Human sample description

Human PRP samples were collected from 26 healthy donors. Seventeen PRP were secondly divided based on the different genotypes in hetero, WT, and LAV (etero: n = 6; WT: n = 8; LAV: n = 3) according to the variant of the protein BPIFB4 owned. All participants signed an informed consent for the management of personal anamnestic data and blood samples.

The collection of human BM biopsy was approved by the internal Ethics Committee of IRCSS MultiMedica: [n°PROT20/2010-Em3, “Rimodellamento e Disfunzione del Midollo Osseo nel Diabete Mellito”].

Animal models

The overall objective of our in vivo study was to investigate the role of platelets in the AVV-LAV-BPIFB4 effects. To investigate this, we used C57BL/6 mice from Jackson Laboratories. Mice were randomly assigned to receive injections of either a control IgG or anti-CD42b to deplete circulating platelets after receiving AAV-LAV-BPIFB4 or EMPTY vector, as control. After 72 h of platelet depletion, mice were harvested, and the blood, lymphoid organs (bone marrow and spleen), and mesenteric aortas were collected. The Institutional Animal Care Use Committee of Neuromed Medical Center approved all animal experiments [n° 327/2023-PR]. Mouse colonies were maintained in the animal facility at IRCCS Neuromed, Pozzilli (IS), Italy. At week 10, mice were randomized into two groups and received either control antibody injections (polyclonal non-immune rat immunoglobulins) or a GPIb- alpha antibody (Emfret) injections to deplete circulating platelets (3 µg/g) through the caudal vein. At the end of treatment, mice were anesthetized with ketamine/xylazine, and euthanized by beheading to collect blood and tissue samples.

In vivo gene therapy

The experimental procedure of both cloning and vector production and purification of BPIFB4 constructs have been detailed elsewhere [6].

Ten-week-old male C57BL/6 mice were anesthetized with 5% isoflurane in 100% O2 (delivery rate, 5L/min), and placed in a dorsal recumbent position on a homoeothermic blanket (N-HB101-S-402) to maintain body temperature at 37 °C. Anesthesia was maintained with 1% isoflurane in 100% O2 at 1.5 L/min, administered by means of a facemask connected to a coaxial circuit (Fluovac anesthetic mask). Vascular surgery was performed with the aid of a microscope at 2–10 × magnification. As previously described [6], femoral arteries were exposed and isolated circumferentially from the inguinal ligament to the knee. To avoid collateral damage to adjacent structures, the artery was carefully separated from associated nerves and veins with fine-tip forceps. After isolation of the arterial segment, a temporary clamp was closed around the proximal end of the artery to temporarily stop the blood flow. Subsequently, the distal end of the femoral artery was permanently occluded to perform a small incision on the superficial wall of the artery, using a syringe connected, through a PE10 – polyethylene tube, with a glass needle tip of 80 microns in diameter, to inject either 50 µl of saline containing AAV-GFP or AAV-LAV-BPIFB4 into the femoral artery. Viral titre was 1 × 1013 GC/kg for each experimental condition. After an incubation of 5 min, before carefully removing the syringe, we performed a permanent ligation just after the incision, and the clamp on the proximal femoral artery was removed to restore femoral blood flow and obtain systemic delivery. As support to the procedure’s success, evaluation of GFP revealed the expression of AAV9 (TBG-promoter) into the liver, thus confirming its systemic delivery. Mice remained anesthetized for 1 h, after which all received 100% O2 until recovery of righting reflex. Animal groups subjected to either control antibody injections (polyclonal non-immune rat immunoglobulins) or a GPIb- alpha antibody (Emfret) injections (3 µg/g), received a blood injection through the caudal vein (tail) after 24 h of AAV- delivery. Mice were sacrificed 96 h after surgery and vascular gene therapy. Mesenteric and femoral arteries were harvested and placed respectively on pressure and wire system for vascular reactivity studies.

Mice tissue sampling and processing

Plasma and Peripheral Blood Mononuclear Cells (PBMCs) were extracted from whole blood of mice by Ficoll density gradient (Histopaque®-1077, Sigma-Aldrich). Plasma was employed to estimate the BPIFB4 levels dosage while PBMCs were stimulated in vitro with lipopolysaccharide (LPS) at a concentration of 1 µg/ml and then analyzed by flow cytometry.

