Deletion of miR-150 Exacerbates Retinal Vascular Overgrowth in High-Fat-Diet Induced Diabetic Mice

Diabetic retinopathy (DR) is the leading cause of blindness among American adults above 40 years old. The vascular complication in DR is a major cause of visual impairment, making finding therapeutic targets to block pathological angiogenesis a primary goal for developing DR treatments. MicroRNAs (miRs) have been proposed as diagnostic biomarkers and potential therapeutic targets for various ocular diseases including DR. In diabetic animals, the expression levels of several miRs, including miR-150, are altered. The expression of miR-150 is significantly suppressed in pathological neovascularization in mice with hyperoxia-induced retinopathy. The purpose of this study was to investigate the functional role of miR-150 in the development of retinal microvasculature complications in high-fat-diet (HFD) induced type 2 diabetic mice. Wild type (WT) and miR-150 null mutant (miR-150-/-) male mice were given a HFD (59% fat calories) or normal chow diet. Chronic HFD caused a decrease of serum miR-150 in WT mice. Mice on HFD for 7 months (both WT and miR-150-/-) had significant decreases in retinal light responses measured by electroretinograms (ERGs). The retinal neovascularization in miR-150-/--HFD mice was significantly higher compared to their age matched WT-HFD mice, which indicates that miR-150 null mutation exacerbates chronic HFD-induced neovascularization in the retina. Overexpression of miR-150 in cultured endothelial cells caused a significant reduction of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels. Hence, deletion of miR-150 significantly increased the retinal pathological angiogenesis in HFD induced type 2 diabetic mice, which was in part through VEGFR2

Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

Dengue virus (DENV) represents a major disease burden in tropical and subtropical regions of the world, and has shown an increase in the number of cases in recent years. DENV is transmitted to humans through the bite of an infected mosquito, typically Aedes aegypti, after which it begins the infection and replication lifecycle within human cells. To perform the molecular functions required for invasion, replication, and spread of the virus, proteins encoded by DENV must interact with and alter the behavior of protein networks in both of these hosts. In this work, we used a computational method based on protein structures to predict interactions between DENV and its human and insect hosts. We predict numerous interactions, with many involved in known cell death, stress, and immune system pathways. Further investigation of these predicted protein-protein interactions should provide targets to combat the clinical manifestations of this disease in humans as well as points of intervention focused within the mosquito vector

Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina

Mitochondrial fission and fusion are dependent on cellular nutritional states, and maintaining this dynamics is critical for the health of cells. Starvation triggers mitochondrial fusion to maintain bioenergetic efficiency, but during nutrient overloads (as with hyperglycemic conditions), fragmenting mitochondria is a way to store nutrients to avoid waste of energy. In addition to ATP production, mitochondria play an important role in buffering intracellular calcium (Ca2+). We found that in cultured 661W cells, a photoreceptor-derived cell line, hyperglycemic conditions triggered an increase of the expression of dynamin-related protein 1 (DRP1), a protein marker of mitochondrial fission, and a decrease of mitofusin 2 (MFN2), a protein for mitochondrial fusion. Further, these hyperglycemic cells also had decreased mitochondrial Ca2+ but increased cytosolic Ca2+. Treating these hyperglycemic cells with melatonin, a multifaceted antioxidant, averted hyperglycemia-altered mitochondrial fission-and-fusion dynamics and mitochondrial Ca2+ levels. To mimic how people most commonly take melatonin supplements, we gave melatonin to streptozotocin- (STZ-) induced type 1 diabetic mice by daily oral gavage and determined the effects of melatonin on diabetic eyes. We found that melatonin was not able to reverse the STZ-induced systemic hyperglycemic condition, but it prevented STZ-induced damage to the neural retina and retinal microvasculature. The beneficial effects of melatonin in the neural retina in part were through alleviating STZ-caused changes in mitochondrial dynamics and Ca2+ buffering

Supplementary Material

<p>Supplemental material, Supp_Figure_(1) for Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors by Janet Ya-An Chang, Liheng Shi, Michael L. Ko, and Gladys Y.-P. Ko in Journal of Biological Rhythms</p

The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses

L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Cav1.2, Cav1.3, and Cav1.4) expressed in the retina. While Cav1.2 is expressed in all retinal cells including the Müller glia and neurons, Cav1.3 and Cav1.4 are expressed in the retinal neurons with Cav1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Cav1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Cav1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Cav1.3 are not associated with severe vision impairment in humans or in Cav1.3-null (Cav1.3−/−) mice. However, a failure to regulate Cav1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Cav1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Cav1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Cav1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Cav1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Cav1.3−/− mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Cav1.3−/− mice retinas. Hence, Cav1.3 plays a more prominent role in retinal physiology and function than previously reported

Overexpression of miR-150 decreased VEGFR2 protein level in endothelial cells.

