CD93 Human

What is the molecular structure of human CD93?

Human CD93 is a type I transmembrane glycoprotein with a canonical protein length of 652 amino acid residues and a molecular mass of approximately 68.6 kDa . It contains a C-type lectin-like domain (CTLD) in its N-terminal region, which is crucial for its binding capabilities . The protein undergoes post-translational modifications, particularly O-glycosylation, which significantly affects its stability and function on the cell surface . Studies have demonstrated that inhibiting glycosylation leads to decreased CD93 expression on the cell surface and increased detection in culture media, indicating that proper glycosylation is essential for maintaining stable cell surface expression .

What are the primary cellular locations of CD93 expression?

CD93 is predominantly expressed in the plasma membrane of various cell types, with highest expression observed in:

• Vascular endothelial cells (primary expression site)

• Cells of myeloid origin (monocytes and neutrophils)

• Platelets

• Also detected in neurons, cytotrophoblast cells, B cells, and natural killer cells

The protein can exist in both membrane-bound and soluble forms (sCD93), with the latter resulting from cleavage of the membrane-bound form . This soluble form has been investigated as a potential biomarker in various cardiovascular conditions and cancer .

How can I differentiate between membrane-bound CD93 and soluble CD93 in experimental settings?

To differentiate between membrane-bound and soluble CD93:

For membrane-bound CD93:

• Flow cytometry using specific anti-CD93 antibodies to detect surface expression

• Immunofluorescence microscopy of intact cells

• Cell surface biotinylation followed by precipitation and western blotting

For soluble CD93 (sCD93):

• ELISA of culture supernatants or biological fluids

• High-throughput proximity extension assays (as used in cardiovascular studies)

• Western blotting of concentrated cell culture supernatants

When designing experiments, consider that inhibition of O-glycosylation (using agents like benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside) can increase shedding, leading to decreased cell surface expression and increased soluble CD93 in experimental settings .

What are the established ligands and binding partners of CD93?

CD93 functions as a receptor or component of receptor complexes for multiple ligands:

Research has demonstrated that the CTLD domain of CD93 specifically binds to CpG oligonucleotides and bacterial DNA, suggesting that CD93 may function like DEC-205 in delivering bacterial DNA to endosomal TLR9 .

How does CD93 regulate endothelial barrier function?

CD93 plays a critical role in maintaining endothelial barrier integrity through:

• Interaction with VE-cadherin: CD93 directly interacts with VE-cadherin and suppresses its phosphorylation and internalization, preserving endothelial junctions .

• Indirect regulation of claudin-5: Despite no direct interaction with claudin-5, CD93 downregulation leads to significant reduction of claudin-5 at cell junctions .

• Prevention of intercellular gap formation: siRNA-mediated knockdown of CD93 in human dermal blood endothelial cells (HDBECs) results in disengagement of junctional proteins and formation of intercellular gaps, disrupting the endothelial barrier .

Methodologically, these functions can be assessed through:

• Co-immunoprecipitation to detect CD93-VE-cadherin interactions

• Immunofluorescence analysis of junctional proteins after CD93 silencing

• In vitro permeability assays using fluorescent dextrans

• Measurement of intercellular gap formation in confluent endothelial monolayers

What methodologies are most effective for studying CD93 role in angiogenesis?

To effectively study CD93's role in angiogenesis, researchers should consider these methodological approaches:

• In vitro angiogenesis assays:

  • Endothelial tube formation assays with CD93 knockdown/overexpression

  • Endothelial cell migration and proliferation assays

  • 3D sprouting assays using spheroids of CD93-manipulated endothelial cells

• Ex vivo approaches:

  • Aortic ring assays comparing wild-type and CD93-deficient tissues

  • Retina explant angiogenesis assays

• In vivo angiogenesis models:

  • CD93 knockout mouse models examining developmental and pathological angiogenesis

  • Zebrafish models with fluorescently tagged vessels for live imaging after CD93 manipulation

  • Matrigel plug assays with CD93-deficient cells or neutralizing antibodies

These methods should be accompanied by molecular analyses of angiogenic signaling pathways to determine the mechanisms by which CD93 promotes angiogenesis in both physiological and pathological conditions .

How does CD93 contribute to cancer progression and immune infiltration?

CD93 has emerged as a significant factor in cancer biology with multiple contributions to tumor progression:

• Promotion of angiogenesis: As one of the top 20 core genes for angiogenesis in human primary tumors, CD93 facilitates tumor vascularization .

