Th female FABP4??mice (both on C57BL/6J background as previously described24) to create the VEGF-TG/FABP4??mouse line. Five- to six-week-old transgenic and wild-typeQuantitative Real-Time PCR AnalysisTracheas were homogenized, and total RNA was isolated with TRIZOL (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized with SuperScript First-Strand Synthesis Program for Real-Time-PCR (Invitrogen) as outlined by the manufacturer’s directions. Real-time PCR reaction was performed in a 20-mL volume with SYBR Green (Bio-Rad, Hercules, CA) together with the use of pooled cDNA samples. The PCR primers are listed in Table 1.ajp.amjpathol.org-The American Journal of PathologyFABP4 in Airway AngiogenesisTable 1 Gene Cyclophilin A Forward Reverse eNOS Forward Reverse FABP4 Forward Reverse IL-b1 Forward Reverse MCP1/CCL2 Forward Reverse SCF Forward Reverse Real-Time PCR Primer Sequences Primer sequences 50 -TCTGAGCACTGGAGAGAAAGGA-30 50 -TATGGCGTGTAAAGTCACCACC-30 50 -ATGCCTACAGCATTGGTTGCAAGG-30 50 -AGCATATGAAGAGGGCAGCAGGAT-30 50 -TCACCATCCGGTCAGAGAGTA-30 50 -GCCATCTAGGGTTATGATGCTC-30 50 -ACGGACCCCAAAAGATGAAG-30 50 -TTCTCCACAGCCACAATGAG-30 50 -GTCCCTGTCATGCTTCTGG-30 50 -GCTCTCCAGCCTACTCATTG-30 50 -TCAAGAGGTGTAATTGTGGACG-30 50 -GGGTAGCAAGAACAGGTAAGG-FABP4 immunoreactivity was observed in endothelial cells of capillaries and little blood vessels in the lamina propria. In VEGF-TG mouse tracheas, FABP4-immunoreactive endothelial cells were detected inside the similar location but had been enhanced in number. In addition, some FABP4?capillaries had expanded to reach an intraepithelial location as previously reported.23 Double-immunofluorescence for CD31, a pan-endothelial cell marker, and FABP4 confirmed the endothelial cell localizationBone Marrow TransplantationVEGF-TG and VEGF-TG/FABP4??mice had been irradiated with 12 Gy in two split doses given 4 hours apart.24 WT and FABP4??mice of matching age have been sacrificed on the very same day, and bone marrow was harvested in the correct tibia and femur under sterile conditions. Two hours following irradiation, VEGF-TG and VEGF-TG/FABP4??mice had been injected with 2 ?106 bone marrow cells in 150 mL of sterile saline from FABP4??and WT mice, respectively, by means of the tail vein. The mice were administered dox-water for 3 or 7 days and euthanized eight to 9 weeks just after bone marrow transplantation (BMT). Tracheas have been harvested and processed as described in Animals and Human Specimens.Statistical AnalysisResults are presented as indicates ?SEMs unless otherwise noted. Statistical significance was determined with KruskalWallis and U-tests for ordinal and Fisher Exact test for nominal variables. P values 0.05 were thought of considerable.ResultsEndothelial Cell FABP4 Expression Is Induced by VEGF inside the Mouse Airway MucosaTo characterize the relation between VEGF and FABP4 in vivo, WT and VEGF-TG mice had been provided dox-water for 2 weeks and sacrificed.Olivetol structure Tracheas have been harvested, and mRNA was isolated and analyzed by reverse transcription real-time PCR for FABP4 expression.Formula of 1538005-13-8 Relative mRNA levels of FABP4 had been substantially increased in VEGF-TG mice than in WT mice (P 0.PMID:24360118 05) (Figure 1A). The localization of FABP4 on tracheal sections was determined by immunohistochemistry in mice offered doxwater for three days (Figure 1B). In manage WT mouse tracheas,Endothelial cell FABP4 expression is induced by VEGF in the airway mucosa. A: WT and VEGF-TG mice were offered dox-water for two weeks (n Z six per group). Tracheas were harvested and snap frozen. RNA was isolated and reverse t.
