Role of Lipid Rafts on LRP8 Signaling Triggered by Anti-ß2-GPI Antibodies in Endothelial Cells.

Antiphospholipid antibody syndrome is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity in association with circulating antiphospholipid antibodies, mainly anti-ß2 glycoprotein 1 antibodies (anti-ß2-GPI antibodies). Previous studies demonstrated that the signaling pathway may involve lipid rafts, plasma membrane microdomains enriched in glycosphingolipid and cholesterol. In this study, we analyzed the signaling pathway of LRP8/ApoER2, a putative receptor of anti-ß2-GPI antibodies, through lipid rafts in human endothelial cells. LRP8, Dab2 and endothelial nitric oxide synthase (e-NOS) phosphorylation were evaluated using Western blot, Nitric Oxide (NO) production with cytofluorimetric analysis, LRP8 enrichment in lipid rafts via sucrose gradient fractionation, and scanning confocal microscopy analysis of its association with ganglioside GM1 was also conducted. The analyses demonstrated that affinity-purified anti-ß2-GPI antibodies induced LRP8 and Dab-2 phosphorylation, together with a significant decrease in e-NOS phosphorylation, with consequent decrease in NO intracellular production. These effects were almost completely prevented by Methyl-ß-cyclodextrin (MßCD), indicating the involvement of lipid rafts. It was supported with the observation of LRP8 enrichment in lipid raft fractions and its association with ganglioside GM1, detected with scanning confocal microscopy. These findings demonstrate that LRP8 signaling triggered by anti-ß2-GPI antibodies in endothelial cells occurs through lipid rafts. It represents a new task for valuable therapeutic approaches, such as raft-targeted therapy, including cyclodextrins and statins.

Lipid rafts mediate multilineage differentiation of human dental pulp-derived stem cells (DPSCs).

Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MßCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MßCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.

Oxidative Stress as a Regulatory Checkpoint in the Production of Antiphospholipid Autoantibodies: The Protective Role of NRF2 Pathway.

Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity. Oxidative stress may affect various signaling pathways and biological processes, promoting dysfunctional immune responses and inflammation, inducing apoptosis, deregulating autophagy and impairing mitochondrial function. The chronic oxidative stress and the dysregulation of the immune system leads to the loss of tolerance, which drives autoantibody production and inflammation with the development of endothelial dysfunction. In particular, anti-phospholipid antibodies (aPL), which target phospholipids and/or phospholipid binding proteins, mainly ß-glycoprotein I (ß-GPI), play a functional role in the cell signal transduction pathway(s), thus contributing to oxidative stress and thrombotic events. An oxidation-antioxidant imbalance may be detected in the blood of patients with APS as a reflection of disease progression. This review focuses on functional evidence highlighting the role of oxidative stress in the initiation and progression of APS. The protective role of food supplements and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) activators in APS patients will be summarized to point out the potential of these therapeutic approaches to reduce APS-related clinical complications.

The Role of Autophagy as a Trigger of Post-Translational Modifications of Proteins and Extracellular Vesicles in the Pathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent joint inflammation, leading to cartilage and bone destruction. Autoantibody production is directed to post-translational modified (PTM) proteins, i.e., citrullinated or carbamylated. Autophagy may be the common feature in several types of stress (smoking, joint injury, and infections) and may be involved in post-translational modifications (PTMs) in proteins and the generation of citrullinated and carbamylated peptides recognized by the immune system in RA patients, with a consequent breakage of tolerance. Interestingly, autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy combines the autophagy machinery with the secretion of cellular content via extracellular vesicles (EVs). A role for exosomes in RA pathogenesis has been recently demonstrated. Exosomes are involved in intercellular communications, and upregulated proteins and RNAs may contribute to the development of inflammatory arthritis and the progression of RA. In RA, most of the exosomes are produced by leukocytes and synoviocytes, which are loaded with PTM proteins, mainly citrullinated proteins, inflammatory molecules, and enzymes that are implicated in RA pathogenesis. Microvesicles derived from cell plasma membrane may also be loaded with PTM proteins, playing a role in the immunopathogenesis of RA. An analysis of changes in EV profiles, including PTM proteins, could be a useful tool for the prevention of inflammation in RA patients and help in the discovery of personalized medicine.

