Laser-induced Choroidal Neovascularization In Rats Research Articles

Metabolic Characterization of Ocular Tissues in Relation to Laser-Induced Choroidal Neovascularization in Rats.

Age-related macular degeneration is a metabolic compromise disorder whose main pathological feature is choroidal neovascularization (CNV) formation. Using untargeted metabolomics analysis, we determined to assess the metabolomic alterations in a CNV rat model to provide an insight into its pathogenesis. In the CNV model, there were 24 significantly changed metabolites in the plasma and 71 in various ocular tissues. Pathway analysis showed that certain metabolic pathways changed in interrelated tissues: for instance, in terms of the altered urea cycle, arginine and proline metabolism were increased in the plasma, while spermidine and spermine biosynthesis activities were increased in the retinal pigment epithelium (RPE)/choroid. The retina and RPE/choroid shared the same changed metabolites of branched-chain amino acid metabolism. Fatty acid metabolism was found to be the significant altered metabolic pathway in the retina of this CNV model. Although the metabolism pattern of different substances is specific for each ocular tissue, there is also a certain material exchange between different tissues. Dysregulated metabolomic profiles in differential tissues may point to an interconnected pathway, oxidative stress response, which may lead to RPE cell degeneration and, ultimately, CNV development.

In vivo evaluation of laser-induced choroidal neovascularization in rats simultaneously using optical coherence tomography and photoacoustic microscopy

Determination of the precise location and the degree of the Choroidal neovascularization (CNV) lesion is essential for diagnosation Neovascular age-related macular degeneration (AMD) and evaluation the efficacy of treatment. Noninvasive imaging techniques with specific contrast for CNV evaluation are demanded. In this paper, two noninvasive imaging techniques, namely Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM), are combined to provide specific detection of CNV for their complimentary contrast mechanisms. In vivo time-serial evaluation of Laser-induced CNV in rats is present at days 1, 3, 5, 7, 14, 21 after laser photocoagulation is applied to the rat fundus. Both OCT and PAM show that the CNV increases to its maximum at day 7 and decreases at day 14. Quantification of CNV area and CNV thickness is given. The dual-modal information of CNV is consistent with the histologic evaluation by hematoxylin and eosin (H&E) staining.

Laser-Induced Choroidal Neovascularization in Rats.

The laser-induced choroidal neovascularization (CNV) model has been widely used for research on wet age-related macular degeneration (wet-AMD) and other ocular neovascular diseases. In this model, the Bruch membrane is perforated by laser injury, resulting in neovascularization formed from the choroidal capillaries. It has become a standard method to evaluate the effect of different treatments on CNV progression in preclinical studies. This protocol can be used in various species, including rat, mouse, pig, and monkey. The rodent laser-induced CNV model is the most commonly used because of the advantages in both cost- and time-efficiency. It takes only 10-15min to complete the whole laser procedure after adequate training and practicing the technique. Peak CNV formation occurs at approximately 2weeks after laser application. The entire protocol may require up to 3weeks to complete the treatment, fundus image acquisition, and tissue collection for histologic analysis. This chapter describes the detailed procedures, protocols, and useful notes on how to induce CNV by laser.

Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats.

To evaluate whether recombinant complement factor B (CFB) short hairpin RNA (shRNA) reduces laser-induced choroidal neovascularization (CNV) in rats. Laser-induced rat CNV model was established, and then the animals underwent fundus fluorescence angiography (FFA) and hematoxylin and eosin (HE) staining. On day 3 and 7 after photocoagulation, the expression of CFB and membrane attack complex (MAC) was detected by immunhischemistry. A recombinant CFB-shRNA plasmid was constructed. CFB and scrambled shRNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment, respectively. On day 7, the incidence of CNV was determined by FFA, and the expression of CFB and vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)/choroidal tissues was detected by immunhischemistry, Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in CFB and scrambled shRNA groups. The possible adverse effects of CFB-shRNA injection were assessed by transmission electron microscopy and electroretinography. FFA and HE results indicated that a laser-induced rat CNV model was successfully established on day 7 after photocoagulation. The expression of CFB and MAC was extremely weak in normal retina and choroid, and increased on day 3 after photocoagulation. However, it started to reduce on day 7. CFB shRNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues. FFA showed CFB knockdown significantly inhibited incidence of CNV in rats. Moreover, CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues. CFB shRNA caused no obvious side effects in eyes. CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF, which is a potential therapy target. The alternative pathway of complement activation plays an important role in CNV formation.

