Advances in the study of microparticles in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the common diabetic microangiopathies, which severely impairs vision in diabetic population. The underlying mechanisms regarding the development of DR are not fully understood, and there is a lack of biomarkers to guide clinical, assessment of disease progression. Recently researchers have found that microparticles (MP) and its bioactive molecules are involved in the development of DR. MP is widely distributed in the circulation and can exert autocrine and paracrine benefits in intercellular signalling, provide a catalytic platform for the thrombospondin complex to promote coagulation, and promote the accumulation of reactive oxygen species to cause endothelial damage. MP interacts with advanced glycosylation end products (AGE) and AGE receptor (RAGE) to activate inflammatory pathways. MP carries a variety of miRNAs that regulate the vascular endothelial growth factor generation pathway. MP has also been applied to the exploration of mesenchymal stromal cell replacement therapy to treat DR. In a word, MP provides new ideas for the study of DR. MP has emerged as a marker to assess the progression of DR. As a potential therapeutic target, MP also has considerable research value.

Efficacy and Safety of Biosimilar QL1207 vs. the Reference Aflibercept for Patients with Neovascular Age-Related Macular Degeneration: A Randomized Phase 3 Trial.

INTRODUCTION: This trial aimed to compare the efficacy and safety between biosimilar QL1207 and the reference aflibercept for the treatment of neovascular age-related macular degeneration (nAMD). METHODS: This randomized, double-blind, phase 3 trial was conducted at 35 centers in China. Patients aged ≥ 50 years old with untreated subfoveal choroidal neovascularization secondary to nAMD and best-corrected visual acuity (BCVA) letter score of 73-34 were eligible. Patients were randomly assigned to receive intravitreous injections of QL1207 or aflibercept 2 mg (0.05 ml) in the study eye every 4 weeks for the first 3 months, followed by 2 mg every 8 weeks until week 48, stratified by baseline BCVA ≥ or < 45 letters. The primary endpoint was BCVA change from baseline at week 12. The equivalence margin was ± 5 letters. The safety, immunogenicity, pharmacokinetics (PK), and plasma vascular endothelial growth factor (VEGF) concentration were also evaluated. RESULTS: A total of 366 patients were enrolled (QL1207 group, n = 185; aflibercept group, n = 181) from Aug 2019 to Jan 2022 with comparable baseline characteristics. The least-squares mean difference in BCVA changes was - 1.1 letters (95% confidence interval - 3.0 to 0.7; P = 0.2275) between the two groups, within the equivalence margin. The incidences of treatment-emergent adverse events (TEAE; QL1207: 71.4% [132/185] vs. aflibercept: 71.8% [130/181]) and serious TEAE (QL1207: 14.1% [26] vs. aflibercept: 12.7% [23]) appeared comparable between treatment groups, and no new safety signal was found. Anti-drug antibody, PK profiles, and VEGF concentration were similar between the two groups. CONCLUSIONS: QL1207 has equivalent efficacy to aflibercept for nAMD with similar safety profiles. It could be used as an alternative anti-VEGF agent for clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05345236 (retrospectively registered on April 25, 2022); National Medical Products Administration of China: CTR20190937 (May 20, 2019).

Effect of sutureless scleral fixed intraocular lens implantation on aphakic eyes: a system review and meta-analysis.

BACKGROUND: Sutureless scleral fixed intraocular lens implantation (SF-IOL) has become one of the mainstream schemes in clinical treatment of aphakic eyes because of its advantages, such as avoiding dislocation of intraocular lens or subluxation caused by suture degradation or fracture and significant improvement of postoperative visual acuity. However, a consensus on the relative effectiveness and safety of this operation and other methods is still lacking. This study aimed to compare the efficacy and safety of sutureless SF-IOL with other methods. Aphakia means that the lens leaves the normal position and loses its original function, including absence or complete dislocation and subluxation of the lens which could cause anisometropic amblyopia, strabismus, and loss of binocular function in children and adolescents. For adults, the loss of the lens could lead to high hyperopia and affect vision. Above all this disease can seriously affect the quality of life of patients. METHODS: Literature about sutureless SF-IOL in PubMed, Cochrane Library, Embase, Web of Science, China National Knowledge Infrastructure, China Technical Journal VIP database, and Wanfang database published from 2000 to 2022 was reviewed. The weighted average difference was calculated by RevMan5.3 software for analysis. Two researchers independently selected the study and used the Cochrane collaboration tool to assess the risk of errors. Cochrane bias risk tool was used to evaluate the quality of evidence. This study is registered on PROSPERO (CRD42022363282). RESULTS: The postoperative IOL-related astigmatism of sutureless SF-IOL was lower than that of suture SF-IOL, and there was statistical difference when we compared the absolute postoperative spherical equivalent after sutureless SF-IOL and suture SF-IOL. Indicating that the degree of refractive error after sutureless SF-IOL was lower. Meanwhile, the operation time of sutureless SF-IOL was shorter than that of suture SF-IOL. The subgroup analysis showed that the absolute postoperative spherical equivalent and astigmatism values in Yamane technique were lower than those in suture SF-IOL. CONCLUSION: Sutureless SF-IOL has the advantages of stable refraction, short operation time, and less postoperative complications. However, high-quality literature to compare these technologies is lacking. Some long-term follow-up longitudinal prospective studies are needed to confirm the findings.