Role of a Novel Heparanase Inhibitor on the Balance between Apoptosis and Autophagy in U87 Human Glioblastoma Cells.

BACKGROUND: Heparanase (HPSE) is an endo-ß-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. METHODS: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. RESULTS: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. CONCLUSIONS: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.

Antiphospholipid antibodies in patients with stroke during COVID-19: A role in the signaling pathway leading to platelet activation.

Background: Several viral and bacterial infections, including COVID-19, may lead to both thrombotic and hemorrhagic complications. Previously, it has been demonstrated an "in vitro" pathogenic effect of "antiphospholipid" antibodies (aPLs), which are able to activate a proinflammatory and procoagulant phenotype in monocytes, endothelial cells and platelets. This study analyzed the occurrence of aPL IgG in patients with acute ischemic stroke (AIS) during COVID-19, evaluating the effect of Ig fractions from these patients on signaling and functional activation of platelets. Materials and methods: Sera from 10 patients with AIS during COVID-19, 10 non-COVID-19 stroke patients, 20 COVID-19 and 30 healthy donors (HD) were analyzed for anti-cardiolipin, anti-ß2-GPI, anti-phosphatidylserine/prothrombin and anti-vimentin/CL antibodies by ELISA. Platelets from healthy donors were incubated with Ig fractions from these patients or with polyclonal anti-ß2-GPI IgG and analyzed for phospho-ERK and phospho-p38 by western blot. Platelet secretion by ATP release dosage was also evaluated. Results: We demonstrated the presence of aPLs IgG in sera of patients with AIS during COVID-19. Treatment with the Ig fractions from these patients or with polyclonal anti-ß2-GPI IgG induced a significant increase of phospho-ERK and phospho-p38 expression. In the same vein, platelet activation was supported by the increase of adenyl nucleotides release induced by Ig fractions. Conclusions: This study demonstrates the presence of aPLs in a subgroup of COVID-19 patients who presented AIS, suggesting a role in the mechanisms contributing to hypercoagulable state in these patients. Detecting these antibodies as a serological marker to check and monitor COVID-19 may contribute to improve the risk stratification of thromboembolic manifestations in these patients.

Advances in the Pathophysiology of Thrombosis in Antiphospholipid Syndrome: Molecular Mechanisms and Signaling through Lipid Rafts.

The pathological features of antiphospholipid syndrome (APS) are related to the activity of circulating antiphospholipid antibodies (aPLs) associated with vascular thrombosis and obstetric complications. Indeed, aPLs are not only disease markers, but also play a determining pathogenetic role in APS and exert their effects through the activation of cells and coagulation factors and inflammatory mediators for the materialization of the thromboinflammatory pathogenetic mechanism. Cellular activation in APS necessarily involves the interaction of aPLs with target receptors on the cell membrane, capable of triggering the signal transduction pathway(s). This interaction occurs at specific microdomains of the cell plasma membrane called lipid rafts. In this review, we focus on the key role of lipid rafts as signaling platforms in the pathogenesis of APS, and propose this pathogenetic step as a strategic target of new therapies in order to improve classical anti-thrombotic approaches with "new" immunomodulatory drugs.

Citrullinated and carbamylated proteins in extracellular microvesicles from plasma of patients with rheumatoid arthritis.

