Novel and robust treatment of pulmonary hypertension with netrin-1 and netrin-1-derived small peptides

Figures

• 

  Fig. 1. Netrin-1 and netrin-1 derived small peptides attenuated elevations in mPAP and RVSP in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. Hemodynamic analyses indicate that increased mPAP and RVSP in hypoxia induced pulmonary hypertensive mice were substantially attenuated by treatment with netrin-1 or netrin-1 derived small peptides V1, V2. or V3. Data are shown as Mean ± SEM. **p < 0.01 vs. Normoxia; #p < 0.05, ##, p < 0.01 vs. Hypoxia.

• 

  Fig. 2. Netrin-1 and netrin-1 derived small peptides attenuated right ventricular hypertrophy in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. Right ventricle hypertrophy as indicated by increased RV/LV + S ratio in hypoxia induced pulmonary hypertensive mice was substantially attenuated by treatment with netrin-1 or netrin-1 derived small peptides V1, V2. or V3. Data are shown as Mean ± SEM. **p < 0.01 vs. Normoxia; ##p < 0.01 vs. Hypoxia.

• 

  Fig. 3. Netrin-1 and netrin-1 derived small peptides alleviated vascular remodeling in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. (A) Representative images of H&E staining (20X magnification) of lung tissue sections of various treatment groups. (B) Medial thickness of pulmonary arterioles (<200 μm in diameter) indicating increased medial thickness by hypoxia exposure, which was substantially alleviated by netrin-1 or netrin-1 derived peptides V1, V2 or V3. (C) Medial thickness in differently sized pulmonary arterioles indicating increased medial thickness by hypoxia exposure, which was substantially alleviated by netrin-1 or netrin-1 derived peptides V1, V2 or V3 in pulmonary arteries categorized by 50–100 and 100–200 μm in diameter. Data are shown as Mean ± SEM. **p < 0.01 or ***p < 0.001 vs. Normoxia; ##p < 0.01, ###p < 0.001 vs. Hypoxia.

• 

  Fig. 4. Netrin-1 and netrin-1 derived small peptides attenuated muscularization and cell proliferation of blood vessels in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. (A, E) Representative images and quantitative data of smooth muscle actin (SMA) expression by immunoflurescent staining indicating increased SMA expression by hypoxia exposure, which was substantilly attenuated by netrin-1 or netrin-1 derived peptides V1, V2 or V3. (B, F) Representative images and quantitative data of SMA expression by immunohistochemical staining using DAB substrate indicating increased SMA expression by hypoxia exposure, which was substantially attenuated by netrin-1 or netrin-1 derived peptides V1, V2 or V3. (C, G) Representative images and quantitative data of proliferative cell nuclear antigen (PCNA) expression by immunoflurescent staining indicating increased PCNA expression by hypoxia exposure, which was markedly attenuated by netrin-1 or netrin-1 derived peptides V1, V2 or V3. D, H) Representative images and quantitative…

• 

  Fig. 5. Netrin-1 and netrin-1 derived small peptides alleviated fibrosis in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. (A, B) Representative images and grouped data of Masson’s Trichrome staining indicating increased collagen deposition (blue; x20 magnification) in peribronchial and perivascular areas of lung sections in hypoxia exposed mice, which was markedly alleviated by netrin-1 or netrin-1 derived small peptides V1, V2 or V3. Data are shown as Mean ± SEM. ***p < 0.001 vs. Normoxia; #p < 0.05, ##p < 0.01 vs. Hypoxia.

• 

  Fig. 6. Netrin-1 and netrin-1 derived small peptides improved NO bioavailability while attenuating ROS production in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. (A, C) Representative images and quantitative data of NO bioavailability by DAF-FM fluorescent imaging indicating NO deficiency in hypoxia exposed mice, which was substantially improved by netrin-1 or netrin-1 derived small peptides V1, V2 or V3. n = 5–7. (B, D) Representative images and quantitative data of ROS production by DHE fluorescent imaging indicating increased ROS production in hypoxia exposed mice, which was markedly attenuated by netrin-1 or netrin-1 derived small peptides V1, V2 or V3. Data are shown as Mean ± SEM. **p < 0.01 or ***p < 0.001 vs. Normoxia; #p < 0.05 or ##p < 0.01 vs. Hypoxia.

• 

  Fig. 7. Netrin-1 and netrin-1 derived small peptides abrogated superoxide production and eNOS uncoupling activity in pulmonary hypertensive mice. Pulmonary hypertension was induced in mice by exposure to normobaric hypoxia (10% O2) for three weeks. Osmotic pump was used to deliver netrin-1 or netrin-1 derived small peptides continuously. Lung tissues were used to measure total superoxide production and eNOS uncoupling activity using electron spin resonance (ESR) as we previously published. (A) Grouped data of total superoxide production determined by ESR indicating increased superoxide production in hypoxia exposed mice, which was substantially attenuated by netrin-1 and netrin-1 derived small peptides V1, V2 or V3. ***p<0.001 vs. Normoxia, ###p<0.001 vs. Hypoxia, $p<0.05 vs. Hypoxia plus Netrin-1 by One-Way ANOVA. (B) Grouped data of superoxide production in the presence or absence of l-NAME for assessment of eNOS uncoupling activity indicating uncoupling of eNOS (reflected by l-NAME sensitive superoxide production) in hypoxic pulmonary hypertensive mice, which was reversed by netrin-1 or netrin-1 derived small peptides V1, V2 or V3. ***p<0.001 by t-test.