The Ablation of VEGFR-1 Signaling Promotes Pressure Overload-Induced Cardiac Dysfunction and Sudden Death

Figures

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  Figure 1. VEGFR-1 signaling regulates the early stage of compensatory LVH. Progression of LVH as followed by echocardiography. (A) Representative short axis view images of LVs at day 0, 6 and 14. (B) LV ejection fraction (EF; n = 6–13/group). (C) LV anterior wall (LVAW) thickness (n = 6–13/group). (D). LV end-systolic diameter (LVESD; n = 6–13/group). (E) LV end-diastolic diameter (LVEDD; n = 6–13/group). (F) LV systolic volume (LV Vol;s; n = 6–13/group). (G) Aortic root diameter (n = 6–13/group). Values are mean ± SEM. Statistical analyses were performed using one-way ANOVA with Bonferroni’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

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  Figure 2. VEGFR-1 TK−/− mice show significantly increased mortality. Mortality of VEGFR-1 TK−/− and control mice during 14-day follow-up after angII infusion (n = 10–13/group). Values are mean ± SEM. Statistical analysis was performed using Kaplan–Mayer with log-rank test for survival curve. *** p < 0.001; ** p < 0.01; * p < 0.05.

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  Figure 3. Deletion of VEGFR-1 signaling leads to QRSp prolongation after pressure overload. (A) Representative electrocardiographs of baseline and day 14 of angII infusion. (B) Electrocardiographic measurements during baseline and LVH (n = 7–13/group). Values are mean ± SEM. Statistical analyses were performed using Student´s t-test. *** p < 0.001; ** p < 0.01; * p < 0.05. Amp indicates amplitude; BPM, beats per minute; HR, heart rate; ms, millisecond; mV millivolt.

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  Figure 4. VEGFR-1 TK−/− mice display cardiomyocyte hypertrophy at day 14. (A) Haematoxylin & Eosin (scale bar is 1000 µm) and laminin (scale bar is 25 µm) stainings of the myocardium at day 14 of angII infusion. (B) Quantification of cardiomyocyte area (n = 8–10/group). (C) LV mass measured with echocardiogram (n = 7–11/group). (D) Relative ANP mRNA expression measured with RT qPCR (n = 5–7/group). (E) Relative BNP expression measured with RT qPCR (n = 5–7/group). (F) Masson’s staining of the myocardium (scale bar is 250 µm). (G) Quantification of the level of fibrosis in the ventricles (n = 6–9/group). (H) Quantification of the level of septal fibrosis (n = 6–9/group). (I) Sirius Red staining of the perivascular fibrosis (scale bar is 100 µm). (J) Quantification of the level of perivascular fibrosis (n = 6–9/group). (K) Relative mRNA expression of Col3a1 measured with RT qPCR (n = 5–7/group). Values are mean ± SEM. Statistical analyses were performed using one-way ANOVA with Bonferroni’s post hoc test and Student´s t-test. *** p < 0.001; ** p < 0.01; * p < 0.05.

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  Figure 5. VEGFR-1 TK signaling does not affect angiogenesis. (A) Representative images of lectin staining of myocardial sections at day 14 of angII infusion (scale bar is 50 µm). (B) Quantification of capillary density in LV (n = 7–9/group). (C) Quantification of capillary area (n = 7–9/group). (D) Representative images of staining for arteries (αSMA; scale bar is 100 µm). (E) Quantification of arterial vessel density (n = 8–10/group). (F) Quantification of αSMA-positive vessel area (n = 8–10/group). (G) Representative images of staining for CD31 to visualise capillary tree at baseline. (H) Quantification of capillary area (n = 6–9/group). (I) Quantification of apoptotic cells at day 14 of angII infusion (n = 8–10/group). Values are mean ± SEM. Statistical analyses were performed using Students t-test. *** p < 0.001; ** p < 0.01; * p < 0.05.

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  Figure 6. LVH increases VEGFR-1 expression in the myocardium. (A) Representative images of VEGFR-1 staining of the myocardium at baseline and after 14-day angII infusion. (B) Relative VEGFR-1 and VEGF-B expression measured with RT qPCR (n = 5–7/group). (C) RNA sequencing of LVs at day 14 of angII infusion. Volcano plot in which red represents significance in VEGFR-1 TK−/− mice compared to littermate controls with the use of a log fold change (logFC) >0.585 and false discovery rate (FDR) value of <0.10 (n = 4/group). (D) Word cloud presentation of differentially regulated genes found in RNA sequencing. Upregulated genes are marked with green and downregulated genes with a red colour. Larger word size correlates with higher fold change (n = 4/group). (E) Western blot analysis of LVH signaling cascades at day 14 of angII infusion. Quantification of VEGFR-2 (F), VEGF-A (G) phosphorylated ERK1/2 (H), Akt (I), mTOR (J) and PKCα (K) expression in LVs (n = 4–5/group). Values are mean ± SEM. Statistical analyses were performed using one-way ANOVA and Student’s t-test. *** p < 0.001; **p < 0.01; * p < 0.05.