Design, construction and in vivo functional assessment of a hinge truncated sFLT01.

Gene therapy for the treatment of ocular neovascularization has reached clinical trial phases. The AAV2-sFLT01 construct was already evaluated in a phase 1 open-label trial administered intravitreally to patients with advanced neovascular age-related macular degeneration. SFLT01 protein functions by binding to VEGF and PlGF molecules and inhibiting their activities simultaneously. It consists of human VEGFR1/Flt-1 (hVEGFR1), a polyglycine linker, and the Fc region of human IgG1. The IgG1 upper hinge region of the sFLT01 molecule makes it vulnerable to radical attacks and prone to causing immune reactions. This study pursued two goals: (i) minimizing the immunogenicity and vulnerability of the molecule by designing a truncated molecule called htsFLT01 (hinge truncated sFLT01) that lacked the IgG1 upper hinge and lacked 2 amino acids from the core hinge region; and (ii) investigating the structural and functional properties of the aforesaid chimeric molecule at different levels (in silico, in vitro, and in vivo). Molecular dynamics simulations and molecular mechanics energies combined with Poisson-Boltzmann and surface area continuum solvation calculations revealed comparable free energy of binding and binding affinity for sFLT01 and htsFLT01 to their cognate ligands. Conditioned media from human retinal pigment epithelial (hRPE) cells that expressed htsFLT01 significantly reduced tube formation in HUVECs. The AAV2-htsFLT01 virus suppressed vascular development in the eyes of newborn mice. The htsFLT01 gene construct is a novel anti-angiogenic tool with promising improvements compared to existing treatments.

Network analysis and the impact of Aflibercept on specific mediators of angiogenesis in HUVEC cells.

Angiogenesis, inflammation and endothelial cells' migration and proliferation exert fundamental roles in different diseases. However, more studies are needed to identify key proteins and pathways involved in these processes. Aflibercept has received the approval of the US Food and Drug Administration (FDA) for the treatment of wet AMD and colorectal cancer. Moreover, the effect of Aflibercept on VEGFR2 downstream signalling pathways has not been investigated yet. Here, we integrated text mining data, protein-protein interaction networks and multi-experiment microarray data to specify candidate genes that are involved in VEGFA/VEGFR2 signalling pathways. Network analysis of candidate genes determined the importance of the nominated genes via different centrality parameters. Thereupon, several genes-with the highest centrality indexes-were recruited to investigate the impact of Aflibercept on their expression pattern in HUVEC cells. Real-time PCR was performed, and relative expression of the specific genes revealed that Aflibercept modulated angiogenic process by VEGF/PI3KA/AKT/mTOR axis, invasion by MMP14/MMP9 axis and inflammation-related angiogenesis by IL-6-STAT3 axis. Data showed Aflibercept simultaneously affected these processes and determined the nominated axes that had been affected by the drug. Furthermore, integrating the results of Aflibercept on expression of candidate genes with the current network analysis suggested that resistance against the Aflibercept effect is a plausible process in HUVEC cells.

Peripheral blood CD163(+) monocytes and soluble CD163 in dry and neovascular age-related macular degeneration.

Macrophages are the main infiltrating immune cells in choroidal neovascularization (CNV), a hallmark of the human wet, or neovascular age-related macular degeneration (AMD). Due to their plasticity and ability to adapt to the local microenvironment in a tissue-dependent manner, macrophages display polar functional phenotypes characterized by their cell surface markers and their cytokine profiles. We found accumulation of hemoglobin-scavenging cluster of differentiation 163 (CD163)(+) macrophages in laser-induced CNV lesions and higher expression of CD163(+) monocytes in the peripheral blood on day 7 post injury in mice. In comparison, CD80(+) macrophages did not differ with laser-injury in young or aged mice and did not significantly change in the peripheral blood of CNV mice. We examined the percentages of CD163(+), CD206(+), and CD80(+) monocytes in the peripheral blood of patients with wet AMD, patients with dry AMD, and in age-matched individuals without AMD as controls. Percentages of peripheral blood CD163(+) monocytes in both dry AMD (P < .001) and wet AMD (P < .05) were higher than in age-matched non-AMD controls, while there was no difference between the groups in the percentages of peripheral CD206(+) and CD80(+) monocytes. Further, serum level of soluble CD163 (sCD163) was elevated only in patients with wet AMD (P < .05). An examination of 40 cytokine levels across the study groups revealed that anti-VEGF treated patients with wet AMD, who showed no exudative signs on the day of blood drawing had a cytokine profile that was similar to that of non-AMD individuals. These results indicate that CD163 could be further evaluated for its potential as a useful marker of disease activity in patients with neovascular AMD. Future studies will address the origin and potential mechanistic role of CD163(+) macrophages in wet AMD pathologies of angiogenesis and leakage of blood components.

Human platelet antigens polymorphisms; association to the development of liver fibrosis in patients with chronic hepatitis C.

