Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis.

OBJECTIVE: To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. METHODS: siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor-reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. RESULTS: The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. CONCLUSIONS: Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF.

Caveolin-1 expression regulates blood-retinal barrier permeability and retinal neovascularization in oxygen-induced retinopathy.

BACKGROUND: Caveolin-1 expression correlates with the permeability of endothelial barriers and angiogenesis. However, the role of caveolin-1 in retinal neovascularization remains unknown. We evaluated the effect of caveolin-1 on the blood-retina barrier and retinal neovascularization in a murine model of oxygen-induced retinopathy. METHODS: Starting at postnatal day 7, mice were exposed to 75 ± 5% oxygen for 5 days and then returned to room air conditions to induce retinal neovascularization. Effects on blood-retina barrier were evaluated by Western blot analysis of extravasated albumin in the retina. Retinal neovascularization morphology was studied by fluorescence angiography and was quantified by counts of the endothelial nuclei that protruded into the vitreous cavity. Reverse transcription-polymerase chain reaction and Western blot analysis was used to examine retinal expression levels of caveolin-1. siRNA against caveolin-1 was injected intravitreally in the oxygen-induced retinopathy models. Effects on caveolin-1 mRNA and protein, and retinal neovascularization were assessed as described above. RESULTS: Caveolin-1 expression was found to increase during hypoxia and overexpression of caveolin-1 correlated with the appearance of extravascular albumin. Caveolin-1 siRNA reduced caveolin-1 mRNA and protein levels by 47.94% and 54.76%, respectively. Furthermore, caveolin-1 siRNA inhibition reduced retinal neovascularization by 51.3% and reduced albumin leakage by 56.32%. CONCLUSIONS: Caveolin-1 may play an important role in induction of retinal neovascularization. SiRNA against caveolin-1 can inhibit experimental retinal hyperpermeability and neovascularization. Therefore, the inhibition of caveolin-1 may be a powerful and novel therapeutic tool for the treatment of ischaemia-induced retinal diseases.

[HIF-1α siRNA reduces retinal neovascularization in a mouse model of retinopathy of prematurity].

OBJECTIVE: To study the inhibition effect of HIF-1α specific siRNA expression vector pSUPERH1-siHIF-1α on retinal neovascularization in a mouse model of retinopathy of prematurity (ROP). METHODS: The mouse model of ROP was prepared by the method Smith described. Forty-eight ROP mice were randomly divided into two groups: an experimental group that was intravitreously injected with pSUPERH1-siHIF-1α and a control group that was injected with pSUPER retro vector. The levels of HIF-1α and vascular endothelia growth factor (VEGF) in the retina were examined by Western blot. The retinal neovascularization was evaluated by angiography using FITC Dextran and quantitated histologically. RESULTS: The levels of HIF-1α and VEGF in the retina in the experimental group were reduced 90% and 65% respectively compared with those in the control group. Meanwhile, the number of retinal neovascular endothelial nucleus outbreaking the inner limit membrane in the experimental group was significantly reduced compared with that in the control group. CONCLUSIONS: The development of retinal neovascularization of ROP can be markedly inhibited by RNA interference targeting HIF-1α.

Morphological Characteristics Predict Postoperative Outcomes After Vitrectomy in Myopic Traction Maculopathy Patients.

