Periodic Reporting for period 1 - PROVE (Pathogenic Triggers and Drivers of Venous Thromboembolism: Intermittent Hypoxia, Intravascular Cells and Extracellular Vesicles)
Periodo di rendicontazione: 2020-09-01 al 2022-08-31
Venous thromboembolism (VTE) is one of the most common cardiovascular diseases, affecting 10 million individuals per year globally, causing severe and long-term suffering. The incidence of VTE is not decreasing despite major efforts in research and clinics, and progress on preventing and predicting the disease is very slow. Several medical conditions including cancer, surgery, hospitalization, immobilization, pregnancy, and obesity are associated with an increased risk of VTE. However, the chain of molecular events leading to VTE is unclear, and for approximately 50% of cases there is no underlying risk factor.
VTE is caused by a thrombus (blood clot) formed in the valves of the deep veins, mostly of the lower limbs. This site harbors several factors thought to contribute to the formation of venous thrombi. These potential ‘VTE triggers’ in the venous valve include low degree of inflammation and alternating levels of oxygen, i.e. intermittent hypoxia. There is evidence supporting that inflammation promotes VTE, however the role of sustained and/or intermittent hypoxia and its potential synergy with inflammation in VTE is unclear.
Project PROVE is dedicated to uncovering novel aspects of VTE initiation on cellular and molecular level with an emphasis on inflammatory and hypoxic triggers and how these may act together to favor VTE. Two cell types are in focus in the study: monocytes and endothelial cells, which are known to have a role in VTE. These cells are exposed to combinations of hypoxia and inflammatory stimuli using a technical platform designed to mimic the pathological milieu in the venous valve. The potential pro-thrombotic effects of VTE triggers on the cells are assessed by studying their RNA and protein expression, as well as their ability to modulate components of the coagulation system.
Last, PROVE applies the acquired knowledge from these experiments to clinical samples by studying extracellular vesicles (EVs) - small ‘nanoparticles’ released from most cells in our bodies. Studying such nanoparticles can reveal the state of the cells they originate from, and help drawing conclusions on biological functions, including disease mechanisms. In addition, EVs have a great diagnostic potential. By stimulating cells in PROVE with VTE-associated triggers, EVs with a presumable pathogenic profile will be released and can be analyzed to find a ‘VTE disease profile’. EVs from VTE patients are then investigated to assess if they have this disease profile which can be used to support VTE diagnostics, but also to advance the understanding of VTE mechanisms.
VTE is caused by a thrombus (blood clot) formed in the valves of the deep veins, mostly of the lower limbs. This site harbors several factors thought to contribute to the formation of venous thrombi. These potential ‘VTE triggers’ in the venous valve include low degree of inflammation and alternating levels of oxygen, i.e. intermittent hypoxia. There is evidence supporting that inflammation promotes VTE, however the role of sustained and/or intermittent hypoxia and its potential synergy with inflammation in VTE is unclear.
Project PROVE is dedicated to uncovering novel aspects of VTE initiation on cellular and molecular level with an emphasis on inflammatory and hypoxic triggers and how these may act together to favor VTE. Two cell types are in focus in the study: monocytes and endothelial cells, which are known to have a role in VTE. These cells are exposed to combinations of hypoxia and inflammatory stimuli using a technical platform designed to mimic the pathological milieu in the venous valve. The potential pro-thrombotic effects of VTE triggers on the cells are assessed by studying their RNA and protein expression, as well as their ability to modulate components of the coagulation system.
Last, PROVE applies the acquired knowledge from these experiments to clinical samples by studying extracellular vesicles (EVs) - small ‘nanoparticles’ released from most cells in our bodies. Studying such nanoparticles can reveal the state of the cells they originate from, and help drawing conclusions on biological functions, including disease mechanisms. In addition, EVs have a great diagnostic potential. By stimulating cells in PROVE with VTE-associated triggers, EVs with a presumable pathogenic profile will be released and can be analyzed to find a ‘VTE disease profile’. EVs from VTE patients are then investigated to assess if they have this disease profile which can be used to support VTE diagnostics, but also to advance the understanding of VTE mechanisms.
The first goal of the project was to setup a platform which would allow us to culture cells in tightly controlled and measurable hypoxic and inflammatory conditions. This was achieved by culturing cells Involved in VTE (monocytes and endothelial cells) in either normoxia, sustained (constant) or intermittent (cycling) hypoxia – with or without inflammatory stimulation.
To assess the effects of the two hypoxia types alone, but also in combination with inflammation, the stimulated cells were investigated by a combination of methodologies including flow cytometry, western blot, ELISA, RT-qPCR and enzymatic assays for tissue factor activity (a pro-coagulant protein). A lipid-dependent coagulation assays was performed to assess the procoagulant activity of the EVs released from the treated cells.