Transscleral sustained ranibizumab delivery using an episcleral implantable device: Suppression of laser-induced choroidal neovascularization in rats.

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.

Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats

ABSTRACTPurpose: Choroidal neovascularization (CNV) is a common vision-threatening complication associated with many fundus diseases. The retinal pigment epithelial (RPE) cell junction barrier has critical functions in preventing CNV, and oxidative stress can cause compromise of barrier integrity and induce angiogenesis. Rap1, a small guanosine triphosphatase (GTPase), is involved in regulating endothelial and epithelial cell junctions. In this work, we explored the function and mechanism of Rap1 in CNV in vivo.Methods: A laser-induced rat CNV model was developed. Rap1 was activated through intravitreal injection of the Rap1 activator 8CPT-2ʹ-O-Me-cAMP (8CPT). At 14 days after laser treatment, CNV size in RPE/choroid flat mounts was measured by fluorescein isothiocyanate-dextran staining. Expression of vascular endothelial growth factor (VEGF) and cell junction proteins in RPE/choroid tissues were analyzed by western blots and quantitative real-time PCR assays. Reactive oxygen species (ROS) in RPE cells were detectedbydichloro-dihydro-fluorescein diacetate assays. The antioxidant apocynin was intraperitoneally injected into rats.Results: Activating Rap1 by 8CPT significantly reduced CNV size and VEGF expression in the rat CNV model. Rap1 activation enhanced protein and mRNA levels of ZO-1 and occludin, two tight junction proteins in the RPE barrier. In addition, reducing ROS generation by injection of apocynin, a NADPH oxidase inhibitor, inhibited CNV formation. Rap1 activation reduced ROS generation and expression of NADPH oxidase 4.Conclusions: Rap1 activation inhibits CNV through regulating barrier integrity and ROS generation of RPE in vivo, and selectively activating Rap1 may be a way to reduce vision loss from CNV.

Expression of CD147 in laser-induced rat choroidal neovascularization model

Background The pathology of choroidal neovascularization (CNV) is not clear.CD147 plays a critical role in angiogenesis through activating matrix metalloproteinases (MMPs) and degradation of extracellular matrix (ECM). However, the study of CD147 in CNV is rare. Objective This study was to investigate the expression of CD147 and vascular endothelial growth factor (VEGF) in laser-induced rat CNV model. Methods Thirty clean BN rats were divided into normal control group (six rats) and laser group (24 rats) according to the random number table.CNV was induced on laser photocoagulation group rats using 532 nm multiple-wave length photocoagulation.The development of CNV was followed up by fundus fluorescence angiography (FFA) on 1 day, 7, 14 and 21 days post-photocoagulation.The expression of CD147 protein in retinal pigment epithelium(RPE)-choroid-sclera complex was examined by Western blot in normal control group and 1 day, 7, 14, 21 days post-photocoagulation group.The vitreous VEGF concentrations of those groups were detected by ELISA. Results Disk like fluorescein leakage was detected on 7 days after photocoagulation, and CNV was formed on 14 days after photocoagulation.The expressions of CD147 protein were 1.00±0.43, 0.97±0.53, 0.99±0.45, 1.56±0.67 and 2.27±0.54, and the concentrations of vitreous VEGF were (72.96±29.95), (79.36±10.46), (103.82±32.94), (166.05±21.54) and (195.64±39.90)pg/ml in normal control group and 1 day, 7, 14, 21 days post-photocoagulation group, with significant differences among the groups (CD147: F=10.95, P<0.01; VEGF: F=304.50, P<0.01). The expressions of CD147 and VEGF were significantly higher in the 14, 21 days post-photocoagulation groups than those in the normal control group (all at P<0.05). Conclusions The expression of CD147 increases as the induction of CNV, in accordance with the expression of VEGF.CD147 may play an important role in the early development of CNV. Key words: Choroidal neovascularization; CD147; Vascular endothelial growth factor; Matrix metalloproteinases

Intravitreal itraconazole inhibits laser-induced choroidal neovascularization in rats.