BACKGROUND AND OBJECTIVES: To provide the surgical indication for patients with myopic traction maculopathy (MTM) by investigating the postoperative outcomes after vitrectomy among different types of morphological characteristic groups. PATIENTS AND METHODS: This was a retrospective cohort study that included patients (37 eyes) diagnosed with MTM at a single institution. All 37 eyes from 37 patients with MTMs were classified into three groups: foveal retinoschisis (FS), lamellar macular hole (LMH), and foveal retinal detachment (FRD). The ratios of anatomic recovery, central retinal thickness (CRT), and best-corrected visual acuity (BCVA) were statistically analyzed among the three groups preoperatively and at 1, 3, 6, and 12 months after vitrectomy. RESULTS: Anatomical recovery could be found in all patients of the FS group at 6 months postoperatively and in the LMH group at 12 months postoperatively. Only 83.33% patients in the FRD group showed anatomic recovery until 12 months. The time taken for CRT to reduce to 200 µm was gradually increased between the FS, LMH, and FRD groups. Postoperative BCVA was better in the FS group than the LMH and FRD groups (P < .05), but the LMH and FDR groups had no difference (P ≥ .05) at any point. The visual acuity was significantly improved in the FS group (P < .01) and FRD group (P = .018), but not in the LMH group (P = .196) at 12 months postoperatively. CONCLUSIONS: The FS group achieved anatomical recovery in the shortest time and had the best postoperative BCVA. FRD patients could get visual gain but need too much time for the anatomical recovery. LMH patients experienced anatomic success with surgery, but not in BCVA. Early surgery might be considered for eyes at FS prior to the occurrence of LMH or FRD. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:574-582.].

Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-1alpha and VEGF.

Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions. Therefore, HIF-1alpha and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF-1alpha and VEGF on the expression of HIF-1alpha and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA-expressing plasmids targeting human HIF-1alpha (HIF-1alpha siRNA) and human VEGF(165) (VEGF siRNA) were constructed. They were transfected and co-transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF-1alpha siRNA and VEGF siRNA specifically downregulated HIF-1alpha and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co-transfection of HIF-1alpha siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF-1alpha siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV.

Suppression of retinal neovascularization by small-interference RNA targeting erythropoietin.

OBJECTIVE: To observe the effect of inhibition of retinal neovascularization by small-interference RNA (siRNA) targeting erythropoietin (EPO). METHOD: Three siRNAs against EPO were designed and synthesized. Then they were transfected to NIH/3T3 cells by liposomes. RT-PCR and Western blot were used to evaluate the efficacy of siRNA in attenuating EPO expression in NIH/3T3 cells. One-week-old C57BL/6J mice were exposed to 75 +/- 2% oxygen for 5 days, then they were returned to room air to induce retinal neovascularization. The siRNA type shown as most powerful in reducing EPO expression in vitro was intravitreally injected in the treatment group. Retinal neovascularization was evaluated by angiography with injection of fluorescein-dextran and quantification of neovascular proliferative retinopathy after 5 days in room air. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of siRNA could affect the expression of EPO in murine retinas. RESULTS: Among the 3 designed siRNAs (named siEPO1-3), siEPO2 is the most efficient in inhibiting EPO expression. In this murine model of oxygen-induced retinopathy, retinal neovascularization in the eyes with siEPO2 injection was significantly reduced compared with that of the contralateral control eyes. Similarly, histological analysis indicates that the number of neovascular nuclei protruding into the vitreous cavity was decreased compared to the control eyes. Furthermore, the expression of EPO in the retinas injected with siEPO2 was dramatically decreased. CONCLUSION: siRNA against EPO could inhibit experimental retinal neovascularization by reducing EPO expression in the retinas of mice. It may provide a powerful and novel therapeutic tool for ischemia-induced retinal diseases.

[Inhibitory effect of interfering RNA targeting HIF-1alpha and VEGF on retinal neovascularization in the mouse].

