(WHS-P2.01) CHARACTERIZING VASCULAR LEAKAGE AFTER ANGIOPOIETIN-1 SIRNA KNOCKDOWN IN DERMAL MICROVASCULAR ENDOTHELIAL CELLS DERIVED FROM POST-BURN HYPERTROPHIC SCAR
Friday, May 17, 2024
10:30 AM – 11:30 AM East Coast USA Time
Recent data demonstrate that dermal microvascular endothelial cells (DMVECs) from hypertrophic scars (HTSs) have increased angiopoietin-1 (ANGPT-1) gene and protein expression compared to normal skin (NS) DMVECs and lower permeability of FIT-C dextran using a transwell assay. This study evaluated the effect of ANGPT-1 knockdown on endothelial cell permeability to characterize the role of endothelial dysfunction in HTS. Full-thickness burns which formed HTSs were created in Duroc pigs. Punch biopsies were taken at day 84 post-burn and stained with Verhoeff Van Geison stain (VVG). HTS and areas of NS were excised and digested in collagenase. Fibroblast-DMVEC co-cultures were saved in cryostorage. Ulex europaeus agglutinin 1 lectin was used to sort for DMVECs by magnetic-activated cell sorting. For the knockdown, DMVECs were grown in 6-well plates with n=3 wells per experimental (treatment, control, scramble) group. Treatment group was treated with siRNA for ANGPT-1. At 24 hours post-transfection, RNA was isolated. Gene expression of ANGPT-1 was quantified using qRT-PCR. In the permeability assay, DMVECs from HTS were seeded onto 12-well transwell plates. Trans-endothelial electrical resistance (TEER) was assessed on day 1 to assess the formation of a monolayer. On day 3 post-seeding, transwells were treated with either siRNA for ANGPT-1 (n=8), H2O as control (n=7), or scramble siRNA (n=7). On day 4, 24 hours post-transfection, FIT-C was added to the apical well and dwelled for 2 hours. FITC-dextran that diffused into the bottom well was measured by spectrophotometry. Student’s t-test was used to evaluate gene expression between knockdown vs control group with p<0.05 considered significant. Transwell concentrations between experimental groups were analyzed using one-way ANOVA with Tukey’s correction for multiple comparisons. Punch biopsies from scars showed significantly increased blood vessel density compared to normal skin on VVG stain (p < 0.01). DMVEC identity was confirmed by immunofluorescence staining for Von-Willebrand factor. ANGPT-1 gene expression showed downregulation >4-fold for siRNA knockdown group compared to control group (p=0.21) while scramble did not show ANGPT-1 knockdown. TEER on day 1 averaged 17.28±4.5 Ω/cm2 for n=12 wells and 28.97±5.3 Ω/cm2 for n=12 wells. siRNA knockdown group showed increased permeability to FIT-C dextran compared to scramble siRNA (-23.39±3.9 vs 4.24±1.6; p=0.0002). Knockdown using siRNA for ANGPT-1 in HTS DMVECs led to an increase in endothelial permeability compared to the scramble group. Additional siRNA targets outside of the angiopoietin group will be tested in future work to identify candidate drug targets for treating endothelial dysfunction in scars.