Research progress of diabetic retinopathy and gut microecology.

According to the prediction of the International Diabetes Federation, global diabetes mellitus (DM) patients will reach 783.2 million in 2045. The increasing incidence of DM has led to a global epidemic of diabetic retinopathy (DR). DR is a common microvascular complication of DM, which has a significant impact on the vision of working-age people and is one of the main causes of blindness worldwide. Substantial research has highlighted that microangiopathy and chronic low-grade inflammation are widespread in the retina of DR. Meanwhile, with the introduction of the gut-retina axis, it has also been found that DR is associated with gut microecological disorders. The disordered structure of the GM and the destruction of the gut barrier result in the release of abnormal GM flora metabolites into the blood circulation. In addition, this process induced alterations in the expression of various cytokines and proteins, which further modulate the inflammatory microenvironment, vascular damage, oxidative stress, and immune levels within the retina. Such alterations led to the development of DR. In this review, we discuss the corresponding alterations in the structure of the GM flora and its metabolites in DR, with a more detailed focus on the mechanism of gut microecology in DR. Finally, we summarize the potential therapeutic approaches of DM/DR, mainly regulating the disturbed gut microecology to restore the homeostatic level, to provide a new perspective on the prevention, monitoring, and treatment of DR.

Serum disease-specific IgG Fc glycosylation as potential biomarkers for nonproliferative diabetic retinopathy using mass spectrometry.

OBJECTIVE: To explore the potential of serum disease-specific immunoglobulin G (DSIgG) glycosylation as a biomarker for the diagnosis of nonproliferative diabetic retinopathy (NPDR). METHODS: A total of 387 consecutive diabetic patients presenting in an eye clinic without proliferative diabetic retinopathy (DR) were included and divided into those with nondiabetic retinopathy (NDR) (n = 181) and NPDR (n = 206) groups. Serum was collected from all patients for DSIgG separation. The enriched glycopeptides of the tryptic digests of DSIgG were detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients were randomly divided into discovery and validation sets (1:1). The differences in glycopeptide ratios between the groups were compared by using Student's t-test or the Mann-Whitney U test. The predictive ability of the model was assessed using the area under the receiver operating characteristic curve (AUC). RESULTS: DSIgG1 G1FN/G0FN, G2N/G2, G2FN/G2N and DSIgG2 G1F/G0F, G1FN/G0FN, G2N/G1N, G2S/G2 were significantly different between NDR and NPDR patients (p < 0.05) in both the discovery and validation sets. The prediction model that was built comprising the seven glycopeptide ratios showed good NPDR prediction performance with an AUC of 0.85 in the discovery set and 0.87 in the validation set. CONCLUSION: DSIgG Fc N-glycosylation ratios were associated with NPDR and can be used as potential biomarkers for the early diagnosis of DR.

Clinical features of retinal amyloid angiopathy with transthyretin Gly83Arg variant.

AIM: To report on the clinical features of patients with retinal amyloid angiopathy (RAA) who were identified to be caused by the transthyretin (TTR) Gly83Arg variant. METHODS: Case series of five patients diagnosed with RAA was collected at Affiliated Hospital of Zunyi Medical University from January 2010 to December 2021. The clinical features, therapeutic strategies, and prognoses of all patients were reviewed. RESULTS: Five patients with a mean age of 52.00±7.23y were diagnosed as RAA. These patients were previously diagnosed with hereditary transthyretin amyloidosis caused by the TTR Gly83Arg variant. Vitreous opacity was found in all 10 eyes, and 7 eyes developed RAA 2 to 20y after the onset of hereditary transthyretin amyloidosis. The clinical manifestations were recurrent vitreous hemorrhage in 2 eyes (29%), neovascular glaucoma in 2 eyes (29%), and iris neovascularization in 1 eye (14%). Microangioma lesions were found in all affected eyes that underwent fundus fluorescein angiography (FFA) in this group of cases, and the incidence of the retinal non-perfusion area was 67%. Although no cases of retinal neovascularization were found, the prognosis of visual acuity was not ideal. CONCLUSION: This is the first report of RAA in patients with the TTR Gly83Arg variant. Complications such as RAA and glaucoma will seriously affect the visual prognosis of patients. Thereafter, regular ophthalmic follow-up of patients with hereditary transthyretin amyloidosis is essential. And FFA after vitrectomy is very important, which can help ophthalmologists detect RAA earlier and treat it in time.

