Platelet-Derived Microparticles From Obese Individuals: Characterization of Number, Size, Proteomics, and Crosstalk With Cancer and Endothelial Cells

Rosalia Grande, Melania Dovizio, Simone Marcone, Paulina B Szklanna, Annalisa Bruno, H Alexander Ebhardt, Hilary Cassidy, Fionnuala Ní Áinle, Anna Caprodossi, Paola Lanuti, Marco Marchisio, Geltrude Mingrone, Patricia B Maguire, Paola Patrignani, Rosalia Grande, Melania Dovizio, Simone Marcone, Paulina B Szklanna, Annalisa Bruno, H Alexander Ebhardt, Hilary Cassidy, Fionnuala Ní Áinle, Anna Caprodossi, Paola Lanuti, Marco Marchisio, Geltrude Mingrone, Patricia B Maguire, Paola Patrignani

Abstract

Rationale: Obesity is a risk factor for atherothrombosis and various cancers. However, the mechanisms are not yet completely clarified. Objectives: We aimed to verify whether the microparticles (MPs) released from thrombin-activated platelets differed in obese and non-obese women for number, size, and proteomics cargo and the capacity to modulate in vitro the expression of (i) genes related to the epithelial to mesenchymal transition (EMT) and the endothelial to mesenchymal transition (EndMT), and (ii) cyclooxygenase (COX)-2 involved in the production of angiogenic and inflammatory mediators. Methods and Results: MPs were obtained from thrombin activated platelets of four obese and their matched non-obese women. MPs were analyzed by cytofluorimeter and protein content by liquid chromatography-mass spectrometry. MPs from obese women were not different in number but showed increased heterogeneity in size. In obese individuals, MPs containing mitochondria (mitoMPs) expressed lower CD41 levels and increased phosphatidylserine associated with enhanced Factor V representing a signature of a prothrombotic state. Proteomics analysis identified 44 proteins downregulated and three upregulated in MPs obtained from obese vs. non-obese women. A reduction in the proteins of the α-granular membrane and those involved in mitophagy and antioxidant defenses-granular membrane was detected in the MPs of obese individuals. MPs released from platelets of obese individuals were more prone to induce the expression of marker genes of EMT and EndMT when incubated with human colorectal cancer cells (HT29) and human cardiac microvascular endothelial cells (HCMEC), respectively. A protein, highly enhanced in obese MPs, was the pro-platelet basic protein with pro-inflammatory and tumorigenic actions. Exclusively MPs from obese women induced COX-2 in HCMEC. Conclusion: Platelet-derived MPs of obese women showed higher heterogeneity in size and contained different levels of proteins relevant to thrombosis and tumorigenesis. MPs from obese individuals presented enhanced capacity to cause changes in the expression of EMT and EndMT marker genes and to induce COX-2. These effects might contribute to the increased risk for the development of thrombosis and multiple malignancies in obesity. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT01581801.

Keywords: cellular cross-talk; microparticles; obesity; platelets; proteomics.

Figures

FIGURE 1
FIGURE 1
Features of MPs released from platelets of obese and non-obese women. (A,B) Density plots of forward scatter height (FSC-H) vs. side scatter height (SSC-H) of a typical MP suspension from non-obese (HS) and obese (OB) individuals, and size distribution. (C,D) Fluorescence intensity of FSC-H and SSC-H parameters reported as arbitrary unit (a.u.) (n = 4 for each group); ∗∗P < 0.01 vs. HS.
FIGURE 2
FIGURE 2
Protein-Protein interaction network of the 214 identified proteins: network nodes represent proteins, network edges indicate the strength of data support (STRING v10.5). Proteins associated with “Platelet activation” pathway are highlighted in red.
FIGURE 3
FIGURE 3
Pathway analysis of 214 proteins showing the top 20 biological processes (A) and molecular functions (B).
FIGURE 4
FIGURE 4
Volcano plot displaying the 47 differential expressed proteins between obese (OB) and healthy control (HC) platelet-derived MPs; the y-axis corresponds to the mean expression value of log10 (p-value), and the x-axis displays the difference values (OB-HC), the red dots represent the differentially expressed proteins (P < 0.05), and the gray dots represent the proteins whose expression levels did not reach statistical significance (P > 0.05).
FIGURE 5
FIGURE 5
(A) STRING network representation of the 47 platelet-derived MP proteins significantly modulated between non-obese and obese individuals: network nodes represent proteins; network edges indicate the strength of data support; significantly modulated proteins associated with “Platelet activation” pathway are highlighted in red. (B) The 20 top-ranked categories (false discovery rate) of KEGG pathways significantly enriched in our dataset.
FIGURE 6
FIGURE 6
Protein-protein interaction network of the 16 mitochondrial proteins identified in platelet-derived MPs; mitochondrial proteins associated with “Oxidation-reduction processes” are highlighted in red.
FIGURE 7
FIGURE 7
Biological processes (A) and pathway analysis (B) of identified mitochondrial proteins.
FIGURE 8
FIGURE 8
Characterization of platelet-derived MPs isolated from healthy (HS) and obese (OB) individuals by flow-cytometry. (A) The count of platelet mitoMP CD41+ was reported as the number of mitoMP/μL. (B) The % of MP CD41+/Annexin V+ and (C) mitoMP CD41+/Annexin V+ are reported. ∗∗P < 0.01 vs. HS (n = 4 for each group).
FIGURE 9
FIGURE 9
Effects of MPs on the expression of gene markers of EMT and EndMT. Co-culture experiments between colon adenocarcinoma cells HT29 (0.25 × 106) (A,B) or human coronary microvascular endothelial cells (HCMEC) (0.25 × 106) (C,D) and MPs (0.25 × 108) from obese (OB) and healthy (HS) individuals for 24 h were reported. Gene expression was evaluated by qPCR and normalized to those of GAPDH as control and expressed as fold-change. Data are reported as mean ± SEM (n = 4, for each experimental condition); ∗P < 0.05 vs. HT29 cultured alone or HCMEC cultured alone.
FIGURE 10
FIGURE 10
(A) Pathway analysis of modulated MP proteins obtained with IPA for “apoptosis and cell death” signaling is reported. IPA analysis showed regulatory relationships between down-regulated (green) and upregulated (red) proteins; the Molecular Activation Prediction tool showed that “apoptosis, cell death, and necrosis” are positively regulated in obese subjects (orange lines); gray line indicates that the effect is not predicted. (B) In co-culture experiment of HCMEC (0.25 × 106) and platelet MPs from OB and HS individuals (0.25 × 108) for 24 h, the gene expression of COX-2 was evaluated by qPCR, normalized to GAPDH levels as control, and expressed as fold-change. Data are reported as mean ± SEM (n = 4 for each experimental condition); ∗P < 0.05 vs. HCMEC cultured alone.

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