Blockade of Jam-B and Jam-C using neutralizing antibodies or soluble Jam-Fc proteins markedly inhibit EC lumen and tube formation from either single or preaggregated ECs suspended in 3D collagen matrices. (A) ECs were seeded within collagen matrices in the presence or absence of blocking antibodies to Jam-B and Jam-C each added at 50 μg/mL, and cultures were fixed, stained, and photographed after 24 hours. Bar equals 50 μm. Arrows indicate EC lumenal structures; arrowheads, individual ECs that do not have lumens. (B) Cultures were established as in panel A under the indicated conditions by adding the antibodies to the collagen matrices. EC lumenal areas were quantitated from the cultures using Metamorph software. Data shown are normalized to the control sample and presents values of lumenal area per HPF ± SD. n = 10, P < .01. (C) Cultures were established as in panel A except that recombinant Jam-Fc proteins were added at the indicated concentrations and conditions for 24 hours. Data shown are normalized to the control sample and presents values of lumenal area per HPF ± SD. n = 10, P < .01. (D) EC-EC aggregates were suspended within 3D collagen matrices in the presence of the indicated neutralizing antibodies (50 μg/mL), the MMP inhibitor GM6001 at 5μM, or PBS control. ECs were preaggregated in a 35-mm polystyrene culture dish for 3 hours before their suspension in the collagen matrix. Arrows indicate lumen structures; arrowheads, EC aggregates with no apparent lumens. Bar equals 100 μm. (E) EC lumens were quantitated by tracing lumen areas using Metamorph software. Data shown are normalized to the control sample and presents values of lumenal area per HPF ± SD. n = 10, P < .01. * indicates a significant decrease.