Advanced quantitative proteomics to evaluate molecular effects of low-molecular-weight hyaluronic acid in human dermal fibroblasts

Hyaluronic acid (HA) is physiologically synthesized by several human cells types but it is also a widespread ingredient of commercial products, from pharmaceuticals to cosmetics. Despite its extended use, the precise intra- and extra-cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. At this regard, the aim of this study is to in-depth identify and quantify proteome's changes in normal human dermal fibroblasts after 24 h treatment with 0.125, 0.25 and 0.50 % LMW-HA (20 1250 kDa) respectively, vs controls. To do this, a label-free quantitative proteomic approach based on high-resolution mass spectrometry was used. Overall, 2328 proteins were identified of which 39 significantly altered by 0.125 %, 149 by 0.25 % and 496 by 0.50 % LMW-HA. Protein networking studies indicated that the biological effects involve the enhancement of intracellular activity at all concentrations, as well as the extracellular matrix reorganization, proteoglycans and collagen biosynthesis. Moreover, the cell's wellness was confirmed, although mild inflammatory and immune responses were induced at the highest concentration. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here provided by an advanced analytical approach and protein networking will be useful to further exploit its features and improve current formulations

Differentially Expressed Proteins in Primary Endothelial Cells Derived From Patients With Acute Myocardial Infarction

Endothelial dysfunction is one of the primary factors in the onset and progression of atherothrombosis resulting in acute myocardial infarction (AMI). However, the pathological and cellular mechanisms of endothelial dysfunction in AMI have not been systematically studied. Protein expression profiling in combination with a protein network analysis was used by the mass spectrometry-based label-free quantification approach. This identified and quantified 2246 proteins, of which 335 were differentially regulated in coronary arterial endothelial cells from patients with AMI compared with controls. The differentially regulated protein profiles reveal the alteration of (1) metabolism of RNA, (2) platelet activation, signaling, and aggregation, (3) neutrophil degranulation, (4) metabolism of amino acids and derivatives, (5) cellular responses to stress, and (6) response to elevated platelet cytosolic Ca2+ pathways. Increased production of oxidants and decreased production of antioxidant biomarkers as well as downregulation of proteins with antioxidant properties suggests a role for oxidative stress in mediating endothelial dysfunction during AMI. In conclusion, this is the first quantitative proteomics study to evaluate the cellular mechanisms of endothelial dysfunction in patients with AMI. A better understanding of the endothelial proteome and pathophysiology of AMI may lead to the identification of new drug targets

Mass Spectrometry-Based Label Free Quantitative Proteomics to study the effect of 3-PO in endothelial cells

The rapid development of label-free quantitative proteomic techniques has provided fast and low-cost measurement of protein expression levels in complex biological samples. In addition, the development of highly reproducible nano-HPLC separation, high resolution mass spectrometer, and delicate computational tools has greatly improved the reliability and accuracy of label-free, comparative LC-MS/MS (1). Commercially available data processing software is able to automatically detect, match, and quantify peptides from hundreds of different LC-MS experiments simultaneously, which provides a high-throughput technique in the drug discovery process. These label-free quantitative approaches have provided powerful tools for analyzing protein changes in large-scale proteomics studies. This approach has been applied for the first time in this study to evaluate the effects of 3PO [3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one] at endothelial level, whose dysfunction has been associated with several cardiovascular diseases including atherosclerosis, acute myocardial infarction and chronic thromboembolic diseases. 3PO has been shown to inhibit the glycolytic flux partially and transiently and to reduce pathological angiogenesis in a variety of disease models (2). In addition, it has been shown that an inflammatory activation of monocytes/macrophages, via Toll-like receptor ligands or pro-inflammatory cytokines, switches the endothelial cell (EC) metabolism from oxidative

Differentially Expressed Proteins in Pulmonary Endothelial Cells in Chronic Thromboembolic Pulmonary Hypertension

Rationale: Dysfunction of endothelial cells is believed to be involved in the development of chronic thromboembolic pulmonary hypertension (CTEPH). The identification of the molecular mechanisms underlying endothelial dysfunction is crucial to the understanding of the disease and to the expansion of novel therapeutic strategies. The objective of this study was to identify the differentially regulated proteins associated with the vascular dysfunctionality of CTEPH. Patient derived endothelial cells provides a unique opportunity to analyze the precise mechanisms involved in the pathophysiology of the disease. Methods: We analyzed endothelial cells isolated from specimens obtained in pulmonary endarterectomy (PEA)(CTEPH-ECs) in 4 patients. Altered protein expression profiling in combination with gene ontology, network and pathway analysis was employed by using the mass spectrometry based label-free quantification approach to explore the molecular proceedings. Moreover, the status of few differentially expressed protein patterns were also determined at mRNA level by using the real-time PCR experiments. The differentially regulated proteins in CTEPH-ECs were identified against the human pulmonary artery endothelial cells (HPAE) as a control. All data were statistically analyzed using the paired t-test and the p value of <0.05 was considered as significance. Results: A total of 1725 proteins were identified and quantified. Significant variations were observed between control and CTEPH-ECs. We identified 46 statistically significant differentially regulated proteins. We found 12 and 4 unique proteins in CTEPH-ECs and HPAE cells, respectively. The gene-annotation enrichment analysis exposed the association of numerous biological processes, cellular component and molecular functions. The analysis using bioinformatic tools revealed the presence of metabolism, interleukin-4 and 13 signaling, biological oxidations, cytokine signaling in immune system, hemostasis and oxidative stress induced senescence pathways in the dysfunctional CTEPH-ECs. The expression pattern of few differentially regulated genes was validated at transcriptome level. Conclusions: This study provided a differentially expressed proteomic profile, biological processes, molecular functions and network pathways associated with the endothelial dysfunction in CTEPH. Further studies should identify the contribution of dysregulated proteins to CTEPH development. This abstract is funded by: H2020 Marie Sk\u142odowska- Curie Actions, Innovative Training Network MOGLYNET, N. 675527. SEPAR (18/2013, 164/2016) SOCAP; Fundaci\uc3\ub3n Contra la Hipertensi\uc3\ub3n Pulmonar and Fondo de Investigaciones Sanitarias (PI15/00582)