Egln1Tie2Cre Mice Exhibit Similar Therapeutic Responses to Sildenafil, Ambrisentan, and Treprostinil as Pulmonary Arterial Hypertension (PAH) Patients, Supporting Egln1Tie2Cre Mice as a Useful PAH Model

Figures

• 

  Figure 1. Effects of chronic treatment of either sildenafil, ambrisentan, or treprostinil on the survival rate of Egln1Tie2Cre mice. (A) At the age of 35 days, separate cohorts of Egln1Tie2Cre mice were treated with either a vehicle (PBS, oral daily), sildenafil (100 mg/kg, oral, daily), ambrisentan (10 mg/kg, oral, daily), or treprostinil (110 ng/kg/min, osmotic pump) until the age of 105 days. The survival rates were recorded. (B) The sildenafil-treated Egln1Tie2Cre mice were treated with sildenafil until the age of 240 days. The survival rates were recorded during this period. ** p < 0.01. log-rank (Mantel–Cox) test.

• 

  Figure 2. Effects of chronic treatment of either sildenafil, ambrisentan, or treprostinil on pulmonary arterial hemodynamics in Egln1Tie2Cre mice. (A) Representative RVSP tracings (1 s). Egln1Tie2Cre mice at the age of 35 days were treated with either a vehicle (PBS, oral, daily), sildenafil (100 mg/kg, oral, daily), ambrisentan (10 mg/kg, oral, daily), or treprostinil (110 ng/kg/min, osmotic pump) until age of 105 days (3.5 months). The RVSP was measured through jugular vein cannulation. (B) The RVSP measurement demonstrated a marked decrease in RVSP by either one of the three drugs compared to the vehicle-treated Egln1Tie2Cre mice. Data are expressed as mean ± SD (n = 5–8). WT = wild-type, Veh = vehicle, Sild = sildenafil, Ambr = ambrisentan, Trep = treprostinil. * p < 0.05; ** p < 0.01. One-way ANOVA with Dunnett’s post-hoc analysis.

• 

  Figure 3. Treatment with FDA-approved drugs had negligible effects on PA function. (A) Egln1Tie2Cre mice at the age of 35 days were treated with either a vehicle (Veh), sildenafil (Sild) (100 mg/kg, oral, daily), ambrisentan (Ambr) (10 mg/kg, oral, daily), or treprostinil (Trep) (110 ng/kg/min, osmotic pump) until the age of 105 days. Echocardiography was carried out to determine PA function indicated by the PA AT/ET ratio. Data are expressed as mean ± SD (n = 5–8). (B) Representative micrographs of pulsed wave doppler of pulmonary arteries of WT mice and the vehicle- or drug-treated Egln1Tie2Cre mice. * p < 0.05. One-way ANOVA with Dunnett’s post-hoc analysis. n.s. = not significant.

• 

  Figure 4. Effects of treatment with either sildenafil, ambrisentan, or treprostinil on RV hypertrophy and function in Egln1Tie2Cre mice. (A) Measurement of RV hypertrophy calculated by the RV/LV + S weight ratio. Egln1Tie2Cre mice at the age of 35 days were treated with either a vehicle (Veh, PBS), sildenafil (Sild) (100 mg/kg, oral, daily), ambrisentan (Ambr) (10 mg/kg, oral, daily), or treprostinil (Trep) (110 ng/kg/min, osmotic pump) until the age of 105 days. (B) Marked improvement in RV contractility by sildenafil but neither ambrisentan nor treprostinil treatment. Echocardiography was carried out to assess RV function by determination of RV fraction area change (RV FAC). (C) Marked improvement of cardiac output (CO) in Egln1Tie2Cre mice treated with either ambrisentan, treprostinil, or sildenafil. Cardiac output was calculated by LV tracing. (D) Representative echocardiography showing the RV and LV chamber area changes at systolic and diastolic. Data are expressed as mean ± SD (n = 5–8). * p < 0.05; ** p < 0.01. One-way ANOVA with Dunnett’s post-hoc analysis. n.s. = not significant.

• 

  Figure 5. Effects of treatment with either sildenafil, ambrisentan, or treprostinil on pulmonary vascular remodeling in Egln1Tie2Cre mice. (A) Representative micrographs of Russell–Movat pentachrome staining of lung sections of WT and Egln1Tie2Cre mice treated with either a vehicle or an FDA-approved drug. Egln1Tie2Cre mice at the age of 35 days were treated with either a vehicle, sildenafil (100 mg/kg, oral, daily), ambrisentan (10 mg/kg, oral, daily), or treprostinil (110 ng/kg/min, osmotic pump) until the age of 105 days. Formalin-fixed lung sections were subjected to Russell–Movat pentachrome staining. Arrows point to vessels. Br = bronchiole. Scale bar, 50 µm. (B) Quantification of pulmonary vascular remodeling. Grade1 (G1), 2× vessel wall thickness/external diameter ratio <30%, indicating non-occlusion; G2, the wall/diameter ratio = 30–50%; G3, the wall/diameter ratio = 50–70% indicating partial occlusion; G4, the wall/diameter ratio ≥70% indicating occlusive lesions. N = 5 mice/group and 25–30 vessels/mouse. (C) Percentage of occlusive vessels. N = 5 mice/group and 25–30 vessels/mouse lung section were assessed for occlusive lesions (≥70% occlusion). * p < 0.05; *** p < 0.001.…

• 

  Figure 6. Summary of the effects of the three drugs on Egln1Tie2Cre mice and PAH patients from clinical trials. (A) Mechanisms of action of sildenafil, ambrisentan, and treprostinil. ET= endothelin-1. (B) The effects of sildenafil, ambrisentan, and treprostinil on survival and PAH phenotypes in Egln1Tie2Cre mice and PAH patients. mPAP = mean pulmonary arterial pressure. Paria 2005 [18]. Scheuemann 2013 [20], Rubin 2011 [21], Barst 2012 [22], Galiè 2008 [23], Blalock 2010 [24], Oudiz 2009 [25], Simonneau 2002 [26], Barst 2006 [27].