Choroidal neovascularization (CNV) is a major cause of severe visual loss in patients with age-related macular degeneration (AMD). Recently, itraconazole has shown potent and dose-dependent inhibition of tumor-associated angiogenesis. We evaluated the anti-angiogenic effect of itraconazole in a rat model of laser-induced CNV. After laser photocoagulation in each eye to cause CNV, right eyes were administered intravitreal injections of itraconazole; left eyes received balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)-choroid tissue complex was stained with Alexa Fluor 594-conjugated isolectin B4 to measure the CNV area and volume. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression was determined 1, 4, 7, and 14 days after intravitreal injection by quantitative RT-PCR or Western blot. VEGF levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Intravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area and volume on flat mounts compared with intravitreal BSS (p = 0.002 for CNV leakage, p<0.001 for CNV area and volume). Quantitative RT-PCR showed significantly lower expression of VEGFR2 mRNA in the RPE-choroid complexes of itraconazole-injected eyes than those of BSS-injected eyes on days 7 and 14 (p = 0.003 and p = 0.006). Western blots indicated that VEGFR2 was downregulated after itraconazole treatment. ELISA showed a significant difference in VEGF level between itraconazole-injected and BSS-injected eyes on days 7 and 14 (p = 0.04 and p = 0.001). Our study demonstrated that intravitreal itraconazole significantly inhibited the development of laser-induced CNV in rats. Itraconazole had anti-angiogenic activity along with the reduction of VEGFR2 and VEGF levels. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.

Inhibition of Laser-Induced Choroidal Neovascularization by Hematoporphyrin Dimethylether-Mediated Photodynamic Therapy in Rats.

This study aimed to investigate the effect of hematoporphyrin dimethylether (HDME)-mediated photodynamic therapy for laser-induced choroidal neovascularization (CNV) in adult Brown Norway rats. HDME was administered via tail vein at 14 d after the laser photocoagulation, and the rats received irradiance with a laser light at 570 nm at 15 min after injection. CNV was evaluated by fundus photography, fundus fluorescein angiography, optical coherence tomography, and hematoxylin and eosin staining. We found that CNV was occurred at 7 d after photocoagulation and reaching peak activity at 14 d after photocoagulation. There is a significant reduction in the total area of the fluorescein leakage and the number of strong fluorescein leakage spots on 7 d after HDME-mediated photodynamic therapy (PDT). The results suggest that HDME-mediated PDT inhibits laser-induced CNV in rats, representing a promising therapy for wet age-related macular degeneration.

Effect of cytokeratin 17 on retinal pigment epithelium degeneration and choroidal neovascularization.

To study the effects of cytokeratin 17 (CK17) on sodium iodate (NaIO3) induced rat retinal pigment epithelium (RPE) degeneration, laser induced rat choroidal neovascularization (CNV), and oxidative stress of human retinal pigment epithelium cells (ARPE-19) and human umbilical vein endothelial cell (HUVEC). Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups, 10 rats in control group treated with solvent alone; 10 rats in NaIO3 group treated with solvent and 35 mg/kg NaIO3 injection through hypoglossal vein and 10 rats in CK17+NaIO3 group treated with 1% CK17 eye drop 3 times a day for 1wk before and 4wk after NaIO3 injection. RPE function was measured with c-wave of electroretinogram (ERG). Another 20 rats were randomly divided into 2 groups. Of them 10 rats in CK17 group were anesthetized to receive Nd:YAG laser and given 1% CK17 eye drop before same as above; 10 rats in control were received Nd:YAG and treated with solvent. The development of choroidal neovascularization (CNV) was determined by fundus fluorescein angiography (FFA) performed on 4wk after laser. Methylthiazoly tetrazolium (MTT) assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC in vitro. Four weeks after NaIO3 injection, the c-wave amplitude of ERG was 0.393±0.02 V in the control group, 0.184±0.018 V in NaIO3 group and 0.3±0.01 V in CK17+NaIO3 group. There was a significant reversal of the c-wave by CK17 as compared to NaIO3 group (P<0.01). Four weeks after laser, the size of the CNV lesion was 2.57±0.27 mm(2) in control group and 1.64±0.08 mm(2) in CK17 group. The lesion size significantly diminished in CK17 group (P<0.01). The in vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 µg/mL and enhanced the injury in HUVECs at different concentrations. CK17 can significantly protect RPE from NaIO3 induced degeneration in vivo and in vitro and also could reverse the various oxidants induced injuries in vitro. It inhibits the development of CNV in rat model, interfered with vascular endothelial cell proliferation in vitro.

Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models

Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.

Subretinal transplantation of retinal pigment epithelium overexpressing fibulin-5 inhibits laser-induced choroidal neovascularization in rats

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Choroidal neovascularization (CNV) is the abnormal angiogenesis that causes severe visual loss in AMD. Fibulin-5 (Fbln5), which functions as an angiogenesis inhibitor, plays an important role in the pathogenesis of AMD. Here, we investigated whether subretinal transplantation of Fbln5-overexpressing retinal pigment epithelial (RPE) cells can inhibit CNV in vivo. Adult Long-Evans rats were used in this study. CNV was induced by laser photocoagulation. One week after laser-induced CNV, RPE cells expressing pZlen-Fbln5-IRES-GFP or the control pZlen-IRES-GFP vectors were transplanted into the subretinal space of the right and left eyes, respectively. CNV was evaluated using fundus photography, fundus fluorescein angiography (FFA), and hematoxylin and eosin staining. We found that CNV occurred at 1 week after photocoagulation, reaching peak activity at 3 weeks and remaining at a high level at 4–5 weeks after photocoagulation. Transplanted RPE cells survived for at least 4 weeks and migrated toward the retina. Subretinal transplantation of Fbln5-overexpressing RPE cells resulted in a significant reduction in the total area of leakage and the number of leakage spots compared with transplantation of RPE cells expressing only green fluorescent protein. Our findings suggest that subretinal transplantation of Fbln5-overexpressing RPE cells inhibits laser-induced CNV in rats and thus represents a promising therapy for the treatment of AMD.

Comparative study of laser-induced choroidal neovascularization in rats by paraffin sections, frozen sections and high-resolution optical coherence tomography

The purpose of this study was to demonstrate the value of a quantitative study concerning experimental choroidal neovascularization (CNV) tissue by correlating the paraffin sections and frozen sections with optical coherence tomography (OCT) images. After OCT imaging was performed, four right eyes from the normal Brown Norway (BN) rats were made into paraffin sections and four left eyes were made into frozen sections. Laser photocoagulation was performed on the right eyes of the other 32 BN rats to induce CNV; these rats then were divided into two groups. After the OCT imaging was performed, four eyes were made into serial paraffin sections in Group A or frozen sections in Group B at 1 week after laser photocoagulation, and weekly for 4 weeks. A quantitative comparison of histopathological images and OCT images was performed. The thickness of the sensory retina and choroid of normal BN rats in paraffin sections was perceptibly less than that in the frozen sections, and the thickness of the sensory retina in the frozen sections was similar to that measured in the OCT images. The maximum thickness of the CNV tissue in the frozen sections was greater than that in the paraffin sections, but similar to that measured in the OCT images from week 1 to week 4. Only the frozen sections accurately matched the high-resolution OCT images because of the processing artifacts in the paraffin sections. In further quantitative studies on experimental CNV, the frozen sections may be more preferable if the thickness of the CNV tissue is to be measured and compared.

Dynamic Contrast-Enhanced MRI for Assessing Therapeutic Response of Choroidal Neovascularization in a Rat Model

We evaluated the potential of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as a noninvasive biomarker of choroidal neovascularization (CNV) and its utility as a tool for monitoring therapeutic response in laser-induced rat CNV models. CNV was induced in the right eyes of 14 rats using a laser. Rats (n = 7) were treated daily for 14 days with a candidate drug (KR-31831, 50 mg/kg of body weight) having antiangiogenic effects, whereas control rats (n = 7) were treated with the vehicle alone (10% cremophor, 10% absolute ethyl alcohol, and 80% saline). DCE-MRI examinations were performed on the day before surgery (D - 1), and 3, 7, and 14 days after surgery (D + 3, D + 7, and D + 14), from which pharmacokinetic parameters (K(trans), v(e), v(p)) were calculated. Angiography was performed to visualize CNV using FITC-labeled high molecular weight dextran after MRI on D + 14. The paired Wilcoxon test and Mann-Whitney U test were performed for statistical analysis. The K(trans) and v(e) values of the CNV-induced right eyes were significantly higher than those of the intact eyes in control rats at D + 14 (P < 0.05). In the CNV-induced eyes, the relative K(trans) and v(e) values of the KR-31831-treated group were significantly lower than those of the nontreated group at D + 14 (P < 0.05). The angiography showed that decreased CNV was observed in rats treated with KR-31831. Quantitative DCE-MRI produces noninvasive biomarker of CNV, thus allowing monitoring of therapeutic response of antiangiogenic drugs in neovascular age-related macular degeneration (AMD).