Von Hipple-Lindau disease complicated with central retinal vein occlusion: a case report.

BACKGROUND: Central Retinal Vein Occlusion (CRVO) is a rare complication of von Hipple-Lindau (VHL) disease. This report presents the first case of VHL disease complicated with CRVO caused by VHL c.208G > A mutation. CASE PRESENTATION: A 20 s man whose left eye visual acuity gradually declined for half a year. The visual acuity of the left eye is counting fingers. Fundus examination revealed that retinal hemangioblastoma was also found in addition to typical CRVO signs such as tortuous expansion of retinal veins and flame-shaped hemorrhage of the retina. Liver tumor, cerebral infarction and erythrocytosis were found during systemic examination, and the diagnosis of polycythemia was confirmed by bone marrow smear. Furthermore, both family history and genetic analysis indicated that the patient had VHL disease caused by VHL c.208G > A. In this patient, a large number of bone marrow erythrocytes proliferated due to VHL disease, which led to the increase of blood viscosity and erythrocyte vascular adhesion, resulting in the obstruction of central retinal vein blood flow, and finally CRVO. For CRVO and its pathogenic factor polycythemia, patient received laser retinal photocoagulation and phlebotomies. After a 1-year follow-up, the vision in the left eye improved to 0.2 logMAR. CONCLUSIONS: This is a rare case of polycythemia complicated by CRVO in patient with VHL disease. It reminds us that the systemic disease factors should be fully considered in the diagnosis of young patients with CRVO, and that treatment requires a coordinated effort of physicians.

The Effects of Combuxil and Leizumab on Retinal Function and Serum Interleukin-17A in Premature Infants with Retinopathy.

Objective: To investigate the effects of Compaximab and raizumab on retinal function and serum interleukin-17A level in retinopathy of prematurity. Methods: Sixty cases of retinopathy of prematurity treated in our hospital from February 2019 to April 2021 were selected. The patients were randomly divided into control group (n = 30) and research group (n = 30). The control group was treated with Compaq and the research group was treated with razumab. The curative effect, retinal function, incidence of complications, intraocular pressure at different time, and the level of serum interleukin-17A were compared. Results: Compared with the two groups, the curative effect of the research group 93.33% (28/30) was greater than that of the control group 66.67% (20/30), and the difference was statistically significant (P < 0.05). Compared with the incidence of complications, the incidence of corneal opacity, lens opacity, preretinal and vitreous hemorrhage, endophthalmitis, and traction retinal detachment in the research group was greatly lower, and the difference was statistically significant (P < 0.05). Following the therapy, the IOP of the two groups decreased at different times. The IOP of 1 min, 10 min, and 30 min in the research group was obviously lower, and the difference was statistically significant (P < 0.05). Following treatments, the levels of serum IL-17A were decreased. Compared with the control group, the level of serum IL-17A in the research group was greatly downregulated, and the difference was statistically significant (P < 0.05). Conclusion: Intravitreal injection of razumab is an effective treatment for retinopathy of prematurity, which can effectively improve the retinal function of infants. The level of serum interleukin-17A can be reduced and intraocular pressure can be regulated, which is safe and effective.

Multimodal imaging of the course of retinal changes in acute posterior multifocal placoid pigment epitheliopathy with bilateral retinal detachment: a case report.

BACKGROUND: To report a rare case of acute posterior multifocal placoid pigment epitheliopathy (APMPPE) with a combination of serous retinal detachment, papilledema, and retinal vasculitis. CASE PRESENTATION: A 19-year-old male complained of floaters in both eyes with decreased vision for 4 days. The best corrected visual acuity of the right eye and the left eye were 1.1 and 0.9 (logMAR), respectively. In both eyes, inflammatory cells can be seen suspended within the vitreous, multiple yellow/white lesions can be seen near the macula, and retinal neuroepithelial detachment. Swelling of the optic disc with blurring of the disc margins, in the left eye. Optical coherence tomography (OCT): showed retinal detachment in both eyes. The patient received oral prednisone treatment. 1 week later, OCT showed absorption of subretinal fluid in the macula of both eyes his binocular vision improved to 0.1 (logMAR). During the subsequent 28-month follow-up, fundus fluorescein angiography and OCT revealed extensive and progressive pigment epithelial atrophy in both eyes, and abnormal retinal vascular perfusion in the right eye due to persistent retinal vasculitis. Although the patient's binocular visual acuity remained at 0.1 (logMAR). CONCLUSIONS: In the present case of APMPPE with a combination of serous retinal detachment, papilledema, and retinal vasculitis, through the multimodal imaging, further confirming that the lesions were located in the choroid, while the pigment epithelial lesions were secondary changes.

