Automated <i>In Vivo</i> Platform for the Discovery of Functional Food Treatments of Hypercholesterolemia

<div><p>The zebrafish is becoming an increasingly popular model system for both automated drug discovery and investigating hypercholesterolemia. Here we combine these aspects and for the first time develop an automated high-content confocal assay for treatments of hypercholesterolemia. We also create two algorithms for automated analysis of cardiodynamic data acquired by high-speed confocal microscopy. The first algorithm computes cardiac parameters solely from the frequency-domain representation of cardiodynamic data while the second uses both frequency- and time-domain data. The combined approach resulted in smaller differences relative to manual measurements. The methods are implemented to test the ability of a methanolic extract of the hawthorn plant (<i>Crataegus laevigata</i>) to treat hypercholesterolemia and its peripheral cardiovascular effects. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052409#s3" target="_blank">Results</a> demonstrate the utility of these methods and suggest the extract has both antihypercholesterolemic and postitively inotropic properties.</p></div

Heart Beat Detection and Area to Volume Conversion.

<p>A. Raw data and automated detection of area (A) of heart during diastole and systole. B. Cardiac waveform generated by automated detection of heartbeat (above) C. Measurement of the volume of chemically arrested hearts D. The C radius was calculated by correlating the volume of five arrested hearts to the cross-sectional areas of those hearts. This gave a relationship between the cross-sectional area and the C radius with the equation: C = (6.8×10⁻⁴) * A+46. Inputting this relationship into the equation for the volume of a prolate spheroid, V = (4/3)*π*x*y*z, where π*x*y = A and z = C, we get the relationship V = (4/3)A*C, where the volume of the ventricle is a function of the area measured. This equation is utilized to transform each area data point in B to volume measurements from which stroke volume (SV), heart rate (HR), cardiac output (CO) and ejection fraction (EF) are calculated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052409#pone-0052409-g004" target="_blank">figure 4</a>).</p

Automated Hypercholesterolemia Screen.

<p>A. Images of Control, 50 µM Ezetimibe treated, and 6.5 mg/mL methanolic hawthorn extract (MHE) treated 5 pf zebrafish embryos B. Quantified results of the automated screen. Bars represent the mean of the mean fluorescence intensity of each individual well (with values of 0, no reading, excluded). Control, 50 µMEzetimibe treated, and between 3.25 mg/mL and 19.5 mg/mL MHE treated groups are show. Ezetimibe served as a positive control C. Dose response curve illustrating the relationship between hawthorn dose and fluorescent output (R² = 0.61). For this experiment n is between 13–30 per group.</p

Waveform Analysis Methodologies.

<p>Volume change over time (top) calculated from area change as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052409#pone-0052409-g003" target="_blank">figure 3</a>. Briefl, area waveform values were input into the equation, C = (6.8×10⁻⁴) * A + 46 from which volume over the heartbeat was calculated according to the equation V = (4/3)**A*C where A is the area of the ventricle during the beat cycle and C is the radius in the Z-direction. A. In the Fourier framework (left), a waveform is transformed to Fourier space in order to extract the amplitude and frequency (f) of the wave. In this case, these values represent ½ of the stroke volume (SV) and theheart rate (HR) respectively. From these parameters, we calculate cardiac output (CO) and ejection fraction (EF). A representative waveform with average diastolic and systolic volumes as calculated by Fourier is presented (bottom left). Notice that thedistance between diastole and systole compared to segmentation approach B. In th segmentation approach (right), the original waveform is transformed to Fourier space. The frequency of the peak of the transform is extracted to determine the period (T) of the waveform which is then utilized as a baseline value on which to base the size of segment for analysis. The algorithm measures maximum and minimum values within each segmen (which is sized at 1.1× T in order to increase the liklihood of capturing the maximum and minimum values) traversing the waveform. Stroke volume is calculated as the mean maximum value – mean minimum value and is represented as average diastole and average systole (bottom right).</p

Hypercholesterolemia Screen Calibrations.

<p>Colors next to images correspond to those covering the area of a representation of a single well in a 38-well plate (upper right). B. In order to determine how a fish' orientation influences the measured fluorescence output, the same fish was measured in 3 different positions. C. Different numbers of slices per z-stack were taken of the same fish in the same position. This was to determine the number of stacks that lead to the least amount of error. Numbers above error bars are the values of the standard error of the mean. Above stack representations is the amount of time the Opera machine would takes to scan each well and an entire 384-well plate at the given number of z-slices per stack, assuming 9 stacks per well (as above).</p

Cardiodynamic Influence of Methanolic Hawthorn Extract (MHE).

<p>A. Fourier transformed data of control and 6.5 mg/mL MHE treated 5dpf zebrafish embryos. Notice the increased amplitude in the MHE treated fish which corresponds to an increased stroke volume (SV) B. and C. SV, heart rate (HR), cardiac output (CO) and ejection fraction (EF) measurements for Fourier method (B) and segmentation method (C). SV and EF were significantly increased according to both measurement paradigms (* indicates P<0.05, n = 22, 26 for Control and MHE treated fish respectively).</p

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

<div><p>Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic <i>KRAS</i> mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model <i>in vivo</i>. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.</p></div

iMDK induced apoptosis in H441 lung adenocarcinoma cells.

<p>iMDK dose-dependently increased apoptosis in H441 cells but not normal NHLF cells. Cells were treated with the indicated concentrations of iMDK for 72 hours and then stained with Hoechst 33342 dye and analyzed under a fluorescence microscope as described in Methods (scale bar shows 50 μm). Apoptosis was induced in H441 cells 48 hours after iMDK treatment at a concentration of 25 nM (upper panel, scale bar shows 200 μm). TUNEL positive cells were increased by iMDK treatment in a dose–dependent manner (bottom panel). TUNEL staining was performed as described in Methods. Statistical significance was defined as p<0.05 (#). <b>A.</b> Cleaved caspase-3, an apoptosis marker, was increased in H441 cells following iMDK treatment. H441 cells were treated for 48 hours with iMDK in the indicated concentrations and harvesting for immunoblot analysis as described in Methods. <b>D.</b> iMDK induced sub-G₀/G₁ DNA content in H441 cells. Cells were treated with iMDK for 72 hours and DNA content was measured by propidium iodide stain and flow cytometric analysis as described in Methods.</p

iMDK induced growth inhibition in MDK-positive non-small cell lung carcinoma cells.

<p><b>B.</b> Growth inhibition by iMDK was increased in the MDK-positive HEK293, H441 and H520 cells but not the MDK-negative A549 cells or normal NHLF cells after 48 hours of treatment. Cell viability was assessed by trypan blue exclusion assay as described in Methods. Statistical significance was defined as p<0.01 (*). Dose-dependent growth inhibition by iMDK was observed morphologically in H441 lung adenocarcinoma cells. Shown are phase-contrast photomicrographs of H441 cells 48 hours after iMDK treatment (scale bar shows 100 μm).</p

iMDK inhibited the PI3K/AKT pathway and influenced the apoptosis pathway.

<p><b>A.</b> Dose-dependently, phosphorylation of PI3K and AKT and the expression of survivin and XIAP, anti-apoptotic factors, were decreased while the expression of BAD, a pro-apoptotic factor, was increased 48 hours after treatment with iMDK. Shown is immunoblot performed as described in Methods. <b>B.</b> Time-dependently, phosphorylation of PI3K and AKT and the expression of survivin and XIAP were decreased while the expression of BAD was increased by treatment with iMDK at a concentration of 50 nM. Immunoblot was performed as described in Methods.</p