CD61 + and CD61- cells were extracted from murine bone marrow through magnetically labeling and separation using CD61 MicroBeads (#130–109-678, MACS Miltenyi Biotec) following manufacturer’s procedures. CD61 positive and negative cells were further used for western blotting analysis.

Cell lines and culture conditions

MEG-01 (ATCC® CRL-2021) were grown in humidified incubator at 37 °C and 5% CO2 in RPMI-1640 (Gibco®, Thermo Fisher Scientific) supplemented with 10% (v/v) fetal serum bovine (FBS, Gibco®, Thermo Fisher Scientific), 1% (v/v) penicillin–streptomycin (Aurogene), 1% (v/v) MEM non-essential amino acids (MEM NEAA, Gibco®, Thermo Fisher Scientific), and 1% (v/v) sodium pyruvate (Aurogene). Differentiated MEG01 were obtained upon 3 days pre-treatment with phorbol 12-myristate 13-acetate (PMA) 5nM and thrombopoietin (TPO) 100 ng/mL. Following the above-mentioned treatment, cells were collected for subsequent assays.

Preparation of PRP from human and murine whole blood

Whole blood collected into vacutainer tubes containing 3.2% sodium citrate solution at the volume ratio 9:1 (BD Vacutainer System) was centrifuged at 2200 g for 30 s at room temperature (RT) to obtain platelet-rich plasma (PRP) by using aggregometer’s centrifuge system (Platelet Aggregation Profiler-PAP8, BIO/DATA CORPORATION, V 2.0 Optics). Platelets were counted using an automatic cell counter (LUNA Automated Cell counter, logos).

Preparation of the monocyte-derived macrophages and platelets co-colture establishment

Peripheral blood mononuclear cells were extracted from the whole blood of a healthy donor by Ficoll density gradient (Cytiva Ficoll-Paque™ PLUS). After separation, PBMCs were washed and collected in MACS buffer (PBS 0.5% BSA, EDTA 2mM). CD14 + cells were harvested by immunomagnetic procedure following manufacturer’s protocol (human CD14 MicroBeads UltraPure, MiltenyiBiotec). Monocytes were seeded at 400.000 cells/mL in ImmunoCult-SF Macrophage Differentiation Medium (Stemcell Technologies) supplemented with 10% heterologous plasma. Monocytes were cultured for 7 days in the presence of autologous platelet-rich plasma at the ratio of monocyte to platelets of 1:10 or 1:50 with or without rhLAV-BPIFB4 (18 ng/mL). During the third day of culture, 50% of medium was replaced with fresh medium, and rhLAV-BPIFB4 (18 ng/mL) was added.

For macrophage conditioning with platelets’ releasate, PRP was diluted with Tyrode’s buffer (v/v 1:1) and centrifuged at 430 g for 15 min to obtain washed platelets. Platelets were resuspended in RPMI-free and stimulated with or without rhLAV-BPIFB4 (18 ng/mL) for 3 h at 37 °C. Platelets’ releasate was harvested after plate centrifugation at 1000 rpm. Monocytes were seeded and conditioned with platelets’ releasate considering monocyte to platelets ratio at 1:100 for 7 days in ImmunoCult-SF Macrophage Differentiation Medium (Stemcell Technologies) supplemented with 10% heterologous plasma.

Production of lentiviral vectors and transfected EMPTY, WT, and LAV MEG-01 cell line

Lentiviral particles (Empty vector, WT- or LAV-BPIFB4) were generated as previously described [6]. Lentiviral particles were concentrated by ultracentrifugation (40,000 rpm for 2 h at 4 °C) and stored at − 80 °C until immediately prior to use. Lentivirus titration was performed by transducing HEK293T cells with concentrated particles in the presence of 4 µg/ml polybrene and measuring GFP expression after 3 days by flow cytometry. 500.000 MEG-01 cells were plated into a 12-well plate in RPMI medium and were infected with empty lentiviral vectors or particles encoding either WT- or LAV-BPIFB4 [at 3 multiplicities of infection (MOI)]. After 72 h, cells were selected with 1 µg/ml puromycin for 48 h.

Immunohistochemistry on bone marrow and human PRP

For immunohistochemistry (IHC) staining, the following antibody was applied on human bone marrow tissue biopsies by using a routine immunoperoxidase technique: GeneTex C20orf186 antibody, rabbit polyclonal to KLH conjugated synthetic peptide derived from human C20orf186.

