Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711
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Browsing Eye / Vision by Author "He, Shaoqing"
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Item Endothelin-1 Induced the Reactivation of Primary Rat Ocular Astrocytes(2016-03-23) Ma, Hai-Ying; Park, Yong; Wang, Junming; Yorio, Thomas; He, ShaoqingPurposes: Astrocytes play a crucial role in cell survival and axon function of retinal ganglion cells (RGCs) by providing the structural support to neurons, secreting neurotrophic factors to regulate apoptosis and maintenance of the extracellular milieu. Endothelin-1(ET-1) and its receptors are found to be involved in the etiology of glaucoma. However, ET-mediated reactivation of astrocytes affecting RGC survival is still not fully understood. This study aimed to investigating the mechanisms by which ET-1 promotes the reactivation of primary rat ocular astrocytes. Methods: The primary astrocytes were isolated from retinas and optic nerve of rats. Immunostaining of glial fibrillary acid protein (GFAP), RNA binding protein with multiple splicing (RBPMS) and alpha smooth muscle actin (α-SMA) was performed on the cultured primary astrocytes to identify the purity of cells. The cultured primary astrocytes were treated with 100nM endothelin-1 for 24 hours followed the protein detection using Western Blot. ET-1-mediated influx of calcium was monitored in astrocytes using Fura-2 AM calcium imaging. Results: GFAP was uniformly stained on the primary astrocytes, and no staining of RBPMS and α-SMA was identified, whereas the staining of α-SMA was identified in NIH3T3 fibroblast cells. The treatment of ET-1 and ET-3 induced the upregulation of GFAP, neural cell adhesion molecule (NCAM), c-Jun, c-Jun N-terminal kinase (JNK) and Ki67 (a protein marker of cell proliferation). Administration of SP600125, an inhibitor of JNK, attenuated the increased GFAP induced by ET-1 in astrocytes. However, BQ788, an antagonist of ETB receptor, didn’t inhibit ET-1-mediated upregulation of GFAP. In addition, ET-1 triggered augment of intracellular calcium in the primary astrocytes, whereas the application of verapamil, an L-type calcium channel blocker, inhibited the influx of calcium. Conclusions: The hallmark of reactive astrocytes, GFAP, is tightly regulated in astrocytes. An increase in protein levels of GFAP and Ki67 induced by ET-1 reflected the reactivation of astrocytes. Meanwhile, other proteins were also found to be upregulated, such as NCAM, c-Jun and JNK. In addition, intracellular of calcium was also promoted with ET-1 treatment. Taken together, the results suggest that calcium-mediated signaling and JNK/c-Jun pathway are involved in reactivation of astrocytes. This reactivation could lead to dysfunction in the optic nerve and affect RGC survival.Item Overexpression of Endothelin A and B Receptors Enhances Calcium Mobilization in Ocular Astrocytes and Ciliary Epithelial Cells(2016-03-23) He, Shaoqing; Ma, Hai-Ying; Park, Yong; Yorio, Thomas; Broyles, Heather V.Purpose: Endothelin-1 (ET-1), a vasoactive peptide, binds ETA receptor and ETB receptor to exert its role in multiple cellular processes. A growing body of evidence suggests that elevated levels of ET-1 and activation of its receptors contributes to neurodegeneration in glaucoma, where reactive astrocytes are found to induce damage of retinal ganglion cells. Overexpression of c-Jun, a transcription factor, has been shown to increase levels of ETB receptor, suggesting that the expression of ETB receptor is regulated by c-Jun. This study attempts to determine if overexpression of ET-1 receptors affect calcium influx in response to ET-1 treatment. Methods: Primary astrocytes were isolated from retina and optic nerve of rat pups postnatal 4-7 days. Calcium imaging using Fura-2-AM fluorescent dye was used to determine calcium influx following treatment of ET-1, in the presence and absence of BQ610 (ETA selective antagonist), or BQ788 (ETB selective antagonist) or no treatment (control). ETA, ETB and c-Jun were also overexpressed in Human Non-Pigmented Epithelial (HNPE) cells using DNA transfection and calcium mobilization was measured. Results: Overexpression of ETA or ETB in HNPE cells significantly increased [Ca2+]i levels compared to control following ET-1 treatment at p2+]i in primary astrocytes. Treatment with either BQ610 or BQ788 in primary astrocytes significantly (p2+]i levels compared to control following ET-1 treatment. Conclusion: This study demonstrated that ETA and ETB can mediate calcium influx in HPNE cells and primary astrocytes. ETA receptor stimulation produced a similar calcium influx in HPNE cells as ETB receptor activation, suggesting that both receptors may be involved in [Ca2+]i signaling. The increase in calcium can result in activation of cell death pathways that may explain the ET-1 neurodegenerative actions.