The Antiangiogenic Effects of Integrin α5β1 Inhibitor (ATN-161) In Vitro and In Vivo

Integrin α5β1 is involved in the development of choroidal neovascularization (CNV). Thus, the inhibition of integrin α5β1 may provide an alternative to the current standard of CNV therapy, which involves inhibition of vascular endothelial growth factor (VEGF) and is not effective in all patients. This study evaluated the antiangiogenic effects of ATN-161, a small peptide inhibitor of integrin α5β1, in human choroidal endothelial cells (hCECs) and in laser-induced CNV in rats. Furthermore, the utility of spectral-domain optical coherence tomography (SD-OCT) for dynamic observation of the development of CNV in animal models was assessed. The antiangiogenic potential of ATN-161 was evaluated in VEFG-stimulated hCECs by MTS proliferation assays, migration assays, and synthetic matrix capillary tube formation assays. To evaluate the antiangiogenic effects of ATN-161 in vivo, CNV was induced in rats by laser photocoagulation. ATN-161, scrambled peptide, or AF564 anti-VEGF antibody, were injected intravitreally immediately after photocoagulation. Eyes were examined by SD-OCT and fluorescein angiography on days 1, 7, and 14 after injection, and the areas of CNV were measured by analysis of choroidal flatmounts at day 14. ATN-161 inhibited VEGF-induced migration and capillary tube formation in hCECs, but did not inhibit proliferation. In vivo, injection of ATN-161 after laser photocoagulation inhibited CNV leakage and neovascularization to an extent similar to AF564. Furthermore, SD-OCT and histologic examinations indicated that ATN-161 significantly decreased the size of laser-induced lesions. Integrin α5β1 inhibition by ATN-161 may be a promising alternative therapy for CNV-related angiogenesis. In addition, SD-OCT technology allows excellent visualization of experimentally induced CNV in vivo.

Cirrus High-Definition Optical Coherence Tomography (OCT) Imaging in normal retina and laser-induced choroidal neovascularization in rats

BACKGROUND: To evaluate the Cirrus High-Definition Optical Coherence Tomography (OCT) features of the retina of rats. MATERIAL AND METHODS: Sixteen eyes of 16 adult BN rats were used. Histologic sections and OCT images of normal retinas and retinas with laser-induced choroidal neovascularization were studied. RESULTS: OCT showed a double-layered structure in the normal retina with a highly reflective layer located in the inner retina and a low reflective layer located in the outer retina. The retinal pigment epithelium (RPE) and choriocapillaris were imaged as a layer with the highest reflection. On the first day after photocoagulation, OCT image showed a disruption of the highly reflective layer corresponded to the RPE and choriocapillaris. Choroidal neovascularisation was seen as a highly reflective area located beneath the subretinal space. The retinal atrophy which appeared 4 weeks after photocoagulation was seen as a thinning reflection of the sensory retina. The retinal layer and thickness maps provided additional information that greatly complemented the OCT images. The histopathological appearance was well correlated with the OCT images. CONCLUSIONS: Cirrus OCT image combined with retinal layer and thickness maps will be a useful tool for the evaluation of animal studies of choroidal neovascularization and other retinal diseases.

Influence of Dll4 via HIF-1α-VEGF Signaling on the Angiogenesis of Choroidal Neovascularization under Hypoxic Conditions

Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis.

Effect of naringenin on NaIO(3)-induced retinal pigment epithelium degeneration and laser-induced choroidal neovascularization in rats.

