Ventricular Ejection Dynamics in Fetuses with Congenital Heart Disease by 2-Dimensional Echocardiography

Abstract

Cyclic stretch and oxygen are key regulators of lung development. Independently, both cyclic stretch and hyperoxia can significantly alter cell viability and proliferation. Relatively little is known however concerning the combined exposure of these factors on the distal lung epithelium. Objective: To study the effects of both cyclic stretch and hyperoxia on cell proliferation and viability using human pulmonary A549 cells. Methods: Cells were plated on collagen-coated flexible membranes and exposed to 1 Hz, 16 kPa cyclic biaxial stretch for 24, 48, and 72 hours in both normoxic (21% O2, 5% CO2) and hyperoxic (95% O2, 5% CO2) conditions. Cell proliferation was determined using manual cell counts and 3 [H] thymidine incorporation. Calcein-AM, LIVE/DEAD staining, and LDH release assays were used to assess viability. Apoptosis was measured with propidium iodide fluorescence together with annexin-V incorporation and caspase-3 activity. The reactive oxygen species, superoxide, was measured using dihydroethidium (DHE). Results: Cyclic stretch under normoxic conditions increased cell number vs. unstretched controls after 24 hours (p = .002). Cell number and 3 [H] thymidine incorporation markedly decreased following exposure to 95% O2 after 48 and 72 hours (p < .001). In contrast, both cell counts and 3 [H] thymidine incorporation following exposure to hyperoxia combined with stretch remained unchanged at 48 hours and were significantly greater vs. the unstretched hyperoxia group at 72 hours (p < .001). Calcein-AM and LIVE/DEAD analysis revealed an increase in cell death with hyperoxia after 72 hours compared to normoxia (p < .001). Cell death from hyperoxia at 72 hours was significantly attenuated when combined with stretch (p < .001). Apoptosis was not detected in any of the study groups using annexin-V and caspase-3. LDH activity was elevated after 72 hours in all the study groups vs. normoxic controls (p < .001) but did not vary between the separate study groups. Increased superoxide levels were detected in cells exposed to hyperoxia vs. normoxia using DHE fluorometric staining. Conclusion: The detrimental effects of hyperoxia on cell proliferation and nonapoptotic cell death in distal lung epithelial cells is significantly minimized when combined with cyclic stretch. The involvement of the mitogen-activated protein kinase pathways in regulating this response is currently being investigated.