Briefly, decalcified human BM biopsy or femoral BM was embedded in paraffin, sectioned on a rotary microtome at 2 µm, and then dried, deparaffinized, and rehydrated. Enzymatic epitope retrieval was performed by microwave, boiled in sodium citrate buffer pH6. For Diaminobenzidine (DAB) reaction, endogenous peroxidase was blocked with H2O2 3%. Dako REAL EnVision/HRP, Rabbit/Mouse (ENV) was used for the detection of primary antibodies. The reactions were revealed by solution (1:50) of Dako REAL DAB + Chromogen and Dako REAL Substrate Buffer. Nuclei were stained with Mayer’s hematoxylin. All staining steps were performed at room temperature.

Morphometric analyses were performed on images captured with a digital camera at a final magnification of 40 × , using an image analysis software (Image proplus 4.0, Media Cybernetics, USA). Fresh human PRP was diluted in cytopath fixative (Diapath), to obtain a solution of 50 × 106 platelets/ml. A monolayer of platelets was deposited on slides, in 20 mm diameter spots, using the semi-automatic instrument CytoPath Processor (Diapath). Samples were analyzed through immunohistochemical staining, using primary antibody anti-BPIFB4 (1:250), incubated at RT for 2 h, and the ultraView Universal DAB Detection Kit (Roche) for the detection. The kit consists of a cocktail of enzyme-labeled secondary antibodies. Negative control was prepared by incubating slides with only the secondary antibodies. Concurrently, slides were colored with GIEMSA staining, in order to underline the presence of platelets. Images were acquired with DMi1 inverted bright-field microscope (Leica), at 10 × magnification.

Platelets count

Differentiated parental, EMPTY-, WT-, and LAV-MEG-01 cells were counted using an automatic cell counter (LUNA Automated Cell counter, logos) and two cell fractions were considered: platelet-like particles (PLPs, Ø 1–3 µm) and megakaryocytes (Ø 16–35 µm). The ratio between MEG-01 cells and PLPs was carried out.

Immunofluorescence on PRP

Tyrode’s buffer (Sigma-Aldrich) was added to the recovered PRP in a 1:200 ratio and centrifuged at 3000 g for 10 min to obtain the washed platelets. Afterwards, the platelets were resuspended in Tyrode’s buffer, loaded into a cytofunnel, and spotted onto the slide through the Cytospin (CENTURION-Scientific Limited) at 1200 rpm for 5 min. Platelets were fixed with 4% formaldehyde for 10 min at room temperature and washed three times with PBS (Gibco®, Thermo Fisher Scientific). Platelets were blocked and permeabilized using PBS with 5% Bovine Serum Albumin (PanReac AppliChem) and 0,005% saponin for 30 min at room temperature. Cells were incubated overnight at 4 °C with the following IgG primary antibodies’ mix: customized rabbit polyclonal anti-BPIFB4 (purchased from CliniSciences S.r.l.-Guidonia Montecelio – Italy; 1:100 in PBS 2.5% goat serum) and mouse polyclonal anti-P-selectin (Santa Cruz, 1:100 in PBS 2.5% horse serum). The next day, three washes of 10 min with PBS were carried out followed by a 30-min incubation at RT in the dark with the following secondary antibodies’ mix: Alexa Fluor 488-conjugate anti-mouse IgG (Vector Laboratories, 1:200 in PBS) and DyLight 549-conjugated anti-rabbit IgG (Vector Laboratories, 1:200 in PBS). Platelets were washed three times with PBS and nuclei were stained with DAPI (1:100 in PBS) for 20 min at room temperature in the dark. After five washes, slides were glycerol-mounted and images were acquired by using a confocal laser-scanning fluorescence microscope TCS SP5 (Leica Microsystems).