The Effect of A2E on the Ca2+-PKC Signaling Pathway in Human RPE Cells Exposed to Blue Light.

Aims: In a model of blue light-induced damage in N-retinylidene-N-retinylethanolamine (A2E)-loaded human retinal pigment epithelial (RPE) cells, we examined the effect of A2E on the calcium (Ca2+)-protein kinase C (PKC) signaling pathway. Methods: Primary human RPE cells were cultured, and the cells in the 4th-6th passages were used in this study. The cells were divided into 5 groups: control cells (no A2E, no blue light), blue light-treated cells, blue light + chloroquine-treated cells, blue light + A2E-treated cells, and blue light + A2E + chloroquine-treated cells. The cells were first treated with chloroquine (15 µM for 12 h) and then loaded with A2E (25 µM for 2 h).The blue light intensity was 2000 ± 500 lux, and the duration was 6 h. After blue light exposure, the cells were cultured for 24 h. Fluo-3/AM staining was used to determine the level of cytoplasmic Ca2+, and the cells were photographed using a laser scanning confocal microscope to analyze the fluorescence intensity. The intracellular levels of inositol triphosphate (IP3) and diacylglycerol (DAG) were measured by enzyme-linked immunosorbent assay (ELISA). Intracellular PKC activity was measured with a nonradioactive nuclide assay. Results: Among all cell groups, the levels of Ca2+, DAG, and IP3 were lowest in the control cells (P < 0.05). The Ca2+, DAG, and IP3 levels in the blue light + A2E-treated cells and blue light + chloroquine-treated cells were higher than those in the blue light-treated cells (P < 0.05). The Ca2+, DAG, and IP3 levels were highest in the blue light + A2E + chloroquine-treated group (P < 0.05). PKC activity was lowest in the control cells (P < 0.05). The PKC activity of the blue light + A2E-treated cells and blue light + chloroquine-treated cells was higher than that of the blue light-treated cells (P < 0.05), and the PKC activity of the blue light + A2E + chloroquine-treated cells was the highest (P < 0.05). Conclusion: Blue light and A2E increased the levels of Ca2+, IP3, and DAG in human RPE cells and enhanced PKC activity, and blue light and A2E had a synergistic effect. Chloroquine further increased the levels of Ca2+, IP3, and DAG and PKC activity in RPE cells or A2E-loaded RPE cells exposed to blue light.

The effect of A2E on lysosome membrane permeability during blue light-induced human RPEs apoptosis.

BACKGROUND: To investigate the effect of N-retinyl-N-retinylidene ethanolamine (A2E) on lysosome membrane permeability (LMP) during blue light-induced human retinal pigment epithelium cells (RPEs) apoptosis. METHODS: By building an A2E and blue light irradiation inducing RPEs damage model, the CCK-8 assay was used to detect RPEs viability loaded with different concentrations of A2E after different culturing time to determine the optimum A2E loading concentration. And the RPEs fluorescence intensity changes were observed by fluorescence microscopy loaded with different concentration of A2E. The RPEs were divided into four groups randomly: control group, A2E-loaded group, blue light irradiation group, and A2E-loaded + blue light irradiation group. Annexin V-FITC/PI and TUNEL/DAPI methods were used to detect RPEs apoptotic rate. Laser scanning confocal microscopy (LSCM) was used to observe RPEs LMP changes stained by acridine orange (AO) method. RESULTS: The CCK-8 result showed a downward trend in cells viability of RPEs loaded with increasing concentration of A2E and extending culturing time. The optimum A2E loading concentration was determined at 25 µmol/L. With increasing A2E loading concentrations, the intensity of fluorescence in RPEs decreased gradually. The RPEs apoptotic rate in blue light irradiation + A2E-loaded group was significantly higher than those in other three groups detected by Annexin V-FITC/PI method, which was similar to TUNEL/DAPI's result. After AO staining, cytoplasmic and nucleolar RNAs emits green fluorescence; lysosomes emit red fluorescence. Through the interference of A2E and blue light on RPEs, red fluorescent leakage from the lysosomes (means LMP increasing) can be observed. The mean red fluorescence intensity was chosen as the statistics indicator to estimate LMP change in RPEs cultured in vitro. Compared with the control group, the red fluorescence intensity decreased in A2E-loaded group, blue light irradiation group, and blue light irradiation + A2E-loaded group. Meanwhile, the mean red fluorescence intensity in blue light irradiation + A2E-loaded group was the lowest. CONCLUSIONS: Both A2E-loaded and blue light irradiation could induce human RPEs apoptosis, and the two factors had a synergistic effect. In addition, both A2E and blue light can lead to LMP increasing, which indicated LMP change might be the upstream part in inducing mitochondrion-dependent apoptotic pathway. These data provided evidence that A2E as the most important auto-fluorescence substance in lipofuscin is an initiator of blue light-mediated damage of RPEs and participate in pathogenesis of retinal degenerative diseases in humans.

