Extracellular Vesicles: Intercellular Communication Mediators in Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL) that cause endothelial injury and thrombophilia. Extracellular vesicles are involved in endothelial and thrombotic pathologies and may therefore have an influence on the prothrombotic status of APS patients. Intercellular communication and connectivity are important mechanisms of interaction between healthy and pathologically altered cells. Despite well-characterized in vitro and in vivo models of APS pathology, the field of extracellular vesicles is still largely unexplored and could therefore provide an insight into the APS mechanism and possibly serve as a biomarker to identify patients at increased risk. The analysis of EVs poses a challenge due to the lack of standardized technology for their isolation and characterization. Recent findings in the field of EVs offer promising aspects that may explain their role in the pathogenesis of various diseases, including APS

Extracellular Vesicles and Antiphospholipid Syndrome: State-of-the-Art and Future Challenges

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thromboembolism, obstetric complications, and the presence of antiphospholipid antibodies (aPL). Extracellular vesicles (EVs) play a key role in intercellular communication and connectivity and are known to be involved in endothelial and vascular pathologies. Despite well-characterized in vitro and in vivo models of APS pathology, the field of EVs remains largely unexplored. This review recapitulates recent findings on the role of EVs in APS, focusing on their contribution to endothelial dysfunction. Several studies have found that APS patients with a history of thrombotic events have increased levels of EVs, particularly of endothelial origin. In obstetric APS, research on plasma levels of EVs is limited, but it appears that levels of EVs are increased. In general, there is evidence that EVs activate endothelial cells, exhibit proinflammatory and procoagulant effects, interact directly with cell receptors, and transfer biological material. Future studies on EVs in APS may provide new insights into APS pathology and reveal their potential as biomarkers to identify patients at increased risk

Molecular profiling of small extracellular vesicles and their originating cells in antiphospholipid syndome

Antifosfolipidni sindrom (APS) je sistemska avtoimunska bolezen, ki se klinično izraža s trombozami v venah, arterijah ali mikrožilju in/ali z zapleti v nosečnosti, laboratorijsko pa s prisotnostjo antifosfolipidnih protiteles (aPL). Vezava aPL na površino celic sproži njihovo aktivacijo, ki vodi v sproščanje zunajceličnih veziklov (EVs po angleškem izrazu »extracellular vesicles«). Tako v plazmi bolnikov z APS kot in vitro so najpogosteje preučevani srednji/veliki vezikli endotelijskega izvora. Ostali podtipi veziklov ter skupina majhnih zunajceličnih veziklov (sEVs) so pri APS slabše raziskani. Namen doktorskega dela je ugotoviti pomen izločanja sEVs in njihovega molekulskega profila pri razlagi patogeneze in heterogenosti APS. V plazmi bolnikov s trombotičnim APS smo pokazali zvišane vrednosti celokupnih in endotelijskih sEVs ter nekaterih podtipov, ki nakazuje na aktivacijo endotelija in trombocitov tudi v odsotnosti akutnega trombotičnega dogodka. Čeprav pri APS nismo zaznali zvišane pojavnosti monocitnih sEVs, je analiza njihovih izvornih celic pokazala na aktivacijo monocitov v polni krvi bolnikov z APS. Zvišano izražanje integrina VLA4 na monocitih je lahko vpleteno v intenzivnejšo interakcijo med monociti in endotelijem. Blokada tega integrina bi lahko v prihodnosti predstavljala alternativo za zdravljenje težjih in na zdravljenje neodzivnih bolnikov z APS. Rezultati raziskav v okviru doktorske naloge kažejo na to, da je profil sEVs, ki jih izločajo različne celice pri bolnikih s trombotičnim APS, spremenjen ter da nakazuje na izvor sEVs in aktivacijski status njihovih izvornih celic. S tem smo pokazali, da sEVs pri APS zagotovo igrajo pomembno vlogo, vendar so potrebne nadaljnje raziskave, ki bodo natančneje opredelile učinke sEVs na endotelijske celice, monocite in trombocite, ter rezultate naših raziskav natančneje umestile v patogenezo APS.Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thromboembolism, obstetric complications, and the presence of antiphospholipid antibodies (aPL). Binding of aPL to the cell surface triggers their activation, leading to the release of extracellular vesicles (EVs). Medium/large vesicles of endothelial origin are most commonly studied in vitro as well as in plasma of APS patients. Other subtypes of vesicles and the group of small extracellular vesicles (sEVs) are less well understood in APS. The aim of this dissertation is to determine the importance of sEVs and their molecular profile in explaining the pathogenesis and heterogeneity of APS. In the plasma of thrombotic APS patients, we found increased levels of total sEVs, endothelial sEVs, and some sEV subtypes, indicating endothelial and platelet activation even in the absence of an acute thrombotic event. Although we did not detect increased levels of monocyte sEVs in APS, analysis of their cells of origin suggests monocyte activation. The increased surface expression of the integrin VLA4 on monocytes could be involved in the enhanced interaction between monocytes and the endothelium. Blocking this integrin could provide an alternative treatment option for more severe and difficult-to-treat APS patients in the future. Taken together, our results suggest that the profile of secreted sEVs is altered in patients with thrombotic APS, indicating their origin and the activation status of the cells of origin. Thus, we have shown that sEVs play an important role in APS, but further research is needed to more precisely define the effects of sEVs on endothelial cells, monocytes, and platelets and to better situate our findings in the pathogenesis of APS

Antiphospholipid Antibody Syndrome-Associated Increased Surface Expression of VLA4 Integrin on Human Monocytes

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis and/or obstetric complications in the presence of antiphospholipid antibodies (aPL). Catastrophic APS (CAPS) is the most severe form of the disease, in which microvascular thromboses develop rapidly, leading to multiorgan failure. Monocytes, along with endothelial cells, are critical players in the pathogenesis of APS. Recruitment of these cells to the site of injury/inflammation involves a series of events, including capture, rolling, adhesion enhancement, and transmigration, which are controlled by surface adhesion molecules. The aim of our study was to investigate the surface adhesion profile of monocytes from APS patients and monocytes stimulated in vitro with aPL from a CAPS patient. The surface expression of the adhesion molecules LFA1, L-selectin, MAC1, PSGL1, and VLA4 was analyzed by flow cytometry. To our knowledge, this preliminary study was the first to show that VLA4 was significantly increased on the surface of monocytes from APS patients. Moreover, in vitro stimulations mimicking CAPS showed an even greater increase in VLA4. Our data suggest that the surface adhesion profile on monocytes is altered in APS and CAPS and may be involved in the thrombotic pathophysiology of the disease by enhancing monocyte adhesion

Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110-170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPL-neg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS

Characterization of plasma-derived small extracellular vesicles indicates ongoing endothelial and platelet activation in patients with thrombotic antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110–170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPLneg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS