In vivo study
We recruited 59 consecutive T2DM, 43 men and 16 women attending the outpatient clinic of the Division of Metabolic Diseases of the University of Padova, from March to November 2017. Inclusion criteria were: type 2 diabetes diagnosis according to the ADA criteria; both genders; age 18–80 years. Exclusion criteria were: type 1 diabetes, clinically relevant diseases, or advanced chronic diabetes complications. According to the values of glycated hemoglobin (HbA1c), T2DM were divided into 2 groups, i.e. with good (mean HbA1c ≤ 7.0%; GGC; n = 28) and poor (mean HbA1c > 7.0%; PGC; n = 31) glycemic control. A control group of matched subjects with normal glucose tolerance (NGT) ascertained by an oral glucose (75 g) tolerance test was included in this study.
The primary demographic and anthropometric data, duration of diabetes, blood pressure values, heart rate, and current therapy were recorded in all the subjects.
A fasting blood sample was drawn by venipuncture from an antecubital vein, in each patient for the determination of glucose, HbA1c (only in T2DM), lipid profile (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides), hematocrit, hemoglobin, red and white blood cell count, platelet count, IL-6, ICAM-1, VCAM-1, and for the assessment and characterization of circulating MPs. Platelets were also collected from T2DM for the in vitro studies.
This study was carried out under the International Ethical Guidelines and the principles of the Declaration of Helsinki and was approved by the local Institutional Review Board of the University of Padova Medical Centre. All subjects signed informed consent.
Plasma glucose, total serum cholesterol, triglyceride, and HDL cholesterol were measured using standard enzymatic methods. LDL cholesterol was calculated by the Friedewald formula. HbA1c was measured via high-performance liquid chromatography. Chromatography was performed using a certified automated HPLC analyzer; the normal range was from 4.25 to 5.9% (23 to 41 mmol/mol).
Red blood cells, hematocrit, hemoglobin, white blood cells, and platelet count were determined by standard methods. According to the manufacturer’s instructions, plasma levels of IL-6, VCAM-1, and ICAM-1 were measured by using a high-sensitivity ELISA assay (BioVision, CA, USA). The intra and inter-assay coefficients of variations were below 10%. All samples were coded for a blinded analysis, and each plasma sample was determined in duplicate.
Circulating microparticles assessment and characterization
Activated endothelial cells MPs (CD62E+), tissue factor-bearing MPs (CD142+), leukocyte-derived MPs (CD45+), and activated platelet-derived MPs (CD62P+, PMP) were determined. Microparticles were prepared from platelet-free plasma (PFP) within 3 h of blood collection by double centrifugation (3000×g for 15 min). One ml of PFP was centrifuged at 18000×g for 40 min at 4 °C to obtain microparticles. MPs were resuspended in 200 µL of phosphate-buffered saline (cat# D8537, PBS, Sigma, USA) and stored at − 80 °C until use. Samples, analyzed only after a single freeze–thaw cycle, were thawed by incubation for 5 min in a water bath at 37 °C immediately before assay.
All assays were performed on a Cytomics FC500 flow cytometer (Beckman Coulter, Miami Florida), as previously described . The MPs gate was established using a blend of mono-dispersed fluorescent beads of three diameters (0.5, 0.9, and 3 μm) (cat# 7801, Megamix, BioCytex, Diagnostica Stago, France) . Twenty microliters (µL) of freshly thawed MPs were directly incubated for 15 min at room temperature in the dark with 2 µL of fluorescent-conjugated monoclonal antibodies against cell-type specific antigens and 2 µL of annexinV-FITC (fluorescein isothiocyanate) (cat# BMS500FI, Bender MedSystems GmbH, Vienna, Austria) for 20 min at 37 °C. Endothelial-derived MPs were identified using CD62E-PE (phycoerythrin) (cat# 336008, BioLegend Europe, The Netherlands) and platelet-derived MPs using CD62P-PC5 (phycoerythrin-cyanin 5.1) (cat# 304908, BioLegend Europe, The Netherlands); leukocyte-derived MPs using CD45-PC5 (cat# 304009, BioLegend Europe, The Netherlands) and Tissue Factor-bearing (TF+ MPs) with CD142-PE, clone HTF-1 (cat# 550312, BD, Biosciences, Milan, Italy). The isotype controls used were IgG1-PC5, clone MOPC-21 (cat# 400118, BioLegend Europe), IgG1-PE, clone MOPC-21 (cat# 556650, BD Biosciences, Milan, Italy); mouse IgG1-FITC, clone MOPC-21 (cat# 400129, BioLegend Europe). The samples were diluted in 500 μL of 0.22 µm filtered Annexin-V kit binding buffer (Bender MedSystems GmbH, Vienna, Austria) before analysis. A total of 20 μL of counting beads with an established concentration (cat# 7547053, Flow Count™ Fluorospheres, Beckman Coulter, Miami Florida) were added to each sample to calculate MPs as absolute numbers per microliter. Patient samples were all processed in the same way by the same experienced operators.