The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light.

We aimed to explore the effect of N-retinylidene-N-retinylethanolamine (A2E) on the uptake and release of calcium in lysosomes and mitochondria by establishing a model of human retinal pigment epithelial (RPE) cell injury induced by exposure to blue light. Primary human RPE cells were cultured from passages 4 to 6 and exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours. After blue light exposure, the culture was maintained for 24 hours. A2E at a final concentration of 25 µM was added to the culture 2 hours before light exposure, and nifedipine at a final concentration of 10-4 M was added 1 hour before light exposure. The levels of Ca2+ in the cytosol (CaTM/2AM), mitochondria (Rhod/2AM), and lysosomes (LysoTracker Red and Fluo-3/AM) were determined. In order to measure the calcium levels in the different organelles, RPE were imaged using a laser scanning confocal microscope. Moreover, changes in the mitochondrial membrane potential were detected by flow cytometry analysis of JC-1-stained cells. The obtained results revealed that blue light illumination increased the calcium fluorescence intensity in the cytoplasm, mitochondria, and lysosomes of human RPE cells when compared with the control cells (P < 0.05). After A2E treatment, the fluorescence intensity of the calcium in the cytoplasm was further increased (P < 0.05), while that in the mitochondria and lysosomes decreased (P < 0.05). In addition, we observed that nifedipine reduced the fluorescence intensity of calcium in the RPE cells. Our results also showed that the mitochondrial membrane potential in the RPE treated with blue light and A2E was lower than that in the control, blue light, and A2E-treated cells (P < 0.05). Blue light increased calcium levels in the cytoplasm, lysosomes, and mitochondria of RPE cells. A2E damages the lysosomal and mitochondrial membranes, resulting in calcium release into the cytoplasm. Finally, our results demonstrated that both blue light and A2E treatments reduced mitochondrial membrane potential, increasing cytosolic Ca2+ levels, which can contribute to the activation of RPE death.

Improved thickness measurement method for choroidal hyperpermeability in central serous chorioretinopathy.

AIM: To observe choroidal thickness changes in the choroidal hyperpermeability area (CHA) in patients with central serous chorioretinopathy (CSC) after photodynamic therapy (PDT) using indocyanine green angiography (ICGA) combined with optical coherence tomography (OCT). METHODS: This was a cohort study of 17 eyes (17 patients) with CSC. In all patients, the range of CHA was determined by ICGA. The patients were divided into two groups based on CHA covered the fovea (group A) or not (group B). All patients received half-dose verteporfin PDT over CHA in ICGA. Choroidal thickness was measured by OCT before, 1, and 3mo after treatment. The choroidal thickness values of the fovea and CHAs were obtained for each measurement. Secondary outcomes were changes in the best-corrected visual acuity (BCVA) and amount of subretinal fluid (SRF). RESULTS: The differences in center choroidal thickness at baseline and at 1 and 3mo post-PDT were statistically significant in group A and all patients (both P<0.001). There was no significant difference in group B (P=0.059). The differences of thickness of CHA and BCVA at baseline and 1 and 3mo post-PDT were statistically significant in group A, group B, and all patients (all P<0.01). All patients showed complete SRF absorption at 3mo post-PDT. CONCLUSION: Center choroidal thickness does not accurately reflect changes in CHA of patients whose CHA does not covered the fovea center. Using CHA as the observation target can make up for this limitation, expand the scope of application, and reduce bias.

A mouse model of a novel missense mutation (Gly83Arg) in a Chinese kindred manifesting vitreous amyloidosis only.

