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Published on: April 7, 2014
Knockout of αA-crystallin inhibits ocular neovascularization
Qiong Xu1, Yujing Bai1, Lvzhen Huang1
1Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
Purpose:
The present study aimed to investigate the effects of αA-crystallin on pathologic ocular neovascularization.
Methods:
Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and αA-crystallin-knockout (CRYAA[-/-]) mice were used for the in vivo study. αA-crystallin was knocked down in HUVECs by using a specific small interfering RNA (siRNA), and the effects of αA-crystallin knockdown on proliferation, migration, tube formation, and apoptosis were evaluated. Enzyme-linked immunosorbent assays were performed to investigate extracellular concentrations of vascular endothelial growth factor (VEGF). In vivo mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) were generated by using CRYAA(-/-) mice. The nonperfused area in the OIR model was measured in flat-mounted retinas, and angiogenesis resulting in CNV was evaluated in retinal sections. Western blot analysis was performed to investigate the phosphorylation status of vascular endothelial growth factor receptor 2 (VEGFR2, high affinity receptor for VEGF), AKT, PLCγ1, FAK, Src, p42/p44MAPK, p38MAPK, caspase-3, and caspase-9 in cultured HUVECs, as well as in the OIR and CNV models.
Results:
The CRYAA siRNA not only induced HUVEC apoptosis but also inhibited exogenous and endogenous VEGF-induced cell activities, including proliferation, migration, and tube formation. Pathologic neovascularization was attenuated in the CRYAA(-/-) murine OIR and CNV models. Both in vitro and in vivo, the inhibition of angiogenesis was mediated by the suppression of VEGF secretion and the inhibition of the VEGFR2 signaling pathway; VEGFR2, AKT, PLCγ1, FAK, Src, p42/p44 MAPK, and p38 MAPK all showed reduced phosphorylation levels. In addition, CRYAA knockout led to increased levels of cleaved caspase-9 and caspase-3.
Conclusions:
Knockout of αA-crystallin inhibited pathologic neovascularization through the VEGF and VEGFR2 signaling pathways both in vitro and in vivo. These results suggest that αA-crystallin could be a novel pharmaceutical target for the prevention of ocular neovascularization.
Insights
Knocking out alphaA-crystallin (αA-crystallin) reduces pathologic ocular neovascularization by inhibiting vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) signaling. This suggests αA-crystallin is a potential therapeutic target for eye diseases.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Pathologic ocular neovascularization is a major cause of vision loss.
- AlphaA-crystallin (αA-crystallin) is a structural protein in the eye lens, but its role in ocular neovascularization is unclear.
Purpose of the Study:
- To investigate the role of αA-crystallin in pathologic ocular neovascularization.
- To determine the molecular mechanisms by which αA-crystallin influences angiogenesis.
Main Methods:
- In vitro studies using human umbilical vein endothelial cells (HUVECs) with αA-crystallin knockdown via siRNA.
- In vivo studies using αA-crystallin-knockout (CRYAA[-/-]) mice in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models.
- Assays included cell proliferation, migration, tube formation, apoptosis, ELISA for VEGF, and Western blot analysis for signaling pathway components.
Main Results:
- αA-crystallin knockdown in HUVECs inhibited proliferation, migration, and tube formation, while promoting apoptosis.
- CRYAA(-/-) mice showed attenuated neovascularization in OIR and CNV models.
- Inhibition of angiogenesis was linked to reduced VEGF secretion and suppressed VEGFR2 signaling pathway, including decreased phosphorylation of VEGFR2, AKT, PLCγ1, FAK, Src, p42/44MAPK, and p38MAPK. CRYAA knockout also increased cleaved caspase-9 and caspase-3 levels.
Conclusions:
- αA-crystallin knockout inhibits pathologic ocular neovascularization in vitro and in vivo.
- The mechanism involves the suppression of VEGF and the VEGFR2 signaling pathway.
- αA-crystallin represents a potential novel therapeutic target for preventing ocular neovascularization.
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