Once conditions were optimized, cells from healthy donors were tested in large-scale experiments and in-depth analyses of the VTE-associated pathological stimulations were conducted by whole transcriptome sequencing. In short, we detected substantial effects to cellular functions, as well as protein and gene expressions that are related to VTE. We found very different patterns of inflammatory modulation by sustained and intermittent hypoxia, and several genes and proteins that were additively elevated when inflammation is induced under hypoxic conditions.
Last, we successfully setup an assay to analyze the profiles of EVs based on eleven different protein markers on vesicles captured from blood samples. The assay was used to analyze samples from a cohort of 200 patients with suspected VTE. The results are in its final stages of analysis, but our preliminary conclusion is that there is a different molecular profile in cases with confirmed VTE – which suggests that vesicle-based blood analysis could help in diagnosing VTE in the future.
Project PROVE studies disease-relevant cells and their EVs. Our findings will be published by the end of, or shortly after, the end of the project period. Findings were however presented at several international meetings: (i) The International society for Thrombosis and Haemostasias (ISTH) 2020 (Italy, and hybrid); (ii) The European Congress on Thrombosis and Haemostasis 2021 (Belgium), and (iii) as an invited presentation at the Nordic coagulation meeting 2021 (Sweden). Currently, three manuscripts are in preparation for publication of the final results.
To assess the effects of the two hypoxia types alone, but also in combination with inflammation, the stimulated cells were investigated by a combination of methodologies including flow cytometry, western blot, ELISA, RT-qPCR and enzymatic assays for tissue factor activity (a pro-coagulant protein). A lipid-dependent coagulation assays was performed to assess the procoagulant activity of the EVs released from the treated cells.
Once conditions were optimized, cells from healthy donors were tested in large-scale experiments and in-depth analyses of the VTE-associated pathological stimulations were conducted by whole transcriptome sequencing. In short, we detected substantial effects to cellular functions, as well as protein and gene expressions that are related to VTE. We found very different patterns of inflammatory modulation by sustained and intermittent hypoxia, and several genes and proteins that were additively elevated when inflammation is induced under hypoxic conditions.
Last, we successfully setup an assay to analyze the profiles of EVs based on eleven different protein markers on vesicles captured from blood samples. The assay was used to analyze samples from a cohort of 200 patients with suspected VTE. The results are in its final stages of analysis, but our preliminary conclusion is that there is a different molecular profile in cases with confirmed VTE – which suggests that vesicle-based blood analysis could help in diagnosing VTE in the future.
Project PROVE studies disease-relevant cells and their EVs. Our findings will be published by the end of, or shortly after, the end of the project period. Findings were however presented at several international meetings: (i) The International society for Thrombosis and Haemostasias (ISTH) 2020 (Italy, and hybrid); (ii) The European Congress on Thrombosis and Haemostasis 2021 (Belgium), and (iii) as an invited presentation at the Nordic coagulation meeting 2021 (Sweden). Currently, three manuscripts are in preparation for publication of the final results.
First and foremost, PROVE has established a technical platform to mimic the complex patterns of intermittent hypoxia associated with VTE, which could be further adjusted and adopted to other cardiovascular and inflammatory diseases. The setup was based on actual measured levels of dissolved oxygen, which is more accurate compared to most published studies where oxygen levels are measured in the incubator (culture cell), rather than the cell culture liquids. Second, we studied both sustained (constant) and intermittent hypoxia in parallel, with and without inflammatory stimuli, which is unique and thus expected to attract attention. Setting up and validating these combinations has been laborious and time consuming, but of critical importance as it may be a milestone in our journey to uncover mechanisms in VTE, as well as other cardiovascular diseases. Further, our preliminary conclusions show that inflammation and hypoxia together promote stronger VTE-favoring effects in cells. This sends the clear and important message that future studies of VTE mechanisms should incorporate multiple triggers to better mimic its pathophysiology.
Third, we believe our assay analyzing EVs from patients with suspected VTE can inspire others to use a similar approach of a broad, but targeted, immunoassay to find a disease profile. EVs have emerged as an important means of communication between cells in health and disease including VTE. Hence, comprehensive analysis of disease-relevant EVs using novel and advanced analytical approaches (as developed here) may be used to shed light on VTE mechanisms and to improve its diagnostic and prediction.
Third, we believe our assay analyzing EVs from patients with suspected VTE can inspire others to use a similar approach of a broad, but targeted, immunoassay to find a disease profile. EVs have emerged as an important means of communication between cells in health and disease including VTE. Hence, comprehensive analysis of disease-relevant EVs using novel and advanced analytical approaches (as developed here) may be used to shed light on VTE mechanisms and to improve its diagnostic and prediction.