Examples of the inhibition of oxygen consumption by MKT-077 and resultant model fits.

<p>A) Change in pO₂ measured in R3230Ac cell suspension before and after addition of different concentrations of MKT-077 (0, 2, or 6 µg/ml) to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the “rate” model (Equations 14 and 17). B) Changes in oxygen consumption, q-q₁, predicted by the “rate” model (Equations 10 and 13).</p

Theoretical relative decrease in oxygen consumption of R3230Ac cells as a function of MKT-077 uptake.

<p>R3230Ac cells were treated with 2, 4, or 6 µg/ml MKT-077 at t = 0 minutes. Percent decrease in consumption, %Δq, was calculated from Equation 26 using the mean fitted parameters (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037471#pone-0037471-t003" target="_blank">Table 3</a>). Points indicate values after 30 minutes (•) or 2 hours (▴) of MKT-077 exposure.</p

Effect of extracellular MKT-077 concentration on cellular uptake.

<p>A) Data from a typical uptake experiment. R3230Ac cells were treated with 4 µg/ml MKT-077 at time 0. Solid line represents total cellular MKT-077 uptake, dashed line represents intracellularly bound or organelle-sequestered drug, and dotted line indicates free drug in the cytoplasm as determined by the model. B) MKT-077 uptake by R3230Ac cells as a function of drug concentration and time. Values are the mean ± SEM. p<0.0001 using a two-way ANOVA. Curves are fits of the mean values to Equation 1. 2 µg/ml: β = 11.1, k = 0.458 min⁻¹ (n = 11); 4 µg/ml: β = 18.0, k = 0.502 min⁻¹ (n = 10); 6 µg/ml: β = 18.0, k = 0.632 min⁻¹ (n = 7). C) Initial drug uptake rate as a function of treatment concentration. Line is calculated from Equation 5 with a slope of 1.44×10⁻⁴ ml/(10⁵ cells min). D) Steady-state MKT-077 uptake as a function of treatment concentration. Line is calculated from Equation 2 with a slope of 0.00845 ml/10⁵ cells. In panels C and D, the points are staggered along the abscissa for clarity, and the horizontal bars represent the mean values. One-way ANOVA with Bonferroni's Multiple Comparison Tests: * p<0.001 vs. 2 µg/ml value, ^# p<0.001 vs. 4 µg/ml value, ^̂ p<0.05 vs. 2 µg/ml value.</p

Fitted and calculated parameters and goodness-of-fit values for the model fits of MKT-077 uptake.

<p><i>Values are means</i> ± <i>SD. Values in italics are the means of the 95% confidence intervals. One-way ANOVA with Bonferroni's Multiple Comparison Tests:</i></p>a<p> <i>p<0.001 vs. 2 µg/ml value,</i></p>b<p> <i>p<0.001 vs. 4 µg/ml value,</i></p>c<p> <i>p<0.05 vs. 2 µg/ml value.</i></p

Effect of MKT-077 on pO₂ and oxygen consumption in MDA-MB-231 cells.

<p>A) Change in pO₂ measured in MDA-MB231 cell suspension before and after addition of different concentrations of MKT-077 (0, 2, or 4 µg/ml) to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the “relative rate” model (Equations 14 and 20). B) Percent decrease in oxygen consumption of MDA-MB231 cells, %Δq, as a function of steady-state MKT-077 uptake. The solid curve is the predicted relationship calculated from Equation 25 using the medians of the fitted parameters: q₁ = 2.91×10⁻⁶ ml O₂/(10⁵ cells min), α_r = 6.04×10⁻⁴ ml O₂ (10⁵ cells)^0.5]/(ng MKT-077)^0.5, and κ = −0.101 min⁻¹. (▪): 2 µg/ml MKT-077; (▴): 4 µg/ml MKT-077; (•): 6 µg/ml MKT-077.</p

Fits of a typical pO₂ data set to the three oxygen consumption models.

