A Simple Method to Assess In Vivo Proliferation in Lung Vasculature with EdU: The Case of MMC-Induced PVOD in Rat

Figure 2

MMC-exposed rat lungs display areas of intense microvascular endothelial cell proliferation. Immunofluorescent staining of frozen rat lung sections and confocal imaging. Red: CD34 (endothelial cells). White: α-smooth muscle actin (smooth muscle cells). Green: click-iT EdU stain (nuclei of proliferating cells). Counterstain = 4′,6-diamidino-2-phenylindole (DAPI). (a) Control rat lung. ((b)–(f)) MMC-exposed rat lung. (a) Control rat lung parenchyma displays very little microvascular endothelial cell proliferation. Note a single endothelial cell in proliferation (arrow). ((b)-(c)) Pulmonary capillary hemangiomatosis like foci from MMC-exposed rats display intense microvascular endothelial cell proliferation. (d) Adventitial fibroblast cells proliferation in small remodeled pulmonary artery. ((e)-(f)) Adventitial fibroblast cells proliferation in small remodeled pulmonary vein. (g) Quantification of the percentage of lung proliferating cells. ((h)-(i)) Exuberant adventitial fibroblast cell proliferation in MMC-exposed rats (i) compared to control rats (h) (Vimentin+EdU+ cells). Adventitial fibroblast cells were stained with antibody against Vimentin (fibroblast marker in red) and proliferative cells with click-it EdU (white nuclei = EdU positive nuclei = proliferating cells). Vessel media and adventitia (Adv) were delimited by the yellow dotted line area. . Red scale bar = 100 μm. White scale bar = 50 μm.