<p>The HUVE cells were transfected with miR-150 (has-miR-150) or a scramble microRNA (Scramble) and cultured for an additional 60 hr. The protein levels of c-Myb and VEGFR2 are significantly lower in HUVE cells with overexpression of miR-150 compared to the scramble (student’s <i>t</i>-test; *<i>p</i> < 0.05). n = 4 for each group. (B) The protein level of VEGFR2 is significantly lower in HRECs transfected with has-miR-150 compared to the ones transfected with scramble (student’s t-test; *p < 0.05). n = 4 for each group. (C) The retinas from WT and miR-150^-/- mice under normal chow diet (Normal) or HFD were isolated and processed for Western blotting. Some retinas were trypsin-digested to obtain the retinal vasculature followed by immunostaining with the VEGFR2 antibody conjugated with Alexa-488. The scale bar = 100 μm.</p

Dark-adapted (scotopic) retinal light responses (Data for Fig 2A, 2B and 2E).

<p>Dark-adapted (scotopic) retinal light responses (Data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.g002" target="_blank">Fig 2A, 2B and 2E</a>).</p

Deletion of miR-150 exacerbates HFD-induced DR neovascularization and microaneurysms.

<p>(A) Upper two rows: the whole mount retinal vasculature was stained with FITC-labeled isolectin-B4. The first row: the fluorescent images from 4 experimental groups were taken at 5X (scale bar = 400 μm). The highlighted regions (yellow square) were magnified at 10X and displayed in the second row. The second row: the fluorescent images from 4 experimental groups were taken at 10X (scale bar = 100 μm). Lower two rows: the mouse retinas were trypsin-digested and the retinal vasculatures were stained with hematoxylin and eosin. The third row: the whole retinal vasculature images are shown (scale bar = 400 μm). The fourth row: magnified images of retinal vasculature (from the third row) are shown (scale bar = 100 μm). White arrowheads indicate the pericytes, the red arrowheads indicate the acellular capillaries, and the white circles indicate the microaneurysm-like (vascular extrusion) structures. (B) Wild type and miR-150^-/- mice fed with HFD have significantly higher (*) vasculature in central and peripheral retinal areas compared to mice fed with normal chow diet (Normal). There is a statistical significant difference in the interaction between miR-150 null mutation and HFD regimen (2-way ANOVA; #). (C) Wild type and miR-150^-/- mice fed with HFD have significantly higher (*) densities of microaneurysm-like structures (microaneurysms per 0.6 mm² retinal area) compared to mice fed with normal chow diet (Normal). There is a statistical significant difference in the interaction between miR-150 null mutation and HFD regimen (2-way ANOVA; #). WT-Normal: n = 6; WT-HFD: n = 8; miR-150^-/--Normal: n = 6; miR-150^-/--HFD: n = 8. <i>p</i> < 0.05 (denoted as *, #).</p

Scotopic and photopic light responses are decreased in WT and miR-150^-/- mice fed with HFD.

<p>All mice were dark adapted for at least 6 hours before ERG recordings. (A) The average scotopic ERG a-wave amplitudes recorded from miR-150^-/- with HFD (miR-150^-/--HFD) are significantly lower compared to the WT fed with normal chow diet (WT-Normal; *) or miR-150^-/- fed with normal chow diet (miR-150^-/--Normal; #). There is no statistical difference between WT fed with HFD (WT-HFD) and miR-150^-/--HFD. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) a-wave amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). The miR-150^-/- mice (both normal chow and HFD groups) had significantly smaller (<b>w</b>) a-wave amplitudes compared to the WT mice (both normal chow and HFD groups). (B) The averaged photopic ERG a-wave amplitudes recorded from WT-HFD are significantly lower than WT-Normal (*) at 3 and 10 cd.s/m² light intensities. The photopic a-wave amplitudes recorded from miR-150^-/--HFD are significantly lower than WT-normal (#) at 3, 10, and 25 cd.s/m² light intensities. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). (C) The average scotopic ERG b-wave amplitudes recorded from miR-150^-/--HFD are significantly lower compared to WT-Normal (*) or miR-150^-/--Normal (#). There is no statistical difference between WT-HFD and miR-150^-/--HFD. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) a-wave amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). The miR-150-/- mice (both normal chow and HFD groups) had significantly smaller (<b>w</b>) a-wave amplitudes compared to the WT mice (both normal chow and HFD groups). (D) The averaged ERG photopic b-wave amplitudes recorded from WT-HFD are significantly lower than WT-Normal (*) and miR-150^-/--Normal (#) at 1 and 10 cd.s/m² light intensities. The photopic b-wave amplitudes recorded from miR-150^-/--HFD are significantly different from the other 3 groups (&) at 25 cd.s/m² light intensities. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). (E) The averaged scotopic oscillatory potential amplitudes [as a summation from OP1 to OP4; Ʃ(OP1-4)] recorded from HFD-mice (both WT-HFD and miR-150^-/--HFD) are significantly lower (*) than mice fed with a normal chow (both WT-Normal and miR-150^-/--Normal) at 0.1, 0.3, 10, and 25 cd.s/m² light intensities. (F) The averaged photopic oscillatory potential amplitudes [Ʃ(OP1-4)] recorded from HFD-mice (both WT-HFD and miR-150^-/--HFD) are significantly lower (*) than mice fed with a normal chow (both WT-Normal and miR-150^-/--Normal) at 25 cd.s/m² light intensities. (A-F) Overall, there is no statistical significance of interaction between two factors: miR-150 null mutation and HFD regimen (2-way ANOVA). <i>p</i> < 0.05 (denoted as *, #, &, v, w). Data of scotopic and photopic ERG a- and b-waves and OPs are listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.t002" target="_blank">2</a>.</p