hyperpigmentation (-34% and -31%, P<0.001), and ptosis (-18% and -22%; P<0.001)]. Results were confirmed using an optical profilometry technique. Products were very well tolerated. CONCLUSION: This clinical study showed the efficacy and value of the RAL-HAFi combination in the management of aging skin in a large cohort of patients.

Eficacia y seguridad del retinaldehido y el Ácido retinoico tópico en el tratamiento del fotoenvejecimiento / Efficacy and safety of retinaldehyde and topical retinoic acid in the treatment of photoaging

El ácido retinoico tópico es eficaz para el tratamiento del fotoenvejecimiento. Sin embargo la irritación cutánea secundaria a su uso es un factor limitante del tratamiento. El retinaldehído es un precursor inmediato del ácido retinoico que posee actividad biológica en la piel, con menos efectos secundarios. Objetivo: comparar la eficacia y tolerancia del retinaldehido y el ácido retinoico tópicos en el tratamiento del fotoenvejecimiento, mediante perfil profilométrico y análisis clínico - fotográfico y determinar los efectos secundarios de estos medicamentos. Métodos: se incluyeron 126 mujeres colombianas (35 a 60 años), quienes no habían recibido tratamientos previos, no estaban embarazadas, ni lactando. En total 119 pacientes completaron las 24 semanas de seguimiento: a 61 pacientes se les aplicó retinaldehído y a 58, ácido retinoico. Se realizó un seguimiento clínico (que evaluó mejoría y tolerancia), en las semanas 4, 8, 16 y 20, y además se realizó profilometría del área de la pata de gallina y seguimiento fotográfico al ingreso y en la semana 24.

Topical retinoic acid has been effectively used to treat photo aging; nevertheless, cutaneous irritation as a side effect is a limiting factor for treatment. Retinaldehyde is an immediate precursor of retinoic acid that has biologic activity in the skin, with few side effects. The objective of this investigation was to compare efficacy and tolerability of topical retinoic acid and retinaldehyde in the treatment of photo aging, by means of profilometry and clinical-photographic analysis and to determine side effects from both medications. Methods: were included 160 colombian women (aged 35 to 60 years), who hadn't had previous treatments and weren't pregnant or breast feeding. A total of 119 patients completed 24 weeks of treatment. Of these, 61 used retinaldehyde and 58 retinoic acid. Clinical evaluation was carried out on weeks 4, 8, 16 and 20, and profilometry of the “crow's feet” area was done at the beginning and at week 24.

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.

Involvement of alcohol and aldehyde dehydrogenase activities on hepatic retinoid metabolism and its possible participation in the progression of rat liver regeneration.

Liver alcohol dehydrogenase (ADH) activity is decreased towards exogenous substrates after partial hepatectomy (PH), probably due to putative endogenous substrates acting as ADH inhibitors. Hence, retinoids could be suitable candidates as such endogenous substrates. Therefore, cytosolic ADH kinetic analysis using several substrates, liver cytosolic and mitochondrial aldehyde dehydrogenase (ALDH) activities, retinal and retinol content, as well as expression of proteins for ADH and CRBPI (a retinol carrier protein) were determined in liver samples, at two stages of liver regeneration (one- or two-thirds PH). The effect of inhibiting in vivo liver ADH by 4-methylpyrazole (4-MP) was also evaluated after 70%-PH. With 70%-PH, in vitro ADH activity towards exogenous alcohols and aldehydes was diminished, but retinol oxidation was increased and retinal reduction was decreased. These activities that be due to the participation of an ADH type which did not correlate with the amount of immunoreactive ADH protein. Cytosolic and mitochondrial ALDH activities oxidized actively retinal, whereas retinol and CBRP-I expression were reduced in these animals. With 30%-PH, these changes were less evident and sometimes opposite to those found with 70%-PH. In addition, retinol readily inhibited ADH-mediated ethanol oxidation. Interestingly, in vivo 4-MP administration inhibited ADH activity in a dose-dependent manner correlating with a progressive inhibition of liver regeneration. In conclusion, PH-induced inhibition of ADH (mainly type I) seems to be related to ADH-mediated retinoid metabolism during liver proliferation. Thus, results suggest a role of ADH in retinoid metabolism, which is apparently required during rat liver regeneration.

Quantum chemical study of electronic and structural properties of retinal and some aromatic analogs.

The electronic and structural properties of retinal and four analogs were studied using semiempirical, ab initio Hartree-Fock, and density functional theory methods with the aim to evaluate the effects caused by some structural modifications in the ring bound to the polyenic chain and compared with the all-E-trans-retinal molecule. Therefore, some properties such as bond lengths, bond angles, atomic charges derived from electrostatic potential charges from electrostatic potential using grid based method as well as frontier orbitals of the polyenic chain were analyzed. Furthermore, the transition energies of the molecules were also calculated using the Zerner's intermediate neglect of differential overlap-spectroscopic, time-dependent Hartree-Fock, and time-dependent density functional theory methods. The results indicate that in spite of the structural modifications of retinal derivatives in comparison with all-E-trans-retinal, their properties seem similar. Thus, these molecules may behave similarly to all-E-trans-retinal and possibly be attempted in the search of novel molecular devices.

