Mice
Female 6- to 7-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were housed under 12 h light- dark cycles in temperature-controlled holding rooms with unlimited access to dry mouse chow and water. Newly received mice were acclimated to the animal facility for at least one week prior to experimental use.
IPSE protein production and labeling
Recombinant H03 H-IPSE, H06 H-IPSE, and an NLS mutant of H03 H-IPSE (the wild type NLS sequence SKRRRKY changed to SAAGAAY) were produced in HEK293-6E cells, and then purified via immobilized-metal affinity chromatography using an 8x His tag in the construct as described (11). H03 H-IPSE was conjugated to Alexa Fluor 488 using a Alexa Fluor 488 antibody labeling kit (Thermofisher Scientific, Waltham, MA) according to the manufacturer’s instructions; however, the pH was kept at 7.4 throughout the reaction to enrich for labeling of the terminal amine (pKa of 7.4). The efficiency of conjugation was confirmed by Nanodrop. The typical level of labeling was one mole of dye per mole of IPSE, which suggested IPSE was only labeled on the terminal amine. Avoiding labelling of lysine or arginine residues in the protein backbone is critical, as each of the positively charged amino acids in the NLS is essential for nuclear translocation (9). Low labeling efficiency thus minimized the potential interference of the dye with IPSE’s functional domains.
S. haematobium egg injection
Bladder wall injections were performed as described previously (14). Female BALB/c mice were anesthetized with isoflurane, a midline lower abdominal incision was made, and the bladder was exteriorized. S. haematobium eggs (3,000 eggs in 50 μl) were injected into the wall of the bladder. Abdominal incisions were subsequently closed with 4-0 Vicryl sutures, and the surgical site was treated once with topical antibiotic ointment.
Histology
Bladder tissues were fixed in neutral buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin. Histology was analyzed by a board-certified pathologist (JIO) in a blinded fashion.
Ex vivo angiogenesis and microvascular leakage microscopy
Microvascular leakage was assessed three weeks following S. haematobium egg or vehicle injection using an established protocol (15). This protocol involves the use of FITC-lectin to gauge vessels that are being perfused while also providing anatomic detail. Briefly, following FITC-lectin injection, 100 µL R50 Fluoro-Max red fluorescent microspheres, 0.048 µm in diameter (Thermo Scientific), were injected through the inferior vena cava. After 3–5 min in circulation, a sternotomy was performed, and the aorta was cannulated via the left ventricle with an 18-gauge angiocatheter and perfused with 1% paraformaldehyde for 3–5 min using a mini pump (Fisher Scientific). Grafts were harvested and mounted as described above. Microvascular permeability was assessed by using confocal microscopy to determine the extent of microsphere extravasation.
Proliferation xCELLigence assay
Cells were seeded at 5,000 cells per well in 200 µl of complete media in E-plates (ACEA Biosciences, San Diego, CA, USA) and grown overnight while monitored with an xCELLigence DP system (ACEA Biosciences) which monitors cellular events in real time by measuring electrical impedance across interdigitated gold micro-electrodes integrated on the bottom of tissue culture plates (16,17). Cells were washed three times with PBS and cultured with 180 µl EGM-2 basal media (no growth factors or supplements) and incubated for a minimum of 6 h before further treatment. Treatments were prepared at 10 × concentrations and added to each well in a total volume of 20 µl. The xCELLigence DP recorded cell index readings every 15 min for 3 days after treatment. Cell index readings were normalized before treatment and cell proliferation ratios were determined from four biological replicates and represent the relative numbers of cells compared to control cells. A two-way ANOVA with Holm–Sidak’s multiple comparisons test was used to compare IPSE treatment to medium-alone control, with P ≤ 0.05 deemed significant.
