Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

Abstract

Background: Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII → ATI trans-differentiation has not been explored.

Method: In a controlled nonvascular environment, an in vitro model of ATII → ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation.

Results: Here, we show that EMAP II significantly blocked ATII → ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA.

Conclusion: Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia.

Figure 1

EMAP II inhibits AT II → AT1 transdifferentiation. Aquaporin 5 (AQP5) expression was examined 8 days following isolation of AT II cells using IgG panning (A - p180 lamellar body positive, cy3; G - surfactant protein C positive, day 0). Immunofluorescence and Western blot analysis for AQP5 indicated that AT II cells transdifferentiate into AT1 cells over an eight-day period (B - FITC, D, E). AT II cells subjected to EMAP II for 8 days in culture have a marked reduction in AQP5 and RAGE expression (C, D, F, G) as compared to control (B, D, F, G). Neither cell population had surfactant protein C (SPC) expression after 8 days in culture (E). DAPI denotes nuclear staining. Scale bar = A - 37.5 μ, B-D-25 μ.

Figure 2

Apoptosis is increased by EMAP II in transdifferentiating cells. Isolated, AT II cells were examined at days 4 and 8 for apoptosis using an immunofluorescent TUNEL assay. Cells treated with EMAP II had a marked increase in apoptotic cells at days 4 and 8 (C, D, and E) as compared to control (A, B, and E). Western blot analysis of the caspase cleaved PARP-1 indicated a marked increase in PARP-1 cleavage in EMAP II treated cells as compared to control (F, G). DAPI denotes nuclear staining. Despite an increase in apoptosis, total cell number remained unchanged between control and EMAP II treated cells (H). Scale bar = 37.5 μ.

Figure 3

EMAP II increases proliferation in non-myofibroblast cells. Proliferation was assessed using immunofluorescence for Ki67 in isolated AT II cells in culture at days 4 and 8. EMAP II significantly increased proliferation at days 4 and 8 (C, D, I) as compared to control (A, B, I). Co-localization of Ki67 with αSMA (E-control, G-EMAP II treated) and F actin (F-control, H-EMAP II treated) indicates that the proliferating cell population was negative for myofibroblast markers. DAPI denotes nuclear staining. Scale bar = 37.5 μ (A-D), 25 μ (E-H).

Figure 4

Decreased Beta-catenin expression corresponds with marked membrane ruffling, increase in nuclear size and nuclear:cytoplasmic ratio in EMAP II treated cells. Immunofluorescent staining of AT II cellular membranes indicate that transdifferentiating AT II cells develop a cobblestone appearance of epithelial like polygonal shaped cells (A, B). Transdifferentiating AT II cells treated with EMAP II lack the polygonal shape (C, D), exhibit membrane ruffling (arrows, D), and have decreased β-catenin expression of at day 4 (E, F). In addition, EMAP II treated cells demonstrated a larger nuclear size and nuclear:cytoplasmic ratio as compared to controls (G, H). DAPI denotes nuclear staining. E-cadherin expression was not significantly different between EMAP II and control cells (I, J) Scale bar = 25 μ.

Figure 5

EMAP II inhibited actin cortical bands and increased actin bundles in transdifferentiating AT II cells. F-actin at days 4 and 8 is distributed in cortical bands in differentiating AT II cells (arrows, A, B and E, F, FITC). Transdifferentiating cells treated with EMAP II lack cortical bands and demonstrate marked increase in actin bundles (stars, C, D and G, H, FITC). G-actin is revealed with cy3 and DAPI denotes nuclear staining. Scale bar = 25 μ.

Figure 6

EMAP II increases Rho-GTPase expression while αSMA and desmin expression are decreased in transdifferentiating AT II cells. Rho-GTPase activity was assessed in AT II cells in culture at 0, 4, and 8 days using a Rhotekin RBD probe to isolate active GTP-Rho. EMAP II treatment increased Rho-GTPase activity as compared to controls (A, B). However total Rho A levels normalized to actin in ATII cells in cultures treated with EMAP II were unchanged as compared to control (A, C). After 8 days in culture, αSMA actin levels were markedly decreased as compared to control (D, E) and desmin was minimally expressed in EMAP II treated cells (D, F). Protein expression was normalized to actin loading controls and whole lung lysate (WLL) was used as an addition loading control (D). Fibronectin expression (FN) was reduced following EMAP II treatment (G, H, normalized to alpha-tubulin loading control).