Contrasting roles of E2F2 and E2F3 in cardiac neovascularization.

Insufficient neovascularization, characterized by poor endothelial cell (EC) growth, contributes to the pathogenesis of ischemic heart disease and limits cardiac tissue preservation and regeneration. The E2F family of transcription factors are critical regulators of the genes responsible for cell-cycle progression and growth; however, the specific roles of individual E2Fs in ECs are not well understood. Here we investigated the roles of E2F2 and E2F3 in EC growth, angiogenesis, and their functional impact on myocardial infarction (MI). An endothelial-specific E2F3-deficient mouse strain VE-Cre; E2F3(fl/fl) was generated, and MI was surgically induced in VE-Cre; E2F3(fl/fl) and E2F2-null (E2F2 KO) mice and their wild-type (WT) littermates, VE-Cre; E2F3(+/+) and E2F2 WT, respectively. The cardiac function, infarct size, and vascular density were significantly better in E2F2 KO mice and significantly worse in VE-Cre; E2F3(fl/fl) mice than in their WT littermates. The loss of E2F2 expression was associated with an increase in the proliferation of ECs both in vivo and in vitro, while the loss of E2F3 expression led to declines in EC proliferation. Thus, E2F3 promotes while E2F2 suppresses ischemic cardiac repair through corresponding changes in EC proliferation; and differential targeting of specific E2F members may provide a novel strategy for therapeutic angiogenesis of ischemic heart disease

The vascular density at infarct border zone is greater in E2F2 KO mice and lower in endothelial E2F3 KO mice than in their WT littermates.

<p>Blood vessels were stained with BS lectin 1 (green), and nuclei were counterstained with DAPI (blue). (<b>A</b>) Representative immunofluorescence images. (<b>B</b>) Quantification of vascular density at the infarct border zone. n = 12 mice per group; *P<0.05 vs. VE-Cre; E2F3^+/+, ^#P<0.05 vs. E2F2 WT; HPF, high power field.</p

Functional recovery of the infarcted heart is enhanced by the loss of E2F2 expression and impaired by the loss of endothelial E2F3 expression.

<p>MI was surgically induced in VE-Cre; E2F3^fl/fl and E2F2 KO mice and their WT littermates, VE-Cre; E2F3^+/+ and E2F2 WT, respectively, and the heart function was assessed with echocardiography at the indicated time points for (<b>A</b>) LV ejection fraction, (<b>B</b>) fractional shortening, (<b>C</b>) end-systolic and (<b>D</b>) end-diastolic volumes. n = 12 mice per group. *P<0.05 vs. VE-Cre; E2F3^+/+, ^#P<0.05 vs. E2F2 WT.</p

The infarct size is smaller in E2F2 KO mice and larger in endothelial specific E2F3 KO mice than in their WT littermates.

<p>Masson Trichrome staining was performed in heart samples 28 days after MI surgery. (<b>A</b>) Representative microphotographs and (<b>B</b>) Quantification of the infarct size. n = 12 mice per group; *P<0.05 versus VE-Cre; E2F3^+/+, ^#P<0.05 versus E2F2 WT; Scale bar = 100 µm.</p

Proliferation is enhanced in E2F2 KO ECs and impaired in E2F3-deleted ECs.

<p><b>(A–B)</b> Immunofluorescent double staining was performed in the ischemic heart sections for CD31 (green) and BrdU (red) to identify ECs (green), proliferating cells (red), and proliferating ECs (yellow). (<b>A</b>) Representative immunofluorescence images and (<b>B</b>) quantification of proliferating ECs in the infarct border zone. n = 12 mice per group; *P<0.05 vs. VE-Cre; E2F3^+/+, ^#P<0.05 vs. E2F2 WT; HPF, high power field. (<b>C</b>) Primary ECs were isolated from the hearts of E2F2 KO, E2F2 WT, and E2F3^fl/fl mice, and the E2F3^fl/fl cells were subsequently transduced with Adenovirus-Cre/GFP or Adenovirus-GFP. EdU incorporation based flow cytometry analyses were performed to assess DNA synthesis. Shown is representative of 3 independent experiments.</p

Supplementary Tables S1 - S5 and Figure S1 from Incubation behaviours of oviraptorosaur dinosaurs in relation to body size

Table S1: Mid- to Upper Cretaceous oviraptorosaur egg and clutch specimens examined in this study; Table S2: Results of regression analyses for clutch volume (mm3, in logarithmic scale) against adult body mass (kg, in logarithmic scale) in living crocodylian and precocial bird species (n = 180) in order to predict adult body mass for the elongatoolithid specimens; Table S3: Outer and inner clutch diameters of various oviraptorosaur egg taxa; Table S4: Eggshell thickness of various oviraptorosaur egg taxa; Table S5: Eggshell porosity and predicted nest type in various oviraptorosaur egg taxa; Figure S1. Comparisons of egg shape in oviraptorosaurs