Diabetic mice 21 days after streptozotocin injection. Mus musculus

We used streptozotocin to induce a model of type I diabetes in transgenic mice that express green fluorescent protein under the control of an endothelial-specific promoter (Tie2 GFP). Three weeks after treatment, endothelial cells were isolated from animals with blood glucose > 350 mg/dl. Age-matched mice received sham intraperitoneal injections. Aortae from the root to the renal bifurcation were rapidly processed by mincing and proteolytic digestion followed by fluorescent activated cell sorting to yield endothelial cell populations of >95% purity. RNA was isolated from >50,000 endothelial cells and subjected to oligodT amplification prior to transcriptional analysis on microarrays displaying the Operon V3 collection of long oligonucleotides representing 32,000 murine transcripts. Dysregulated transcripts were confirmed by real-time PCR, and included glycam1, angptl4, slc36a2, cidea, adam5, ces3, adipsin and adiponectin. By comprehensively examining cellular gene responses in vivo in a whole animal model of type I diabetes, we have identified novel regulation of key endothelial transcripts that likely contribute to the metabolic, angiogenic and pro-inflammatory responses which accompany diabetes. Overall design: Mice homozygous for the Tie2-GFP transgene (Tg[TIE2GFP]287Sato, stock number 003658) were obtained from Jackson labs (Bar Harbor, ME) and bred for these experiments. Males were used at 6-10 weeks of age. Controls were siblings of the treated animals. All procedures were approved by the Institutional Animal Care Committee of the University of Hawaii. Induction of Diabetes Following an overnight fast, a single 180 mg/kg dose of streptozotocin (STZ, Sigma, St. Louis, MO) was injected into the peritoneum. Control animals received injections of sterile saline. Animals were maintained on normal chow and cages changed daily. At the time of sacrifice, glucose levels in cardiac blood were assessed by glucometer (Accucheck, Becton Dickinson). Diabetic, STZ-treated animals (with a glucose level >350 mg/dL) and controls were processed on the same day. Two independent experiments both performed 21 days after STZ-injection. Animals were euthanized by CO2 asphyxiation. The aortas from the iliac bifurcation to the aortic root were excised by dissection and freed of any adherent tissue. The luminal blood was removed and the aortas were sliced into 2 mm segments. The aortic segments pooled from 3-4 animals were suspended in 5 ml of Dulbecco’s PBS with dextrose 2 mg/ml. The suspension was combined with 5 ml of prewarmed PBS to contain 5mg/ml type II collagenase (Worthington), and deoxyribonuclease I (300 units), agitated continuously at 37°C on a shaking platform, and triturated 10 times every 10 minutes for a total digestion period of 40min to generate a single cell suspension. The cell suspension was maintained at 0-4ºC throughout the remainder of the isolation, which lasted 2-3 hours total. The suspension was combined with 10 ml of 10% FBS in DMEM and cells were collected by centrifugation and resuspended in 10 ml of PBS. This suspension was then filtered through a sterile 40 um mesh filter to remove undigested tissue fragments. Following centrifugation, the pellet was resuspended in 3.5 ml PBS containing 0.5 mM EDTA, 30U/ml DNAse I, 3% FBS, and 2 mg/ml dextrose. The suspension was once again filtered through a 40 um mesh filter. Aortic cell suspensions were sorted using a MoFlo from Dako Cytomation (Carpinteria, CA) or the FacsAria from Becton Dickinson. Cells were excited by a 488 nm laser and GFP signals collected via the FL1 channel (510 to 550 nm). Positive cells (were collected directly into Trizol (Invitrogen, Carlsbad CA) and processed according to the manufacturer’s protocol. Glycogen was added to facilitate precipitation of the sub-microgram quantities of RNA thus obtained. RNA was purified further using MicroRNEasy system (Qiagen,) according to the manufacturer’s protocol. The Qubit system (Invitrogen) was used to fluorometrically quantify the resultant RNA.Up to 20 ng RNA isolated from cells was then subjected to two cycles of amplification as described [9] using the Message Amp kit (Ambion, Austin, TX) according to manufacturer’s instructions. This produced approximately 5-30 ug of amplified cRNA after two rounds of amplification. Microarray Analysis 2 ug of cRNA each from diabetic and control were labeled directly with Cy3 and Cy5 fluorophores with the MicroMax labeling kit (Perkin Elmer) according to the manufacturer’s instructions and hybridized overnight to microscope slides containing Operon V3 long oligo array. Following stringency washing, slides were scanned using the Molecular Devices 4000B (CA). Replicate hybridizations were performed (with Cy3-Cy5 dye-swapped) and were analyzed with Genepix and Acuity software from Molecular Devices.