Suppressed Vascular Leakage and Myocardial Edema Improve Outcome From Myocardial Infarction

FIGURE 1. Improved cardiac function following MI in Vegfr2Y949F/Y949F mice. (A–C) Heart function assessed using echocardiography; (A) ejection fraction, (B) fractional area change (FAC), (C) cardiac output (CO) in Vegfr2Y949F/Y949F mice and WT littermates in baseline condition (n = 8–11 mice/strain), and at 24 h following myocardial infarction (n = 12–13 mice/strain, conducted at three different occasions). (D,E). Heart weights before infarction (D; n = 4 mice/strain) and induced infarct size at 24 h after ligation (E) (n = 12–13 mice/strain) in WT and Vegfr2Y949F/Y949F mice. Data in (A–E) are shown as mean ± SEM. Statistical significance was determined using Mann–Whitney test, *p < 0.05. (F) Survival after large infarct (n = 11–12/strain). Chi-squared test; *p < 0.05. For all panels, n refers to biological replicates.

FIGURE 2. Reduced edema and fibrinogen leakage in Vegfr2Y949F/Y949F MI hearts. (A) Left ventricular (LV) wall mass (mg) in WT and Vegfr2Y949F/Y949F hearts at baseline condition (n = 8–11 mice/strain) and 24 h after MI (n = 12–13 mice/strain). Mann–Whitney test, *p < 0.05. (B) Hematoxylin/Eosin (H&E) and fibrinogen immunofluorescent staining on total WT (top) or Y949F (bottom) heart sections. Scale bar, 1500 μm. The infarct area detected using echocardiography, is indicated by stippled lines in black or red. (C) Fibrinogen (gray) and CD31 (green) immunofluorescent staining of WT and Vegfr2Y949F/Y949F hearts at 3 days after MI; sections from the infarcted area. Scale bar, 100 μm. Hoechst (blue) shows nuclei. (D) Quantification of fibrinogen area normalized to CD31 area in randomly selected regions in total WT and Vegfr2Y949F/Y949F hearts at 3 days after MI (n = 17–21 fields/section from 6 to 7 individual hearts/strain and 2–3 sections/heart). Mann–Whitney test, *p < 0.05. For all panels, n refers to biological replicates.

FIGURE 3. Inflammation, neutrophil infiltration and MPO levels in conjunction with MI. (A) Immunostaining for CD45 (green) and CD31 (red) to visualize infiltrating leukocytes and the vasculature, respectively, in the infarcted areas of WT and Vegfr2Y949F/Y949F hearts at 3 days post-MI. DAPI (blue) shows nuclei. Scale bar, 100 μm. (B,C) Quantification of CD45-positive and CD31-positive area/field (n = 4–5 individual hearts/strain, 5 fields/section, and 3 sections/heart) in the infarcted part of the heart shows no significant differences using Mann–Whitney test. (D) Myeloperoxidase (MPO) protein levels in heart lysate measured using a multiplex inflammatory protein biomarker panel (n = 10 individual heart lysates/strain). Mann–Whitney test *p < 0.05. (E) Immunostaining for Ly6G (yellow) and CD45 (magenta) in the infarcted area of WT and Vegfr2Y949F/Y949F hearts at 3 days after MI. Hoechst (blue) shows nuclei. Scale bar, 100 μm. (F) Quantification of Ly6G area normalized to CD31 area in the infarcted part of the heart (n = 6–7 individual hearts/strain, 5 fields/section, and 3 sections/heart) shows no significant differences using Mann–Whitney test. For all panels, n refers to biological…

FIGURE 4. VEGF-induced VE-cadherin pY685 accumulation in hearts. (A) Immunoblot for pY1173 VEGFR2 and total VEGFR2, and pY685 VE-cadherin and total VE-cadherin on heart lysates from WT and Vegfr2Y949F/Y949F mice. Mice were injected with PBS or VEGFA in the tail-vein, followed by circulation for different time periods; PBS was injected and left to circulate for 1, 2 or 5 min (denoted P1, P2, P5). Alternatively VEGFA was injected and left to circulate for 1, 2, 5 min etc. (denoted V1, V2, V5 etc.). Heart lysates were immunoblotted for 3-phosphate dehydrogenase (GAPDH) as a loading control. (B) Quantification of pY685 VE-cadherin normalized to total VE-cadherin in samples shown in (A). (C) Immunoblotting for pY418 c-Src and total c-Src, pT308 Akt and total Akt, pT202/Y204 Erk1/2 and total Erk1/2. (D–F) Quantification of phosphorylated protein bands normalized to the corresponding total protein. n = 3; i.e., three independent replicates of experiments as shown in panel (A) with one mouse/time point for each strain and nine mice in total/strain for each experiment. Wilcoxon test, *p < 0.05.

FIGURE 5. VEGFA/VEGFR2 regulates edema and tissue damage in myocardial infarction. Activation of VEGFR2 on endothelial cells leads to phosphorylation on Y949 and subsequently, phosphorylation of VE-cadherin on Y685, leading to dismantling of adherens junctions and leakage, edema, neutrophil infiltration, MPO release, and tissue damage. Mutation of Y949 in VEGFR2 halts edema and improves survival after myocardial infarction.