OBJECTIVE: To evaluate the inhibitory effect of small interfering RNA (siRNA) on the expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in retinal neovascularization in the mouse. METHODS: HIF-1alpha siRNA recombinant plasmid was constructed. Liposome mediated the expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 complex was injected into the vitreous of C57BL/6J mice. The expression of GFP was observed in retinal flat-mounts one day after injection. Randomized controlled trial was performed. There were totally ninety (seven-day-old) C57BL/6J mice in which seventeen mice were chosen as normal group and seventy-three mice were divided into five groups randomly including control model group, vector group and gene therapy group (HIF-1alpha siRNA group, VEGF siRNA group and co-transfection group), in which retinal neovascularization was induced by hypoxia. Liposome with vector plasmid, HIF-1alpha siRNA and VEGF165 siRNA were injected into the vitreous in the vector group, HIF-1alpha siRNA group and VEGF siRNA group respectively one day before mice were moved out to room air from the cabin. Liposome with HIF-1alpha siRNA and VEGF165 siRNA was injected in the co-transfection group at the same time point. Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. HIF-1alpha and VEGF levels in retinas were measured by reverse transcriptase-polymerase chain reaction and Western blot. Significant differences between groups were evaluated by one-way analysis of variance, followed by a least-significant difference analysis. RESULTS: The GFP expression in retinal cells was observed one day after injection of liposome mediated pEGFP-N1 complex. Neovascular tufts and fluorescein leakage were decreased in gene therapy group especially in co-transfection group compared to the control model group. The neovascular nuclei were decreased in gene therapy group [HIF-1alpha siRNA group (27.73 +/- 2.33), VEGF siRNA group (15.43 +/- 3.23), co-transfection group (8.70 +/- 2.88)] compared to the other three groups (F = 3016.537, P < 0.01). The expression of HIF-1alpha mRNA and protein in retinas were increased in control model group (1.08 +/- 0.06, 0.383 +/- 0.009) and vector group (1.09 +/- 0.05, 0.386 +/- 0.010) as compared with normal group (0.81 +/- 0.07, 0.035 +/- 0.003), while decreased 57.4% and 52.5% respectively in the HIF-1alpha siRNA group (0.46 +/- 0.06, 0.182 +/- 0.008) as compared with control model group (F = 139.804, 2686.001; P < 0.01). The expression of VEGF mRNA and protein in retinas were increased significantly in control model group (1.53 +/- 0.07, 0.340 +/- 0.004) and vector group (1.59 +/- 0.06, 0.337 +/- 0.009) as compared with normal group (0.27 +/- 0.08, 0.051 +/- 0.008), while decreased significantly in gene therapy group especially co-transfection group (decreased 85.6% and 80.9% respectively) as compared with control model group (F = 421.423, 2513.583; P < 0.01). CONCLUSIONS: HIF-1alpha siRNA and VEGF165 siRNA can inhibit retinal neovascularization in the mouse effectively. Co-transfection of these two siRNAs shows the greatest inhibitory effect.

[Study on suppression of hypoxia inducible factor-1 alpha and vascular endothelial growth factor expression by siRNA targeting hypoxia inducible factor-1 alpha].

OBJECTIVE: To evaluate suppression efficiency of hypoxia inducible factor-1 alpha (HIF-1 alpha) specific siRNA derived from recombinant plasmid (pSUPER(H1-siHIF-1 alpha)) on both HIF-1 alpha mRNA and protein expression and concomitant downregulation of expression of downstream angiogenic factor vascular endothelial growth factor (VEGF). METHODS: Stable pSUPER(H1-siHIF-1 alpha) expression cell lines were constructed by transient transfection of pSUPER(H1-siHIF-1 alpha) eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER(H1-siHIF-1 alpha) cell line with highest HIF-1 alpha inhibition efficiency determined by reverse transcription-polymerase chain reaction (RT-PCR) was cultured under normoxia (20% O(2)) and hypoxia conditions (1% O(2)) together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER(H1-siHIF-1 alpha) on HIF-1 alpha and VEGF expression. RESULTS: Compared to the control cells, both mRNA and protein level of HIF-1 alpha and VEGF were dramatically decreased by pSUPER(H1-siHIF-1 alpha) under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1 alpha mRNA level was downregulated by pSUPER(H1-siHIF-1 alpha), but pSUPER(H1-siHIF-1 alpha) did not cause suppression of VEGF expression. CONCLUSIONS: pSUPER(H1-siHIF-1 alpha) could decrease HIF-1 alpha expression under both normoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER(H1-siHIF-1 alpha) might be a powerful tool for the inhibition of retinal neovascularization.

Effect of pyridone agent on blood-retinal barrier in diabetic mice.

