Src contributes to the Ang II-induced MLC phosphorylation and contractility in human VSMCs.

<p>The human coronary artery SMCs in culture were treated with SU6656 (5 uM) or Vehicle (–) for 30 min, then stimulated with Ang II (0.1 nmol/L) or Vehicle (–) for additional 10 min. (<b>A</b>) Representative and (<b>B</b>) quantification of Western blotting analyses of phosphorylated Src and MLC. Levels of the phospho-proteins were normalized to total proteins and expressed as fold differences relative to the values in the Vehicle group. n = 5/group, **p<0.01, ***p<0.001, Ang II vs. Vehicle; #p<0.05, ##p<0.01, Ang II+SU6656 vs. Ang II. (<b>C</b>) Immunofluorescent staining of Actin. Results are representative of 3 independent experiments.</p

Inhibition of SFK lowers the BP level in Ang II-treated mice.

<p>Eight week-old male C57BL/6 mice were administered Ang II (1.4 mg/kg/d) or Vehicle via a subcutaneous minipump (14-day release), and starting from 13^th day, received daily i.p. injection of SU6656 (8 mg/kg/d) or Vehicle for 2 days. The systolic <b>(A)</b>, diastolic <b>(B)</b> and mean <b>(C)</b> BP were measured in mice by a tail-cuff method at day 0 (prior to Ang II treatment), day 12 (prior to SU6656 treatment), and day 14. n = 10 per group; **p<0.01, Ang II vs. Vehicle; #p<0.05, Ang II+SU6656 vs. Ang II.</p

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

Data_Sheet_1_Phosphatidylinositol-4,5-Bisphosphate Binding to Amphiphysin-II Modulates T-Tubule Remodeling: Implications for Heart Failure.docx

BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.</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