The purpose of this study is to establish a mouse model of transthyretin (TTR) Gly83Arg gene mutation by the technique of gene targeting for research on hereditary vitreous amyloidosis (HVA) and to confirm whether this point mutation is a genetic feature of HVA. A vector (pBR322-MK-TTR) was constructed to target ES cells. The successfully transfected ES cells were used for blastocyst injection, thus generating F0. F0 and Flp mice were mated to generate F1 (TTR+/-, Flp +/-) mice that lacked the neo gene but carried the Flp gene. F1 mice were mated with C57BL/6N wild type mice to generate F2 (TTR+/-) mice. F3 homozygous and heterozygous mice were generated by mating F2 mice with each other. PCR and sequencing were performed for F3 mice. Amyloid was detected using Congo red stain and polarized light. Immunohistochemistry was used to detect the expression of TTR in the tissues. Quantitative fluorescent PCR and Western blotting were used to detect the expression of TTR mRNA and TTR protein, respectively. Two F0-generation, 2 F1-generation and 15 F3-generation mice were obtained. The gene sequencing of F3 mice showed TTR Gly83Arg mutation. When examined with Congo red and polarized light, the vitreous of TTR Gly83Arg mutant mice tested positive for amyloid. The hearts, livers, brains and kidneys of the experimental group and control group were all negative by Congo red staining. Immunohistochemical staining showed that the vitreous of TTR Gly83Arg mutant mice and the livers of the control mice were positive, but the kidneys, hearts and brains of both groups were negative. Quantitative fluorescent PCR showed that the mRNA expression of mutant mice was significantly lower than that of wild-type mice (F = 0.295, P = 0.023). Western blotting showed that the expression of TTR protein was significantly lower in the model mice than in the wild-type mice (t = 3.224, P = 0.018). TTR gene mutation is indeed a molecular characteristic of HVA and manifest in the eye disease only. A C57BL/6 mouse line carrying the TTR Gly83Arg gene mutation was successfully established. This strain of mice can be used for the study of HVA.

[The correlation between the concentrations of VEGF and PEDF and Ca2+-PKC signaling pathways in human retinal pigment epithelial cells cultured in vitro after exposuring to blue light].

OBJECTIVE: To investigate the concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), inositol triphosphate (IP3) and diacylglycerol (DAG) in human retinal pigment epithelium (RPE) cells after exposuring to blue light, and to explore the relationship with Ca2+-PKC signaling pathways, to evaluate the role of Ca2+-PKC signaling pathways of blue-light irradiation induced apoptosis in RPE cells. METHODS: The fourth generation human RPE cells in vitro were exposured to blue light (2000±500 lux) for 6 hours, 24 hours prolongation of post-exposure culture. The concentrations of VEGF, PEDF, IP3 and DAG were assayed by enzyme linked immunosorbent assay (ELISA). Cells were randomly divided into 6 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+PMA), group D (exposure to blue light+Calphostin C), group E (exposure to blue light+Nifedipine), group F (exposure to blue light+Calphostin C+Nifedipine). Flow cytometry was used to detect the apoptosis rate of human RPE cells in A, B and F group. RESULTS: Comparing with group A (584.38±10.66), the concentration of VEGF in group B (700.70±5.88), group C (698.21±6.66) and group E (648.30±4.91) was higher, the difference was statistically significant (P=0.002, 0.002, 0.016). Comparing with group B (700.70±5.88), the concentration of VEGF in Group D (623.87±3.12) and E (648.30±4.91) was lower (P=0.001, 0.002). Comparing with group A (75.96±1.70), the concentration of PEDF in Group B (71.82±1.67) and C (72.43±0.58) was lower (P=0.004, 0.011), but the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group B (71.82±1.67), the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group A (7.70±0.29), the ratio of VEGF to PEDF in Group B (9.85±0.34) and Croup C (9.64±0.02) was higher (P=0.008, 0.027) Comparing with group B, The ratio of VEGF to PEDF in Group D (7.23±0.08) and E (6.92±0.06) was lower (P=0.016, 0.015). Comparing with group A (108.42±0.75, 995.47± 13.61), the concentration of IP3 and DAG in Group B (117.24±1.06, 1070.10±10.07), C (137.12±2.71, 1046.40±7.90), D (139.17±1.40, 1041.13±9.76) and E (149.61±0.77, 1273.14±10.89) was higher, the difference was statistically significant (P=0.003, 0.007, 0.000, 0.000, 0.000, 0.000, 0.000, 0.000). Comparing with group B, the concentration of IP3 in Group C, D and E was higher (P=0.011, 0.000, 0.000). Comparing with group B, the concentration of DAG in Group C and D was lower (P=0.021, 0.007). Comparing with group B, the concentration of DAG in Group E was higher (P=0.000). Comparing with group A (10.27±1.88), the apoptosis rate of RPE cells in Group B(25.07±2.66) and F(19.37±3.23) was higher, the difference was statistically significant (P=0.001, 0.009). Comparing with group B (25.07±2.66), the apoptosis rate of RPE cells in Group F (19.37±3.23) was lower (P=0.038). CONCLUSIONS: (1) After exposuring to blue light, the concentrations of VEGF, IP3 and DAG are increased and the ratio of VEGF to PEDF is also increased and the concentration of PEDF is decreased in human RPE cells. (2) L-Type Calcium Channels and Ca2+-PKC signaling pathways may be regulate the concentrations of VEGF, PEDF, IP3 and DAG in RPE cells after exposuring to blue light by feedback regulation. (3) The application of Calphostin C combined with Nifedipine may be restrain the apoptosis of RPE cells after exposuring to blue light.

