Pulmonary hypertension (PH) is a disabling disease characterized by higher prevalence in females [
Thirty eight 6-week-old athymic RNU-rats (Crl:NIH-
All procedures were performed under general anesthesia combining buprenorphine (0.5 mg/kg, SC) with inhaled isoflurane (2% mixed with 0.5 L/min 100% oxygen for induction, 1% to 1.5% for maintenance). Rats were then placed in the supine position (head up) onto a warmed plate to help maintain the rat core temperature at 37°C.Cardiac echography (Vevo 2100 system, VisualSonics, Toronto, Canada) was performed 21 days after PH induction to assess the mid-right-ventricular end-diastolic diameter (RVEDD) and the tricuspid annular plane systolic excursion (TAPSE). The ultrasound transducer (13–24 MHz, MS250, VisualSonics) was manually immobilized on the shaved area overlying the heart with a 45° angle. A complete two-dimensional (2D) 4-chamber view was obtained by adjusting the position of the probe parasternally over the cardiac apex. TAPSE was measured as the maximal motion toward the apex of the lateral tricuspid annulus (Figure
Representative cardiac echo imaging in rats at 3 weeks. The tricuspid annular plane systolic excursion (TAPSE) is measured on a 4-chamber apical view (2D-mode) as the difference in length between the tricuspid annulus and a fixed point at the end-diastolic (a) and end-systolic times (b). On the same view, the right ventricular end-diastolic diameter (RVEDD) is measured transversally from the midinterventricular septum to the mid-right-ventricular free wall (c, 2D-mode). In SU_M at 3 weeks, pericardial effusion can be observed (d, parasternal short axis, 2D-mode). RV: right ventricle; RA: right atrium; LV: left ventricle.
Representative pressure-volume loops from SU_M and RNU_M at 4 weeks. At baseline, the pressure-volume loop is in red. During occlusion of the inferior vena cava, the right ventricular end-diastolic volume decreases, shifting the following loops (green) toward the left. Similarly the right ventricular end-systolic pressure decreases, shifting the loops to the bottom. The linear end-systolic relationship is represented in blue, linking all end-systolic points from the successive pressure-volume loops during transient occlusion of the inferior vena cava. The slope of this line defines the end-systolic elastance of the ventricle (
Animals were sacrificed at day 28 for heart and lung procurement. To assess RV hypertrophy, the Fulton index was calculated as the weight ratio of the right ventricle and left ventricle + septum. After one-day fixation in 4% buffer formalin solution, 5
To study gender differences in smooth muscle cells (SMCs) plasticity, paraffin-embedded lungs underwent heat-induced antigen retrieval with Dako antigen retrieval solution (Dako, Glostrup, Denmark) in a steamer for 20 min. After blocking with Image-iT FX signal enhancer (Invitrogen, Carlsbad, Ca) for 30 minutes, slides were incubated with primary antibodies against smooth-muscle-heavy-chain (SM heavy chain; EPR5335, Abcam, Cambridge, UK) and embryonic smooth muscle myosin heavy chain (SMemb; 3H2, Yamasa, Tokyo, Japan) for 16 h at 4°C. After washing with PBS, sections were incubated for 1 h at 37°C with the corresponding secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen). DAPI was used to counterstain cell nuclei. Images were obtained by a Nikon Eclipse TiE microscope (Nikon, Tokyo, Japan) equipped with the Perkin Elmer UltraVIEW VoX confocal imaging system (Perkin Elmer, Waltham, MA).
All variables were reported as mean value ± standard deviation. One-way analysis of variance was performed for between-group comparisons with a 95% confidence interval using the Prism software (GraphPad, La Jolla, CA). Only animals who survived until day 28 were included in the statistical analysis.
Among the 11 semaxanib-treated males, 3 died before day 28 (on days 21, 21, and 26) and 3 developed marked pleuropericardial effusion. All females survived until day 28 without any thoracic effusion. Induction of experimental PH resulted in a significant enlargement of the right ventricle after 3 weeks in males compared to females (RVEDD:
Right heart hemodynamics at 4 weeks.
