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. 2023 Aug 31;43(10):1967–1989. doi: 10.1161/ATVBAHA.123.319925

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© 2023 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 3. — The impact of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) on endothelial function and mesenchymal activation. A, Effects of hnRNP H1 knockdown on endothelial and mesenchymal marker expression. hnRNP H1 was knocked down by siRNA transfection (48 hours) followed by RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR) analysis of selected marker gene expression. Expression normalized to Gapdh, n=3, each in triplicate, data shown as average±SEM. Normality was tested by a Shapiro-Wilk test, significance of normally distributed data was assessed by unpaired Student t test (transforming growth factor [TGF]-β2 expression was tested by Mann-Whitney U test). Significance values of <0.05 are shown as 3 significant figures. B, Effects of Csde1 knockdown on endothelial and mesenchymal marker expression. Csde1 was knocked down by siRNA transfection (48 hours) followed by RNA isolation and RT-qPCR analysis of selected marker gene expression. Expression normalized to Gapdh, n=3, each in triplicate, data shown as average±SEM. Normality was tested by a Shapiro-Wilk test, significance of normally distributed data were assessed by unpaired Student t test (Sma and Col5a1 expression was tested by Mann-Whitney U test). Significance values of <0.05 are shown to 3 significant figures. C, Effects of hnRNP H1 and Csde1 knockdown on tubule formation. hnRNP H1 and Csde1 were knocked down (siRNA, 48 hours) in increasing concentrations of TGF-β (24 hours). Cells were plated onto Matrigel matrix for 24 hours; BCECF was added and tubule formation was assessed by fluorescent microscopy. Quantifications represent average total tubule length and number of complete loops per visible field. Representative images (n=3 triplicates/condition, scale bar 100 µm). Data shown as average±SEM, normality was assessed by Shapiro-Wilk test, and significance was assessed by 1-way ANOVA with Dunnett test for multiple comparisons. Significance values shown to 3 significant figures. D, Effects of hnRNP H1 and Csde1 on endothelial cell migration. Mouse cardiac endothelial cells (MCECs) were incubated with si-hnRNP H1 or siCsde1 for 48 hours in the presence and absence of TGF-β stimulation (10 ng/mL, 24 hours). Migration was assessed 24 hours after scratching. Representative images, n=3 triplicates/condition, data shown as average±SEM, normality was assessed by Shapiro-Wilk test, and significance assessed by 1-way ANOVA with Dunnett test for multiple comparisons. Significance values shown to 3 significant figures. E, Effects of hnRNP H1 and Csde1 knockdown on LDL (low-density lipoprotein) uptake. hnRNP H1 and Csde1 were knocked down (siRNA, 48 hours)±TGF-β stimulation (10 ng/mL, 24 hours). Cells were incubated in fluorescently labeled LDL and uptake was assessed by fluorescence microscopy. Representative images (n=3, scale bar 50 µm). Data shown as average±SEM. n=3 (10 quantifications per replicate), normality was assessed by Shapiro-Wilk test, and significance was assessed by 1-way ANOVA with Dunnett test for multiple comparisons. Significance values are shown to 3 significant figures. F, Effects of hnRNP H1 and Csde1 knockdown on Smad2/3 phosphorylation. MCECs were incubated with si-hnRNP H1 or siCsde1 for 48 hours in the presence and absence of TGF-β stimulation (10 ng/mL, 24 hours). Smad 2/3 phosphorylation was assessed by Western blot (relative to total Smad 2/3 expression). Representative blot. n=3, data shown as average±SEM. Normality was assessed by Shapiro-Wilk test, and significance was assessed by 1-way ANOVA with Dunnett test for multiple comparisons (no significance). G, Validation of TGF-β driven changes in RNA-binding in primary human cardiac microvascular endothelial cells (HCMECs). HCMECs were incubated with TGF-β (10 ng/mL) for 24 hours followed by RNA interactome capture (RIC) and Western blot analysis. RBPs (RNA-binding proteins) abundance was normalized to input lysate for each replicate. n=3, data shown as average±SEM, normality was confirmed by Shapiro-Wilk test and significance assessed by unpaired Student t test, significance values are shown to 3 significant figures. H, Validation of changes in mesenchymal maker gene expression after hnRNP H1 and Csde1 knockdown in HCMECs. hnRNP H1 and Csde1 were knocked down in HCMECs by siRNA transfection (48 hours) followed by RNA isolation and RT-qPCR analysis of selected marker gene expression. n=3, each in triplicate, data were tested for normality by a Shapiro-Wilk test and significance assessed by 1-way ANOVA with Dunnett multiple comparison test, data shown as average±SEM, significance values of <0.05 are indicated to 3 significant figures. BCECF indicates 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; siControl, control siRNA; siCsde1, siRNA targeting Csde1; sihnRNP H1, siRNA targeting hnRNP H1; siRNA, small interfering RNA; Smad, suppressor of mothers against decapentaplegic; and pSmad, phosphorylated-Smad.