[The effect of blue light on human retinal pigment epithelium cells α1D subunit protein expression and vascular endothelial growth factor and basic fibroblast growth factor secretion in vitro].

OBJECTIVE: To investigate the effect of blue light on human retinal pigment epithelium (RPE) α1D subunit protein expression and its relationship with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion in vitro. METHODS: The fourth generation cultured human RPE cells in vitro were randomly divided into 4 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+nifedipine), group D [exposure to blue light+(-)Bay K8644]. Cells were exposed to blue light (2 000 ± 500) lx for 6 hours, and cells culture completed 24 hours later. VEGF and bFGF concentration were assayed by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction was used to analysis L-type calcium channel α1D subunit mRNA expression. Western blot was used to examine the protein expression of L-type calcium channel α1D subunit. Analysis of variance was used to compare the difference of α1D subunit mRNA and protein expression, VEGF and bFGF concentration between groups. Correlation analysis was used to show the relationship between α1D subunit protein expression and concentration of VEGF and bFGF. RESULTS: (1) There is significant statistically difference in the population mean of VEGF and bFGF concentration in four groups (F = 99.441, 21.310, P = 0.000,0.000) . VEGF and bFGF concentration in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) and group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) was higher than it was in group A (2 401.09 ± 228.07, 1 232.42 ± 65.41) , which was statistically different (P = 0.000, 0.000, 0.019, 0.000). And it was higher in group D (3 808.01 ± 94.01, 1 485.82 ± 108.97) compared with group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.006). While, it was lower in group C (1 927.28 ± 143.11, 1 149.39 ± 62.99) than it was in group B (3 281.51 ± 251.73, 1 346.81 ± 62.27) (P = 0.000, 0.000) . (2) The mean of mRNA expression of α1D subunit between four groups was statistically significant (F = 50.320, P = 0.000) . It was higher in group B (210 ± 23) , group D (478 ± 80) and group C (232 ± 14) than group A (100 ± 20). It was statistically significant different (P = 0.023, 0.006, 0.010). And it was higher in group D (478 ± 80) than group B (210 ± 23) and C (232 ± 14) (P = 0.032, 0.039). (3) There was statistically significant difference in the expression of L-type calcium channel α1D subunit protein in four groups (F = 1 940.363, P = 0.000) . It was significantly higher (P = 0.000,0.000) in group B (0.974 2 ± 0.014 7) and group D (0.654 9 ± 0.005 0) than group A (0.503 2 ± 0.007 5). And it was higher in group D (0.654 9 ± 0.005 0) than it was in group C (0.413 9 ± 0.008 8) (P = 0.000). (4) There was positive correlation between the L-type calcium channel α1D subunit protein expression and VEGF concentration secreted by retinal pigment epithelium cells (r = 0.674, F = 8.333, P = 0.016), but there was no correlation with bFGF concentration (r = 0.537, F = 4.061, P = 0.072). CONCLUSIONS: L-type calcium channel α1D subunit may be involved in blue light induced damage on human retinal pigment epithelial cells. Blue light exposure can induce the mRNA and protein expression of α1D subunit, VEGF and bFGF concentration in retinal pigment epithelium cells Increased. And there was a positive correlation between α1D subunit protein expression and the VEGF concentration.

Profiling of human leukocyte antigens in Eales disease and tuberculosis.

