Irisin effects on angiogenesis in human endothelial cells

PRIMARY OUTCOME

angiogenic capacity and gene expression

EXPECTED OUTCOMES

## Expected Outcomes ### Primary Outcomes 1. **Proliferation:** Dose-dependent increase with EC50 ~30-50 ng/mL irisin 2. **Migration:** 30-50% increase in transwell migration at 50 ng/mL 3. **Tube formation:** 40-80% increase in total tube length at optimal irisin concentration 4. **AMPK activation:** Rapid phosphorylation within 15-30 min, sustained up to 2 hours ### Secondary Outcomes - Upregulation of VEGFA, ANGPT1, ANGPT2 gene expression - Enhanced VE-cadherin organization at cell junctions - Cytoskeletal reorganization (F-actin stress fiber formation) ### Null Result Interpretation - If no effect, verify irisin purity and bioactivity (positive control VEGF should work) - Check FNDC5 receptor expression on HUVECs (integrin αVβ5) - Consider higher concentrations (up to 500 ng/mL)

SUCCESS CRITERIA

## Success Criteria ### Primary - [ ] HUVEC identity confirmed: CD31+, VE-cadherin+ by flow cytometry - [ ] Cell viability > 90% at all concentrations (no cytotoxicity) - [ ] Tube formation: ≥40% increase at 50 ng/mL vs control - [ ] Migration: ≥30% increase vs control - [ ] AMPK phosphorylation: significant increase (p < 0.05) at 30 min ### Secondary - [ ] Dose-response curve: EC50 calculated for each assay - [ ] Inhibitor reverses irisin effects (AMPK or PI3K inhibitor) - [ ] VEGF receptor expression confirmed (KDR/Flk-1) ### Technical Quality Gates - [ ] Matrigel polymerization verified (control tubes form within 4 hours) - [ ] Serum-free conditions confirmed for migration assay - [ ] Positive control (VEGF) shows expected response - [ ] ≥3 independent biological replicates (different HUVEC lots)

PROTOCOL

## Protocol: Irisin Effects on Angiogenesis in Human Endothelial Cells ### Study Design In vitro study examining the effects of irisin (FNDC5/myokine) on angiogenesis using human umbilical vein endothelial cells (HUVECs). Assess proliferation, migration, tube formation, and signaling pathway activation. ### Cell Culture 1. HUVECs (Lonza, pooled donor, Passage 2-5) 2. Culture in EBM-2 basal medium + EGM-2 MV SingleQuots supplements 3. Maintain at 37°C, 5% CO2, 95% humidity 4. Verify endothelial phenotype: VE-cadherin+, CD31+ by flow cytometry ### Experimental Groups 1. **Control:** EBM-2 + 0.1% FBS 2. **Irisin treatment:** EBM-2 + 0.1% FBS + recombinant human irisin (10-100 ng/mL) 3. **VEGF control:** EBM-2 + 0.1% FBS + VEGF-A (50 ng/mL) as positive control 4. **Irisin + inhibitor:** Pre-treat with PI3K inhibitor (LY294002) or AMPK inhibitor (compound C) ### Angiogenesis Assays **Proliferation Assay:** 1. Seed 5,000 cells/well in 96-well plates 2. Starve in 0.1% FBS EBM-2 for 6 hours 3. Treat with irisin (10, 50, 100 ng/mL) for 24, 48, 72 hours 4. Measure proliferation: CellTiter-Glo luminescence or BrdU incorporation **Migration Assay (Transwell):** 1. Seed 10,000 cells in upper chamber (serum-free) 2. Add irisin (50 ng/mL) to lower chamber with 5% FBS as chemoattractant 3. Incubate 6 hours, fix with 4% PFA, stain with crystal violet 4. Count migrated cells (5 fields per filter) **Tube Formation Assay:** 1. Coat 96-well plates with 50 µL Matrigel (10 mg/mL) per well 2. Seed 15,000 HUVECs/well 3. Treat with irisin (10, 50, 100 ng/mL) 4. Image at 4 hours (10x): quantify total tube length, junctions, branches (ImageJ Angiogenesis Analyzer) ### Signaling Pathway Analysis 1. **Western blot:** Phospho-AMPK, phospho-AKT, phospho-ERK1/2, phospho-FAK 2. **Immunofluorescence:** VE-cadherin organization, F-actin stress fibers 3. **AMPK activity assay:** Commercial kit or phospho-AMPK/total AMPK ratio 4. **Gene expression (qPCR):** VEGFA, ANGPT1, ANGPT2, KDR, PECAM1 ### Controls - **Negative control:** No irisin, 0.1% FBS only - **Positive control:** VEGF-A (50 ng/mL) for tube formation - **Vehicle control:** PBS for irisin dilution - **Inhibitor controls:** LY294002 and compound C alone (no toxicity confirmation) ### Expected Outcomes 1. **Increased proliferation:** 20-40% increase at 50-100 ng/mL irisin 2. **Enhanced migration:** 30-50% increase in transwell migration 3. **Enhanced tube formation:** 40-80% increase in total tube length 4. **AMPK/AKT activation:** Phosphorylation increased within 15-30 minutes 5. **Synergy with VEGF:** Irisin may potentiate VEGF-mediated angiogenesis ### Success Criteria - [ ] HUVEC identity confirmed (CD31+, VE-cadherin+) - [ ] Cell viability > 90% at all irisin concentrations (LDH release assay) - [ ] Dose-response curve generated (EC50 for proliferation/migration) - [ ] Tube formation: ≥40% increase at 50 ng/mL irisin vs control - [ ] AMPK phosphorylation significantly increased (p < 0.05) - [ ] PI3K/AMPK inhibitor reverses irisin effects (confirms pathway specificity) - [ ] Minimum 3 biological replicates per condition

LINKED HYPOTHESES