1. Anesthetize mouse by injecting pentobarbital (i.p., 120 mg/kg) and appropriate anesthesia was verified by checking for lack of tactile response when pinching footpads.

2. Restrain mouse on surgical pad under microscope with ventral side facing up.

3. Make a midline vertical cervical skin incision and expose the ventral side of trachea by dissecting the midline raphe which separates fatty tissue on the left and right sides.

4. Once the trachea was clearly seen, hooks were inserted on both sides of the skin incision to open the wound and enhance visualization of the trachea.

5. Insert a cannula into the trachea through a small cut with the bent tip tilted toward the ventral surface of the mouse. Make sure that the cannula is placed below the vocal cords but well above the carina. Suture should be used to stabilize the cannula inside the trachea.

6. Slowly inject 1× PBS (5–10 ml) into the RV using a 22-G needle to superfuse the lungs. After perfusion, the lungs frequently turned to white.

7. Inject 3-ml 10% formalin (using a syringe) via the tracheal cannula to inflate the lungs. When lungs were fully inflated, the cannula was gently removed from the trachea and suture was used to tie up the trachea securely to prevent the formalin release.

8. Dissect the lungs out of the body and place the lungs in formalin for 24 h (at 1:10 ratio) to fix the lungs. The fixed lung tissues were dehydrated, embedded into paraffin, and then sectioned.

9. Process the lung tissue slice for hematoxylin and eosin (H&E) staining and generate tissue slides.

10. Deparaffinize sections twice in xylene and dehydrate sections with alcohol at a series of concentrations (twice in 100%, once in 95% and 70% alcohol).

11. Stain sections with hematoxylin and counterstain in eosin. Dehydrate sections once in 95% and twice in 100% alcohol.

12. Clear sections twice in xylene, mount slides with xylene-based mounting medium, and dry slides overnight in the hood.

13. Take images on the stained samples or lung tissue slides by an Eclipse Ti2 inverted microscope (Nikon, Japan) and process the lung tissue images using an NIS-Elements software (version 5.21.00).

14. Carefully highlight the total area of the whole PA (AT, including intraluminal area) and the area of the PA wall (APA) using the tool menu in Photoshop software (v. 13.1.2) (see Fig. X).

15. Measure and record the highlighted whole PA area (AT) and the highlighted PA wall area (APA) by ImageJ Analysis menu.

16. Calculate the PA wall thickness as the ratio of the area of PA wall to the area of whole PA, that is, PA wall thickness = APA/AT, where AT = APA+intraluminal area (AIA).

17. Measure and record the outer diameter (OD) of each PA section highlighted and used to calculate PA wall thickness.

18. Categorize the recorded PAs, based on OD, into three groups: (a) large PA (OD > 100 µm, (b) medium-sized PA ( OD = 50–100 µm), and (c) small PA (OD < 50 µm).