To analyze the association between Eales disease, histocompatibility leukocyte antigen alleles (HLA-A/B, HLA-DRB/DQB) and tuberculosis infection, and to explore susceptible genes and protective genes associated with Eales disease and tuberculosis infection in a population of Han nationals from Zunyi City in Guizhou Province, China. The subjects were analyzed by a case-control study consisting of three groups--Eales disease group (47 cases), pulmonary tuberculosis group (36 cases) and normal control group (100 cases). The Eales disease group was divided into four parts. Part one consisted of 12 patients who had suffered from pulmonary tuberculosis. Part two consisted of 27 patients who had not suffered from pulmonary tuberculosis. Parts three and four consisted of 27 patients with a positive purified protein derivative test and 12 patients with a negative test, respectively. DNA samples from 47 patients with Eales disease, 36 patients with pulmonary tuberculosis and 100 healthy people were detected by polymerase chain reaction with sequence-specific primers. The 59 HLA-A/B and HLA-DRB/DQB alleles from Eales disease were compared with those from tuberculosis and normal control, and a correlativity test of common susceptible genes was performed to analyze the potential relationship between Eales disease and pulmonary tuberculosis. The frequency distribution of HLA-A*02 alleles (OR 9.719, OR 95 % CI 4.377-21.580, P = 0.000) and HLA-B*07 (OR 11.605, OR 95 % CI 2.397-56.191, P = 0.001) in the Eales disease group was higher than in the normal control group, but the HLA-A*11 alleles (OR 0.495, OR 95 % CI 0.245-1.000, P = 0.048) were lower than in the normal control group, showing a significant difference. Compared with parts two and four, the frequency distribution of HLA-A*02, HLA-A*11 and HLA-B*07 alleles in parts one and three showed no significant difference (P > 0.05). HLA-A*A02, HLA-A*24, HLA-B*07 and HLA-DRB*16 alleles between the Eales disease, pulmonary tuberculosis and normal control group showed statistical significance (P < 0.05). HLA-A*24 alleles in the pulmonary tuberculosis group were lower than the Eales disease group (χ(2) 7.289, P = 0.007), but HLA-A*02 alleles showed no significant difference (P > 0.05) between the two groups. The results show that HLA-A*02 and HLA-B*07 may be genetic predisposing genes, but HLA-A*11 alleles may be protective genes of Eales disease, the HLA-A*02 allele may be a common susceptible gene of Eales disease and pulmonary tuberculosis. HLA-A*11 and HLA-A24 alleles are protective genes of Eales disease and pulmonary tuberculosis, respectively. In summary, the frequency distribution of susceptible genes of Eales disease and pulmonary tuberculosis in a population of Han nationals from Zunyi City in Guizhou Province, China showed no significant difference.

[Experimental research of endostatin microcapsules by periocular injection].

OBJECTIVE: To detect the release of endostatin in microcapsules of calcium alginate gel by emulsification-internal gelatification technology, to observe the distribution of Endostatin microcapsules in the eye by periocular injection and biocompatibility. METHODS: The calcium alginate gel microcapsules encapsulating endostatin were prepared by emulsification-internal gelatification technology, the release of endostatin in microcapsules in vitro was examined by uv-Spectrophotometry. 32 SD mice were divided into randomly four groups: group A, periocular injection of endostatin microcapsules 20 microl (2.4 g/L); group B, periocular injection of endostatin protein 20 microl (2.5 g/L); group C, periocular injection of null-Microcapsules 20 microl; group D, periocular injection of saline 20 microl. Western blot, immunohistochemical and enzyme-linked immunosorbent assay were used. Protein expression and pathology of the heart, liver, spleen, lung, kidney and periocular tissue were examined. Daily activities and eating condition were observed.t-test was used to analyze the data. RESULTS: SDS polyacrylamide gel electrophoresis showed strap of ES protein. Concentration of ES protein in superior schedule fluid gradually increased at 3, 7, 14, 21 days. A group: ES protein expression were observed in inner nuclear layer, outer nuclear layer and RPE lamina after periocular injection 7 or 14 days. B group: ES protein expression were observed in above tissues at 7 days, but not at 14 days. C and D group: ES protein expression were not observed in above tissues at 7 or 14 days. The results showed that Endostatin microcapsules was able to pass through the sclera and spread out in various retinal tissues. Concentration of endostatin in the homogenates of eyeball was (63.16 +/- 7.64) microg.L(-1).eye(-1) and (33.2 +/- 5.77) microg.L(-1).eye(-1) respectively after one and two weeks in A group, but (33.2 +/- 2.89) microg.L(-1).eye(-1) and (15.73 +/- 2.08) microg.L(-1).eye(-1). The concentration of endostatin was significant difference in two group, A group was obviously larger than B group (t = 6.364 and 4.920, P = 0.003 and 0.008 respectively). Periocular administration. Daily activities and eating condition were normal. The abnormality in important organs were not detected by pathologic examination. CONCLUSIONS: Endostatin microcapsules is able to release persistently, to pass through the sclera and spread out in various retinal tissues. There were good biocompatibility and not ill effect remarkably.