Inhibitory effect of YC-1,3-(5 '-hydroxymethyl-2 '-furyl)-1-benzylindazole, on experimental choroidal neovascularization in rat
ABSTRACT It was the aim of this study to evaluate the effects of YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, one of the hypoxia-inducible factor 1 (HIF-1) inhibitors, on laser-induced choroidal neovascularization (CNV) in rats.
Thirty female Brown Norway rats underwent laser photocoagulation to induce CNV. Twenty of them (treatment group) were treated with a single intravitreal injection of 5 microg YC-1, and the remaining 10 (control) were sham-treated with a single intravitreal injection of 2.5 mg/ml dimethyl sulfoxide 2 weeks after laser photocoagulation. The expression of HIF-1alpha and vascular endothelial growth factor (VEGF) in CNV was evaluated by immunofluorescence staining. Fluorescein angiography was performed 2 weeks before and 2 weeks after single intravitreal YC-1 (5 microg) and dimethyl sulfoxide (2.5 mg/ml) injection, grading fluorescein leakage from 0 to 3. The size of the CNV was measured on histologic sections.
Both HIF-1alpha and VEGF were expressed in CNV lesions in the control group 4 weeks after laser photocoagulation, whereas the expressions of HIF-1alpha and VEGF were not observed in the intravitreally YC-1-treated group. The mean fluorescein leakage score decreased from 2.56 +/- 0.49 to 0.79 +/- 0.71 in the intravitreally YC-1-injected group and from 2.62 +/- 0.49 to 1.58 +/- 0.60 in the control group. Sixty-eight (71.6%) out of 95 CNV lesions of intravitreally YC-1-treated eyes (71.4%) and 12 (21.8%) out of 55 lesions in DMSO-treated eyes showed a decreased fluorescein leakage score of 2 or more. The mean difference of fluorescein leakage scores between the intravitreally YC-1-treated group and the control group was significant (p = 0.004). The mean thickness of the CNV lesions in the intravitreally YC-1-treated group (27.30 +/- 6.47 microm) was smaller than that of the control group (64.36 +/- 8.26 microm, p < 0.001). There was no ocular inflammation, retina hemorrhage or systemic toxicity induced by YC-1 treatment.
These results suggest that YC-1 inhibits the HIF-1 expression after photocoagulation and suppresses the development of laser-induced CNV formation.
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ABSTRACT: The survival of retinal ganglion cells (RGCs) is a hallmark of many optic neurodegenerative diseases such as glaucoma. YC-1, a potential anticancer drug, is known to be able to decrease the stability and protein expression of hypoxia-inducible factor (HIF)-1α that is triggered by hypoxia and related to RGC survival. We hypothesized that YC-1 may alter RGC cell viability through the down-regulation of HIF-1α. Cell viability of the RGC-5 cell line was measured with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometry, a LIVE/DEAD viability assay, and high-content screening (HCS) with MKI67 (K(i)-67) monoclonal antibodies were used to detect cell death and cellular proliferation. We found that cells treated with 20 µM YC-1 for 24 h decreased the HIF-1α level in an RGC-5 cell line using immunoblotting and reduced the live cell number in an MTT assay. Results of flow cytometry and HCS demonstrated that reducing the cell proliferation of RGC-5 cells, not cell death, led to the decreased level in the MTT assay. Our findings demonstrate that YC-1-induced down-regulation of HIF-1α might reduce RGC cell proliferation and viability under normoxia, which implies a role of YC-1 in the neuroprotective effect for further clinical applications.Molecular vision 06/2012; 18:1594-603. · 2.25 Impact Factor
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ABSTRACT: To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms. C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)-1α in the RPE-choroid was determined by Western blotting. Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF-1α (P<0.05). Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF-1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.PLoS ONE 12/2012; 7(12):e53329. DOI:10.1371/journal.pone.0053329 · 3.53 Impact Factor
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ABSTRACT: 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.Investigative ophthalmology & visual science 04/2009; 50(4):1873-9. DOI:10.1167/iovs.08-2591 · 3.66 Impact Factor