• Immune microenvironment modulation: CD93 expression levels strongly correlate with immune infiltration in various cancer types. Specifically, CD93 shows:

  • Positive correlation with cancer-associated fibroblasts (CAFs), endothelial cells, myeloid dendritic cells, hematopoietic stem cells, macrophages, and neutrophils

  • Negative correlation with Th1 cells, myeloid-derived suppressor cells (MDSCs), natural killer cells, and T-cell follicular helpers

• Prognostic implications: Increased CD93 gene expression is associated with poor prognosis in most cancer types, correlating with markers such as mismatch repair (MMR), tumor mutational burden (TMB), microsatellite instability (MSI), and immune checkpoints .

Methodologically, CD93's role in cancer can be studied through:

• Multi-parameter flow cytometry of tumor tissues

• Single-cell RNA sequencing to map CD93-expressing cells in the tumor microenvironment

• Spatial transcriptomics to visualize CD93 expression relative to immune cell infiltration

• CD93 inhibition studies using knockdown approaches or neutralizing antibodies in tumor models

What is the significance of CD93 in hematological malignancies, particularly chronic myeloid leukemia (CML)?

CD93 plays a crucial role in regulating stemness and proliferation of leukemic stem cells (LSCs) in CML:

• Regulation of LSC self-renewal: CD93-signaling induces the expression of genes associated with stemness and proliferation in both human and murine CML LSCs .

• Effect on clonogenic potential: Colony formation and re-plating capacity of human CD34+CD38- LSCs is significantly impaired when CD93 signaling is inhibited by metoclopramide at pharmacological concentrations (0.1mM) .

• Targetable pathway: CD93 expression by LSCs represents a promising novel target for CML treatment, potentially offering therapeutic options beyond conventional tyrosine kinase inhibitors .

Experimental approaches to study CD93 in CML include:

• Colony formation assays of sorted CD34+CD38- LSCs with CD93 inhibition

• Serial transplantation assays in immunodeficient mice to assess LSC self-renewal capacity

• Gene expression profiling after CD93 inhibition to identify downstream signaling pathways

• Pharmacological screening for compounds that effectively target CD93-expressing LSCs

What is the current evidence for CD93 as a biomarker in cardiovascular diseases?

The role of CD93 (particularly soluble CD93) as a biomarker in cardiovascular diseases has been investigated with mixed results:

• Heart Failure (HF):

  • Patients who experienced cardiovascular mortality, HF-hospitalization, heart transplantation, or left ventricular assist device implantation showed higher sCD93 levels at both baseline and follow-up

  • The adjusted hazard ratio per 0.1 standard deviation of the annual slope of CD93 was 1.43 (CI 1.13–1.92, p = 0.002)

  • In heart failure with preserved ejection fraction (HFpEF), sCD93 was associated with coronary microvascular dysfunction in men but not in women

• Ischemic Stroke:

  • CD93 transcript levels were 2-times higher in patients with ischemic stroke compared to control subjects (p = 0.03)

  • sCD93 was significantly associated with increased mortality at 90 days after ischemic stroke (OR 4.30, CI 1.99–9.30; p = 0.0002)

• Genetic associations:

  • CD93 ranked among the top 10 key driver genes potentially affecting numerous genes involved in cardiovascular disease and type 2 diabetes pathways

What are the most reliable methods for detecting CD93 expression in human tissues and cells?

Several complementary techniques offer reliable detection of CD93 expression:

When selecting antibodies for CD93 detection, consider that CD93 has multiple synonyms (C1qR(P), C1qRP, CDw93, ECSM3, MXRA4, dJ737E23.1, C1QR1) . Validate specificity using appropriate positive controls (endothelial cells, monocytes) and negative controls.

How can I effectively manipulate CD93 expression in experimental models?

Several approaches can be used to manipulate CD93 expression:

• siRNA knockdown:

  • Multiple effective siRNAs targeting different regions of CD93 mRNA have been validated in endothelial cells

  • Transfection efficiency should be monitored using fluorescently labeled control siRNAs

  • Expression levels should be confirmed by qRT-PCR and western blotting

• shRNA for stable knockdown:

  • Lentiviral or retroviral delivery systems for long-term studies

  • Selection with appropriate antibiotics to generate stable cell lines

• CRISPR-Cas9 genome editing:

  • Complete knockout of CD93 gene

  • Generation of specific mutations in functional domains (e.g., CTLD)

  • Knock-in of reporter genes for live cell imaging

• Overexpression systems:

  • Transfection of CD93 expression vectors (consider using cell-type specific promoters)

  • Inducible expression systems for temporal control

  • Domain-specific constructs to study structure-function relationships

• Pharmacological approaches:

  • Metoclopramide (0.1mM) has been shown to inhibit CD93 signaling in leukemic stem cells

  • Glycosylation inhibitors like benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (BAG) affect CD93 stability and surface expression

What are the best experimental systems to study CD93 interactions with bacterial DNA and TLR9?

