Now showing 1 - 6 of 6
  • Publication
    Platelet-Derived Microparticles From Obese Individuals: Characterization of Number, Size, Proteomics, and Crosstalk With Cancer and Endothelial Cells
    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.
      434Scopus© Citations 43
  • Publication
    A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids
    Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.
      397Scopus© Citations 38
  • Publication
    Identification of β2-microglobulin as a urinary biomarker for chronic allograft nephropathy using proteomic methods
    Chronic allograft nephropathy (CAN) remains the leading cause of renal graft loss after the first year following renal transplantation. This study aimed to identify novel urinary proteomic profiles, which could distinguish and predict CAN in susceptible individuals. Experimental Design: The study included 34 renal transplant patients with histologically proven CAN and 36 patients with normal renal transplant function. High-throughput proteomic profiles were generated from urine samples with three different ProteinChip arrays by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Following SELDI a biomarker pattern software analysis was performed which led to the identification of a novel biomarker pattern that could distinguish patients with CAN from those with normal renal function. Results: An 11.7 kDa protein identified as β2 microglobulin was the primary protein of this biomarker pattern, distinguishing CAN from control patients (ROC = 0.996). SELDI-TOF-MS comparison of purified β2 microglobulin protein and CAN urine demonstrated identical 11.7 kDa protein peaks. Significantly higher concentrations of β2 microglobulin were found in the urine of patients with CAN compared to the urine of normal renal function transplant recipients (p<0.001). Conclusions and clinical relevance: Whilst further validation in a larger more diverse patient population is required to determine if this β2 microglobulin protein biomarker will provide a potential means of diagnosing CAN by non-invasive methods in a clinical setting, this study clearly shows a capability to stratify control and disease patients.
      1064Scopus© Citations 24
  • Publication
    Examining gender effects in different types of undergraduate science assessment
    The optimum assessment structure measures student knowledge accurately and without bias. In this study, the performance of the first-year undergraduate science students from the University College Dublin was evaluated to test the gender equality of the assessment structure in place. Results of male and female students taking three life science modules were analysed, for two academic years, with assessment structure based on a combination of three types of evaluation: continuous assessment and multiple choice questions (MCQ) exam scored with/without negative marking. We found no significant gender effect associated with performance in continuous assessment, or MCQ exams scored without negative marking. However, a significant bias against females was consistently observed for the same cohort of students in the MCQ exams with negative marking of 0.25 points. This bias was at least partially linked to a gender difference in willingness to guess and preliminary data suggest that it disappears after removal of negative marking from the MCQ exams. Our results support the view of a diverse assessment structure being fairer to the students. Moreover, caution is advised while using negative marking, and regular reviews of assessment strategy should be implemented by higher education institutions to ensure gender-bias free evaluation of students’ performance.
      289Scopus© Citations 5
  • Publication
    Thrombin generation correlates with disease duration in multiple sclerosis (MS): Novel insights into the MS-associated prothrombotic state
    Background: Thrombin is well recognised for its role in the coagulation cascade but it also plays a role in inflammation, with enhanced thrombin generation observed in several inflammatory disorders. Although patients with multiple sclerosis (MS) have a higher incidence of thrombotic disease, thrombin generation has not been studied to date. Objectives: The aim of this study was to characterise calibrated automated thrombography parameters in patients with relapsing–remitting MS (RRMS) and primary progressive MS (PPMS) in comparison to healthy controls (HCs). Methods: Calibrated automated thrombography was performed on platelet poor plasma from 15 patients with RRMS, 15 with PPMS and 19 HCs. Results: We found that patients with RRMS generate thrombin at a significantly faster rate than the less inflammatory subtype, PPMS or HCs. In addition, the speed of thrombin generation was significantly correlated with time from clinical diagnosis in both subtypes. However, in RRMS the rate of thrombin generation was increased with increased time from clinical diagnosis, while in PPMS the rate of thrombin generation decreased with increased time from clinical diagnosis. Conclusions: These data likely reflect the differential active proinflammatory states in each MS subtype and provide novel mechanistic insights into the clinically relevant prothrombotic state observed in these patients.
      305Scopus© Citations 11
  • Publication
    Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype
    Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1–CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.
      389Scopus© Citations 85