AIM: To evaluate the therapeutic effect of fluorofenidone on disrupted blood-retinal barrier in the diabetic mice and uncover its underlying mechanism. METHODS: db/db mice were randomly chosen for treatment with daily doses of fluorofenidone or placebo at 5-week-old, treatment continued until mice reach 24-week-old. Then, expression of transcriptiona factor insulin gene enhancer binding protein-1 (Islet-1) and vascular endothelial growth factor (VEGF) in murine retinas were evaluated. Retinal vascular permeability was assessed by examining the level of albumin in db/db murine retinas. Furthermore, the retinal vessel tight junction was estimated by checking the level of occludin in the murine retinal tissues. RESULTS: After occurrence of diabetic retinopthy in db/db mice, expressions of transcritpional factor Islet-1 was found to be upregulated in db/db murine retinas compared with non-diabetic controls. Similar to expression pattern of Islet-1, VEGF were also demonstrated to be increased in retinas of db/db mice, which was accompanied by increased retinal vascular leakage and decreased tight junction protein level. Systemetic administration of fluorofenidone repaired broken retinal vascular tight junction by restoring occludin expression in db/db retinal tissue. Consequently, retinal vascular premeability were indicated to be reduced by examining the transudative albumin level in diabetic retinal tissues. Both Islet-1 and VEGF expression were inhibited in the retinas of db/db mice after treatment with fluorofenidone. CONCLUSION: Fluorofenidone significantly protectes retinal tight junction and reduces retinal vascular leakage. The phenomenon can be partially attributed to reducing overexpression of Islet-1 and VEGF in diabetic retinal tissues.

Treatment of myopic foveoschisis via macular buckling and vitrectomy.

The aim of the present study was to evaluate the efficacy and safety of the treatment of myopic foveoschisis patients using the macular buckling with L-shaped titanium plate and silicon sponge combined with vitrectomy. The data of the patients who underwent macular buckling combined with vitrectomy was collected. The study recorded the following parameters: best corrected visual acuity (BCVA), axial length, intraocular pressure, central macular thickness, and the position of the titanium plate. Following the surgery, the BCVA of the included patients were improved, whereas the axial lengths were reduced followed by resolution of the foveoschisis compared with that noted prior to the operations. All patients had orbital CT examination and the results indicated that the titanium plates were appropriately placed and were not in contact with the optic nerve. Therefore, it is effective to treat myopic foveaschisis by macular buckling using the L-shaped titanium plate and silicon sponge in the presence of vitrectomy.

Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function.

Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function.

HIF-1α siRNA reduces retinal neovascularization in a mouse model of retinopathy of prematurity.

OBJECTIVE: To explore the effect of HIF-1α specific siRNA expression vector pSUPERH1-siHIF-1α on retinal neovascularization in a mouse model of retinopathy of prematurity (ROP). METHODS: Forty-eight newborn C57BL/6J mice were randomly divided into the control and experimental groups (n=24 apiece) to create the model of ROP following the methods described by Smith et al. Twelve days after birth, the experimental group received intravitreal injection with pSUPERH1-siHIF-1α; meanwhile, mice in the control group were injected with empty vectors. The expressions of HIF-1α and vascular endothelia growth factor (VEGF) in the retina were examined by Western blotting in both groups. The differences in the neovascular endothelial cell count were compared based on the FITC-Dextran fluorescence stretched preparation/sections. RESULTS: Compared with the control group, the expressions of HIF-1α and VEGF significantly decreased in the experimental group (P<0.01). Meanwhile, the number of retinal neovascular endothelial nuclei that had protruded the internal limiting membrane was significantly lower in the experimental group than in control group (P<0.01). CONCLUSIONS: RNA interference targeting HIF-1α can effectively inhibit the retinal neovascularization of ROP.

Activation of the ERK 1/2 and STAT3 signaling pathways is required for 661W cell survival following oxidant injury.

