Lower expression of TLR4, CCR5 and CD80 in ECs from young donors
To confirm the RNA-seq and qRT-PCR results and to further explore probable immunoregulatory mechanisms of ECs, we determined the expression of TLR4, CCR5, and costimulatory molecules such as CD80 and CD86 in young and old BM ECs by flow cytometry after 4 hours of LPS stimulation. Fig. 5 shows the representative gating strategy for the expression of TLR4, CCR5, CD80 and CD86 (left panel) and their expressed frequencies (right panel) in ECs from the young and old donor groups. The frequencies of TLR4+ ECs (Fig. 5A, 4.16% ±0.82% vs. 10.39% ± 1.32%, P =0.0001), CCR5+ ECs (Fig. 5B, 4.8% ±0.89% vs. 10.05% ± 1.47%, P =0.001), and CD80+ ECs (Fig. 5C, 5.62% ±0.78% vs. 10.67% ± 1.25%, P =0.002) in young donors were markedly lower than those in old donors. The percentage of CD86+ ECs (Fig. 5D, 13.51% ±0.98% vs. 16.53% ± 1.22%, P =0.08) was lower in young donors than in old donors, although the result was not statistically significant.