Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and/or obstetric events together with persistent high titers of circulating antiphospholipid antibodies (aPL) (Miyakis et al., 2006). Thrombosis in APS can involve different components of the vascular bed in any tissue or organ, such as arteries (coronary artery disease, ischemic stroke, and transient ischemic attack), veins (deep venous thrombosis of lower limbs or pulmonary embolism), or small vessels (catastrophic APS with episodes of thrombosis in small vessels of multiple organs causing a systemic dysfunction). Pregnancy-related morbidity in APS may include early or late gestational losses, intrauterine growth restriction, fetal demise, preterm labor, or preeclampsia. In addition to the aforementioned clinical diagnostics defined by the Sapporo criteria (Miyakis et al., 2006), there are other clinical presentations not included. These manifestations can be hematologic (thrombocytopenia and hemolytic anemia), cardiac (heart valve disease), cutaneous (livedo reticularis), renal (nephropathy), or neurologic (cognitive dysfunction not associated with stroke) (Ziporen et al., 1996; Asherson et al., 2003; Garcia and Erkan, 2018; Turrent-Carriles et al., 2018; Kolitz et al., 2019). While APS is still considered a relatively rare disorder, our understanding of its diagnosis and management is continuously advancing (Tektonidou et al., 2019). Some recent studies have estimated that the prevalence of APS is 50 per 100,000 people, and the incidence is 2.1 per 100,000 person-years without differences between men and women (Duarte-Garcia et al., 2019). The estimated frequency of aPL in thrombotic complications was reported to be 9.5% for deep vein thrombosis, 11% for myocardial infarction, and 13.5% for stroke (Andreoli et al., 2013), the latter being more associated in patients under 50 years of age (Petri, 2000). The prevalence of obstetric complications was reported to be between 6 and 50% (Andreoli et al., 2013; Alijotas-Reig et al., 2015; Cervera et al., 2015; Esteve-Valverde et al., 2016).

The prevalence of aPL in the general population ranges between 1 and 5%. However, only a minority of these individuals will develop APS (Gomez-Puerta and Cervera, 2014). Pathological aPL are a heterogeneous population of autoantibodies mainly directed against phospholipid-binding proteins such as cardiolipin (CL) and/or β2-glycoprotein I (β2GPI) (Di Simone et al., 2007). Anti-CL and anti-β2GPI aPL, in combination with lupus anticoagulant (LA), constitute the current laboratory criteria for diagnosis. However, there is a group of aPL classified as non-criteria including antithrombin, anti-phosphatidylserine, and anti-phosphatidylethanolamine antibodies which are associated with APS (Bertolaccini et al., 2011). Several studies have described patients who lack the classical clinical manifestations of APS but who present consistently with high aPL positivity, and these cases are known as “non-criteria APS” (Tektonidou et al., 2019). In contrast, patients with clinical manifestations fulfilling APS classification criteria but who are consistently negative for aPL tests are classified as “Seronegative APS” patients (Hughes and Kamashta, 2003; Jara et al., 2017; Conti et al., 2019; Hughes and Khamashta, 2019).

