NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4.

BACKGROUND: The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study. METHODS: Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy. RESULTS: The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbß3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls. CONCLUSION: These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface.

Dimerization of the C-type lectin-like receptor CD93 promotes its binding to Multimerin-2 in endothelial cells.

Blocking the signaling activated by the plasma membrane receptor CD93 has recently been demonstrated a useful tool in antiangiogenic treatment and oncotherapy. In the proliferating endothelium, CD93 regulates cell adhesion, migration, and vascular maturation, yet it is unclear how CD93 interacts with the extracellular matrix activating signaling pathways involved in the vascular remodeling. Here for the first time we show that in endothelial cells CD93 is structured as a dimer and that this oligomeric form is physiologically instrumental for the binding of CD93 to its ligand Multimerin-2. Crystallographic X-ray analysis of recombinant CD93 reveals the crucial role played by the C-type lectin-like and sushi-like domains in arranging as an antiparallel dimer to achieve a functional binding state, providing key information for the future design of new drugs able to hamper CD93 function in neovascular pathologies.

Bioinformatics Approaches to Predict Mutation Effects in the Binding Site of the Proangiogenic Molecule CD93.

The transmembrane glycoprotein CD93 has been identified as a potential new target to inhibit tumor angiogenesis. Recently, Multimerin-2 (MMRN2), a pan-endothelial extracellular matrix protein, has been identified as a ligand for CD93, but the interaction mechanism between these two proteins is yet to be studied. In this article, we aim to investigate the structural and functional effects of induced mutations on the binding domain of CD93 to MMRN2. Starting from experimental data, we assessed how specific mutations in the C-type lectin-like domain (CTLD) affect the binding interaction profile. We described a four-step workflow in order to predict the effects of variations on the inter-residue interaction network at the PPI, based on evolutionary information, complex network metrics, and energetic affinity. We showed that the application of computational approaches, combined with experimental data, allowed us to gain more in-depth molecular insights into the CD93-MMRN2 interaction, offering a platform for developing innovative therapeutics able to target these molecules and block their interaction. This comprehensive molecular insight might prove useful in drug design in cancer therapy.

Synthesis of Unsymmetrical Squaramides as Allosteric GSK-3ß Inhibitors Promoting ß-Catenin-Mediated Transcription of TCF/LEF in Retinal Pigment Epithelial Cells.

The glycogen synthase kinase 3ß (GSK-3ß) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3ß potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3ß of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce ß-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 µM.

Untangling the Extracellular Matrix of Idiopathic Epiretinal Membrane: A Path Winding among Structure, Interactomics and Translational Medicine.

Idiopathic epiretinal membranes (iERMs) are fibrocellular sheets of tissue that develop at the vitreoretinal interface. The iERMs consist of cells and an extracellular matrix (ECM) formed by a complex array of structural proteins and a large number of proteins that regulate cell-matrix interaction, matrix deposition and remodelling. Many components of the ECM tend to produce a layered pattern that can influence the tractional properties of the membranes. We applied a bioinformatics approach on a list of proteins previously identified with an MS-based proteomic analysis on samples of iERM to report the interactome of some key proteins. The performed pathway analysis highlights interactions occurring among ECM molecules, their cell receptors and intra- or extracellular proteins that may play a role in matrix biology in this special context. In particular, integrin ß1, cathepsin B, epidermal growth factor receptor, protein-glutamine gamma-glutamyltransferase 2 and prolow-density lipoprotein receptor-related protein 1 are key hubs in the outlined protein-protein cross-talks. A section on the biomarkers that can be found in the vitreous humor of patients affected by iERM and that can modulate matrix deposition is also presented. Finally, translational medicine in iERM treatment has been summed up taking stock of the techniques that have been proposed for pharmacologic vitreolysis.

SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters.

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Allele-specific silencing by RNAi of R92Q and R173W mutations in cardiac troponin T.

Autosomal dominant mutations in sarcomere proteins such as the cardiac troponin T (TNNT2) are the main genetic causes of human hypertrophic cardiomyopathy and dilated cardiomyopathy. Allele-specific silencing by RNA interference (ASP-RNAi) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. Here, we propose ASP-RNAi as a possible strategy to specifically knockdown mutant alleles coding for R92Q and R173W mutant TNNT2 proteins, identified in hypertrophic and dilated cardiomyopathy, respectively. Different siRNAs were designed and validated by luciferase reporter assay and following analysis in HEK293T cells expressing either the wild-type or mutant TNNT2 alleles. This study is the first exploration of ASP-RNAi on TNNT2-R173W and TNNT2-R92Q mutations in vitro and gives a base for further application of allele silencing as a therapeutic treatment for TNNT2-mutation-associated cardiomyopathies.

Xanthopsin-Like Systems via Site-Specific Click-Functionalization of a Retinoic Acid Binding Protein.

