Platelet HYAL2 regulates trans-endothelial PBMC migration. (A) Representative microvessels present in colon tissues isolated from non-IBD control subjects, patients with IBD, and WT or HYAL2 KO mice subjected to DSS-induced colitis were evaluated for HA-HC by immunostaining for HA (green) and the HCs of IαI (red) and counterstained with 4′,6-diamidino-2-phenylindole for nuclei. Arrows indicate HA glycocalyx structures extending from the vessel surface. (B) Quantitative real-time polymerase chain reaction analysis of HYAL2 expression of HIMECs isolated from non-IBD and IBD patient surgical specimens (n = 8 each; **P < .01). (C) Representative immunoblots analyzing HYAL2 levels in HIMECs isolated from non-IBD and IBD patient surgical specimens (n = 8 each). Lysates were normalized to total protein (25 µg), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (D) HIMECs were cultured with or without TNF‐α for 16 hours and evaluated for HYAL2 by using immunoblot over multiple passages. (E) HIMECs were seeded on permeable supports (3 μm pore size) placed into a 24-well plate, and grown to confluence. HIMECs were treated with or without TNF‐α for 16 hours at 37°C to promote leukocyte-adhesive HA-HC formation. Following activation, HIMECs were incubated in the presence or absence of nonactivated freshly isolated human platelets (100 × 10⁶ per well) for 1 hour at 37°C. In some experiments, HIMECs were treated with Streptomyces hyaluronidase or with platelets preincubated with an HYAL2-blocking antibody as indicated. CCL5 (100 ng/mL) was added to the bottom well, and Calcein AM–labeled PBMCs (1 × 10⁶ per well) were added to the upper chamber. (F) HIMECs were cultured with or without TNF-α to induce HA-HC formation on the cell surface. Cultures were then washed and incubated in the presence or absence of platelets as indicated. HA released into the media was measured by using an enzyme-linked immunosorbent assay–like method, and the data represent 3 independent experiments. (G) TNF-α–stimulated HIMECs were pretreated with platelets isolated from IBD platelets or non-IBD control subjects before PBMC trans-endothelial migration. Data are reported as mean ± SEM; n = 4 independent experiments of at least 6 patients each. Values with different alphabetical superscripts are significantly different from each other (P < .05); *P < .05. Image, detection, and software details: TCS SP5 II confocal/multi-photon high-speed upright microscope, HCX PL APO ×40/1.25NA oil immersion objective, HyD system detector, and LAS AF software (all Leica Biosystems). Scale bar: 25 µm. Data acquisition details: Calcein AM–labeled PBMCs were detected in the lower wells with an automated Leica DM inverted microscope at 20× magnification set to well-scan mode. PBMCs were enumerated on the basis of fluorescence and size by using Image-Pro Plus acquisition software.