Several studies have described the mechanisms by which aPL lead to prothrombotic and proinflammatory states. In endothelial cells, these mechanisms include alterations in the coagulation cascade and platelet activation; increased production of reactive oxygen species (ROS) and pro-inflammatory cytokines; and decreased nitric oxide production (Hidalgo, 2014; Mulla et al., 2018; Schreiber et al., 2018). Anti-β2GPI antibodies are recognized as the most pathogenic subset of aPL. Among them, the anti-domain I β2GPI antibodies have a strong correlation with thrombosis and with pregnancy morbidity (PM) (Iwaniec et al., 2017; Liu et al., 2020), which has been used as a predictor tool for patients with late PM (Chighizola et al., 2018). Studies have demonstrated that anti-β2GPI antibodies can activate receptors such as toll-like receptor (TLR) 4, TLR2, and Apolipoprotein E receptor E2 (APOER2) expressed on the surface of endothelial cells (Ramesh et al., 2011; Benhamou et al., 2014; Raschi et al., 2014), and this can lead to the activation of the nuclear factor kappa B (NFκB), p38 mitogen-activated protein kinase (p38 MAPK), and the phosphatidylinositol 3-kinase (PI3K) signaling pathways (Meroni et al., 2014; Chighizola et al., 2015). Another mechanism involved in APS pathophysiology is oxidative stress (Alves and Grima, 2003). It was recently demonstrated that monocytes and neutrophils, from patients with APS, display increased ROS production, increased expression of pro-inflammatory and prothrombotic molecules, and a loss of mitochondria function (Perez-Sanchez et al., 2012; Lopez-Pedrera et al., 2016). This mitochondrial dysfunction was also described in a mouse model of systemic lupus erythematosus (SLE) and was associated with activation of the PI3K pathway and mammalian target of rapamycin (mTOR) (Oaks et al., 2016), a kinase that modulates cellular growth, proliferation, and apoptosis (Magnuson et al., 2012). Activation of mTOR was also increased in renal endothelial cells from patients with APS samples (Canaud et al., 2014; Chighizola et al., 2015). In addition to cell growth and survival, mTOR activation is associated with anabolic mechanisms at the intracellular level (Magnuson et al., 2012), which leads to inhibition of catabolic processes like autophagy. However, the mTOR and autophagic pathways may both be activated under conditions associated with oxidative stress and inflammation (Chen et al., 2011, 2016). Despite evidence of alterations in these pathways in other autoimmune diseases such as SLE (Lui et al., 2008; Oaks et al., 2016), less is known about the relationship between cellular metabolism and homeostasis in the context APS, and in particular, how aPL may disrupt the balance in endothelial cells. Therefore, the objective of this study was to investigate the impact of aPL on endothelial cell mitochondrial function, activation of the mTOR and autophagy pathways, and cellular growth.

Endothelial cells are responsible for maintaining vascular homeostasis and play an important role in the development of thrombosis in patients with APS (Poredos and Jezovnik, 2018). These cells are the target of several bioactive circulating factors that can cause a generalized dysfunction through deregulations in metabolism (Possomato-Vieira and Khalil, 2016; Bierhansl et al., 2017). MMP plays a key role in the maintenance of mitochondrial homeostasis (Zorova et al., 2018). MMP of a cell can change depending on the microenvironment, access to nutrients, cellular stress, and metabolic activity (Hirata and Sahai, 2017). aPL can induce a perturbation of the MMP in trophoblast cells, hepatocytes, and lymphocytes, leading to depolarization or hyperpolarization (Lai et al., 2015; Oaks et al., 2016; Alvarez et al., 2017). Since both depolarization and hyperpolarization involve dissociation of the electron transport chain and an increase of oxygen consumption, these conditions can be harmful to the cell through the induction of oxidative or reductive stress (Guo et al., 2011; Oaks et al., 2016; Zorova et al., 2018). We previously reported that serum from women with PM/VT increased the intracellular and mitochondrial production of ROS in HUVECs (Velásquez et al., 2019), the same group of patients whose IgGs induced the high MMP (hyperpolarization) in this study. Taken together, these findings suggest a mechanism by which aPL from women with PM/VT induced HUVEC oxidative stress. In fact, oxidative stress has been associated with endothelial cell dysfunction in preeclampsia, a clinical manifestation of obstetric APS (Rodríguez-Almaraz et al., 2018). Moreover, in the trophoblast, aPL recognizing CL and β2GPI bind to the mitochondria and induce ROS production (Zussman et al., 2020).

