Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) were washed and lysed with RIPA buffer (Sigma‐Aldrich). Protein concentrations were determined by using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc). Aliquots of cell lysates were loaded into 10% SDS‐polyacrylamide gels, electrophoresed and transblotted onto PVDF membranes (Bio‐rad). The blots were blocked with 10× blocking buffer (Sigma‐Aldrich) for 1 h and incubated with primary antibodies (1–1000 dilution) at 4°C overnight.²⁴ The antibodies included phosphorylated p38 mitogen‐activated protein kinase (p‐p38), total p38, phosphorylated c‐Jun N‐terminal kinase (p‐JNK), total JNK, phosphorylated extracellular signal‐regulated kinase (p‐ERK) and total ERK from Cell Signalling Technology. The other antibodies included PP2A from Arigo Biolaboratories. The other antibodies of loading control included GAPDH and α‐tubulin from Thermo Fisher Scientific Inc. and β‐actin from Sigma‐Aldrich. The blots were further incubated with horseradish peroxidase‐conjugated secondary antibodies (1–10,000 dilution, Jackson ImmunoResearch) for 1 h at room temperature. Immunoreactivity was visualized using VisioGlo (Amresco) according to the manufacturer's instructions. The radiographs were subject to VisionWorks software (Ultra‐Violet Products Ltd.). To normalize the expression level, blots were stripped with stripping buffer (69 mmol/L SDS, 100 mmol/L 2‐mercaptoethanol, 93.75 mmol/L Tris‐HCl, pH 6.8) at 56°C and incubated with anti‐β‐actin, GAPDH or α‐tubulin antibody as internal control.²⁴

Circulating endothelial cells (CEC), including circulating endothelial mature cells (CMECs) and circulating endothelial progenitor cells (CEPCs) were separated from blood samples obtained from SSPs 48 to 72 h after admission to the ICU, and from HVs. Blood samples were collected in a 3 ml vacutainer tube containing EDTA as anticoagulant. Collection of blood samples and isolation of cells and their analysis were carried out by double-blinded personnel, who were totally blinded to the patient group corresponding to the collected sample, as well as clinical characteristics or further outcome of the patients.
The CMECs and CEPCs were isolated by magnetic bead-based immunoseparation as described previously [48 (link)–50 (link)]. Briefly, after blood samples were obtained, total mononuclear blood cell fraction was isolated from blood by Ficoll-Histopaque (Sigma-Aldrich, USA) gradient separation. The mononuclear cell fraction was washed by centrifugation with phosphate-buffered saline solution. Then, the mononuclear blood cell fraction was subjected to immunomagnetic bead capture (IBC) using a bead-conjugated CD133 monoclonal antibody and magnetic cell separation system (Miltenyi Biotec). The captured cells corresponded to an enriched CEPC sample (positive selection, CD133⁺), while the cells in the eluted solution contained CMECs (negative selection, CD133⁻). To directly isolate CMECs, eluted fluid was subsequently subjected to a second step of IBC positive selection using a bead-conjugated CD146 monoclonal antibody (Miltenyi Biotec), obtaining an enriched CMECs sample (CD146⁺ and CD133⁻). CMEC and CEPC quantification was performed by flow cytometry. Compensation particles (BD CompBeads) and amine polymer microspheres (Becton Dickinson) were used for compensation [48 (link)–50 (link)]. Fluorescently conjugated antibodies against VE-Cadherin⁺ and CD31⁺ and against VEGFR-2⁺ and CD34⁺ were used for the detailed phenotype characterization of CMECs and CEPCs, respectively.