Online Supplement
A Role for Low Density Lipoprotein Receptor-related Protein 1 in the Cellular Uptake of
Tissue Plasminogen Activator in the Lungs
Swan Lin1, Jennifer Racz1, Melissa F. Tai1, Kristina M. Brooks1,
Phillip Rzeczycki2, Lauren J. Heath1, Michael Newstead3, Theodore J. Standiford3,
Gus R. Rosania2 and Kathleen A. Stringer1
1Department
3Division
of Clinical Sciences; 2Department of Pharmaceutical Sciences, College of Pharmacy;
of Pulmonary and Critical Care Medicine, School of Medicine, University of Michigan,
Ann Arbor, MI, USA
Methods
Western Blot of Human tPA in Mouse Lung Homogenate
The lungs of a post-mortem C56Bl/6 male mouse were perfused blood-free and homogenized in the
absence of protease inhibitors as previously described (1). Briefly, the sample was homogenized
(Tissuemiser, Fisher Scientific, Pittsburg, PA) on ice in the presence of tissue extraction reagent
(Pierce, Rockford, IL). Residual red blood cells were lysed by the addition of red blood cell lysing
buffer (1 mL; Sigma, St. Louis, MO). Homogenate was transferred to a microcentrifuge tube and
clarified by centrifugation (13,000 x g, 4°C) for 20 min. A known amount of htPA (5 µg) was added to
the homogenate (1 mL) and it was incubated (37°C). Over time, aliquots (50 µL) were removed from
the primary sample and transferred to the freezer (-80°C). Upon completion of the experiment, an
equal volume (30 µL) of each sample was subjected to 12.5% SDS-PAGE. Following electrophoresis,
proteins were transferred to PVDF and the membrane was probed for human tPA (1:10,000 dilution
of a rabbit anti-human tPA antibody; Molecular Innovations). Protein was detected using a secondary
fluorescent antibody (goat anti-rabbit IgG cy5, 1:5000 dilution) and an image of the blot was acquired
using a Typhoon Variable Mode Imager (GE Healthcare).
Image Acquisition and Analysis Workflow
Digital images of stained and unstained lung and liver sections at 40X magnification were acquired on
a Ti Eclipse inverted microscope (Nikon Instruments, Inc., Melville, NY, USA) using bright field optics
under Kohler illumination. To acquire and quantify DAB stained images, a red filter (655 ±30 nm),
which corresponds to the absorbance peak of the background staining, and a blue filter (480 ± 30
nm), which corresponds to the absorbance peak of DAB, were used (Figure E1). The density of DAB
staining is indicative of LRP1 receptor density. To quantify DAB staining density all images were
analyzed with ImageJ. The red filter image was first converted to grey scale (Figure E2). Then, a
threshold was applied to this image to select the pixels corresponding to the tissue region. This
threshold was based on a visual inspection of the tissue sample, capturing pixel intensities that
corresponded to the tissue itself. This was performed on five control lung and liver images, allowing
for an average background tissue signal to be determined for each organ. The average threshold
value, as determined from the untreated tissue samples, was applied to the corresponding blue
image, selecting the pixels corresponding to the darkly stained DAB positive regions. The selected
pixels for the red image was used as a measure of the total area of the tissue region, and the
selected pixels for the blue were used as a measure of the tissue area with DAB signal. Dividing the
pixel area of DAB signal by the total tissue area was then used to calculate the % area stained
measurement.
For the comparative analysis of lung and liver LRP1, the area and area fraction (%area)
measurements were acquired. The area captures the total image in square pixels while the %area
measures the percentage of pixels that fall within the set threshold over the total area. The ratio of
LRP1 positive staining was determined by dividing the pixel area of DAB signal by the total tissue
area which was used to calculate the % area stained measurement. Any nonspecific background
staining removed from the true LRP1 signal, by subtracting the background % tissue area with LRP1
staining measured with a negative control (unstained) set of images from the % tissue area with
LRP1 staining from the experimental (DAB stained) set of images (Figure E3).
Figure S1: Western blot of human tPA (htPA) that was added to homogenized mouse lung and
shows the rapid degradation of the parent protein (MW ~68kDa; lane 2). This is further illustrated by
the increased signal intensities of smaller molecular degradation products. This is apparent as early
as 5 min after the addition of htPA (5µg) to lung homogenate (lane 1) and continues to be evident at
15 (lane 3) and 30 min (lane 4). MWM= molecular weight marker.
Figure S2: Bright field images with blue filter show greater intensity of staining than red filter images.
A representative image of an LRP1 stained mouse lung (40X) section in the (A) bright field; (B) with
the red filter; and (C) the blue filter. Scale bar is 50µm.
Figure S3: Threshold applied images from bright field red and blue filters. The red filtered image
captures all tissue positive areas on the slide. The blue filtered image captures only stained area on
the tissue. A representative bright field image (A) of stained mouse lung section (40X) with the
threshold-applied red filter image (B) and the threshold-applied blue filter image (C). Scale bar is
50µm.
Figure S4: Sections of LRP1 stained and unstained sections of mouse liver and lung (40X).
Representative (A) LRP1 stained liver; (B) unstained liver; (C) LRP1 stained lung and (D) unstained
lung. Scale bar is 50µm.
Figure S5: As in the mouse, the ratio of LRP1 staining in macaque monkey liver and lung sections
were similar. Representative color light micrographs of (A) liver and (B) lung sections (40X) stained
for LRP1 and 3’3’-diaminozenzidene (DAB) that is depicted by the brown staining. (C) Quantitation of
the ratio of LRP1 staining in liver and lung sections was not different between the two groups
(p=0.969 by unpaired Student’s t-test). Data are mean (±S.E.M.) of 7 sections/group. Scale bar is
50µm.
1.
Lackowski NP, Pitzer JE, Tobias M, Van Rheen Z, Nayar R, Mosharaff M, Stringer KA. Safety
of prolonged, repeated administration of a pulmonary formulation of tissue plasminogen activator in
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