Inhibition of Src attenuates Ang II-induced MLC phosphorylation in mesenteric vessels.

<p>After last BP measurements (at day 14) in mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127891#pone.0127891.g001" target="_blank">Fig 1</a>, the mice were euthanized, and their mesenteric vessels were isolated and subjected to Western blotting analyses for the phosphorylated Src and MLC. (<b>A</b>) Representative and (<b>B</b>) quantification of Western blotting analyses in mice treated with Vehicle (–), Ang II, SU6656, or Ang II+SU6656. The intensities of the phospho-protein bands were quantified densitometrically, normalized to the total proteins, and expressed as fold differences relative to the values in the Vehicle group (“SU6656[–] Ang II [–])”. n = 5/group; *p<0.05, **p<0.01, Ang II vs. Vehicle; # p<0.05, Ang II+SU6656 vs. Ang II.</p

A novel mechanism by which Ang II induces smooth muscle contraction.

<p>Interactions between Ang II and AT1R triggers SFK phosphorylation, which in turn, via tyrosine kinase activity and currently-unknown enzymatic substrates, results in MLCK phosphorylation at Serine 19 and activation of smooth muscle contractile machinery.</p

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

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

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

Ros increases circEPCs and promotes endothelial lineage differentiation.

<p>(<b>A</b>) C57BL/6 male mice received daily <i>s.c.</i> injections of different doses of Ros for 7 days. Twenty-four hours after last injection, PB MNCs were collected, and circEPCs were evaluated with a culture assay. One group received an intramuscular injection of 200 ug pVEGF165 plasmid as positive control. n = 4, **P<0.001 vs. Saline. (<b>B</b>) The kinetics of PB circEPCs within 24 h after <i>s.c.</i> injection of a single dose of Ros (0.1 mg/kg) or Sim (0.2 mg/kg). n = 4, ***P<0.001 vs. Saline. (<b>C–D</b>) Mice were <i>s.c.</i> injected with a mixture of 300 uL growth factor-reduced Matrigel and 2×10⁶ Tie2/LacZ BM MNCs and then s.c. injected with Ros (0.1 mg/kg) or saline daily for 5 days. The Matrigel plugs were then removed, fixed, and stained in X-gal solution. (<b>C</b>) Representative images. (<b>D</b>) β-gal positive cells were quantified and expressed as percentage of total cells, n = 4, **P<0.01 vs. Saline.</p

Ros increases BM-derived EPCs in neovascularization.

<p>BM MNCs isolated from Tie2/LacZ mice were used to transplant lethally irradiated syngeneic FVB/NJ mice. One month later, HLI was induced in the recipient mice, and Ros (0.1 mg/kg) was <i>s.c.</i> injected daily. (<b>A–C</b>) At day 14 after HLI, mice were injected with BS lectin and 10 min later, euthanized. The ischemic tissues were stained immunofluorescently with BS lectin (FITC, green) and β-gal (PE, red) to indicate vasculature and BM-derived cells, respectively. (<b>A</b>) Representative images of immunofluorescent double staining. White arrows indicate BS lectin and β-gal double positive cells. 400× original magnification. (<b>B</b>) Quantification of BM-derived EPCs incorporated in the neovasculature (i.e., double positive cells) in the ischemic limb. (<b>C</b>) Quantification of capillary densities (BS lectin-FITC positive cells). n = 6, *P<0.05, **P<0.01 vs. Saline. (<b>D</b>) Blood flow was monitored with LDPI at days 3, 5, 7, 10, 14, and 16 following HLI. *P<0.05, **P<0.01 vs. Saline.</p

Ros enhances ischemic neovascularization.

<p>(<b>A</b>) C57BL/6 male mice were rendered surgical hindlimb ischemia. Ros at low dose (0.1 mg/kg), high dose (5 mg/kg), or saline was injected daily, and blood flow recovery was monitored by LDPI at days 3, 5, 7, 14, 21, and 28 after the surgery. n = 8, *P<0.05, **P<0.01 vs. Saline. (<b>B</b>) Representative micrographs of BS lectin staining (<i>left panel</i>, 200× original magnification) and quantification of capillary densities (<i>right panel</i>) in the limb tissues at day 14. n = 8, **P<0.01 vs. Saline.</p

The Ros-mediated enhancement of EPC mobilization and neovascularization is dependent on eNOS expression.

<p>(<b>A–C</b>) eNOS^−/− mice were rendered surgical HLI and <i>s.c.</i> injected daily with Ros (0.1 mg/kg) or saline. (<b>A</b>) The rate of limb loss, n = 18. (<b>B</b>) Blood flow assessments with LDPI in mice without limb loss at day 7, 14, 28, and 42 after the surgery. (<b>C</b>) In a separate experiment, capillary density in the ischemic limb of day 14 was evaluated with BS lectin, n = 6. (<b>D</b>) eNOS^−/− mice received daily s.c. injections of Ros (0.1 mg/kg) for 7 d. PB MNCs were collected 24 h after the last injection, and circEPCs were assayed with the culture assay, n = 4. (<b>E</b>) EPCs were cultured from BM MNCs of WT mice for 7 days, and then treated with different doses of Ros for 30 min. The cells were lysed and the levels of phospho-Akt, Akt, phospho-eNOS, and eNOS were analyzed by Western blotting. <i>Left panel,</i> representative Western blotting. <i>Right panel</i>, the levels of phospho-Akt and phospho-eNOS were normalized to Akt and eNOS, respectively, and expressed relative to the values in saline-treated group. n = 4, *P<0.05, **P<0.01 vs. Saline.</p