Oxidative cellular stress resulting from excessive metabolic ROS production can lead to the activation of rescue mechanisms such as autophagy. Autophagy is an intracellular degradation pathway that traffics substrates that could otherwise cause cytotoxicity (Chen et al., 2009; Ryter et al., 2019) through a catabolic system of double-membrane vesicles called autophagosomes, which are then fused with lysosomes (Bento et al., 2016; Qian et al., 2017). While some studies have reported altered autophagy in experimental models of APS (Mulla et al., 2018; Mu et al., 2020), less is known about how aPL impact endothelial cell autophagy. Endothelial cells control vascular homeostasis (Badimón and Martínez-González, 2002), and the mTOR pathway plays a major role in regulating cell metabolism, growth, and survival. There is also cross talk between the mTOR and autophagic pathways (Ryter et al., 2019). Classically, activation of the mTOR pathway has been associated with inhibition of autophagy through phosphorylation of ULK1 protein at Ser757 (Kim et al., 2011). However, in other contexts, such as tumor growth, there is coexistence between mTOR and autophagy activation. A similar behavior was observed in this study since the mTOR activation (determined by RPS6 phosphorylation) and concomitant autophagy activity (determined by a loss of ULK1 phosphorylation and increased LC3-II/LC3-I expression) were induced by IgG from patients with PM/VT and also IgG from patients with VT. In addition, IgG from women of the VT group induced lysosomal acidification, the ability of these aPL to trigger the final step of this degradative/recycling pathway.

Augmented endothelium cell survival has been associated with pathological conditions since it can lead to hyperplasia resulting in the occlusion of vessels and thus, generate a prothrombotic environment (Rajendran et al., 2013; Widlansky and Malik, 2015). aPL-induced proliferation was previously demonstrated in trophoblast cells (Alvarez et al., 2017). Another study showed increased cell proliferation in vascular endothelial cells exposed to aPL, which was associated with mTOR activation; and this activation was also observed in renal microvasculature of patients with APS nephropathy. In addition, patients who received rapamycin showed decreased vascular proliferation and no recurrence of vascular lesions (Canaud et al., 2014). This study highlights the relationship between cell survival pathways and cell proliferation under aPL conditions. Our findings of elevated cell proliferation in response to IgG from patients with VT only are, in part, in agreement with this report and indicate a link between the thrombotic features of that group of patients and the aPL-induced vascular effects.

This study aimed to establish a link between endothelial cell mitochondrial dysfunction, mTOR activation, and autophagy in the context of aPL, although it was performed in a descriptive way. Our findings have shown endothelial cell mitochondrial dysfunction in association with activation of the mTOR pathway and concurrent autophagic activity in response IgG from patients with PM/VT, while IgG from patients with VT only induced endothelial autophagy and cell proliferation in the absence of elevated mTOR activity or mitochondrial dysfunction. This underscores the heterogeneity of aPL. As expected, IgG from the control group PM/aPL- did not induce any responses in the endothelial cells. IgG from the patient group with clinical features of PM but non-criteria aPL (SN-OAPS) also did not induce any responses in the endothelial cells, thus revealing specific mechanisms triggered by the classical pathological aPL present in patients with PM/VT and VT. We previously showed several in vitro effects induced by aPL from women with PM/VT when compared with aPL from women with PM alone, suggesting that these aPL are distinct and could be triggering other pathways, which leads to different and/or more complex clinical manifestations (Alvarez et al., 2015, 2017). These subtle differences among the mechanisms triggered by aPL subpopulations have been suggested from prior studies (Ripoll et al., 2018; Alvarez et al., 2021). We also highlight that our cellular model was FBS-free, and since we did not performed any recombinant β2GPI addition, it is possible that cellular responses observed here were induced by aCL rather than aB2GPI antibodies. In conclusion, aPL from women with PM and vascular events induce cellular stress evidenced by mitochondrial hyperpolarization and increased activation of the mTOR and autophagic pathways, which may play a role in the pathogenesis of obstetric APS. These pathways may provide us with pharmacological targets to study further since compounds such as rapamycin and chloroquine that inhibit mTOR and autophagy have been used in experimental models of APS (Xia et al., 2017; Liu et al., 2019; Miranda et al., 2019).

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee from the Medical Investigations Institute from the School of Medicine (University of Antioquia). The patients/participants provided their written informed consent to participate in this study.

AA and AC responsible for obtaining the funds and designed this study. CRú and AC were responsible for the recruitment of patients. CRo and MV-B performed the experiments and analyzed the data. CRú performed and analyzed the hematological tests. CRo, AA, AC, MV, and VA wrote and performed a critical review of the manuscript. All the authors contributed to interpreting the results and revising the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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