Genetic Deletion of the LINC00520 Homolog in Mouse Aggravates Angiotensin II-Induced Hypertension

Figures

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  Figure 1. Leene-KO mice showed higher BP with AngII infusion. (A) Design of the Angiotensin-II (AngII)-induced hypertension (HTN) model: BP was measured every 3 days before and after osmotic pump implantation for 4 weeks. (B,C) Systolic and diastolic BP in 2-month-old mice with Ang II infusion (n = 8–9 mice per group). * p < 0.05 based on two-tailed Student’s t-test between WT and KO mice at the same time point. # p < 0.05 between WT and KO based on repeated-measures t-test. (D) Kaplan–Meier survival analysis in WT and KO mice receiving AngII (n = 24 vs. 38 per group). (E) Representative images of the heart obtained from AngII-treated WT and leene-KO littermates. (F,G) Heart weight and heart weight/body weight (mg/g) of WT and KO mice (n = 5–8 mice per group). Scale bar = 1 cm. Data are represented as mean ± SEM. * p < 0.05 between WT and KO based on Student’s t-test.

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  Figure 2. Leene-KO mice show impaired heart function in AngII model. (A) Representative M-mode images of echocardiography from WT and leene-KO mice with Ang II infusion on Day 27. (B–D) Fractional shortening (FS), ejection fraction (EF), and heart rate (n = 4–5 mice per group). (E,F) Representative images of H&E staining of heart. Quantification of the size of cardiomyocytes (n = 3 mice per group). (G–J) Representative images and quantification of Masson Trichrome staining (G,H) and immunohistochemical (IHC) staining of CD31 (I,J) in the heart. Three fields of view were randomly selected per animal, and three mice per group were quantified. (K) qPCR analysis of mRNA levels of genes as indicated in the heart, with 36B4 detected as the internal control (n = 3–6 mice per group). Scale bars = 100 μm (E,I) and 250 μm (G). Data are represented as mean ± SEM. * p < 0.05 based on two-tailed Student’s t-test.

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  Figure 3. Leene-KO mice show aggravated kidney damage in AngII model. (A,B) Representative images of H&E staining of kidney and quantification of glomerular size. (C,D) Representative images of PAS staining and quantification of positive PAS staining area in the kidney. (E,F) Representative images of Masson Trichrome staining and quantification of the positive staining area in the kidney. Three fields of view were randomly selected per mouse and five mice per group were analyzed. Scale bar = 50 μm (A,C,E). Data are represented as mean ± SEM. * p < 0.05 based on two-tailed Student’s t-test.

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  Figure 4. LEENE regulates pathways in ECs relevant to BP modulation. (A–D) WT and KO mice were infused with AngII for two weeks, and lung ECs were isolated from three mice per group for RNA-seq. (A,B) Top enriched biological pathways of upregulated (A) and downregulated (B) DEGs in lung ECs revealed by RNA-seq. (C) Heatmap showing the Z-scaled expression of select genes, including the upregulated DEGs involved in the immune and inflammatory pathways (indicated in red in A) and downregulated DEGs involved in flow response and atherosclerosis-related pathways (marked in green in B). (D) Downregulated DEGs involved in three select pathways. (E) qPCR analysis of indicated RNAs in lung ECs isolated from AngII-infused KO mice (for 2 weeks) and then treated with Ad-GFP/LEENE. (F,G) Aortic rings were isolated from AngII-infused WT mice and then treated with Ad-GFP/LEENE. Representative images are shown in (F), and qPCR analyses of indicated RNAs are shown in (G). n = three mice per group in (E,G). Scale bar = 1 mm. Data are represented as mean ± SEM. * p < 0.05 based on two-tailed Student’s t-test.

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  Figure 5. Effect of TKIs on LEENE levels in ECs. (A) Representative images of HUVECs treated with DMSO (vehicle), axitinib (1 µM) or lapatinib (5 µM) for 24 h. Scale bar = 100 μm. (B) qPCR analysis of indicated genes in HUVECs treated with DMSO, axitinib, or lapatinib. (C) qPCR analysis of indicated RNA level in WT aortic rings treated with DMSO, axitinib or lapatinib for 24 h (n = three mice per group). Data are represented as mean ± SEM from three independent experiments. * p < 0.05 between the indicated drug and DMSO group based on ANOVA followed by Tukey’s test.