Cell lines
3B-11 cells were obtained from ATCC (Manassas, Virginia). ATCC uses morphology, karyotyping, and PCR based approaches to confirm the identity of human cell lines and to rule out both intra- and interspecies contamination. These include an assay to detect species specific variants of the cytochrome C oxidase I gene (COI analysis) to rule out inter-species contamination and short tandem repeat (STR) profiling to distinguish between individual human cell lines and rule out intra-species contamination. HCV-29 cells were obtained from Dra. Monica Botelho. These cells have undergone STR profiling using the following markers: amelogenin, D8S1179, D18S51, D21S11, FGA, TH01, and vWA. The 3B-11 cells and the HCV-29 cells were Mycoplasma-free as established using the Lookout Mycoplasma PCR detection kit (Sigma-Aldrich)
Tubule formation assay
Growth factor-reduced Matrigel (Corning, Corning, NY, USA) was plated into a 96-well μ-angiogenesis plate (ibidi, Planegg, Germany) at 10 μl/well, and incubated at 37°C in 5% CO2 in air for 60 min as described (18). 3B-11 cells were detached using Trypsin/EDTA and resuspended in DMEM (Gibco), and seeded at 30,000 cells/well in medium supplemented with 10 µM sulforaphane (SFPH, Sigma) (negative control), or 1.8 μg/mL or 3.6 μg/mL IPSE. The ibidi plate was incubated for 5 h in a humidified atmosphere of 5% CO2 in air at 37°C in a microscope stage top incubator (OKOLAB, Pozzuoli, Naples, Italy). At intervals, photomicrographs of cells and nascent and developed tubules were collected using a Leica DMi8 automated platform microscope under bright field at 2.5 × magnification, and LASX software (Leica).
Analysis of tubule formation
Automated angiogenesis assessment was performed on TFA 4902 pixel images by ImageJ (NIH) with the phase-contrast Angiogenesis Analyzer plugin tool as described (19,20). Settings used were as follows: 10 pixel minimum object size; 25 pixel minimum branch size; 2,500 pixel artefactual loop size; 25 pixel isolated element size threshold; 30 pixel master segment size threshold; with iteration number of 3. The four output metrics (mesh count, segment count, segment length, and junction count) were either plotted directly or as a percentage relative to the medium-alone blank treatment (treatment measure divided by medium-alone measure). A two-way ANOVA with Holm–Sidak’s multiple comparisons test was used to compare IPSE treatment against medium-alone blank control for the four metrics with P ≤ 0.05 deemed significant.
Combining the four metrics into a single evenly weighted variable was accomplished through the calculation of Z standardized scores that were based on population values (21). The formula below generates the Z score and represented the distance between the raw score and the population mean in units of the SD. Population values were estimated from 39 treatment replicates.
Z=(treatment metric value−metric population mean)/population standard deviationZ=(treatment metric value−metric population mean)/population standard deviation
The combined robust Z score (Z*) was generated for each replicate from the median Z score of the four metrics. Z* scores were plotted, and IPSE treatments compared to medium-alone blank control using one-way ANOVA with Holm–Sidak’s multiple comparisons test, P ≤ 0.05 was considered to be statistically significant.
Cell cycle analyses and CFSE assay
The human bladder epithelium (urothelium) cell line HCV-29 was grown in T-75 tissue culture flasks in complete DMEM media (Gibco) under 5% CO2 at 37°C. For cell cycle assays, 1x105 urothelial cells were co-incubated with IPSE. Following 48 hours of culture, the cells were fixed and stained with propidium iodide for cell cycle analysis. For CFSE assays to assess cell proliferation, cells were stained with the CFSE dye prior to stimulation with IPSE and cultured for 48 hours. The CFSE dye was evaluated post-culture by flow cytometry using the FITC channel. The intensity of CFSE dye, which halves with each cell cycle, was used to track the generations of urothelial cells.
Endocytosis assays
HCV-29 human derived urothelial cells [see (22)] were grown in MEM (Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). For internalization assays, floating cells and adherent cells (released via 0.12% trypsin (Sigma-Aldrich, St. Louis, MO) without EDTA) were washed in fresh medium, and aliquoted into 24 well plates at 200,000 cells/mL in 1 mL. The cells were incubated with Alexa 488-labeled H03 at 1 μg/mL (H03 was conjugated to Alexa 488 using a kit from Thermofisher Scientific, Waltham, MA) for 16 hours at 37° C. Cells were released via 0.12% trypsin without EDTA and washed 3 times with PBS (Sigma-Aldrich, St. Louis, MO). 0.4% trypan blue (Thermofisher Scientific, Waltham, MA) was added to the cells (1:4) to quench extracellular Alexa 488 signal. The cells were analyzed by flow cytometry (Beckman Coulter, CytoFLEX) to isolate the intracellular Alexa 488 signal. Data were analyzed using FlowJo and GraphPad.
Statistical analysis
Statistical analyses were performed using GraphPad software. Except where noted otherwise, the Mann-Whitney U test and Student’s t test were used to evaluate statistical significance for nonparametrically and parametrically distributed data, respectively. P values of <0.05 were defined as significant.