Calcium Oscillations in Human Pulmonary Artery Smooth Muscle Cells: Developing a Hierarchical Clustering Method for Analysis and the Effects of IL‐1b

Abstract

RationaleAcute respiratory distress syndrome (ARDS) is characterized by lung inflammation, edema, and severe hypoxemia. In previous work, we showed that IL‐1b is required for the development of severe hypoxemia in a murine model of acute lung injury, independent of measures of inflammation and edema. We hypothesized that IL‐1b is altering the regulation of pulmonary blood flow to cause severe V/Q mismatching in acute lung injury. To explore possible mechanisms for this, we studied the effects of IL‐1b on calcium flux in human pulmonary artery smooth muscle cells (hPASMCs) in response to the vasoconstrictor endothelin‐1 (ET‐1).MethodshPASMCs were grown on glass bottom dishes to confluence and some dishes were treated with IL‐1b (10ng/mL) for 3h. Cells were loaded with 5mM Cal‐520 and stimulated with ET‐1 (10nM) during fluorescent imaging on a Zeiss LSM confocal microscope. All cells from each field were individually selected using Fiji image analysis software. By visual inspection, we noticed that a marked number of cells showed oscillations in calcium flux after ET‐1. To quantify oscillatory from non‐oscillatory responses, we used a mixture of information theoretic and machine learning algorithms and developed a custom classifier tool. We applied this tool to our control and IL‐1b‐treated data (Figure 1) and created a contingency table for the effects of IL‐1b on the induction of oscillatory responses. χ2 confidence intervals and statistical significance were computed by bootstrap simulation with 105 replicates using custom scripts in R(v3.4.1).ResultsIn control dishes (n = 6), there were a total of 444 non‐oscillators cells and 379 oscillator cells. In dishes treated with IL‐1b (n = 2), there were 50 non‐oscillator cells and 211 oscillators total. A bootstrap simulation based χ2 test was performed and a strong relationship was found between IL‐1b treatment and the presence of oscillatory responses, χ2 = 96.7 (71.2, 125.5; 95% C.I.), p < 10−5.Support or Funding InformationNat'l Heart, Lung & Blood Inst.K08HL125806This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.