Patterns of retinal light absorption related to retinitis pigmentosa mutants from in silico model structures of rhodopsin.

Changes induced by mutations in rhodopsin that are associated with the degenerative visual disease retinitis pigmentosa result in an altered pattern of light absorption according to quantum mechanical simulations and reference experimental works. Eleven single-point mutations associated with retinitis pigmentosa at and in the proximity to the retinal binding pocket of rhodopsin have been modeled in silico and their spectra calculated with the NDOL (Neglect of Differential Overlap accounting L azimuthal quantum number) a priori method. The altered pattern of absorption found would lead to cumulative consequences in energy dissipation with aging. Different energy balances in the case of mutants at the very molecular level, compared to native nonmutated rhodopsin, can cause permanent cellular stress and would play a role in the progression of the retine degenerative process. It could explain the worsening of the pathological condition mostly in adults and suggests the probable beneficial effects of using quenching drugs and protection devices against excess of light in the early stages of life for avoiding or reducing potential damage.

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products.

Epidemiological studies testing the effect of beta-carotene in humans have found a relative risk for lung cancer in smokers supplemented with beta-carotene. We investigated the reactions of retinal and beta-apo-8'-carotenal, two beta-carotene oxidation products, with 2'-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N(2)-etheno-2'-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with beta-carotene or beta-carotene oxidation products, significantly increased levels of 1,N(2)-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of beta-carotene oxidation products.

Citoquinas y patogénesis del edema macular diabético / Cytokines and pathogenesis of edema diabetic macular

Diabetic macular edema (DME) is the leading cause of visual loss in diabetic patients and is characterized by accumulation of extracellular fluid in the retina. The most important mechanism involved is breakdown of the blood-retinal barrier (BRB), which is likely to be due to changes of tight junction proteins, including occludin and the zonula occludens proteins. Hyperglycemia and retinal ischemia stimulate the synthesis and secretion of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6). Both the aqueous humor and vitreous fluid levels of VEGF and IL-6 are significantly elevated in patients who have DME when compared with non-diabetic patients. Vascular permeability factors, including VEGF and IL-6, may act directly on endothelial cell tight junctions to decrease their protein content or increase phosphorylation. Either or both of these changes may lead to increased paracellular permeability. The specific molecules that are allowed to move through intercellular junctions may depend on the particular vascular permeability factor involved, as well as its concentration, duration of action, and interaction with other factors. Defining the molecular mechanisms of DME will be critical for the development of specific therapy.

Induction of erythrophagocytic activity in cultured mouse trophoblast cells by phorbol myristate acetate and all-trans-retinal.

Phorbol myristate acetate (PMA) and all-trans-retinal (retinal) were evaluated as possible phagocytic stimulators of cultured, post implantation, trophoblast cells. Ectoplacental cones dissected from 7.5 day-old mouse embryos provided the source of trophoblastic cells. Co-cultures were performed using stimulated and non-stimulated trophoblast cells and erythrocytes under standard conditions. Phagocytic activity was expressed as the total number of phagocytic cells per ectoplacental cone, and as phagosomic vacuoles per trophoblast giant cell, either in the presence or absence of the stimulators. Both chemical agents had similar effects, less than 12 h after stimulation, statistically significant numbers of erythrophagosomes appear in the trophoblast giant cells (TGC). These findings demonstrate that TGC, like neutrophils and macrophages, can be activated to phagocytosis by exogenous factors. This enhanced activity may result from the generation and release of reactive oxygen species. In conclusion, our data suggest that, because stimulation was provided, the remarkable in vivo phagocytic activity of the trophoblast can be maintained under in vitro conditions, allowing study of the pathways and regulatory steps involved in this process.

Post-implantation mouse embryos have the capability to generate and release reactive oxygen species.

The capability of the mouse embryo to generate reactive oxygen species (ROS) was examined. Post-implantation embryos were carefully harvested on Day 8 of pregnancy and the production of ROS was quantified using luminol-sensitized chemiluminescence. The embryos were stimulated with either phorbol myristate acetate (PMA) or all-trans-retinal (retinal) and the reaction kinetics were followed over 10 min. ROS secretion was directly proportional to the number of embryos and was suppressed 56% by superoxide dismutase (SOD), 25% by mannitol and as little as 16% by catalase. Embryos deprived of trophoblast showed no light emission suggesting that the source of ROS generation is the trophoblast. Dihydronicotinamide adenine dinucleotide (NADH)-dependent oxidase activity in the plasma membrane of the trophoblast surface was demonstrated by cytochemical methods. The release of ROS into the extracellular medium during the phagocytic process has been related to the cytolytic effect exhibited by these molecules and, perhaps by this means, the trophoblast can play an active role in the phagocytosis of maternal cells during the process of embryo implantation.