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RNA-Seq Provides Insights into VEGF-Induced Signaling in Human Retinal Microvascular Endothelial Cells: Implications in Retinopathy of Prematurity [RNA-seq_1]
The pathophysiology of retinopathy of prematurity (ROP) is postulated to first involve delayed intraretinal vascularization, followed by intravitreal neovascularization (IVNV). Although intravitreal agents that reduce the bioactivity of vascular endothelial growth factor (VEGF) are used to treat IVNV, concerns exist regarding their effects on intraretinal vascularization. In an experimental ROP model, VEGF receptor 2 (VEGFR2) knockdown in retinal endothelial cells reduced IVNV and promoted intraretinal vascularization, whereas knockdown of a downstream effector, signal transducer and activator of transcription 3 (STAT3) in retinal endothelial cells only reduced IVNV. In this study, we tested the hypothesis that the different pathways involved in VEGF-triggered VEGFR2 signaling and VEGF-triggered STAT3 signaling in retinal endothelial cells would allow us to delineate signaling pathways involved in IVNV from those involved in intraretinal vascularization in ROP. To address our hypothesis, we used RNA-sequencing and pathway enrichment analysis to determine changes in the transcriptome of cultured human retinal microvascular endothelial cells (HRMECs). Of the enriched pathways, inactivation of oncostatin M signaling was predicted by either KDR or STAT3 knockdown in the presence of VEGF. Activation of kinetochore metaphase signaling was predicted by KDR knockdown, whereas inactivation was predicted by STAT3 knockdown in the presence of VEGF. Inactivation of signaling by the Rho family of GTPases was predicted by KDR knockdown, but activation was predicted by STAT3 knockdown in the presence of VEGF. Taken together, our data identified unique signaling pathway differences between VEGF-triggered VEGFR2 and VEGF-triggered STAT3 in HRMECs that might have implications in ROP. Overall design: Human retinal microvascular endothelial cells (HRMECs, Cell Systems, Krikland, WA, USA), passages 3–5, were cultured in attachment factor (Cell Systems) coated cultureware with the Endothelial Growth Medium BulletKit (EGM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS). Cultured HRMECs were transfected with equal concentrations of control small interfering RNA (siRNA) or KDR siRNA oligos (Thermo Fisher Scientific, Waltham, MA, USA) using Targefect Solution A and Virofect (Targeting Systems, El Cajon, CA, USA). Twenty-four hours after transfection, HRMECs were serum starved in Endothelial Basal Media (EBM, Lonza) for eight hours and then treated with either VEGF (25 ng/mL, R&D Systems, Minneapolis, MN, USA) or an equal volume of phosphate-buffered saline (PBS) as vehicle control for four hours. Overall, this study had four experimental groups and 3 biological replicates per group for a total of 12 samples from one independent experiment.
RNA-Seq Provides Insights into VEGF-Induced Signaling in Human Retinal Microvascular Endothelial Cells: Implications in Retinopathy of Prematurity [RNA-seq_2]
The pathophysiology of retinopathy of prematurity (ROP) is postulated to first involve delayed intraretinal vascularization, followed by intravitreal neovascularization (IVNV). Although intravitreal agents that reduce the bioactivity of vascular endothelial growth factor (VEGF) are used to treat IVNV, concerns exist regarding their effects on intraretinal vascularization. In an experimental ROP model, VEGF receptor 2 (VEGFR2) knockdown in retinal endothelial cells reduced IVNV and promoted intraretinal vascularization, whereas knockdown of a downstream effector, signal transducer and activator of transcription 3 (STAT3) in retinal endothelial cells only reduced IVNV. In this study, we tested the hypothesis that the different pathways involved in VEGF-triggered VEGFR2 signaling and VEGF-triggered STAT3 signaling in retinal endothelial cells would allow us to delineate signaling pathways involved in IVNV from those involved in intraretinal vascularization in ROP. To address our hypothesis, we used RNA-sequencing and pathway enrichment analysis to determine changes in the transcriptome of cultured human retinal microvascular endothelial cells (HRMECs). Of the enriched pathways, inactivation of oncostatin M signaling was predicted by either KDR or STAT3 knockdown in the presence of VEGF. Activation of kinetochore metaphase signaling was predicted by KDR knockdown, whereas inactivation was predicted by STAT3 knockdown in the presence of VEGF. Inactivation of signaling by the Rho family of GTPases was predicted by KDR knockdown, but activation was predicted by STAT3 knockdown in the presence of VEGF. Taken together, our data identified unique signaling pathway differences between VEGF-triggered VEGFR2 and VEGF-triggered STAT3 in HRMECs that might have implications in ROP. Overall design: Human retinal microvascular endothelial cells (HRMECs, Cell Systems, Krikland, WA, USA), passages 3–5, were cultured in attachment factor (Cell Systems) coated cultureware with the Endothelial Growth Medium BulletKit (EGM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS). Cultured HRMECs were transfected with equal concentrations of control small interfering RNA (siRNA) or STAT3 siRNA oligos (Thermo Fisher Scientific, Waltham, MA, USA) using Targefect Solution A and Virofect (Targeting Systems, El Cajon, CA, USA). Twenty-four hours after transfection, HRMECs were serum starved in Endothelial Basal Media (EBM, Lonza) for eight hours and then treated with either VEGF (25 ng/mL, R&D Systems, Minneapolis, MN, USA) or an equal volume of phosphate-buffered saline (PBS) as vehicle control for four hours. Overall, this study had four experimental groups and 3 biological replicates per group for a total of 12 samples from one independent experiment.
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