Cells were removed using trypsin/EDTA, washed with DMEM containing 10% FBS, centrifuged at 400 for 5 min, and suspended at 2105cells/ml in EC growth medium without serum
September 20, 2021
Cells were removed using trypsin/EDTA, washed with DMEM containing 10% FBS, centrifuged at 400 for 5 min, and suspended at 2105cells/ml in EC growth medium without serum. Cyp1B1-mediated EC function 27. In addition, microarray studies show dramatic up-regulation of Cyp1B1 by arterial levels of shear stress in cultures of human EC 20. These results suggest an important role for Cyp1B1 in vascular development and homeostasis. However, expression of Cyp1B1 in perivascular supporting cells, including PC, and its deficiency on PC function remains to be explored. Much investigation into the interactions between EC and PC has revealed that these two vascular cell types are interdependent, and that primary defects in one cell-type may have obligatory consequences on the other 28C29. However, the expression and function of Cyp1B1 in PC that invest the microvessels requires further investigation. Using transgenic mice that carry an interferon–inducible temperature-sensitive large T antigen, we isolated PC from and mice. Here we demonstrate that Cyp1B1 is constitutively LRP1 expressed in PC, and its deficiency leads to increased oxidative stress, sustained NF-B p65 activation, and altered production of the matricellular proteins including increased expression Schisandrin C of thrombospondin-2 (TSP2). These cells Schisandrin C also exhibited alterations in the rate of proliferation and apoptosis, migration, adhesion to various extracellular matrix proteins, as well as their receptor expression, and decreased expression of vascular endothelial growth factor (VEGF). Together our results suggest that the expression of Cyp1B1 in retinal PC is essential for maintaining the physiological function and integrity of the vasculature. MATERIAL AND METHODS Experimental Animals All experiments were carried out in accordance to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin School of Medicine and Public Health. Immortomice expressing a temperature-sensitive simian virus (SV) 40 large T antigen (Charles River Laboratories, Wilmington, MA) were backcrossed into C57BL/6jmice in our laboratory, and further crossed with mice, and generated in a C57BL/6j background. The immorto -mice were identified by PCR analysis of DNA isolated from tail biopsies. The PCR primer sequences were as follows: immorto forward: Schisandrin C 5-CCT CTG AGC TAT TCC AGA AGT AGT G-3, immorto reverse: 5-TTA GAG CTT Schisandrin C TAA ATC TCT GTA GGT AG-3; Neomyacin forward: 5-TTG GGT GGA GAG GCT ATT CGG CTA TGA-3, Neomycin reverse: 5-GGC GCG AGC CCC TGA TGC TC-3; Cyp1B1 forward: 5-CTG AGT TGG ACC AGG TTG TGG-3; Cyp1B1 reverse: 5-CAT GGA TTC TAA ACG ACT AGG-3. Tissue Preparation and Culture of Retinal Pericytes Pericytes were isolated from mouse retinas by collecting retinas from one litter (6C7 pups, 4 wk old) using a dissecting microscope. Twelve to fourteen retinas were rinsed with serum-free Dulbeccos Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA), pooled in a 60-mm dish, minced, and digested for 45 min with collagenase type II (1 mg/ml, Worthington, Lakewood, NJ) with 0.1% BSA in serum-free DMEM at 37C. Cells were rinsed in DMEM containing 10% fetal bovine serum (FBS) and centrifuged for 5 min at 400 PC. Confluent cultured PC from 60 -mm culture plates were rinsed with phosphate buffered saline (PBS) containing 0.04% EDTA and incubated with 1.5 ml of cell dissociation solution (Tris-buffered saline [20 mM Tris-HCl and 150 mM NaCl; pH 7.6] TBS containing 2 mM EDTA and 0.05% BSA). Cells were rinsed from plates with DMEM containing 10% FBS, washed once with 5 ml of TBS, and blocked in 0.5 ml of TBS with 1% goat serum for 20 min on ice. Cells were centrifuged 5 min at 400 retinal PC at 1104 in triplicate per time point in 60-mm tissue culture dishes. Cell numbers were counted every other day in triplicate for seven days and fed on days they were not counted. The rate of DNA synthesis was measured using Click-iT? EdU Alexa Fluor 488 kit as recommended by the supplier (Invitrogen). The assay measures incorporation of 5-ethynyl-2-deoxyuridine (EdU), a nucleoside analogue of thymidine, during cell proliferation. and retinal PC were plated at 5105cells on 60 -mm tissue culture dishes and were incubated with 10 M EdU in PC medium for 2 h at 33C. DNA synthesis was analyzed by measuring incorporated EdU using the FACSscan Caliber flow cytometer (Becton-Dickinson). Ten thousand cells were analyzed for each sample and three independent experiments were performed with two different isolation of PC. Real Time-PCR Analysis PC were allowed to reach 90% confluence, rinsed twice with PBS, scraped from 60-mm tissue culture plates and transferred to Eppendorf tubes. Cells were centrifuged, immediately.