Recently, human platelet antigens (HPAs) polymorphisms are found to play a role in susceptibility to hepatitis C virus (HCV) infection and fibrosis progression. The aim of the current study was to evaluate the possible association between the HPAs polymorphisms with liver fibrosis progression in HCV patients. HPAs polymorphisms genotyping was performed in HCV patients (n = 71) by Sequence-specific primers-polymerase chain reaction. Fibrosis progression was evaluated using the Metavir scoring system and liver biopsy, and the patients were assigned to two groups, namely, G1 (n = 35) that included patients with F1 (portal fibrosis without septa) or F2 (few septa) and G2 (n = 36) that comprised patients with F3 (numerous septa) or F4 (cirrhosis). The data analyses were performed using Pearson's χ2 test. The genotype frequency of HPA-3ab was significantly higher in G1 patients than in G2 patients (P = 0.015). No statistically significant differences were found between the patient groups (G1 and G2) regarding the distributions of the allelic and genotypic frequencies of the HPA-1, -2, -4, -5, and -15 systems. Multivariate logistic regression showed an independent association between the genotype HPA-3aa/BB and severe fibrosis (F3-F4), when compared with genotype HPA-3ab, independent of the viral genotype, high alanine transaminase, sex, age, time of infection, diabetes, and high cholesterol as risk factors. The present study suggested that the HPA-3ab genotype could be noticed as a potential protecting factor against hepatic fibrosis. Therefore, the antigenic variation of integrins might be considered as a part of the coordinated inflammatory process involved in the progression of liver fibrosis.

The influence of rAAV2-mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study.

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells.

AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases.

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc.

Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.

Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies.

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.

PURPOSE: Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. METHODS: Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 µg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography. RESULTS: Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. CONCLUSIONS: Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.

Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (P<0.01), compared to normal controls. More than 80% of the VEGFR-2 in the diabetic retina was in the capillaries, compared to 47% in normal controls (P<0.01). Angiography in rabbit retinas revealed microvascular capillaries to be the location for VEGF-A-induced leakage, as expressed by significantly higher rate of fluorophore spreading with VEGF-A injection when compared to vehicle control (26±2 vs. 3±1 µm/s, P<0.05). Immunohistochemistry showed VEGFR-2 expression in capillaries of diabetic animals but not in normal controls. Macular vessels from diabetic patients (n=7) showed significantly more VEGFR-2 compared to nondiabetic controls (n=5) or peripheral retinal regions of the same retinas (P<0.01 in both cases). Here we introduce a new approach for early diagnosis of DR and VEGFR-2 as a molecular marker. VEGFR-2 could become a key diagnostic target, one that might help to prevent retinal vascular leakage and proliferation in diabetic patients.

In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

PURPOSE: Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. METHODS: RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. RESULTS: Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. CONCLUSIONS: This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

Methylenetetrahydrofolate reductase C677T mutation and risk of retinal vein thrombosis.

BACKGROUND: Elevated plasma homocysteine (Hcy) level has been established as a significant risk factor for venous thrombosis and cardiovascular disease. Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T mutation has been associated with elevated plasma Hcy concentration and may contribute to retinal vein thrombosis (RVT) development. The aim of the present study was to investigate whether the hyperhomocysteinemia and/or homozygosity for the MTHFR C677T mutation are associated with an increased risk for RVT. MATERIALS AND METHODS: Our study population consisted of 73 consecutive patients (50-78 years old) with RVT and 73 control subjects (51-80 years old), matched for age and sex. Genotyping for the MTHFR C677T mutation was performed by polymerase chain reaction-restriction fragment length polymorphism technique and Hcy level was determined by an enzyme immunoassay kit. RESULTS: The prevalence of 677TT genotype was higher in patients than control subjects, but the difference in frequency didn't reach a significant value (P = 0.07). The frequency of the 677T allele was 26% and 21.2% in patients and controls, respectively and did not differ significantly between the two groups (odds ratio = 1.3, 95% confidence interval (0.75-2.24), P = 0.33). Fasting plasma total Hcy level was significantly higher in patients than controls (P = 0.001). CONCLUSION: Our study demonstrated that hyperhomocysteinemia, but not the MTHFR C677T mutation, is associated with RVT.

microRNA-96 targets the INS/AKT/GLUT4 signaling axis: Association with and effect on diabetic retinopathy.

Background: miR-96-5p is a highly expressed microRNA in the retina of subjects with diabetes. The INS/AKT/GLUT4 signaling axis is the main cell signaling pathway of glucose uptake in cells. Here, we investigated the role of miR-96-5p in this signaling pathway. Methods: Expression levels of miR-96-5p and its target genes were measured under high glucose conditions, in the retina of streptozotocin-induced diabetic mice, in the retina of AAV-2-eGFP-miR-96 or GFP intravitreal injected mice and in the retina of human donors with diabetic retinopathy (DR). MTT, wound healing, tube formation, Western blot, TUNEL, angiogenesis assays and hematoxylin-eosin staining of the retinal sections were performed. Results: miR-96-5p expression was increased under high glucose conditions in mouse retinal pigment epithelial (mRPE) cells, in the retina of mice receiving AAV-2 carrying miR-96 and STZ-treated mice. Expression of the miR-96-5p target genes related to the INS/AKT/GLUT4 signaling pathway was reduced following miR-96-5p overexpression. mmu-miR-96-5p expression decreased cell proliferation and thicknesses of retinal layers. Cell migration, tube formation, vascular length, angiogenesis, and TUNEL-positive cells were increased. Conclusions: In in vitro and in vivo studies and in human retinal tissues, miR-96-5p regulated the expression of the PIK3R1, PRKCE, AKT1, AKT2, and AKT3 genes in the INS/AKT axis and some genes involved in GLUT4 trafficking, such as Pak1, Snap23, RAB2a, and Ehd1. Because disruption of the INS/AKT/GLUT4 signaling axis causes advanced glycation end product accumulation and inflammatory responses, the inhibition of miR-96-5p expression could ameliorate DR.

PlGF gene knockdown in human retinal pigment epithelial cells.

BACKGROUND: To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. METHODS: Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). RESULTS: At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. CONCLUSIONS: Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

Evaluation of RPE65, CRALBP, VEGF, CD68, and tyrosinase gene expression in human retinal pigment epithelial cells cultured on amniotic membrane.

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.