Diabetic retinopathy remains the leading vascular-associated cause of blindness throughout the world. Its treatment requires a multidisciplinary interventional approach at both systemic and local levels. Current management includes laser photocoagulation, intravitreal steroids, and anti-vascular endothelial growth factor (VEGF) treatment along with systemic blood sugar control. Anti-VEGF therapies, which are less destructive and safer than laser treatments, are being explored as primary therapy for the management of vision-threatening complications of diabetic retinopathy such as diabetic macular edema (DME). This review provides comprehensive information related to VEGF and describes its role in the pathogenesis of diabetic retinopathy, and in addition, examines the mechanisms of action for different antiangiogenic agents in relation to the management of this disease. Medline (Pubmed) searches were carried out with keywords “VEGF”, “diabetic retinopathy”, and “diabetes” without any year limitation to review relevant manuscripts used for this article.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

BackgroundMitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).Methodology/Principal FindingsCybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.Conclusion/SignificanceOur findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other 'modifiers' may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt-nuclear interactions.

cHerpes simplex virus 1 (HSV-1) and HSV-2 are medically significant pathogens. The development of an effective HSV vaccine remains a global public health priority. HSV-1 and HSV-2 immunodominant "asymptomatic" antigens (ID-A-Ags), which are strongly recognized by B and T cells from seropositive healthy asymptomatic individuals, may be critical to be included in an effective immunotherapeutic HSV vaccine. In contrast, immunodominant "symptomatic" antigens (ID-S-Ags) may exacerbate herpetic disease and therefore must be excluded from any HSV vaccine. In the present study, proteome microarrays of 88 HSV-1 and 84 HSV-2 open reading frames(ORFs) (ORFomes) were constructed and probed with sera from 32 HSV-1-, 6 HSV-2-, and 5 HSV-1/HSV-2-seropositive individuals and 47 seronegative healthy individuals (negative controls). The proteins detected in both HSV-1 and HSV-2 proteome microarrays were further classified according to their recognition by sera from HSV-seropositive clinically defined symptomatic (n ‫؍‬ 10) and asymptomatic (n ‫؍‬ 10) individuals. We found that (i) serum antibodies recognized an average of 6 ORFs per seropositive individual; (ii) the antibody responses to HSV antigens were diverse among HSV-1-and HSV-2-seropositive individuals; (iii) panels of 21 and 30 immunodominant antigens (ID-Ags) were identified from the HSV-1 and HSV-2 ORFomes, respectively, as being highly and frequently recognized by serum antibodies from seropositive individuals; and (iv) interestingly, four HSV-1 and HSV-2 cross-reactive asymptomatic ID-A-Ags, US4, US11, UL30, and UL42, were strongly and frequently recognized by sera from 10 of 10 asymptomatic patients but not by sera from 10 of 10 symptomatic patients (P < 0.001). In contrast, sera from symptomatic patients preferentially recognized the US10 ID-S-Ag (P < 0.001). We have identified previously unreported immunodominant HSV antigens, among which were 4 ID-A-Ags and 1 ID-S-Ag. These newly identified ID-A-Ags could lead to the development of an efficient "asymptomatic" vaccine against ocular, orofacial, and genital herpes.

PURPOSE. The authors recently reported that Foxp3(+)CD4(+) CD25(+(Bright)) "natural" regulatory T cells (nT(reg) cells) are abundant in rabbit conjunctiva and suppress herpes simplex virus (HSV)-1-specific CD4(+) and CD8(+) effector T cells (T(eff) cells). However, little is known about the overall regulatory mechanisms of these nT(reg) cells. The authors investigate the regulation of conjunctiva-resident nT(reg) cells through Toll-like receptors (TLRs) and their effect on ocular mucosal T(eff) cell immunity. METHODS. CD4(+)CD25(+) nT(reg) cells were purified from naive rabbit conjunctivas, and their TLR expression profile was determined. The effects of TLR engagement on nT(reg) cell-mediated suppression of CD4(+) T(eff) cells were determined in vitro and in vivo. RESULTS. The authors found that conjunctiva-resident nT(reg) cells express high levels of TLR2 and TLR9; exposure to the TLR2 ligand lipoteichoic acid (LTA) led to the increased activation and proliferation of nT(reg) cells, and the addition of autologous APCs further increased nT(reg) cell expansion; in contrast, the TLR9 ligand CpG(2007) inhibited the proliferation of nT(reg) cells, and the addition of autologous APCs had no effect on such inhibition; nT(reg) cells treated with LTA, but not with CpG(2007), expressed IFN-γ and IL-10 mRNA, but not TGF-β; consistent with in vitro data, rabbits immunized by topical ocular drops of HSV-gD peptides + TLR2 ligand (LTA) displayed enhanced CD4(+)CD25(-) T(eff) cell immune responses when compared with HSV-gD peptides + TLR9 ligand (CpG(2007)). CONCLUSIONS. Although conjunctiva-resident CD4(+)CD25(+) nT(reg) cells express high level of TLR2 and TLR9, their suppressive function is more significantly reversed after the administration of TLR2 ligand (LTA; P < 0.005) than of TLR9 ligand (CpG(200); P > 0.005). These findings will likely help optimize the topical ocular administration of immunotherapies.

BackgroundIt has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation.Methodology/Principal FindingsCybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids.Conclusion/SignificanceIn cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.

Purpose:Our goal was to identify the cellular and molecular effects of 2-ethylpyridine (2-EP, a component of cigarette smoke) on human retinal pigment epithelial cells (ARPE-19) in vitro.Materials and Methods:ARPE-19 cells were exposed to varying concentrations of 2-EP. Cell viability (CV) was measured by a trypan blue dye exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was detected with a 2’,7’-dichlorodihydrofluorescein diacetate dye assay. The JC-1 assay was used to measure mitochondrial membrane potential (ΔΨm). Mitochondrial redox potential was measured using a RedoxSensor Red kit and mitochondria were evaluated with Mitotracker dye.Results:After 2-EP exposure, ARPE-19 cells showed significantly decreased CV, increased caspase-3/7 and caspase-9 activities, elevated ROS/RNS levels, decreased ΔΨm value and decreased redox fluorescence when compared with control samples.Conclusions:These results show that 2-EP treatment induced cell death by caspase-dependent apoptosis associated with an oxidative stress and mitochondrial dysfunction. These data represent a possible mechanism by which smoking contributes to age-related macular degeneration and other retinal diseases and identify mitochondria as a target for future therapeutic interventions.