<p>A) Change in pO₂ measured in R3230Ac cell suspension before and after addition of 6 µg/ml MKT-077 to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the following models: dq/dC_C = −α₀ (“constant”, dotted line); dq/dC_C = −α₁C_C (“uptake”, dashed line); dq/dC_b = −α₂ (dC_C/dt) (“rate”, solid line) [see Equations 14–17]. The inset shows the same data from −20 to 15 minutes. B) Corresponding changes in oxygen consumption, q, predicted by the three models (Equations 10–13).</p

HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in <i>Pseudomonas aeruginosa</i> Keratitis

<div><p>Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with <i>Pseudomonas (P.) aeruginosa</i>. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in <i>P. aeruginosa</i>-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.</p></div

Comparison of the fits of all 20 data sets to the three different models of oxygen consumption change.

<p><i>Values are means</i> ± <i>SD. Repeated Measures One-Way ANOVA: Goodness-of-fit values (r² and RMS error) - p<0.0001. Bonferroni's Multiple Comparison Tests:</i></p>a<p> <i>p<0.001 vs. “uptake” model value,</i></p>b<p> <i>p<0.01 vs. “uptake” model value. Paired t-tests:</i></p>c<p> <i>p<0.02 vs. “constant” model value,</i></p>d<p><i>p<0.002 compared to q₁ values determined from linear fit of pre-injection pO₂ data (t≤0) {q₁ = </i>4.37±1.02×10^−<i>6</i><i> [ml O₂/(10⁵ cells min)], mean ± SD, n = 20}.</i></p

In vitro effects of HIF-1α inhibition on PMN function.

<p>Phaygocytosis (A), as measured by uptake of GFP⁺<i>P. aeruginosa</i> 19660 by PMN, was not affected by DMAG-induced HIF-1α inhibition. Confocal laser scanning images documented intracellularly located GFP⁺ bacteria (green) with no DMAG treatment (positive control) (B) and after DMAG treatment (10 µM) (C). The negative control (no bacteria, no DMAG) (D) shows no GFP⁺ bacteria associated with the PMN, but stained positive for SYTOX Orange nuclear stain only. Intracellular killing by PMN (E) was analyzed by enumerating viable CFUs in the cell lysates. Significantly more viable bacteria were detected after DMAG treatment and affects were dose-dependent. Apoptotic and necrotic cells were measured (F) after HIF-1α inhibition and indicated that DMAG treatment significantly decreased apoptosis/increased cell necrosis in a dose-dependent response compared to positive controls (bacteria only, no DMAG). Apoptotic and necrotic cells were measured (G) after HIF-1α inhibition (10 µM) using both 19660 (a cytotoxic strain) and PAO1 (an invasive strain) and revealed no significant differences between cell viability, apoptosis and necrosis between the two bacterial strains. Data represent three independent experiments. *<i>P</i><0.05, ** <i>P</i><0.01 for apoptotic cell counts – (+) control versus DMAG treated; # <i>P</i><0.01 for necrotic cell counts – (+) control versus DMAG treated. B, C, D magnification = 1,000×. NS = Not Significant.</p

Relative change in oxygen consumption as a function of extracellular and intracellular MKT-077 concentration.

<p>A) Percent decrease in oxygen consumption of R3230Ac cells, %Δq, as a function of [MKT-077] in medium. The points are staggered along the abscissa for clarity, and the horizontal bars represent the mean values. The %Δq values were significantly dependent on dose (ANOVA, p = 0.003). The dashed line is a linear regression: %Δq = 6.99C_M0+32.2, r = 0.706, p = 0.0005, n = 20. The solid curve is the predicted relationship calculated from Equation 24 using the mean parameters. *p<0.01 compared to the 2 µg/ml group using one-way ANOVA with Bonferroni's Multiple Comparison Test. B) Percent decrease in oxygen consumption of R3230Ac cells, %Δq, as a function of steady-state MKT-077 uptake. The dashed line is a linear regression: %Δq = 0.664C_C,∞+41.7, r = 0.621, p = 0.004, n = 20. The solid curve is the predicted relationship calculated from Equation 25 using the mean parameters. (▪): 2 µg/ml MKT-077, n = 7; (▴): 4 µg/ml MKT-077, n = 7; (•): 6 µg/ml MKT-077, n = 6.</p