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.

Role of CD93 in Health and Disease.

CD93 (also known as complement protein 1 q subcomponent receptor C1qR1 or C1qRp), is a transmembrane glycoprotein encoded by a gene located on 20p11.21 and composed of 652 amino acids. CD93 can be present in two forms: soluble (sCD93) and membrane-bound (CD93). CD93 is mainly expressed on endothelial cells, where it plays a key role in promoting angiogenesis both in physiology and disease, such as age-related macular degeneration and tumor angiogenesis. In fact, CD93 is highly expressed in tumor-associated vessels and its presence correlates with a poor prognosis, poor immunotherapy response, immune cell infiltration and high tumor, node and metastasis (TNM) stage in many cancer types. CD93 is also expressed in hematopoietic stem cells, cytotrophoblast cells, platelets and many immune cells, i.e., monocytes, neutrophils, B cells and natural killer (NK) cells. Accordingly, CD93 is involved in modulating important inflammatory-associated diseases including systemic sclerosis and neuroinflammation. Finally, CD93 plays a role in cardiovascular disease development and progression. In this article, we reviewed the current literature regarding the role of CD93 in modulating angiogenesis, inflammation and tumor growth in order to understand where this glycoprotein could be a potential therapeutic target and could modify the outcome of the abovementioned pathologies.

Diagnostic and Prognostic Role of CD93 in Cardiovascular Disease: A Systematic Review.

INTRODUCTION: Cluster of Differentiation (CD) 93 (also known as complement protein 1 q subcomponent receptor C1qR1 or C1qRp) is a transmembrane glycoprotein that can also be present in a soluble (sCD93) form. Recent studies have investigated the role of this protein in cardiovascular disease (CVD). The present systematic review aims to assess the associations between CD93 and cardiovascular (CV) risk factors and disease at both the proteomic and genomic levels. METHODS: We conducted systematic searches in the PubMed, EMBASE, and Web of Science databases to identify all human studies since inception to February 2023 that investigated the role of CD93 in CV risk factors, CVD, and CV-associated outcomes. The data collection and analysis have been independently conducted by two reviewers. The search terms included: cardiovascular, heart failure, acute stroke, myocardial infarction, stroke, peripheral artery disease, cardiovascular death, MACE, hypertension, metabolic syndrome, hyperuricemia, diabetes, cd93, c1qr, C1qR1, complement protein 1 q subcomponent receptor. RESULTS: A total of 182 references were identified, and 15 studies investigating the associations between CD93 protein levels or CD93 genetic polymorphisms and the development or prevalence of CV risk factors (i.e., hypertension, dyslipidemia, and obesity) and CVD (i.e., heart failure, coronary artery disease, and ischemic stroke) were included. Although promising, the quality and dimension of the analyzed studies do not allow for a definitive answer to the question of whether CD93 may hold diagnostic and prognostic value in CVD.

Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

CD93 and dystroglycan cooperation in human endothelial cell adhesion and migration adhesion and migration.

CD93 is a transmembrane glycoprotein predominantly expressed in endothelial cells. Although CD93 displays proangiogenic activity, its molecular function in angiogenesis still needs to be clarified. To get molecular insight into the biological role of CD93 in the endothelium, we performed proteomic analyses to examine changes in the protein profile of endothelial cells after CD93 silencing. Among differentially expressed proteins, we identified dystroglycan, a laminin-binding protein involved in angiogenesis, whose expression is increased in vascular endothelial cells within malignant tumors. Using immunofluorescence, FRET, and proximity ligation analyses, we observed a close interaction between CD93 and ß-dystroglycan. Moreover, silencing experiments showed that CD93 and dystroglycan promoted endothelial cell migration and organization into capillary-like structures. CD93 proved to be phosphorylated on tyrosine 628 and 644 following cell adhesion on laminin through dystroglycan. This phosphorylation was shown to be necessary for a proper endothelial migratory phenotype. Moreover, we showed that during cell spreading phosphorylated CD93 recruited the signaling protein Cbl, which in turn was phosphorylated on tyrosine 774. Altogether, our results identify a new signaling pathway which is activated by the cooperation between CD93 and dystroglycan and involved in the control of endothelial cell function.