To study the effects of naringenin eye drops on NaIO(3)-induced retinal pigment epithelium (RPE) degeneration and laser-induced choroidal neovascularization (CNV) in rat eyes. The 35mg/kg NaIO(3)-induced RPE degeneration was prevented by 10g/L naringenin eye drops 3 times a day for 7 days in advance of NaIO(3) injection, and then 2 to 4 weeks thereafter, RPE function was measured with C-wave of electroretinogram (ERG). The laser-induced CNV rats were treated with laser to break the Bruch's membrane and the CNV formation was prevented by 10g/L naringenin eye drops instilled 3 times a day for 2 to 4 weeks. The CNV formation was measured with fluorescein angiography (FA) and flat mount. Two weeks after NaIO(3) injection, the amplitude of ERG C-wave fell markedly in NaIO(3) group to 53% of normal group (P<0.01). No apparent difference was observed in naringenin+NaIO(3) group. Four weeks later, the NaIO(3) group fell to 37% of normal group (P<0.01), while the naringenin+ NaIO(3) group fell to only 57% of normal group (P<0.01). There was a 52% reversal of the ERG C-wave by naringenin as compared to NaIO(3) treated group (P<0.05). Two weeks and four weeks after laser treatment, naringenin reduced the CNV formation to 53% and 49% of control group (100%) measured by FA (P<0.01). Four weeks after laser treatment, naringenin reduced the CNV formation by 47% as compared to control group measured with flat mount (P<0.01). Naringenin can significantly protect RPE from NaIO(3) induced degeneration and also prevent CNV formation.

Development of Laser-Induced Choroidal Neovascularization in Rats after Retinal Damage by Sodium Iodate Injection

Purpose: To determine the role of the retinal pigment epithelium (RPE) in the development of laser-induced choroidal neovascularization (CNV) by selective damage with sodium iodate (SI). Methods: In group 1, diode laser photocoagulation was applied to the right eye of Long-Evans rats (n = 11). In group 2 (n = 11), SI was administered intravenously at a dose of 40 mg/kg of body weight 2 days after photocoagulation. In group 3 (n = 10), SI was injected 2 days before laser was applied. In group 4 (n = 10), SI was injected immediately after photocoagulation. Results: CNV ingrowth developed in 42 of 93 burns (45%) of the laser group and in 46 of 75 burns (61%) when SI was administered 2 days after laser (p = 0.036). No CNV developed in the other groups. Conclusions: The angiogenic effect of the RPE is essential during the first hours after the onset of the laser-induced CNV process. When the RPE was damaged before or at the time of photocoagulation, CNV was not induced. These results suggest that RPE integrity is a decisive element in CNV formation.

Role of PI3K/Akt and MEK/ERK in Mediating Hypoxia-Induced Expression of HIF-1α and VEGF in Laser-Induced Rat Choroidal Neovascularization

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis and is activated during hypoxia by stabilization of the subunit HIF-1alpha. Hypoxia plays an important role in the development of choroidal neovascularization (CNV). Expression of HIF-1alpha has been demonstrated in CNV. Vascular endothelial growth factor (VEGF) is one of the most well-characterized angiogenic factors in CNV, which is under the regulation of HIF-1. The aim of the present study was to explore the upstream signaling pathways involved in regulating hypoxia-induced expression of HIF-1alpha and VEGF in laser-induced rat CNV. A well-established rat model of CNV and cultured human retinal pigment epithelium (hRPE) was used to investigate the role of PI3K/Akt and MEK/ERK pathways in regulating HIF-1alpha and VEGF expression in CNV in rat and hRPE under hypoxia by immunohistochemistry, Western blot analysis, real-time PCR, and ELISA. pAkt, pERK, HIF-1alpha, and VEGF were upregulated in vivo and in vitro. PI3K inhibitor (Ly294002) significantly decreased pAkt activity and HIF-1alpha and VEGF expression in vivo and in vitro, whereas MEK inhibitor (PD98059) reduced ERK phosphorylation and the expression of VEGF but had no effect on HIF-1alpha. LY294002 and PD98059 severely inhibited the formation of CNV. The PI3K/Akt pathway was required for hypoxia-induced expression of HIF-1alpha and VEGF, whereas the MEK/ERK pathway was required only for VEGF in laser-induced rat CNV.