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells.

During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.

LRG1 Expression Is Elevated in the Eyes of Patients with Neovascular Age-Related Macular Degeneration.

Leucine-rich a-2-glycoprotein 1 (LRG1) is a candidate therapeutic target for treating the neovascular form of age-related macular degeneration (nvAMD). In this study we examined the expression of LRG1 in eyes of nvAMD patients. Choroidal neovascular membranes (CNVMs) from patients who underwent submacular surgery for retinal pigment epithelium-choroid graft transplantation were collected from 5 nvAMD patients without any prior intravitreal anti-VEGF injection, and from six patients who received intravitreal anti-VEGF injections before surgery. As controls free of nvAMD, retina sections were obtained from the eyes resected from a patient with lacrimal sac tumor and from a patient with neuroblastoma. CNVMs were immunostained for CD34, LRG1, and α-smooth muscle actin (α-SMA). Aqueous humor samples were collected from 58 untreated-naïve nvAMD patients prior to the intravitreal injection of anti-VEGF and 51 age-matched cataract control patients, and LRG1 concentration was measured by ELISA. The level of LRG1 immunostaining is frequently high in both the endothelial cells of the blood vessels, and myofibroblasts in the surrounding tissue of CNVMs of treatment-naïve nvAMD patients. Furthermore, the average concentration of LRG1 was significantly higher in the aqueous humor of nvAMD patients than in controls. These observations provide a strong experimental basis and scientific rationale for the progression of a therapeutic anti-LRG1 monoclonal antibody into clinical trials with patients with nvAMD.

The C-type lectin CD93 controls endothelial cell migration via activation of the Rho family of small GTPases.

Endothelial cell migration is essential to angiogenesis, enabling the outgrowth of new blood vessels both in physiological and pathological contexts. Migration requires the activation of several signaling pathways, the elucidation of which expands the opportunity to develop new drugs to be used in antiangiogenic therapy. In the proliferating endothelium, the interaction between the transmembrane glycoprotein CD93 and the extracellular matrix activates signaling pathways that regulate cell adhesion, migration, and vascular maturation. Here we identify a pathway, comprising CD93, the adaptor proteins Cbl and Crk, and the small GTPases Rac1, Cdc42, and RhoA, which we propose acts as a regulator of cytoskeletal movements responsible for endothelial cell migration. In this framework, phosphorylation of Cbl on tyrosine 774 leads to the interaction with Crk, which acts as a downstream integrator in the CD93-mediated signaling regulating cell polarity and migration. Moreover, confocal microscopy analyses of GTPase biosensors show that CD93 drives coordinated activation of Rho-proteins at the cell edge of migratory endothelial cells. In conclusion, together with the demonstration of the key contribution of CD93 to the migratory process in living cells, these findings suggest that the signaling triggered by CD93 converges to the activation and modulation of the Rho GTPase signaling pathways regulating cell dynamics.

Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats' eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

MicroRNA Expression in the Aqueous Humor of Patients with Diabetic Macular Edema.

We identified and compared secreted microRNA (miRNA) expression in aqueous humor (AH) and plasma samples among patients with: type 2 diabetes mellitus (T2D) complicated by non-proliferative diabetic retinopathy (DR) associated with diabetic macular edema (DME) (DME group: 12 patients); T2D patients without DR (D group: 8 patients); and non-diabetic patients (CTR group: 10 patients). Individual patient AH samples from five subjects in each group were profiled on TaqMan Low Density MicroRNA Array Cards. Differentially expressed miRNAs identified from profiling were then validated in single assay for all subjects. The miRNAs validated in AH were then evaluated in single assay in plasma. Gene Ontology (GO) analysis was conducted. From AH profiling, 119 mature miRNAs were detected: 86 in the DME group, 113 in the D group and 107 in the CTR group. miRNA underexpression in the DME group was confirmed in single assay for let-7c-5p, miR-200b-3p, miR-199a-3p and miR-365-3p. Of these four, miR-199a-3p and miR-365-3p were downregulated also in the plasma of the DME group. GO highlighted 54 validated target genes of miR-199a-3p, miR-200b-3p and miR-365-3p potentially implied in DME pathogenesis. Although more studies are needed, miR-200b-3p, let-7c-5p, miR-365-3p and miR-199a-3p represent interesting molecules in the study of DME pathogenesis.

Anethole Dithiolethione Increases Glutathione in Kidney by Inhibiting γ-Glutamyltranspeptidase: Biochemical Interpretation and Pharmacological Consequences.

AIMS: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. RESULTS: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. CONCLUSION: s. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

The Binding of CD93 to Multimerin-2 Promotes Choroidal Neovascularization.

