Researchers discovered that an antibody called LOB7, which binds to the protein vimentin, can boost the formation of capillary-like tubes by human endothelial cells — a key process in angiogenesis (the creation of new blood vessels).
They used phage display antibody technology to screen for antibodies binding proteins on ageing endothelial cells. 🧫
One antibody, LOB7, stood out for enhancing tube formation — the way endothelial cells mimic tiny blood vessels in lab assays!
| Finding | Meaning |
|---|---|
| LOB7 binds extracellular vimentin | Vimentin has roles outside cells |
| Tube formation ↑ 21% | Vimentin interaction promotes angiogenesis |
| No change in migration/proliferation | Effect likely structural |
| Binds coil 2 domain | New functional region identified |
| First such result under normoxia | Expands understanding of extracellular vimentin |
Vimentin is linked to:
Understanding how antibodies like LOB7 modulate vimentin could reveal new therapeutic targets — but also warns that blocking or activating vimentin might have unintended angiogenic effects. ⚠️
Mathias Lindh Jørgensen et al., Aarhus University, Denmark 🇩🇰 Published in Scientific Reports (2017).
Meaning: A technique that uses an antibody to “pull down” its binding protein from a complex mixture (like a cell lysate). Why used: The authors used the LOB7 antibody to “fish out” its target protein from HUVEC lysates. Purpose in paper: To confirm what protein LOB7 binds to — mass spectrometry showed the pulled-down protein was vimentin.
Meaning: An antibody that recognizes and binds specifically to the protein vimentin. Why used:
Yes. Previous studies showed anti-vimentin peptides only enhanced tube formation under hypoxia (low oxygen). This study found that LOB7 promotes angiogenesis even under normoxic (normal oxygen) conditions. → This shows that vimentin’s angiogenic role doesn’t depend on oxygen stress, broadening its physiological relevance.
Meaning: The ability of cells to move — critical in tissue repair, immune responses, and metastasis. Why mentioned: The authors checked if LOB7 affected cell migration (motility) using a scratch assay. It didn’t. → So the increased tube formation isn’t from faster movement but likely from improved cell–matrix adhesion.
Vimentin is part of the cytoskeleton — it gives cells shape and helps them reorganize.
Meaning: One of the three main cytoskeletal systems in cells (along with actin filaments and microtubules). Function: Provides mechanical support and resilience. Vimentin’s role: It’s the major intermediate filament in mesenchymal cells, linking organelles and maintaining cell integrity.
Meaning: A method to discover new antibodies by displaying them on bacteriophage (virus that infects bacteria) surfaces. How it works:
Meaning: Standard oxygen levels (around 21% O₂), like normal body tissues. Why mentioned: The authors highlight that LOB7 promotes angiogenesis even without hypoxia, unlike prior peptides. → Indicates that extracellular vimentin can drive angiogenesis under physiological oxygen.
Meaning: A post-translational modification where the amino acid arginine → citrulline by the enzyme PAD (peptidylarginine deiminase). Effect: Changes protein charge and folding, often affecting immune recognition. Why mentioned: Vimentin can be citrullinated — these modified forms are implicated in rheumatoid arthritis autoantigens.
Meaning: Enzymes like caspases (active during apoptosis) and calpain (calcium-dependent protease) cut vimentin into fragments. Why important:
Meaning: Tagging cell-surface proteins with biotin while the cells are still intact (“in vivo” here means within living cells in culture). Why: Biotin allows easy purification later using streptavidin beads. Purpose: To selectively label membrane proteins for antibody screening — ensuring LOB7 targets something on the cell surface.
Meaning: Proteins that are pulled out of a solution (e.g., by antibody-bead binding) and separated by centrifugation or magnet. Why mentioned: These were the proteins bound by LOB7 in immunoprecipitation; they were analyzed by SDS-PAGE + silver staining + MS.
Meaning: Caspases are enzymes that execute apoptosis by cutting specific proteins. Connection: Caspases cleave vimentin during apoptosis, changing its size and weakening the cytoskeleton — seen as smaller fragments on Western blot.
Meaning: A human endothelial cell line used in the lab (like HUVEC). Why used: The authors observed that LOB7 binds more strongly to old ASF-2 cells than young ones → suggesting vimentin expression increases with cell age.
Meaning: A lysate (cell extract) containing biotin-tagged surface proteins. Purpose: These biotin-labeled proteins were used to capture antibody-binding targets during the phage selection process.
Meaning: “Fixing” locks proteins into the gel (using ethanol and acid) before staining. Why:
Meaning: A key extracellular matrix protein forming the basement membrane. Why included: Used as a negative control in ELISA — to ensure LOB7 binds specifically to vimentin, not to other structural proteins like laminin.
Find and characterize an antibody that affects tube formation in endothelial cells and identify its target.
| Step | Method | What They Did | What It Showed |
|---|---|---|---|
| 1️⃣ Screening for antibodies | Phage display antibody technology | Created a large library of phages displaying single-chain antibody fragments (scFv). These were exposed to biotin-labeled membrane proteins from endothelial cells (HUVEC, ASF-2). Non-binders washed away. | Found one interesting antibody, LOB7, that bound strongly to aged endothelial cells. |
| 2️⃣ Identify the target of LOB7 | Immunoprecipitation + Silver stain + Mass spectrometry | Used LOB7 attached to beads to “pull down” its binding partner from cell lysate → separated proteins by SDS-PAGE → silver-stained → sent bands for MS. | Identified the pulled-down protein as vimentin. |
| 3️⃣ Confirm the target | Western blot (with V9 control) | Compared bands detected by LOB7 vs known anti-vimentin antibody (V9) in HUVEC lysate and Matrigel. | Both detected the same-sized protein → confirmed LOB7 binds vimentin. |
| 4️⃣ Test binding specificity | Phage-ELISA | Coated plates with recombinant vimentin, laminin, and milk (negative controls). Added LOB7 phage. | Strong signal only with vimentin → specific binding. |
| 5️⃣ Map where LOB7 binds on vimentin | Fragment mapping using recombinant vimentin pieces | Produced seven vimentin fragments (F1–F7). Tested binding with LOB7 and V9. | LOB7 bound coil 2 region (aa 268–396); V9 bound tail region (aa 405–466). |
| 6️⃣ Test biological effect | 2D Matrigel tube-formation assay | Grew HUVEC on Matrigel with or without 0.5 mg/mL LOB7 or control antibody. | After 5 h: +21 % tube length with LOB7; effect lasted ≈10 h, gone by 20 h → pro-angiogenic activity. |
| 7️⃣ Rule out indirect effects | Scratch (migration) assay + BrdU (proliferation) assay | Tested if LOB7 made cells move or divide faster. | No difference → LOB7 enhances tube organization, not migration or growth. |
Picture a flow diagram with arrows:
Phage library ↓ (biotin-labeled endothelial proteins) Selection → LOB7 antibody ↓ Immunoprecipitation + Silver stain + MS → Target = Vimentin ↓ ELISA + Western blot → Confirms specificity to vimentin ↓ Fragment mapping → Binding site = Coil 2 domain ↓ Matrigel tube-formation assay → 21 % ↑ tube length (angiogenesis) ↓ Migration & proliferation tests → No effect → direct structural role
Color-code it if you draw it:
Together, they proved:
“Binding extracellular vimentin at its coil 2 region by LOB7 promotes endothelial tube formation under normal oxygen conditions.”