OBJECTIVES: To investigate the expression of citrullinated and carbamylated proteins in extracellular microvesicles (EMVs) from RA patients. METHODS: We enrolled 24 RA naïve for biological therapy and 20 healthy donors (HD), matched for age and sex. For each patient, laboratory and clinical data were recorded and clinical indexes were measured (Clinical Disease Activity Index, Simplified Disease Activity Index, DAS28). EMVs in RA patients and HD were purified from plasma and measured by nanoparticle tracking analysis (NanoSight). Further, EMVs were incubated with anti-citrullinated/carbamylated proteins antibodies and processed by flow cytometry and western blot to evaluate the expression of citrullinated/carbamylated antigens. RESULTS: NanoSight revealed a significant increase of EMVs in RA compared with HD. Moreover, cytofluorimetric analysis showed a significative higher expression of citrullinated antigens on EMVs' surface in RA than donors, while no substantial difference was found in the expression of carbamylated antigens. These data were confirmed by western blot which identified vimentin, glycolytic enzyme alpha-enolase 1 and collagen type II as the main citrullinated and carbamylated proteins carried by EMVs. Finally, a relevant correlation between the expression of citrullinated antigens and disease activity was found. CONCLUSIONS: The results of this study suggest an involvement of EMVs in the pathogenesis of RA by inducing autoimmunity.

Gangliosides and Their Role in Multilineage Differentiation of Mesenchymal Stem Cells.

Gangliosides (GGs) are a glycolipid class present on Mesenchymal Stem Cells (MSCs) surfaces with a critical appearance role in stem cell differentiation, even though their mechanistic role in signaling and differentiation remains largely unknown. This review aims to carry out a critical analysis of the predictive role of gangliosides as specific markers of the cellular state of undifferentiated and differentiated MSCs, towards the osteogenic, chondrogenic, neurogenic, and adipogenic lineage. For this reason, we analyzed the role of GGs during multilineage differentiation processes of several types of MSCs such as Umbilical Cord-derived MSCs (UC-MSCs), Bone Marrow-derived MSCs (BM-MSCs), Dental Pulp derived MSCs (DPSCs), and Adipose derived MSCs (ADSCs). Moreover, we examined the possible role of GGs as specific cell surface markers to identify or isolate specific stem cell isotypes and their potential use as additional markers for quality control of cell-based therapies.

Relationship Between Gender Differences and Clinical Outcome in Patients With the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS), characterized by artherial and/or venous thrombosis, pregnancy morbidity and "antiphospholipid" antibodies (aPLs), is more common in women than in men, with a female to male ratio of about 3.5:1. Only few studies have investigated the clinical differences between male and female patients with APS. Therefore, this study was aimed to analyze the differences of clinical manifestations and laboratory tests, at diagnosis, between female and male APS patients and the clinical outcome. We enrolled 191 consecutive APS patients (125 with primary APS, PAPS, and 66 with secondary APS, SAPS) with a female predominant ratio of approximately 3:1 (142 vs 49). The prevalence of PAPS was higher in males than females (p<0.001). The analysis of aPL profile revealed that high IgM anti-cardiolipin (aCL) and high-medium IgG aCL titers were more frequent in males. In thrombotic APS peripheral arterial thrombosis was more common in male than female patients (p=0.049), as well as myocardial infarction (p=0.031). Multivariate analysis to correct for cardiovascular risk factors, high titer of aPLs and triple positivity for aPLs, revealed that the odds ratio for myocardial infarction in male was 3.77. Thus, APS may be considered as a disease in which serological (IgM titer) and clinical profiles are influenced by gender.

HMGB1 in Pediatric COVID-19 Infection and MIS-C: A Pilot Study.

Objective: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, a novel syndrome known as a multisystem inflammatory syndrome in children (MIS-C) was reported in previously healthy children. A possible pro-inflammatory molecule, high-mobility group box 1 (HMGB1), may be assumed to play an important role in the pathogenesis and clinical presentation of MIS-C. We described the clinical picture of patients with MIS-C and we also aimed to test and compare HMGB1 serum levels of MIS-C patients with those of patients with previous SARS-CoV2 infection and healthy children. Study design: We determined HMGB1 levels by Western blot in 46 patients and divided them into three groups, namely, five patients with MIS-C (median age: 8.36 years), 20 children with a history of SARS-CoV-2 infection (median age: 10.45 years), and 21 healthy children (controls) (median age: 4.84 years), without evidence of respiratory infection in the last 3 months. Results: The median level of HMGB1 in the serum of five patients with MIS-C was found to be significantly higher compared with both patients with a recent history of COVID-19 (1,151.38 vs. 545.90 densitometric units (DU), p = 0.001) and control (1,151.38 vs. 320.33 DU, p = 0.001) groups. The HMGB1 level in MIS-C patients with coronary involvement had a slightly higher value with respect to patients without coronary dilatation (1,225.36 vs. 1,030.49 DU, p = 0.248). In two of the five children with MIS-C that performed a follow-up, the HMGB1 value decreased to levels that were superimposable to the ones of the control group. Conclusion: The significantly high level of HMGB1 protein found in the serum of COVID-19 and patients with MIS-C supports its involvement in inflammatory manifestations, suggesting HMGB1 as a potential biomarker and therapeutic target in patients with severe illness.