Purpose: The purpose of this study was to investigate the involvement of CD93 and Multimerin-2 in three choroidal neovascularization (CNV) models and to evaluate their contribution in the neovascular progression of age-related macular degeneration (AMD). Methods: Choroidal neovascular membranes collected during surgery from AMD patients were analyzed by microscopy methods. Laser-induced CNV mouse models and choroid sprouting assays (CSAs) were carried out using the CD93 knockout mouse model. An original ex vivo CSA of vascular angiogenesis, employing choroid tissues isolated from human donors, was developed. Results: In contrast to healthy choroid endothelium, hyperproliferative choroidal endothelial cells (ECs) of AMD patients expressed high levels of CD93, and Multimerin-2 was abundantly deposited along the choroidal neovasculature. CD93 knockout mice showed a significant reduced neovascularization after laser photocoagulation, and their choroidal ECs displayed a decreased ability to produce sprouts in ex vivo angiogenesis assays. Moreover, the presence of an antibody able to hamper the CD93/Multimerin-2 interaction reduced vascular sprouting in the human CSA. Conclusions: Our results demonstrate that CD93 and its interaction with Multimerin-2 play an important role in pathological vascularization of the choroid, disclosing new possibilities for therapeutic intervention to neovascular AMD.

Heat Shock Protein 90 Involvement in the Development of Idiopathic Epiretinal Membranes.

Purpose: This work was aimed to further characterize cells of idiopathic epiretinal membranes (iERMs). We wanted to determine the contribution of 90-kDa heat shock protein (HSP90) to sustain the transforming growth factor-ß (TGF-ß)-mediated signal transduction pathway in iERM. Methods: Immunofluorescence and confocal microscopy were carried out on deplasticized sections from 36 epiretinal membranes processed for electron microscopy and on frozen sections from five additional samples with antibodies against α-smooth muscle actin (αSMA), vimentin, glial fibrillary acidic protein (GFAP), SMAD2, HSP90α, type-II TGF-ß1 receptor (TßRII), type-I collagen, and type-IV collagen. In addition, Müller MIO-M1 cells were transfected with HSP90 and challenged with TGF-ß1. Results: Double and triple labeling experiments showed that a variable number of TßRII+ cells were present in 94.1% of tested iERMs and they were mostly GFAP-/αSMA+/vimentin+/HSP90α+. In almost half of the cases these cells contained type-I collagen, suggesting their involvement in matrix deposition. HSP90 overexpressing MIO-M1 cells challenged with TGF-ß1 showed increased levels of TßRII, SMAD2, SMAD3, and phosphor-SMAD2. Nuclear SMAD2 staining could be observed in HSP90α+ cells on frozen sections of iERMs. Conclusions: Cells in iERMs that express TßRII are also HSP90α+ and show the antigenic profile of myofibroblast-like cells as they are GFAP-/αSMA+/vimentin+. HSP90α-overexpressing MIO-M1 cells challenged with TGF-ß1 showed an increased activation of the SMAD pathway implying that HSP90α might play a role in sustaining the TGF-ß1-induced fibrotic response of iERM cells.

The small GTPase Rab5c is a key regulator of trafficking of the CD93/Multimerin-2/ß1 integrin complex in endothelial cell adhesion and migration.

BACKGROUND: In the endothelium, the single-pass membrane protein CD93, through its interaction with the extracellular matrix protein Multimerin-2, activates signaling pathways that are critical for vascular development and angiogenesis. Trafficking of adhesion molecules through endosomal compartments modulates their signaling output. However, the mechanistic basis coordinating CD93 recycling and its implications for endothelial cell (EC) function remain elusive. METHODS: Human umbilical vein ECs (HUVECs) and human dermal blood ECs (HDBEC) were used in this study. Fluorescence confocal microscopy was employed to follow CD93 retrieval, recycling, and protein colocalization in spreading cells. To better define CD93 trafficking, drug treatments and transfected chimeric wild type and mutant CD93 proteins were used. The scratch assay was used to evaluate cell migration. Gene silencing strategies, flow citometry, and quantification of migratory capability were used to determine the role of Rab5c during CD93 recycling to the cell surface. RESULTS: Here, we identify the recycling pathway of CD93 following EC adhesion and migration. We show that the cytoplasmic domain of CD93, by its interaction with Moesin and F-actin, is instrumental for CD93 retrieval in adhering and migrating cells and that aberrant endosomal trafficking of CD93 prevents its localization at the leading edge of migration. Moreover, the small GTPase Rab5c turns out to be a key component of the molecular machinery that is able to drive CD93 recycling to the EC surface. Finally, in the Rab5c endosomal compartment CD93 forms a complex with Multimerin-2 and active ß1 integrin, which is recycled back to the basolaterally-polarized cell surface by clathrin-independent endocytosis. CONCLUSIONS: Our findings, focusing on the pro-angiogenic receptor CD93, unveil the mechanisms of its polarized trafficking during EC adhesion and migration, opening novel therapeutic opportunities for angiogenic diseases.