Figure Legends
Fig 1. Comparable frequencies but lower ROS levels in BM ECs
from young donors than from old donors. Representative gating strategy
for BM ECs
(CD34+CD309+CD133+)
from healthy donors (A) . Frequencies of BM ECs in
BMMNCs(B) and intercellular ROS levels in BM ECs (C) among
young, middle-aged and old donors. The data are showed as the mean ±
standard error of the mean (SEM).
Fig 2. Better migration and tube formation functions of BM ECs
from young donors than from old donors. Representative tube formation
images and tube length of 7-day primary cultured BM ECs (magnification
×4) in the young and old donor groups (A) . Typical figures of
the
transwell
migration assay and
counts
of the migrated BM ECs (magnification ×10) from young and old donors(B) . Representative images of double-positive-stained ECs
(yellow) with DiI–Ac-LDL uptake (red) and FITC–UEA-I binding (green)
and the double-positive-stained cell numbers
(magnification
×10) in the young and old donor groups (C) .
Fig 3. ECs from young donors regulated T cells to produce fewer
proinflammatory cytokines. Schematic diagram of the study design of the
coculture of ECs and CD3+ T cells and the measurement
of effector T subsets after 3 days of coculture (A) . The
frequencies of BM Th1
(CD3+CD8−IFN-γ+)(B) , Th2
(CD3+CD8−IL-4+)(C) , Tc1
(CD3+CD8+IFN-γ+)(E) , Tc2
(CD3+CD8+IL-4+)(F) , Th17
(CD3+CD8−IL-17+)(I) and Tregs
(CD3+CD8−CD25+Foxp3+)(J) in young and old donors. The ratios of BM Th1/Th2(D) , Tc1/Tc2 (G) ,
CD4+/CD8+ T cells (H) in
young and old donors.
Fig 4. Upregulated genes related to the cytokine-mediated
signalling pathway and the regulation of T cell activation in BM ECs
from old donors. Schematic diagram of the study design for RNA-seq
analysis of ECs from donors (A) . Volcano plots of the
downregulated genes (red) and upregulated genes (green) in the old group
and comparable genes (blue) between the two groups (B) . GO
enrichment analysis shows the top 10 biological processes enriched by
upregulated genes in ECs from young and old donors (C) .
Heatmaps show genes expression involved in the regulation of T cell
activation and cytokine−mediated signalling pathways, including TLR1,
TLR2, TLR4, CD80, CD86, CXCL10, CCR2, CCR5, PRF1, and NLRP3, in young
and old ECs (scaled by row) (D) . The relative mRNA expression
levels of
aforementioned
genes in ECs from young and old donors tested by qRT-PCR (E) . *P <0.05, ** P <0.005.
Fig
5. Lower expression of TLR4, CCR5 and CD80 in ECs from young donors than
from old donors. The representative gating strategy and frequencies of
TLR4+ ECs (A) , CCR5+ ECs(B) , CD80+ ECs (C) , and
CD86+ ECs (D) from the young and old donors
with LPS stimulation.
Fig 6. Graphical abstract of the current study. BM ECs
from young donors had lower ROS levels, better functions, and more
balanced T cell modulating effects than ECs from old donors. Lower TLR
signaling and costimulatory molecules, and less inflammatory and
chemotaxis cytokine secretion in ECs from young donors might cause young
ECs to induce more balanced T cell subset differentiation than old ECs.