Crosstalk of Autophagy and Apoptosis.

Autophagy and apoptosis represent two fundamental pathophysiological mechanisms of cell fate regulation. However, the signaling pathways of these processes are significantly interconnected through various mechanisms of crosstalk. Indeed, autophagy/apoptosis crosstalk is still an emerging field, in which an increasing number of molecules are involved, including, for example, PINK1 and ERLINs. On the other hand, this crosstalk involves signal transduction pathways which are strongly dependent on Ca2+. Interestingly, crosstalk between autophagy and apoptosis impacts several pathologies, including multiple rheumatic diseases. The purpose of this Special Issue is also to investigate the bioactive properties of drugs with antitumor activity, focusing particularly on the role of anthraquinone derivatives in the regulation of cell death and autophagy crosstalk. This Special Issue of Cells brings together the most recent advances in understanding the various aspects of crosstalk between autophagy and apoptosis and the interconnected signaling pathways, implying therapeutic perspectives for the utility of its modulation in an anti-cancer setting.

Hypoxia Induces DPSC Differentiation versus a Neurogenic Phenotype by the Paracrine Mechanism.

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

Anti-ß2-GPI Antibodies Induce Endothelial Cell Expression of Tissue Factor by LRP6 Signal Transduction Pathway Involving Lipid Rafts.

In this study we analyzed whether anti-ß2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-ß2-GPI antibodies. Both LRP6 and ß-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-ß2-GPI antibodies induced LRP6 phosphorylation with consequent ß-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-ß-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-ß2-GPI antibodies effect, indicating that the anti-ß2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between ß2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-ß2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-ß2-GPI binding triggers ß-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).

Carbamylation of ß2-glycoprotein I generates new autoantigens for antiphospholipid syndrome: a new tool for diagnosis of 'seronegative' patients.

OBJECTIVES: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-ß2-glycoprotein I (ß2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ('seronegative APS', SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-ß2-GPI (Carb-ß2-GPI) as a new autoantigen of APS. METHODS: ß2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived dendritic cell (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with RA, 20 non-APS thrombosis and 50 healthy donors were analysed for anti-Carb-ß2-GPI by ELISA. RESULTS: Carb-ß2-GPI is able to activate moDCs, inducing upregulation of CD80, CD86 and CD40, activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB, and IL-12p70 release. Serological results showed that both 37/114 SN-APS (32.46%) and 23/60 APS (38.33%) patients resulted positive for anti-Carb-ß2-GPI. Interestingly, SN-APS patients who tested positive for anti-Carb-ß2-GPI showed a higher prevalence of thrombocytopenia (P = 0.04, likelihood positive ratio of 3.9). CONCLUSION: Data obtained from both functional tests on moDCs and immunological approaches prompted identification of Carb-ß2-GPI as a 'new' antigenic target in APS. In particular, anti-Carb-ß2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients who tested positive for anti-Carb-ß2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Prions and Neurodegenerative Diseases: A Focus on Alzheimer's Disease.

Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The misfolded proteins are involved in prions, amyloid-ß (Aß), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aß and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aß oligomers (AßOs) and PrPC. Also, we studied the role of PrPC as an AßO receptor that initiates an AßO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aß and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AßOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.