AAA formation is altered in the presence of a TAFI-inhibitor.

<p>Mice were treated with saline control, NaCl 0.9%, (n = 23), Ang II (n = 35), Ang II and MA-TCK26D6 (n = 12) or Ang II and UK-396082 (n = 12) for 28 days. Panel A Incidence of AAA in each group. Panel B AAA size. Data is shown as mean±standard deviation. * p<0.05 compared to Angiotensin II alone by Chi-Squared testing.</p

Aortic distensibility decreases with AAA formation, but is not affected by TAFI-inhibition, in the Angiotensin II model of AAA.

<p>The aorta was imaged in longitudinal section using the Vevo2100 scanner, and the distensibility measured using ECG-gated images and VevoVasc software. Panel A shows aortic wall distensibility in all mice with AAA (MA-TCK26D6 treated and sham treated). One week following initiation of Ang II infusion, mice received either MA-TCK26D6 or control (NaCl 0.9%) as an IV injection via the femoral vein. Panel B and C There was no difference in the change of distensibility by week 3 in mice receiving treatment with MA-TCK26D6 at 1 week compared with controls (data shown is change in distensibility between week 1 and week 3). Data is shown as mean±standard deviation, *** p = 0.001 compared to baseline by student t-test.</p

The effect of TAFI inhibition on mortality in the Angiotensin II model of AAA.

<p>Panel A and B Blood pressure (BP) and heart rate (HR) measurements taken in all groups of mice in the second week of the experimental period using the CODA non-invasive BP device. Panel C and D Mortality of mice treated with Ang II, compared with NaCl 0.9% or Ang II plus MA-TCK26D6 or UK-396082. Data is shown as mean±standard deviation. Mortality in this model of AAA typically occurred early (Days 3–8 post initiation of Ang II infusion). * p<0.05 compared to Angiotensin II alone by Chi-Squared testing.</p

There is no effect of TAFI inhibition of the growth of an established AAA in the Angiotensin II model.

<p>AAA were induced in hyperlipidaemic mice by infusion of Ang II 750 ng/kg/min. After 1 week, mice were treated with a single injection of either MA-TCK26D6 or NaCl control. AAA progression in both groups was evaluated at 2 weeks post injection using Vevo2100 pre-clinical ultrasound scanning, and 3D reconstructions of the aortic segment at risk of AAA formation were created. Panel A Aortic diameter, Panel B Aortic volume, Panel C The process of creating the 3D aortic reconstruction. Panel D Example 3D reconstruction showing AAA progression over time (0, 1 and 3 weeks post Angiotensin II infusion). Data is shown as mean±standard deviation.</p

A Heat-Shock Protein Axis Regulates VEGFR2 Proteolysis, Blood Vessel Development and Repair

<div><p>Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration <em>in vitro</em>. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair <em>in vivo</em> consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.</p> </div

HSP90 inhibition perturbs mouse arterial repair and endothelial cell migration.

<p>(A) A representative view of femoral artery re-endothelialization in control mice (DMSO treated). (B) A representative view of femoral artery re-endothelialization in geldanamycin-treated mice. (C) Quantification of arterial re-endothelialization (blood vessel repair) using Evans Blue staining. Error bars denote ±SEM (n = 5), **<i>p</i><0.01 using Student’s t-test. (D) Quantification of HUVEC migration across a growth factor gradient in the presence of either VEGF-A or basic FGF. Error bars denote ±SEM (n = 3), **<i>p</i><0.01 using one-way ANOVA.</p

HSP70 requirement for geldanamycin-stimulated VEGFR2 degradation.

<p>(A) HUVECs pre-treated with siRNA duplex ‘a’ to HSP70 alone or with geldanamycin (4 h) were analyzed by immunoblotting (IB) for proteins as indicated. Arrowhead indicates mature VEGFR2. (B) Quantification of mature VEGFR2 levels under conditions in panel A. (C) Quantification of HSP70 levels in HUVECs pre-treated with either of two different synthetic siRNA duplexes ‘a’ or ‘b’ to HSP70±geldanamycin. (D) Quantification of VEGFR2 levels in HSP70-depleted HUVECs (duplex ‘a’ or ‘b’) ±geldanamycin (4 h). Error bars denote ±SEM (n≥3), *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005 using one-way ANOVA. (E) HUVECs pre-treated with siRNA duplexes for HSP70 or control siRNA were treated ±geldanamycin (4 h) ±VEGF-A (5 min) and analyzed for signaling events by immunoblotting. (F) Quantification of phospho-PLCγ1 levels from panel E. Error bars denote ±SEM (n≥3), *<i>p</i><0.05 using one-way ANOVA.</p

HSP70 overexpression and co-distribution with VEGFR2.

<p>(A) Lentiviral transduced HUVECs expressing human HSP70-FLAG were labeled for VEGFR2 (green) and HSP70-FLAG (red), which were detected using labeled secondary antibodies and co-distribution shown (yellow). Bar, 10 µm. (B) Quantification of VEGFR2 co-distribution with HSP70-FLAG under control or HSP70 overexpression conditions. Error bars denote ±SEM (n = 15), ***<i>p</i><0.005 using Student’s t-test. (C) HEK-293T cells were transfected with VEGFR2 and/or HSP70-FLAG and processed for immunoblotting (IB) using anti-VEGFR2 antibody. Arrowhead denotes mature transfected VEGFR2. (D) HUVECs were treated ±geldanamycin (4 h) and immunoprecipitation (IP) carried out for control protein (Goat IgG) and VEGFR2 as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048539#pone-0048539-g002" target="_blank">Figure 2</a>. Isolated protein complexes were processed for immunoblotting (IB) using anti-VEGFR2 and anti-CHIP antibodies. Arrrowhead denotes mature VEGFR2. (E) HUVECs were treated with either control siRNA or siRNA duplex targeted against CHIP and treated ±geldanamycin (4 h). Cells were lysed and processed for imunoblotting (IB). Representative immunoblots shown. CHIP, carboxy terminus of HSP70-interacting protein.</p

Inhibition of HSP90 stimulates HSP70-VEGFR2 interaction.

<p>(A) HUVECs pre-treated with geldanamycin for 4 h were lysed and control protein (goat IgG) or VEGFR2 immuno-isolated complexes (IP) were analyzed by immunoblotting (IB) for HSP70 or HSP90β. Arrowhead indicates mature VEGFR2. (B) Quantification of HSP70 and HSP90β association with VEGFR2 under control conditions without (−) or with (+) geldanamycin; error bars denote ±SEM (n = 6), ***<i>p</i><0.005 using one-way ANOVA.</p

Clathrin-regulated endocytosis is a requirement for geldanamycin-stimulated VEGFR2 degradation.

<p>(A) HUVECs subjected to CHC17 knockdown were followed by geldanamycin treatment (4 h) and immunoblot analysis (IB) of indicated proteins. (B) Quantification of mature VEGFR2 levels from the experiments shown in panel A. Error bars denote ±SEM (n≥3), *<i>p</i><0.05 using one-way ANOVA. CHC17, clathrin heavy chain.</p