Role of IL-8 and TIMP-2 on term canine trophoblast migration, invasion, and proliferation Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/kp78gk50x

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  • Placental pathologies are not uncommon in dogs. For example, in primiparous bitches, the reported incidence of subinvolution of placental sites is 21% - 100%. Despite years of research in multiple species, the mechanisms regulating late gestation trophoblast behavior in the dog remain unexamined. Therefore, the objective of this study was to characterize normal in vitro term canine trophoblast (TCT) physiology, with respect to migration, invasion, and proliferation. In addition, the effects of interleukin-8 (IL-8) and tissue inhibitor of metalloproteinase-2 (TIMP-2) on trophoblast physiology were examined. Following isolation of primary TCT, the cell suspension was seeded into the migration (wound-healing) assay, invasion (Matrigel) assay, and proliferation (MTT) assay. Cells were cultured under the same conditions at 37ºC with 5% CO₂ for each assay. Recombinant human IL-8 (200-08M, Peprotech, Rocky Hill, NJ, USA) was used at a concentration of 10 ng/ml for each assay. Recombinant human TIMP-2 (410-02, Peprotech) was used at a concentration of 0.5 μg/ml for each assay, respectively. For the migration assay, cells were suspended in medium supplemented with 10% fetal bovine serum (10% FBS). Primary TCT (1000X10³ cells/well) were cultured in 12-well tissue culture plates until a confluent monolayer was formed. A wound was added with a sterile pipette tip, the monolayer was rinsed, and 10% FBS with no factor (control), with IL-8 or TIMP-2 was then added. Photomicrographs of the wound were taken with phase-contrast microscopy (100X) after 8 h in culture and the area of the wound was measured using ImageJ v.1.34 software. The experiment was performed in triplicate (n=5 dogs). The invasion assay was performed using Matrigel invasion chambers (#354480, BD Biosciences). Primary TCT (250X10³) were suspended in protein-free medium (PFM) with no factor (control), with IL-8 or TIMP-2 and seeded onto Matrigel-coated filter membranes in the upper well. Chemoattractant (10% FBS) was placed in the lower well and the 24-well chambers were cultured for 22 h. Non-invading cells were removed from the filters that were then fixed and stained. Cells were counted under light microscopy (400X). The experiment was performed in triplicate (n=5 dogs). For the proliferation assay, cells were suspended in PFM. For the proliferation assay (CGD-1, Sigma, St. Louis, MO, USA) primary TCT (100X10³ cells/well) were suspended in PFM with no factor (control) or with IL-8 and cultured in 96-well tissue culture plate for 28 h. Cells were then incubated with MTT for 4 h, which was later replaced with 1-propanol and the plates were vigorously shaken. Absorbances were measured with a microplate reader at 570 nm and 690 nm (to remove background). Measurements were analyzed with SoftMax Pro (5.2 program SoftMax® Pro Data Acquistion & Analysis Software, Sunnyvale, CA). The experiment was performed in quadruplicate (n=4 dogs). Statistical analysis of the data ws performed using Repeated Measures ANOVA (migration), one-way ANOVA (invasion), or two-way ANOVA (proliferation) in PROC MIXED using SAS (Version 9.2, SAS Institute Inc., Cary, NC). For the migration assay, the mean of the control wound area at 0 h was set to 0% wound closure and the data were expressed as the percent wound closure of the control for each dog. For the invasion assay and proliferation assay, the mean of the controls was set to 100% and the data was expressed as the percentage of the control for each dog. Significance was defined as P<0.05. Analysis showed that IL-8 increased cell migration by 35% compared to the control (P<0.01). TIMP-2 had no significant effect on cell migration (P=0.38). There was no significant effect of IL-8 on cell invasion compared to the control (P=0.42), whereas TIMP-2 decreased cell invasion by 57% compared to the control (P<0.05). Also, for the proliferation assay IL-8 had no significant effect on cell proliferation (P=0.18). This study was the first to investigate term canine trophoblast physiology. Future studies should compare differences between normal term trophoblasts and those from cases of subinvolution of placental sites. It would also be of interest to compare differences in trophoblast physiology at different stages of gestation.
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