Figure 2. Adventitial fibroblasts activate macrophages.

(A) Transwell experiment depicting mRNA expression in mouse bone marrow-derived macrophages (mouse BMDMs) exposed to soluble factors generated by distal pulmonary artery (dPA) explants from calves with chronic hypoxia-induced PH. dPAs were isolated such that an intact piece of PA tissue (intact dPA), a PA tissue piece from which the adventitia had been removed (dPA w/o adv.), and the removed adventitia piece itself (dPA adventitia) were incubated in the upper chamber of a 0.4-µm Transwell in the presence of naïve mouse BMDMs (lower chamber) for 16 h prior to RNA isolation and qRT-PCR (as depicted in the diagram). Displayed is the fold-induction (normalized to basal expression) of a representative PCR triplicate (average +/− SEM) from one of two calves. Three dPA segments were tested from each animal.. *P < 0.05 by one-way ANOVA. (B) Gene expression in BMDMs or monocytes exposed to conditioned media (CM) from dPA adventitial fibroblasts (PH-Fibs). Left panels top and bottom: CM from ex vivo-cultured bovine PH-Fibs (cells isolated from the dPA of calves with PH), and not from controls (CO-Fib CM) promotes gene expression of Cd163 (top) and Cd206 (bottom) in naïve mouse and bovine BMDMs; Right panels top and bottom: CM from bovine and human PH-Fibs induce expression of Cd163 and Cd206 in naïve human THP1 monocytes after 16 hrs of exposure. Relative mRNA levels are presented as mean±SEM of PCR triplicates after normalization to Hprt1 expression and relative to gene expression in untreated macrophages/monocytes (basal) and are representative of at least n=5 experiments with CM from at least 3 different PH-Fibs and CO-Fibs populations isolated from at least 3 different animals/patients and using BMDMs from at least 3 different animals. *P < 0.05 by unpaired two-tailed Student’s t-test.