Fig. 5

HYAL1 enzymatic activities and perturbation of glycocalyx hyaluronan in PCV endothelial cells. a Western blot analyses of cell lysates from 4 PCV and 3 normal BOEC lines, subjected to 24 h of heterogeneous flow, showing HYAL1 detection at 51 kDa and Actin as the loading control at 42 kDa. Right panel shows densitomeric quantification of HYAL1 bands normalized against Actin. Box and whiskers plots showing median with minimum and maximum. *p < 0.05 (two-tailed t-test). b Enzymatic activity of HYAL1 was evaluated according to the workflow (upper panel). Conditioned media (S/N, supernatant) was obtained from BOEC cultures rotated for 24 h. Box and whiskers plots showing median with minimum and maximum. n indicates number of cell lines while p value is from two-tailed t-test, *p < 0.05. c ELISA was used to detect HYAL1 in BOEC conditioned media after 24 h heterogeneous flow. Box and whiskers plot shows median with minimum and maximum. n indicates number of cell lines while p value is from two-tailed Mann-Whitney test, **p < 0.01. d ELISA detection of HYAL1 in PCV patient plasma and vitreous humor. Box and whiskers plot shows median with minimum and maximum. Every point represents an average of duplicates or triplicates of an individual patient. n indicates number of patients while p value from two-tailed Mann-Whitney test, **** p < 0.0001. e HA in the glycocalyx was detected using biotinylated HABP before detection with FITC-Neutravidin (green). Nuclei were stained blue with DAPI. Left panel shows maximal intensity projections of representative frames from each cell line evaluated. Cells were subjected to heterogeneous flow for 24 h. Three image frames were taken for each well region. Right panel shows total HA-stained areas summed across z-stacks per image frame. Box and whiskers plot showing median with minimum and maximum. n indicates number of cell lines while p value is from two-tailed t-test, ** p < 0.01