Cd31 cells

Primary BMVECs were isolated from E18 PAE and SAC cortices (pooling all cortices from each dam) following removal of the meninges using the Magnetic Activated Cell Sorting (MACS®) system with microbeads for negative selection of CD45+ cells and positive selection of CD31+ cells (Miltenyi Biotec). Immortalized bEnd.3 murine BMVECs were purchased from Sigma-Aldrich and were grown in Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (Neuromics) and 1% Penicillin–Streptomycin (Thermo Fisher Scientific). Downregulation or overexpression of miR-150-5p was achieved by transfecting bEnd.3 BMVECs with 50 nM miR-150-5p LNA™ mimics miR-150-5p LNA™ inhibitors, negative control mimics, or negative control inhibitors (Qiagen). Overexpression of Vezf1 was achieved by transfecting bEnd.3 BMVECs with 130 ng (96-well plate) or 4 μg (6-well plate) of pCMV6 Vezf1 (Origene) or pcDNA3.1 control vector (Thermo Fisher Scientific). Transfections were performed using antibiotic-free media using Lipofectamine™ 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific).

Primary murine lung EC isolation was performed as we previously did^{12 (link)–14 (link)}. Briefly, 14–18wk old mice were euthanized using 100% CO₂ inhalation followed by cervical dislocation. The chest was immediately opened through a midline sternotomy. The left ventricle was identified and the ventricular cavity was entered through the apex with a 27-gauge needle. The right atrium was identified and an incision was made to exsanguinate the animal and to allow the excess perfusate to exit the vascular space. The animal was perfused with 20 ml of cold PBS. The lung tissue was collected and minced finely with scissors. The tissue fragments were digested in DMEM medium containing 1 mg/mL Collagenase D/Dispase (Roche, Switzerland) and 25 U/mL DNase (Sigma, St. Louis, MO) at 37C for 2hr with shaking, after which the suspension was homogenized by triturating. The homogenate was filtered through a 70μm nylon mesh (BD Biosciences, San Jose, CA) and pelleted by centrifugation (400g for 5 min). Cells were first depleted for CD45⁺ cells (MiltenyiBiotec) and then positively selected for CD31⁺ cells (Miltenyi Biotec) using magnetically labeled microbeads according to the manufacturer’s protocol. Isolated ECs (CD45^-CD31⁺) were cultured in EC culture medium with no medium change for the first 72hrs to allow EC attachment followed by medium change about every 3 days.

Primary murine EC isolation was performed as we previously described²⁷. Briefly, mice were euthanized, the lung, which is an organ with hematopoietic function especially thrombopoiesis,[28 (link)] or spleen tissue was collected and minced finely with scissors. The tissue fragments were digested in DMEM medium containing 1 mg/mL Collagenase D (Roche, Switzerland), 1 mg/mL Collagenase/Dispase (Roche) and 25 U/mL DNase (Sigma) at 37°C for 1 hr (for spleen) or 2 hr (for lung) with shaking, after which the suspension was homogenized by triturating. The homogenate was filtered through a 70μm nylon mesh (BD Biosciences, San Jose, CA) and pelleted by centrifugation (400 g for 5 min). Cells were first depleted for CD45⁺ cells (Miltenyi Biotec, SanDiego, CA) and then positively selected for CD31⁺ cells (Miltenyi Biotec) using magnetically labeled microbeads according to the manufacturer’s protocol. Isolated ECs (CD45⁻CD31⁺) were cultured in EC culture medium with no medium change for the first 72 hrs to allow EC attachment followed by medium change every 2–3 days. Cells were re-selected for CD31⁺ cells when they reach >70–80% confluence (usually after 3–4 days of culture).