Group |
RNU_M |
RNU_F |
SU_M |
SU_F |
---|---|---|---|---|
Semaxanib (mg/kg) | 0 | 0 | 40 | 40 |
Heart rate (beat/min) | 269 ± 37 | 311 ± 35 | 296 ± 48 | 280 ± 73 |
RVESP (mm Hg) | 21 ± 2.5 | 24.3 ± 8.1 | 66.3 ± 8.9 |
29.5 ± 13.4 |
RVEDP (mm Hg) | 1.5 ± 3.5 | 3 ± 3.6 | 6.2 ± 2.8§ | 2.1 ± 4.3 |
CO (mL/min) | 134 ± 4 | 122 ± 5 | 58 ± 24 |
89 ± 17 |
PVR (dyn⋅s⋅cm−5) | 0.013 ± 0.002 | 0.016 ± 0.006 | 0.103 ± 0.06 |
0.027 ± 0.01 |
|
1173 ± 161 | 1311 ± 414 | 2636 ± 495 |
1374 ± 320 |
|
−1318 ± 37 | −871 ± 232 | −2370 ± 912 |
−1072 ± 415 |
|
0.19 ± 0.05 | 0.21 ± 0.15 | 0.12 ± 0.06 |
0.18 ± 0.05 |
|
0.04 ± 0.01 | 0.06 ± 0.03 | 0.33 ± 0.12 |
0.09 ± 0.02 |
|
4.75 ± 0.63 | 3.5 ± 0.45 | 0.36 ± 0.17 |
2.1 ± 0.34 |
RVESP: right ventricular end-systolic pressure; RVEDP: right ventricular end-diastolic pressure; CO: cardiac output; PVR: pulmonary vascular resistance;
Fulton index (RV/(LV + S)) in rats at 4 weeks. The right ventricle of Su_M was significantly hypertrophied compared to Su_F, RNU_F, and RNU_M, as illustrated by the elevated Fulton index in this group. # explains
Morphometry of the pulmonary arteries and of the right ventricle at 4 weeks. (a) Pulmonary artery muscularization, defined by medial thickening over 10% of the cross-sectional diameter, was remarkable in male rats, while females did not develop significant medial hypertrophy in response to semaxanib injection (black arrows show the medial layer). (b) Similarly, cardiomyocytes hypertrophy was lower among females exposed to experimental PAH. Scale bars represent 50
Immunofluorescence analysis of the pulmonary arteries at 4 weeks. (a) In male rats, SMemb, the embryonic form of smooth muscle myosin heavy chain and a marker for dedifferentiated SMCs, was increased within the pulmonary arteries, indicating SU-induced SMC-phenotype switch. (b) In contrast, confocal immunofluorescence revealed differentiated SMC-phenotype in female pulmonary arteries. (Blue: nucleus staining with DAPI; green: smooth-muscle-heavy-chain (SM heavy chain); pink: embryonic smooth muscle myosin heavy chain (SMemb); scale bars represent 33
Athymic female rats developed less severe PH in response to vascular endothelial growth factor (VEGF) receptor blockade. As a consequence, RV function and RV-PA coupling were preserved only in females in this experimental descriptive study. We hypothesize a protective role of endogenous estrogens against PH in semaxanib-treated rats under normoxic conditions. Our findings are in accordance with previous experimental studies showing that ovariectomized rats develop more severe PH in response to chronic hypoxia or to monocrotaline [
Effects of estrogens on the pulmonary vasculature are complex and not clearly understood. Sex differences have been poorly investigated in PH. Jacobs et al. recently reported in a retrospective clinical study worse outcome in men, suggesting gender difference in cardiac adaptation to chronic PH [
Female athymic rats developed less severe pulmonary vascular damage as males in response to angioproliferative stress. Moderate pulmonary artery muscularization and the lack of phenotype switch of smooth muscle cells from contractile to dedifferentiated state was associated with preserved ventricular-arterial coupling and RV efficiency in semaxanib-treated females. Control of smooth muscle cells plasticity may be a promising therapeutic approach to reverse established vascular remodeling in PH patients.
Pulmonary hypertension.
The authors have no conflict of interests.
Julien Guihaire performed the experiments, analyzed the data, and wrote the paper. Tobias Deuse designed the studies and wrote the paper. Dong Wang performed the experiments related to gender differences in SMC plasticity and edited the paper. Elie Fadel and Hermann Reichenspurner designed the studies and edited the paper. Sonja Schrepfer designed the studies, secured the funding, analyzed the data, and wrote the paper.
The authors thank Christiane Pahrmann for her technical assistance. They also thank UKE Imaging Facility (UMIF, Bernd Zobiak) and the UKE Animal Facility. This study was funded by the “Fédération Française de Cardiologie” (Paris, France: Julien Guihaire), the “Association Chirurgicale pour le Développement et l’Amélioration des Techniques de Dépistage et de Traitement des Maladies Cardio-vasculaires” (Suresnes, France: Julien Guihaire), the German Research Foundation (Deutsche Forschungsgemeinschaft; DFG: SCHR992/3-1 and SCHR992/4-1; Sonja Schrepfer), and the International Society for Heart and Lung Transplantation (ISHLT; Dong Wang and Sonja Schrepfer).