In vitro study
Platelet preparation and platelet-derived microparticle (PMP) production
Platelets were isolated from fresh human blood by centrifugation at 200×g for 20 min at 20 °C to obtain platelet-rich plasma (PRP). Washed platelets were isolated from PRP after centrifugation and resuspended in calcium and magnesium-free HEP buffer with prostaglandin E1 (1 µM, pH 7.4). Platelet counts were determined with a cell counter (TC20™, Biorad, USA). In vitro, platelet-derived microparticles were generated from platelets (500 × 103 platelets/µL), incubated for 30 min at 37 °C with thrombin (1 U/mL), or with PAR-1 (20 µM), or PAR-4 (200 µM) agonists, and collagen (10 μg/mL) co-stimulus, or with calcium ionophore A23187 (10 μmol/L) in Tyrode’s buffer (1 mM MgCl2, 2 mM CaCl2, 3 mM KCl), as previously described . Activation was stopped by the addition of 2.0 mmol/L of EDTA. Platelets and debris were removed with centrifugation for 10 min at 3000×g and PMP were obtained after centrifugation and analyzed by flow cytometer, as described above.
Platelet cytosolic Ca
Ca2+ measurement was determined as previously described . Briefly, platelets (1.5 × 107 cells/μL) were loaded with the fluorescent probe, 2.5 μM Fura-2/AM, for 30 min at 37 °C. After recovery, levels of cytosolic calcium (Cacyt2+) were measured by Shimadzu spectrofluorometer. The baseline fluorescence was obtained by alternating the excitation wavelength between 340 and 380 nm and recording the 510 nm emission intensity. [Ca2+]I was calculated from the fluorescence ratio recordings according to the standard formula: [Ca2+]I = Kd [(R − Rmin)/(Rmax − R)](Sf2/Sb2). The dissociation constant (Kd) was taken as 224 nmol/L; Rmax (340/380 ratio under Ca2+-saturating conditions), Rmin (340/380 ratio under Ca2+-free conditions;), and Sf2/Sb2 (ratio of baseline fluorescence) were calculated by a calibration curve with buffers containing different Ca2+ concentrations; PAR-1 agonist (20 µM) or PAR-4 agonist (200 µM) were added when baseline fluorescence was stable. Basal Cacyt2+ levels were reported after a 60 s recording period. All determinations were performed in duplicate for each patient.
Platelets were lysed in RIPA buffer containing protease inhibitors. Proteins were separated by 10% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane in a semidry blotter. Blots were incubated for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 5% skimmed milk to block residual protein binding sites. Membranes were incubated overnight with specific antibodies against anti-PAR-1 (1:1000; cat# ab233741, Abcam, Cambridge, UK), anti-PAR-4 (1:1000; cat# 2328S, Cell Signaling Tech. MA, USA), and anti-β actin (1:5000; cat# 3700, Cell Signaling Tech. MA, USA). Detection was achieved using an enhanced chemiluminescence system (cat# EMP011005, Euroclone, Italy). The blots were scanned and quantified using a chemiluminescence molecular imaging system (Versa Doc 3000. Bio-Rad, Hercules, CA, USA). The results were expressed relative to the control on the same blot, defined as 100%, and by the protein of interest/β actin densitometric ratio. Protein concentration was determined by BCA’s method (cat# EMP014500, Euroclone, Italy).
Calpain activity assay
Calpain activity was determined by Calpain-Glo™ Protease Assay (cat# G8502, Promega, Madison, USA) according to the manufacturer’s instructions. Briefly, washed platelet (400 to 500 × 109 platelets/L) were stimulated with PAR-4 agonist peptide (AY-NH2) for 30 min at 37 °C in the presence and in the absence of ALLN, a calpain inhibitor. Then, all conditions were centrifuged (10 min at 650×g) and the pellets were resuspended in lysis buffer (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and Protease Inhibitor Cocktail) for 30 min at 4 °C. Lysates (50 μL) were mixed with 50 μL of Calpain-Glo™ Reagent (with 2 mM CaCl2 for calpain activation) incubated for 30 min at room temperature and finally the luminescent readings performed in an EnSight™ multimode plate reader (Perkin Elmer, Milan, Italy). All the results were expressed as relative luminescence units per microgram of protein lysate.
PMP staining and incorporation into macrophages
The THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Sigma Aldrich, Milan, Italy) supplemented with 10% FBS, 1% l-glutamine, and 1% antibiotic solution in a humid atmosphere containing 5% CO2 at 37 °C. For the induction of macrophage differentiation, cells (1–2 × 106 per mL) were seeded with 100 nM phorbol 12-myristate 13-acetate (PMA, cat# 79346, Sigma Aldrich, Milan, Italy) for 72 h. After incubation, nonattached cells were removed by aspiration, and the adherent cells were washed and cultured in serum-reduced RPMI-1640 medium (3% FBS). Subsequently, THP-1 transformed macrophages were treated with PMP (1000 MPs/µL). To assess the uptake of PMP in THP-1, PMP were stained with 10 µL of Calcein-AM for 40 min (20 µM; cat#17783, Sigma Aldrich. Milan, Italy), washed and resuspended in PBS for the incorporation into macrophages (0.5 × 106 cells), as previously described. Calcein-AM is non-fluorescent until enters into intact MPs to be activated and becomes fluorescent (λex 496 nm; λem 516 nm ± 5 nm). After 4 h, cells were washed once with PBS, fixed with paraformaldehyde 4% and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (cat# D9542, Sigma Aldrich. Milan, Italy; λex 340 nm; λem 488 nm). Images were captured using a Zeiss microscope (Oberkochen, Germany) with Apotome upgrade for confocal imaging (630×).
