🧫
VE-cadherin control of reelin secretion in vitro and in vivo
active
experiment
Created: 2026-04-06T12:34:46
By: etl-v1-backfill
Quality:
50%
✓ SciDEX
ID: exp-cdf8b84b-bc12-4a1f-8aa9-957d3d54eee3
🧫 Experiment Protocol
ExploratoryCDH5lymphatic endothelial cells and animal modelsproposed
This experiment investigated the novel finding that VE-cadherin plays a requisite role in controlling the secretion of reelin, a lymphangiocrine glycoprotein. The researchers conducted both in vitro cell culture experiments and in vivo studies to demonstrate this regulatory relationship. Reelin is a recently appreciated factor with important roles in governing heart development and injury repair. The experiments likely involved measuring reelin secretion levels under different conditions of VE-cadherin expression or function, potentially using techniques such as ELISA, Western blotting, or immunofluorescence. The in vivo component may have involved animal models to validate the physiological relevance of this regulatory mechanism.
PRIMARY OUTCOME
demonstration of VE-cadherin requirement for reelin secretion
EXPECTED OUTCOMES
## Primary Outcomes
**In Vitro VE-cadherin Requirement**: VE-cadherin knockdown reduces Reelin secretion by ≥65% vs. control siRNA (p < 0.001, n≥4 biological replicates). Intracellular domain deletion (ΔICD) abolishes rescue capability (<20% restoration), confirming catenin-dependent signaling requirement.
**In Vivo Phenotype**: Endothelial-specific Cdh5-CreER;Reln-flox mice show: (a) ≥40% reduction in lymphatic vessel Reelin content (immunofluorescence, p < 0.01), (b) ≈50% reduction in serum soluble Reelin (ELISA, p < 0.01), and (c) reduced lymphatic pumping frequency (-30% vs. controls, p < 0.05).
## Secondary Outcomes
**Mechanistic Insight**: VE-cadherin deletion disrupts Reelin secretion by mislocalizing Reelin-containing vesicles from the cell periphery to a perinuclear accumulation pattern, suggesting impaired vesicular trafficking rather than Reelin protein stability.
SUCCESS CRITERIA
## Primary Success Criteria
**Secretion Dependency**: siRNA-mediated VE-cadherin knockdown must reduce Reelin secretion by ≥50% in at least 3 independent HMVEC-LLy donor preparations. Rescue with WT VE-cadherin but not ΔICD mutant must restore secretion to ≥70% of control levels.
**In Vivo Confirmation**: VE-cadherin deletion in adult mice must produce ≥30% reduction in lymphatic Reelin content and altered lymphatic function (either reduced contractile frequency or impaired response to shear stress) within 4 weeks of induction.
## Secondary Success Criteria
**Specificity**: Non-endothelial cells (e.g., HeLa, HEK293T) must not show Reelin secretion changes upon VE-cadherin manipulation, confirming endothelial-specific mechanism.
**Temporal Requirement**: Acute VE-cadherin knockdown (72h) is sufficient to impair Reelin secretion, indicating rapid signaling dynamics rather than developmental adaptation.
PROTOCOL
# VE-cadherin Control of Reelin Secretion In Vitro and In Vivo Protocol
## Phase 1: In Vitro siRNA Knockdown in Lymphatic Endothelial Cells (Days 1-14)
**Cell Culture**: Culture human lymphatic microvascular endothelial cells (HMVEC-LLy, Lonza #CC-2810) in EGM-2MV medium on gelatin-coated plates (0.1% gelatin, 1 hour, RT). Maintain at 37°C, 5% CO₂. Use cells between passages 4-7.
**VE-cadherin Knockdown**: Transfect subconfluent HMVEC-LLy with VE-cadherin (CDH5) siRNA (Dharmacon #J-011459-09-0005, 50 nM) or non-targeting control siRNA (D-001810-10-05) using RNAiMAX (Life Technologies). Knockdown efficiency assessed at 48h and 72h via qRT-PCR (target: ≥75% mRNA reduction) and western blot (target: ≥60% protein reduction).
**Reelin Secretion Assay**: At 72h post-transfection, wash cells and replace with serum-free EGM-2MV for 24 hours. Collect conditioned medium (CM), concentrate via Amicon Ultra-15 (10 kDa cutoff). Lyse cells for total protein normalization. Measure Reelin in CM via ELISA (ALCOS #27711, 1:100 dilution).
## Phase 2: VE-cadherin Overexpression Rescue (Days 15-21)
**Rescue Construct**: Clone human CDH5 (NM_001795) into pCMV-3xFLAG vector. Generate truncation constructs: ΔICD (lacks intracellular domain), ΔECD (lacks extracellular domain), and ΔCatenin-binding (mutates β-catenin binding motif Y781A). Sequence-verify all constructs.
**Rescue Experiment**: Co-transfect HMVEC-LLy with VE-cadherin siRNA + FLAG-VE-cadherin-WT or mutant constructs. At 72 hours, assess Reelin secretion. Rescue defined as ≥70% restoration of Reelin secretion vs. non-targeting siRNA control levels.
## Phase 3: In Vivo Validation in Conditional KO Mice (Days 22-42)
**Mouse Model**: Cross Cdh5-CreER (Cdh5<tm1(cre/ERT2)Rgm>) with Reln-flox mice (B6;129-Reeln<tm1.1Gsq>/J). Administer tamoxifen (75 mg/kg, i.p., 5 consecutive days) at 8 weeks of age to induce endothelial-specific VE-cadherin activation + Reelin deletion.
**Tissue Collection**: At 4 weeks post-tamoxifen, collect: (a) mesenteric lymphatic vessels for Reelin immunohistochemistry (anti-Reelin, 1:200, Millipore #MAB5364), (b) serum for soluble Reelin ELISA, (c) popliteal lymph nodes for lymphatic pumping assessment (intravital microscopy).
**Functional Assessment**: Measure lymphatic contractility via ex vivo tissue bath: record spontaneous rhythmic contractions (frequency, amplitude) of isolated mesenteric lymphatic vessels in response to shear stress and noradrenaline (1 μM).
Source: PMID 39232006 ↗
🧫 Experiment Extras
PATHWAY
reelin secretion pathway
MARKET PRICE
$0.50
STATUS
proposed
▸Metadataorigin_type: v1_polymorphic_backfill
| origin_type | v1_polymorphic_backfill |
| source_table | experiments |
| _schema_version | 1 |
📊 Evidence Profile
Evidence Balance
+0%
Certainty
0%
Debates
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Incoming
0
Outgoing
0
0 supporting
0 contradicting
0 neutral
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