AIM: To evaluate the influence of hydrogen peroxide (H(2)O(2)) on mouse photoreceptor-derived 661W cell survival and to determine the effect of PD98059, an inhibitor for MEK1 (the direct upstream activator of ERK1/2), and S3I201, a STAT3- specific inhibitor on 661W cell survival after H(2)O(2) exposure. METHODS: The mouse photoreceptor-derived 661W cells were cultured. 661W cells were treated for 12 hours with different concentrations (0, 0.25, 0.50, 0.75, 1mmol/L) of H(2)O(2) and cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ) (MTT) assay. 661W cells were treated with different concentrations H(2)O(2) (0, 5, 10, 50, 500, 1000 µmol/L) for 15 minutes or 1mmol/L H(2)O(2) for different time points (0,5,10,15,30 minutes), and p-Tyr705-STAT3, STAT3, Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2 were surveyed by immunoblot analysis. After treatment with 50µmol/L PD98059, or S3I201 for 1 hour, the inhibition efficiency of cell signal pathways was analyzed by immunoblot analysis and the effects of inhibitors on cell viability were determined by MTT. RESULTS: After treating with different concentrations of H(2)O(2) for 12 hours, the cell viability of 661W cells decreased in concentration-dependent manner (P<0.05). Moreover, H(2)O(2) induced phosphorylation of ERK1/2 and STAT3 in 661W cells (P<0.05). After pretreatment with 50µmol/L PD98059 or S3I201 for 1 hour, H(2)O(2)-induced phosphorylation of ERK1/2 or STAT3 was suppressed separately (P<0.05). Using PD98059 or S3I201 to inhibit ERK1/2 or STAT3 signal pathway, the cell viability of 661W cells decreased significantly (P<0.05). CONCLUSION: We demonstrated that the exposure of 661W cells to H(2)O(2) increased the activation of ERK1/2 and STAT3 signal pathways. Activation of these pathways is required for 661W cell survival following oxidant injury.

Significance of outer blood-retina barrier breakdown in diabetes and ischemia.

PURPOSE: The outer blood-retina barrier (BRB) separates the neural retina from the choroidal vasculature, which is responsible for approximately 80% of blood supplies in the eye. To determine the significance of outer BRB breakdown in diabetic retinopathy, the outer BRB-specific leakage of macromolecules in diabetic and ischemic rodents was investigated. METHODS: Diabetes and ischemia were induced in rodents by streptozotocin and oxygen-induced retinopathy, respectively. Diabetic and ischemic rodents were injected intravenously with fluorescein isothiocyanate (FITC)-dextran. The outer BRB-specific leakage in diabetic and ischemic rodents was visualized by fluorescent microscopy. RESULTS: A microscopic imaging assay was developed to examine outer BRB breakdown. The outer BRB-specific leakage of fluorescent macromolecules was visualized in diabetic and ischemic rodents. Substantial leakages of macromolecules through the outer BRB in diabetic and ischemic rodents were detected with this assay. The number of severe outer BRB leakage sites is inversely proportional to the size of macromolecules. Significant depletion of occludin in the RPE of ischemic and diabetic rodents was also observed. CONCLUSIONS: For the first time, a microscopic imaging assay for directly visualizing macromolecules leaked through the outer BRB in rodents was developed. Using this assay, the authors demonstrated the significance of outer BRB breakdown in diabetes and ischemia, which will have implications to the understanding, diagnosis, and treatment of diabetic macular edema and other ocular diseases with outer BRB defects. The microscopic imaging assay established in this study will likely be very useful to the development of drugs for macular edema.

Expression of FLT4 in hypoxia-induced neovascular models in vitro and in vivo.

AIM: To investigate the expression of FLT4 in retina with oxygen induced retinopathy (OIR) and in brain endothelial cell lines (bEnd3) under hypoxia conditions in mice. METHODS: Fifty-two one-week-old C57BL/6J mice were divided into control group and hypoxia group. The mice of hypoxia group were exposed to 75% oxygen for 5 days and then returned to the room air to induce retinal neovascularization. Mice in control group were raised in the environment of room air at the same time. The expressions of FLT4 mRNA and protein were checked with RT-PCR and Western Blot analysis at postnatal day 14, 17 and 21 ( P14, P17 and P21) respectively. 125mmol/L CoCl(2) were added to the culture medium of bEnd3 cell, proteins were extracted in 12, 24, 48 and 72 hours and FLT4 levels were examined by Western Blot analysis. RESULTS: The mRNA and protein level of FLT4 expressed in P14 and P17 OIR mice retina statistically up-regulated as compared with those in control group, but there was no statistical difference between OIR group and control group at P21. FLT4 levels increased significantly in 12, 24 and 48 hours hypoxia intervened bEnd3 cells, its levels in 72 hours increased mildly but showed no significance. CONCLUSION: FLT4 levels increase in OIR mice retinas and bEnd3 cells in hypoxia. It may play an important role in endothelial cells proliferation in hypoxia and retinal neovascularization in OIR mice.