Moreover, THP-1 cells were incubated with no labelled PMP (1000 MPs/µL) generated from PAR-4 treated platelets of T2DM with PGC (in the presence and in the absence of ALLN, a calpain inhibitor) and with GGC. Treatment with unstimulated PGC-PMP and TNFα (10 ng/mL) on THP-1 were performed as a negative and positive control, respectively. After 24 h, THP-1 cells and their culture medium were collected to measure the gene expression and release of IL-6, and NF-kB acetylation .
Cell viability assay
At the end of each treatment, the number of live and total cells was counted with trypan blue staining (Sigma Aldrich. Milan, Italy). Cell viability was assessed by calculating the percentage of live cells using trypan blue exclusion.
Total RNA was isolated from THP-1 transformed macrophages by RNeasy Mini kit (cat# 74104, Qiagen, Hilden, Germany), following the manufacturer’s instructions. RNA was treated with DNase I (Roche) before reverse transcription (RT). RNA was quantified using the NanoDrop 2000C (Thermo Scientific, USA). cDNA was synthesized with 500 ng of RNA extracted using iScript cDNA synthesis kit (cat#1708891, Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction assay was performed in a Bio-Rad CFX96 Real-Time PCR detection system. The PCR reaction was performed in a 25 µL final reaction volume containing 200 nmol of each primer and SsoFast EVAGreen SuperMix (cat# 5201, Bio-Rad, USA). All the reactions were performed in 96-well plates, in triplicate. Primers were designed from sequences derived from the GenBank database using Primer 3 (Whitehead Institute, Massachusetts, USA) and Operon's Oligo software (Operon, California, USA). They were purchased from Eurofins MWG (Ebersberg, Germany). The specific primers were (Eurofins): IL-6, Forward AGTCCTGATCCAGTTCCTGC and reverse CTACATTTGCCGAAGAGCCC; β-actin, as a housekeeping gene, Forward AGAGCTACGAGCTGCCTGAC and reverse GGATGCCACAGGACTCCA. Data analyses were performed with the Bio-Rad CFX Manager. The comparative cycle threshold method (∆∆Cq) was used to obtain the relative fold change of gene expression.
Interleukine-6 (IL-6) levels were quantified in cell culture supernatants, using a commercially available enzyme-linked immunosorbent assay (Raybio Human IL-6 ELISA kit; RayBiotech Norcross, GA, USA), following the manufacturer's protocols.
NF-kB-acetylation was carried out by incubating 2 μg of anti-NF-kB antibody with 1 mg of cell lysate overnight, followed by 30 μg of EZ viewTM Red Protein A Affinity Gel (cat# P6486, Sigma Aldrich. Milan, Italy) for 4 h at 4 °C. After washing, immunoprecipitates were boiled in SDS-PAGE loading buffer, subjected to SDS-PAGE, transferred on to nitrocellulose filters and probed with the specified primary antibody against acetylated lysine (cat# 9814, Cell Signaling Tech. MA, USA) and the appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Illinois, USA). Results were expressed relative to the control, on the same blot, and the values were expressed as fold increase after normalization with total NF-kB.
PAR-1 and PAR-4 agonists
PAR-1 agonist peptide (TRAP-6, [serine-phenylalanine-leucine-leucine-arginine-asparagine amide], cat# 3497) and PAR-4 agonist peptide (AY-NH2, H2 [alanine–tyrosine–proline–glycine–lysine–phenylalanine amide], cat# 1487) were purchased from Tocris. Calpain Inhibitor I (ALLN, cat# A6185) was purchased by Sigma Aldrich.
We used previous data , to calculate the sample size needed in order to estimate the statistically significant difference between PMP in subjects with type 2 diabetes and non-diabetic subjects. Since there are no data about differences in patients either in good or poor metabolic control, we assumed no difference between normal subjects and T2DM in good metabolic control. Considering an α error level of 5%, 16 subjects per group will allow for an estimate of the difference between groups with a power equal to 90%.
Continuous variables are expressed as mean ± SEM and categorical variables as percentages. Data were tested for significance using a Student’s t-test for two normally distributed groups. Variable normality distribution was performed by the Shapiro–Wilk test. Data from three or more groups were analyzed by one-way ANOVA test followed by a Bonferroni post hoc test. Categorical data were analyzed with a Chi-squared test. To determine the association between MPs type and studied variables, univariate analyses were run. Statistical significance was accepted at p < 0.05. SPSS (IBM SPSS Statistics for Windows, version 26 Bologna, Italy) and GraphPad (vers. 0.8.3 for Mac, La Jolla, CA) were used for statistical analysis.