HUVECs were freshly isolated from umbilical cords essentially as previously described . Cells were pooled from two or more donors - to minimize variability associated with cells derived from a single male or female newborn donor - and used at passages 1-5. HUVECs were routinely grown in 199 medium supplemented with 20% fetal bovine serum (FBS), 25 µg/ml endothelial cell growth supplement (ECGS), and 50 mg/ml heparin on 0.1% gelatin-coated surfaces. All the experiments were performed on ECs or conditioned media collected after an overnight incubation in the absence of serum or in the presence of 2% FBS, unless otherwise indicated.
Label-free mass spectrometry (LC-MSE) analysis
Secretome samples for proteomic analysis were desalted, concentrated and digested as previously described . After lyophilization, the secreted protein pellets were dissolved in 25 mmol/L NH4HCO3 containing 0.1% RapiGest (Waters Corporation), sonicated, and centrifuged at 13,000g for 10 min. Samples were then incubated 15 min at 80°C and reduced with 5 mmol/L DTT at 60°C for 15 min, followed by carbamidomethylation with 10 mmol/L iodoacetamide for 30 min at room temperature in the darkness. Then, sequencing grade trypsin (Promega) was added to each sample (1 mg every 50 mg of proteins) and incubated overnight at 37°C. After digestion, 2% TFA was added to hydrolyze RapiGest and inactivate trypsin. Tryptic peptides were used for label-free mass spectrometry analysis, LC-MSE, performed on a hybrid quadrupole-time of flight mass spectrometer coupled with a nanoUPLC system and equipped with a Trizaic source (Waters Corporation) as previously described in details [23,26]. Statistical analysis has been performed by means of Progenesis QIP v 4.1 (Nonlinear Dynamics) using a Uniprot human protein sequence database (v2017-1). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner repository with the dataset identifier PXD020375 and 10.6019/PXD020375.
Gene Ontology analysis
Data were analyzed with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 11.0) database  as previously described  to identify enriched gene ontology (GO) terms in the biological process, molecular function or cellular component categories. We specifically employed the enrichment widget of STRING, which calculates an enrichment P-value based on the Hypergeometric test and the method of Benjamini and Hochberg for correction of multiple testing (P value cut-off of <0.05).
Measurement of secreted PTX3
Conditioned media (2.0-2.5 ml/flask) were collected after an overnight incubation from confluent HUVEC monolayers cultured in 25 cm2-gelatin coated flasks. After a 10-min centrifugation (300g at 4°C) to remove cellular debris, media were aliquoted and stored at -80°C. The concentration of PTX3 was measured using the Human Pentraxin 3/TSG-14 Immunoassay (R&D Systems Inc).
Photographs of HUVECs were acquired at a 10x magnification with an Olympus U-CMAD3 phase contrast microscope equipped with an Olympus digital camera.
Measurement of adherent cells
The number of adherent cells was measured by crystal violet on HUVECs plated at a density of 2.0x104 cells/well in 0.1% gelatin-coated 96-well microplates the day before the experiment. Crystal violet binds to DNA and proteins, thus allowing the detection of adherent cells . Briefly, cells were fixed with ice-cold 100% methanol for 10 min and stained with crystal violet (0.5% in 20% methanol) for about 15-20 min. After multiple washes with deionized water, the plate was air-dry, and crystal violet stain was solubilized in DMSO (100 µl/well) before the measurement of optical density at 595 nm by a multiplate reader (Victor™, PerkinElmer).
Measurement of Reactive Oxygen Species (ROS)
ROS were detected as previously described  on HUVECs plated at a density of 2.0x104 cells/well in 0.1% gelatin-coated black 96-well microplates the day before the experiment. Cells were loaded for 30 min at 37°C in the dark with the fluorescent dye 5(6)-Carboxy-2′7′-dichlorofluorescein diacetate (CM-DCFDA, 10 µM) in HBSS buffer (Hepes 25 mM pH 7.4, NaCl 120 mM, KCl 5.4 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 15 mM) containing 1% FBS. Afterwards, the cells were washed, and fluorescence was assessed after 30 min by a multiplate reader (Victor™, PerkinElmer) with excitation and emission wavelengths of 485 and 530 nm, respectively. The protein content of each well was quantified by the BCA assay (Pierce) to normalize sample-to-sample variation.
