Heparan sulfate-dependent transport of CCL2 across an in vitro model of the human blood-brain barrier

Abstract Background: Transport of immune-active substances across the blood-brain barrier (BBB) is an important mechanism of neuroimmune regulation. CCL2 is among the chemokines known to cross the intact BBB in the blood-to-brain direction and is supported to do so in mice through interactions with heparan sulfate (HS)-containing components of the endothelial glycocalyx. The goal of this study was to characterize blood-to-brain transport mechanisms of human CCL2 in a human induced pluripotent stem-cell (iPSC)- derived in vitro model of the BBB. Methods: Human brain endothelial-like cells (iBECs) were differentiated using established methods and then changed to heparin-free medium. All experiments were conducted 9 days after seeding differentiated iBECs on permeable culture inserts or tissue culture plates. Human recombinant CCL2 and bovine serum albumin (Alb) as a leakage tracer was labeled with 125 I and 131 I, respectively, and their flux across the monolayer was quantified by calculating the permeability-surface area coefficient. Transport of 125 I-CCL2 and 131 I-Alb was evaluated at baseline, in the presence of a CCR2 inhibitor and heparin, following treatment with heparinases, and following treatment with the heparan sulfate synthesis inhibitor GalNaz to evaluate HS-dependent mechanisms of transport. We further determined the mechanism of 125 I-CCL2 transcytosis using inhibitors of clathrin, caveolae, and dynamin. Results: We found that iBECs have a functional blood-to-brain transport system for CCL2. Similar to our previous findings in mice, heparin inhibited CCL2 transport whereas the CCR2 inhibitor did not. We further showed that both heparinase treatment and treatment with GalNaz inhibited CCL2 transport across the BBB, supporting the requirement for HS in CCL2 transport. CCL2 transcytosis was clathrin-independent and caveolae and dynamin-dependent. Conclusions: Our findings support that human CCL2 is transported across the human BBB in vitro by a mechanism that was HS-dependent, caveolae and dynamin-dependent, and clathrin-independent. Our findings underscore the utility of iBECs for the study of mechanisms of heparan sulfate/glycocalyx interactions in the transport of substances across the BBB.