Enzyme-linked immunosorbent assay (ELISA)

The BPIFB4 release was analyzed both on human PRP and plasma from mouse models. Human PRP in resting condition and upon stimulation with CaCl2 (22 mM for 40 min at 37 °C) or rhLAV-BPIFB4 (18 ng/mL for 40 min at 37 °C) were centrifuged at 13000 rpm for 5 min to collect the supernatants. BPIFB4 levels were determined using the Human Long palate, lung, and nasal epithelium carcinoma-associated protein 4 (C20orf186) ELISA kit (Cusabio CSBYP003694HU) following the manufacturer’s protocol. Briefly, supernatants were incubated for 2 h at 37 °C in the assay-coated microplate. After removing any unbound substances, a biotin-conjugated antibody specific for C20orf186 was added to the wells and incubated for 1 h at 37 °C. After washing, avidin-conjugated horseradish peroxidase (HRP) was added to the wells and incubated for 1 h at 37 °C. Following a wash, substrate solution was added and the consequent color development was stopped. Optical density was measured at 450 nm.

Western blotting

CD61 + and CD61- cells from mouse bone marrow, platelets from human and murine PRP, and MEG-01 cells were washed with PBS (Gibco®, Thermo Fisher Scientific), harvested, and lysed in ice-cold RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/mL aprotinin) as also detailed by Ciaglia et al. After centrifugation at 13,000 rpm for 20 min at 4 °C, in order to remove cell debris, proteins were quantified. About 30 µg of proteins were separated on 10% SDS-PAGE at 90 V for 1 h and at 120 V for another hour and then transferred to a nitrocellulose membrane. After blocking with 5% non fat dried milk powder (PanReacAppliChem) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated overnight with the following primary antibodies: customized BPIFB4 (purchased from CliniSciences S.r.l.-Guidonia Montecelio – Italy) and β-actin (Abcam #4990 mouse mAb 1:50000). Immunodetection of specific proteins was carried out with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad), using the enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific) according to the manufacturer’s instructions and then exposed to X-ray films (Thermo Fisher Scientific). Western-blot data were analyzed using Photoshop software to determine the optical density (OD) of the bands. The OD readings of protein were expressed as a ratio relative to β-actin.

Flow cytometry analysis

PBMCs of treated mice were stained with mAb against mouse CD86 (PO3.3; Miltenyi Biotec; 1:10), CD206 (C068C2; Biolegend; 1:50), and Ly6C (REA796; Miltenyi Biotec; 1:50). Human PBMCs were stained with mAb against human CD86 (REA968; Miltenyi Biotec; 1:50), CD206 (Miltenyi Biotec; 1:10), CD14 (HCD1-4; Biolegend; 1:50), CD163 (REA812; Miltenyi Biotec; 1:50). After 30 min incubation at 4 °C in the dark, cells were washed with staining buffer (PBS 2% fetal serum bovine, 0.01% sodium azide), centrifuged at 1800 rpm for 5 min, and resuspended in staining buffer for the FACS analysis. Instead, MEG-01 cells were incubated for 30 min at 4 °C in the dark with a primary customized anti-BPIFB4 (purchased from CliniSciences S.r.l.-Guidonia Montecelio – Italy, 1:100); then cells were washed with staining buffer and incubated with secondary PE donkey anti-rabbit IgG (Poly4064; Biolegend; 1:1000) for 30 min at + 4 °C in the dark. At the end of the incubation, the cells were washed as before and then resuspended in staining buffer for the FACS analysis. For each test, cells were analyzed using FACS Verse Flow Cytometer (BD Biosciences).

Vascular reactivity study

Vascular reactivity studies were performed on second-order branches of the mesenteric artery, as previously described [6]. Quantification of vasomotor response was performed by a second individual who was blind to the genotype of the animal and/or the hypothesis that was being tested for each group. Vessels were isolated and dissected from fat and connective tissue in ice-cold Krebs solution and gassed with 95% O2 and 5% CO2. Subsequently, arteries were mounted on a pressure myograph in organ chambers with Krebs solution and treated with increasing concentrations of U46619 (10–9 to 10–6 M) to obtain a similar level of precontraction in each ring (80% of initial KCl-induced contraction). Caution was taken to avoid damage to the endothelium. Vascular r effect of LAV treatment was evaluated by assessment of the vasodilation response to acetylcholine (10–9 to 10–6 M). Vasorelaxation was expressed as a percentage reduction of U46619-induced contraction.

Statistical analysis

In all experiments shown, statistical analysis was performed using the GraphPad Prism 6.0 software package for Windows (GraphPad software). For each type of assay or phenotypic analysis, the data obtained from multiple experiments were calculated as mean ± SD and analyzed for statistical significance using appropriate tests. In the analysis of variance (ANOVA) for multiple comparisons, p-values < 0.05 were considered significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.