Figure 2

In vitro transwell assay to measure endothelial permeability to adiponectin complexes. A) Murine endothelial cell lines MS1, bEnd.3 (Bend), and EOMA used in transwell assays to assess adiponectin flux exhibit different morphologies, but all demonstrate VE-Cadherin (green) positive junctions that inhibit paracellular large molecule transport (DAPI, blue; Bar?=?20 μm). B) 70 kDa and 3 kDa dextran flux across MS1-seeded transwells over time normalizes concentrations above and below the cells in approximately 8 hours. 6 hours was chosen for transport studies. C) Effective permeability (P_eff) of LMW was significantly greater than HMW adiponectin across MS1 cells; HMW transport was greater across EOMA as compared to MS1 at 37°C. D) Ratios of adiponectin permeability to that of 3 kDa dextran demonstrated increased LMW permeability as compared to HMW in MS1 and Bend cells at 37°C. E) The P_eff of globular adiponectin (gAPN) is significantly greater than that of full-length adiponectin oligomers. F) The permeability of Bend and EOMA cells to LMW adiponectin was increased at 4°C, suggesting a loss of active barrier function for LMW adiponectin; HMW transport was unchanged. G) As a ratio to dextran, LMW adiponectin transport was greatly enhanced at 4°C. H) Inhibition of dynamin by Dynasore greatly enhanced full-length adiponectin transport in MS1 and Bend cells. No response was measured in EOMA cells. All analyses were by two-way (cell, treatment) ANOVA with Bonferroni posthoc analysis. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to HMW. #p < 0.05, ##p < 0.01 and ###p < 0.001 compared to MS1 cells.