RPE barrier breakdown in diabetic retinopathy: seeing is believing.

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in working-age Americans. DR is traditionally regarded as a disorder of blood-retina barriers, and the leakage of blood content is a major pathological characteristic of the disease. While the breakdown of the endothelial barrier in DR has been investigated extensively, the vascular leakage through the retinal pigment epithelium (RPE) barrier in the disease has not been widely acknowledged. As the blood content leaked through the RPE barrier causes excessive water influx to the retina, the breakdown of the RPE barrier is likely to play a causative role in the development of some forms of diabetic macular edema, a major cause of vision loss in DR. In this article, we will discuss the clinical evidences of the diabetes-induced RPE barrier breakdown, the alteration of the RPE in diabetes, the molecular and cellular mechanism of RPE barrier breakdown, and the research tools for the analysis of RPE barrier leakage. Finally, we will discuss the methodology and potential applications of our recently developed fluorescent microscopic imaging for the diabetes- or ischemia-induced RPE barrier breakdown in rodents.

Suppression of retinal neovascularization by shRNA targeting HIF-1alpha.

PURPOSE: To investigate whether vector-based HIF-1alpha -targeted shRNA expression system (pSUPER(siHIF-1alpha)) can inhibit HIF-1alpha and VEGF expression in vitro and suppress retinal neovascularization in the murine model of oxygen-induced retinopathy. METHODS: pSUPER(siHIF-1alpha) from which siRNA targeting HIF-1alpha could be generated was constructed and transfected to human umbilical vein endothelial cell lines (HUVECs). Then the expression levels of HIF-1alpha and VEGF in the cultured cells were measured by RT-PCR, immunoblot, and ELISA assays. Subsequently, pSUPER(siHIF-1alpha)was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of pSUPER(siHIF-1alpha)could affect the expression levels of HIF-1alpha and VEGF in murine retinas. RESULTS: HIF-1alpha and hypoxia-induced vascular endothelial growth factor (VEGF) increase in cultured cells were greatly abolished by pSUPER(siHIF-1alpha). Meanwhile, retinal neovascularization in the eye with pSUPER(siHIF-1alpha)injection was significantly reduced compared with that of the contralateral control eye. Histological analysis indicates that neovascular nuclei protruding into the vitreous cavity were decreased by nearly 65%. Furthermore, HIF-1alpha and VEGF expression levels were down-regulated in the murine retinas treated with pSUPER(siHIF-1alpha). CONCLUSIONS: RNAi targeting HIF-1alpha could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1alpha and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.

[mRNA expression of transforming growth factor-beta 1 in the cornea after photorefractive keratectomy in rabbits].

OBJECTIVE: To investigate the roles of transforming growth factor beta 1 (TGF-beta 1) during healing after photorefractive keratectomy (PRK). METHODS: Twenty of 24 rabbits underwent bilateral 193 nm excimer laser photorefractive keratectomy to correct 10 diopters of myopia, and the other 4 rabbits were taken as the normal group. The mRNA levels of TGF-beta 1 in the corneas of the rabbits were determined by in situ hybridization with a Digoxigenin labled probe. RESULTS: 1. Corneal haze appeared in operated eyes from the 7th day, and peaked on the 28th day after PRK. The most severe haze reached Grade 3. 2. In the normal group, a low level of mRNA expression of TGF-beta 1 was observed in the epithelium, and no positive result in the stroma. On the 7th, 14th, and 28th days after the operation, the mRNA levels of TGF-beta 1 in the epithelium and stroma of the operation group were significantly higher than those in the normal group. CONCLUSION: TGF-beta 1 may involve in corneal wound healing after PRK and may increase haze by promoting the synthesis of the extracellular matrix and the proliferation of keratocytes.