Quantification of apoptosis/necrosis was performed by Annexin V-FITC conjugate and propidium iodide (PI) staining (Immunological Sciences) followed by fluorescence activated cell sorting (FACS) performed with a FACScalibur flow cytometer equipped with a 488 nm argon laser (Becton Dickinson). The collected data were evaluated by Cell Quest software. In addition, active caspase-3 and PARP-1 proteins were detected by conventional western blot on total cell lysates prepared in SDS-PAGE sample buffer (62 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.04% bromophenol blue). Densitometric analyses of immunoblots were performed using the National Institute of Health (NIH) Image J software package. Full length unedited blots are shown in the Additional file 2.
Small interfering RNA (siRNA) transfection
To silence PTX3 expression, HUVECs were transfected with the Hs_PTX3_1 FlexiTube siRNA duplexes against human PTX3 (SI00695947, Qiagen). An AllStars Negative Control FlexiTube siRNA (Qiagen) was used as control. Both siRNAs were individually transfected at a 5 nM concentration using the PepMute transfection reagent according to the manufacturer’s instructions (Signa Gen Laboratories). For in vitro efferocytosis assays, cells were transfected 48h before calcein-AM loading and overnight serum-deprivation. The knockdown of PTX3 expression was analyzed by reverse transcription and quantitative real time PCR (RT-qPCR).
Total RNA extraction and RT-qPCR
Total cellular RNA was extracted with the Total RNA Purification Kit (Norgen Biotek Corp) and reverse transcribed (1 μg) as previously described . RT-qPCR was performed in triplicate with 2.5 μL of cDNA incubated in 22.5μL IQ Supermix containing primers and SYBRGreen fluorescence dye (Bio-Rad Laboratories) using the iCycler Optical System (Bio-Rad Laboratories). The sequences of PTX3 primers were: forward, 5’- TGC GAT TCT GTT TTG TGC TC-3’; reverse, 5’- TGA AGA GCT TGT CCC ATT CC-3’. GAPDH was used as normalizer. The sequences of GAPDH primers were: forward, 5′- ACG GAT TTG GTC GTA TTG GGC-3′; reverse, 5′- CTC CTG GAA GAT GGT GAT GG-3′.
RAW 264.7 macrophages were plated at a density of 5.0×104 cells/well in 100 µl/well of DMEM + 5% FBS in a black 96-well microplate. HUVECs were loaded for 30 min with calcein-AM (2 µM) in HBSS before overnight incubation in 2% FBS-containing medium. After detachment and re-suspension in the conditioned media, HUVECs were collected by centrifugation and overlaid on the RAW 264.7 cells at a 1:1 ratio of macrophages:HUVECs in a 100 µl volume of 20% FBS-containing 199 medium . A 100 µl aliquot of HUVEC suspension was concurrently plated in empty wells to measure the total fluorescence added to macrophages. Following 2h of incubation, cells were washed 3 times with HBBS - to remove cells that were not engulfed – and the Raw 264.7-associated fluorescence was assessed by a multiplate reader (Victor™, Perkin Elmer). Data are expressed as percent (%) of engulfed cells, that is the RAW 264.7 cell-associated fluorescence divided by the total fluorescence multiplied by 100.
Unless otherwise indicated, data are expressed as mean ± s.e.m. of at least 3 independent experiments performed on different cell preparations. Statistical analysis was carried out using unpaired Student's t-test or 2-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison test. In the 2-way ANOVA analyses, we considered as row factor the sex of cells, and as column factor the treatment (20 vs 2% FBS in Figs. 2D and 3A/B, and ctrl vs PTX3 siRNA in Fig. 5C). P-values of <0.05 were considered significant. All the analyses were performed using the GraphPad Prism software (version 8.4.2).
Reagents and antibodies
All tissue culture reagents were from Euroclone, except ECGS and heparin (Sigma Aldrich). Crystal violet was from Sigma Aldrich; CM-DCFDA and calcein-AM from Cayman Chemicals. Primary antibodies used were mouse monoclonals anti-caspase 3 (Enzo Life Sciences, ALX-804-305) and anti-actin (BD Biosciences, 612656), and rabbit monoclonal anti-cleaved PARP-1 (Cell Signaling, #5625). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Dako (#P0260 and #P0399 for rabbit anti-mouse and swine anti-rabbit antibodies, respectively). Proteins in western blot were detected by the LiteAblot Turbo Extra-Sensitive Chemiluminescent Substrate (Euroclone).