Assessment of pulmonary vascular remodeling

Immunohistochemical staining with anti-α-smooth muscle actin antibody (Santa Cruz, CA, USA) was performed for additional evaluation of pulmonary vascular remodeling. All small intrapulmonary arteries (≤50 μm in external diameter) adjacent to the alveolar ducts were analyzed. Each vessel was categorized as nonmuscularized (actin staining <25% of the circumference), partially muscularized (actin staining 25–75% of the circumference) and fully muscularized (actin staining >75% of the circumference). The fully muscularized vessels were calculated and expressed as the percentage of total small intrapulmonary arteries. All morphometric measurements were performed by 2 independent researchers operating in a blinded manner. Interobserver difference in the measurements was <5%.