To study CD93's role in binding and delivering bacterial DNA to TLR9, consider these experimental approaches:

• Binding assays:

  • ELISA-based binding assays between purified CD93-CTLD and CpG oligonucleotides

  • Tryptophan fluorescence studies to measure direct binding of bacterial DNA to CD93

  • Surface plasmon resonance to determine binding kinetics and affinity

• Cellular models:

  • Cell lines expressing CD93 (e.g., IMR32 neuroblastoma cells with CD93 expression)

  • Primary cells naturally expressing CD93 (endothelial cells, monocytes)

  • Comparative studies between CD93-expressing and control cells

• Functional assays:

  • Measurement of cytokine responses (e.g., IL-6) after stimulation with CpG ODN in CD93-expressing versus control cells

  • Tracking of fluorescently labeled CpG ODN in CD93-expressing cells

  • Co-localization studies of CD93, CpG ODN, and TLR9 using confocal microscopy

• Signal transduction analysis:

  • Analysis of TLR9-dependent signaling pathways (NF-κB, IRF) in the presence or absence of CD93

  • Phosphorylation studies of signaling intermediates

These approaches should be complemented with appropriate controls, including:

• Use of non-CpG DNA as negative control

• Blocking antibodies against CD93 to confirm specificity

• Chloroquine treatment to inhibit endosomal acidification and TLR9 signaling

How do glycosylation patterns affect CD93 function in different pathological contexts?

CD93 O-glycosylation is crucial for its stability at the cell surface, with inhibition of glycosylation causing rapid release of CD93 into culture supernatants . Advanced research questions include:

• Glycosylation heterogeneity: Do different tissues or pathological states exhibit distinct CD93 glycosylation patterns? This can be investigated using:

  • Mass spectrometry-based glycoproteomic analysis

  • Lectin arrays to profile glycan structures

  • Site-directed mutagenesis of glycosylation sites

• Functional consequences: How do specific glycoforms affect:

  • Ligand binding properties (particularly bacterial DNA and complement components)

  • Protein-protein interactions with VE-cadherin and other partners

  • Susceptibility to proteolytic cleavage and generation of sCD93

• Therapeutic implications: Could targeting specific glycoforms provide selective approaches for:

  • Cancer therapy (targeting tumor-specific CD93 glycoforms)

  • Reducing pathological inflammation while preserving homeostatic functions

  • Modulating endothelial barrier function in vascular disorders

Research methodologies should include comparative glycomic profiling of CD93 across healthy tissues, inflammatory conditions, and malignancies, combined with functional studies using glycosylation inhibitors and glycoform-specific antibodies.

What is the molecular basis for CD93's dual role in endothelial function and immune regulation?

CD93's expression across both endothelial and immune cell populations suggests complex multifunctional roles that warrant further investigation:

• Structural determinants: Which domains of CD93 mediate endothelial versus immune functions?

  • Domain-specific deletion constructs to map functional regions

  • Cryo-EM or X-ray crystallography of CD93 complexes with various ligands

  • Identification of cell-type specific binding partners through proteomics

• Signaling integration: How does CD93 integrate signals from the endothelial barrier and immune activation?

  • Phosphoproteomic analysis after specific stimuli in different cell types

  • Temporal dynamics of CD93-dependent signaling using biosensors

  • Identification of adaptor proteins that may differ between cell types

• Soluble versus membrane-bound functions: How do these forms differentially affect:

  • Angiogenesis and vascular permeability

  • Immune cell recruitment and activation

  • Cancer progression and metastasis

This research direction requires sophisticated approaches combining single-cell analyses, spatially resolved proteomics, and advanced imaging techniques to understand the contextual regulation of CD93 function across different microenvironments.

How might CD93-targeted therapies be developed for cancer and inflammatory diseases?

Based on its roles in cancer progression, immune regulation, and endothelial function, CD93 represents a potential therapeutic target:

• Therapeutic approaches:

  • Neutralizing antibodies against specific CD93 domains

  • Small molecule inhibitors of CD93-ligand interactions

  • RNA therapeutics to modulate CD93 expression in specific cell types

  • CAR-T cells targeting CD93-overexpressing tumors

• Disease-specific considerations:

  • For cancer: targeting CD93's angiogenic function while preserving barrier integrity

  • For inflammatory conditions: modulating CD93's role in bacterial DNA sensing

  • For leukemia: specifically inhibiting CD93 signaling in leukemic stem cells

• Biomarker-guided therapy:

  • Using sCD93 levels to identify patients most likely to benefit from therapy

  • Monitoring treatment response through changes in sCD93 or tissue CD93 expression

  • Developing companion diagnostics for CD93-targeted therapeutics

Research should include high-throughput screening for CD93 modulators, development of humanized mouse models with CD93 variants, and detailed toxicology studies to understand potential side effects, particularly on normal vascular and immune function.