Source

Abstract

Rationale

Acute 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).

Methods

hPASMCs 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. $\chi^2$ confidence intervals and statistical significance were computed by bootstrap simulation with $10^5$ replicates using custom scripts in R(v3.4.1).

Results

In 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 $\chi^2$ test was performed and a strong relationship was found between IL-1b treatment and the presence of oscillatory responses, $\chi^2 = 96.7(71.2, 125.5; 95\% C.I.)$, $p<10^{-5}$.

Support or Funding Information

Nat’l Heart, Lung & Blood Inst. K08HL125806

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.