Mechanistic insights into the role of EGLN3 in pulmonary vascular remodeling and endothelial dysfunction

Figures

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  Fig. 1. EGLN3 elevation in PAECs correlates with PAH severity. A Differentially expressed proteins in the GSE163827 data set, red represents up-regulated expression, green represents down-regulated expression B The lungs of mice with hypoxia-induced PH EGLN3 mRNA expression. C The lungs of rat with MCT-induced PH EGLN3 mRNA expression. D EGLN3 mRNA expression in PH patients' lungs. E The lungs of mice with hypoxia-induced PH EGLN3 protein expression. F The lungs of rat with MCT-induced PH EGLN3 protein expression. G EGLN3 protein expression in PH patients’ lungs. H Quantification of EGLN3 and VWF signaling localization in the pulmonary vasculature. Scale bar, 5 μm. I Quantification of EGLN3 levels in hypoxia-induced PH. J Quantification of EGLN3 levels in MCT-induced PAH. K and L The association between EGLN3 mRNA levels (normalized to β-actin) in lung tissues and RVSP during PH development is demonstrated by Pearson comparison analysis. *P < 0.05, **P < 0.01,.***P < 0.001

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  Fig. 2. Knockdown of EGLN3 reduces PAEC proliferation and migration. A Shows the expression levels of EGLN3 in PAECs subjected to various treatment conditions. B Confirms the efficiency of EGLN3 knockdown. C Presents the proliferation curves of cells across different treatment conditions. D Demonstrates the cell proliferation capabilities under these conditions, with a greater number of red dots indicating enhanced cell proliferation ability. Bar 200 μm. E Displays representative images of cell migration distances at 0 and 12 h across different treatment conditions. Bar 100 μm. F Provides representative images depicting the number of migrating cells under various treatment conditions. Bar 50 μm. *P < 0.05, **P < 0.01,.***P < 0.001

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  Fig. 3. EGLN3 regulates the PI3K/AKT and MEK/ERK signaling pathways. A RNA-seq technology is employed to detect differential mRNA expression between the hypoxia + shNC group and the hypoxia + shEGLN3 group. B Several biological processes are identified where differential proteins are predominantly enriched. C Differential proteins are primarily concentrated in specific cellular components. D Various signaling pathways are highlighted where differential proteins show significant enrichment. E Activation levels of the PI3K/AKT signaling pathway are compared across different groups. F The activation of the MEK/ERK signaling pathway is also assessed among the various groups. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 4. EGLN3 binds HUR to positively regulate the stability of EGFR. A The expression levels of various receptor tyrosine kinases (RTKs) mRNA following the knockdown of EGLN3 are presented. B The protein expression of EGFR post-EGLN3 knockdown is shown. C The efficiency of EGFR knockdown is verified. D The activation of the PI3K/AKT and MEK/ERK signaling pathways was assessed under different treatment conditions. E The effects of various treatment conditions on cell proliferation were evaluated. F Cell migration was measured following different treatment conditions. Bar 50 μm. G The expression of EGFR mRNA at various time points under distinct treatment conditions reflects the changes in EGFR mRNA stability. H An EGLN3 antibody was utilized to pull down the protein, allowing for the detection of HUR expression. I The HUR antibody was used to pull down the protein to assess the expression of EGLN3. J Under varying treatment conditions, HUR antibodies or IgG were employed to pull down the mRNA, followed by PCR amplification to detect EGFR mRNA expression. K The stability of EGFR mRNA was evaluated under different treatment conditions. *P < 0.05, **P < 0.01,.***P < 0.001

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  Fig. 5. JUN binds to the EGLN3 promoter region to promote EGLN3 transcription under hypoxic conditions. A The JASPAR and PROMO databases predict the transcription factors that can bind to the EGLN3 promoter region, and the intersection of these predictions is analyzed. B Following various treatment conditions, the activation of EGFR, EGLN3, and associated signaling pathways was assessed. C The DNA binding motif of the JUN protein is presented. D Sequences predicted to bind JUN within the EGLN3 promoter region are detailed. E Different vectors were constructed and co-transfected into endothelial cells, allowing for the detection of fluorescent protein expression in each experimental group. F The specific binding of EGLN3 to the promoter region was confirmed using CHIP-qPCR technology. *P < 0.05, **P < 0.01,.***P < 0.001

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  Fig. 6. Endothelium-specific knockout of EGLN3 alleviates the progression of PH. A The timeline for establishing the mouse model of PH is presented. B Verification of EGLN3 and EGFR protein expression levels in various mouse groups is shown. C Multiple staining of VWF and PCNA was conducted to assess the proliferation capability of PAECs across the different groups. Bar 20 μm. D HE staining, along with immunofluorescence staining, was employed to evaluate vascular remodeling among the groups. Bar 50 μm. E HE staining was used to assess right ventricular wall thickening across the different groups. Bar 1 mm. F and G Differences in RVSP and RVHI among the groups were measured. H and J Differences in the percentage of WT% and WA% among the groups were analyzed. J The distance traveled by mice in various groups on the treadmill was recorded to reflect their cardiac function.*P < 0.05, **P < 0.01,.***P < 0.001

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  Fig. 7. Overexpression of EGLN3 aggravates the progression of PH A The timeline for establishing the rat model of PH is presented. B Verification of EGLN3 and EGFR protein expression levels in various rat groups is shown. C Multiple staining of VWF and PCNA was conducted to assess the proliferation capability of PAECs across the different groups. Bar 20 μm. D HE staining, along with immunofluorescence staining, was employed to evaluate vascular remodeling among the groups. Bar 50 μm. E HE staining was used to assess right ventricular wall thickening across the different groups. Bar 1 mm. F and G Differences in RVSP and RVHI among the groups were measured. H and J Differences in the percentage of WT% and WA% among the groups were analyzed. J The distance traveled by rats in various groups on the treadmill was recorded to reflect their cardiac function.*P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 8. Graphical Abstract Under hypoxic conditions, JUN enhances the transcription of EGLN3, which subsequently binds to HUR to positively regulate the stability of EGFR mRNA. This interaction activates the PI3K/AKT and MEK/ERK signaling pathways, ultimately promoting the proliferation and migration of endothelial cells