Loss of endothelial mir-126 drives age-related reduction of hematopoietic activity Free

Aging hematopoietic stem cells (HSCs) expand but their functional capacity declines. To prioritize the relative contributions of intrinsic versus (vs) extrinsic factors to HSC aging, we transplanted BM HSCs (Fit3⁻CD150⁺CD48⁻ LSK) from young (2-3 months old) and aged (18-24 months old) CD45.2 B6 mice into age-matched young and aged CD45.1 B6 recipients (200 cells/mouse). Transplanted HSCs from aged mice had reduced self-renewal and reconstitution capacities compared with transplanted HSCs from young mice, regardless of the recipient age (aged vs young HSC engraftment at 24 weeks: 5.5% vs 41.3%, P<0.0001 in young recipients and 1.167% vs 25.93%, P=0.001 in aged recipients). We also noticed that long-term engraftment of donor HSCs, regardless of age, were consistently lower in the aged vs young recipients (24 weeks: 25.9% vs 45.2% for young donor HSCs, P=0.0034; 1.17% vs 5.53% for aged donor HSCs, P=0.01). Thus, we concluded that hematopoiesis and regenerative capacity depend on the age of both HSCs and BM niche.

The vascular compartment of the BM niche reportedly affects HSC functionality, leading us to postulate a role of vascular remodeling in BM aging. Thus, we compared vessels and endothelial cells (ECs) in tibias from aged vs young mice. Notably, we noticed a significant decrease in CD31⁺Sca-1^high EC-lined arterioles by confocal microscopy and in CD31⁺Sca-1^high ECs by flow cytometry and increase in vessel permeability by intravital confocal microscopy in the aged vs young mice. Similar results were obtained in genetically modified mice carrying tamoxifen-induced double-fluorescent reporter [Tie2-CreER/TdTomato/Tg(Ly6a-GFP)] that allowed to track ECs. Thus, we concluded that aging BM niche loses arterioles. Of note, inflammaging (aging-associated inflammation) reportedly impacts HSC function. We previously reported that in pathologic conditions, leukemic blast-derived cytokines (i.e., TNFα) suppresses EC miR-126, leading to arteriolar loss. MiR-126 is a microRNA that is highly expressed in ECs, regulates angiogenesis and in turn HSC quiescence and stemness. Herein, we observed higher levels of pro-inflammatory cytokines, BM expansion of myeloid cells expressing high TNFα levels, and decrease in BM EC miR-126 and arteriolar density in the aged vs young mice. Treatment with TNFα recapitulated these findings in young mice; co-treatment with synthetic miR-126 mimic rescued these changes in aged mice. These results suggest that inflammaging leads to downregulation of EC miR-126 and loss of arterioles. ECs express a log-fold higher level of miR-126 than HSCs and supply miR-126 to HSCs. Thus, TNFα-induced decline in miR-126^higharterioles in the aged BM niche ultimately reduces EC-derived miR-126 supply to HSCs and impact their self-renewal activity. Accordingly, while levels of pri- and pre-miR-126 were similar in young and aged BM HSCs, mature miR-126 levels were significantly reduced in the aged HSCs, which despite being expanded, had lower self-renewal capacity. These features were remarkably similar to those we observed in young EC-miR-126 KO mice (Mir126^f/fTie2-cre+) that, like aged wt mice, had low miR-126 levels by virtue of their genotype, reduction of arterioles and expansion of BM HSCs, and yet, despite their young age, showed a significantly impaired long-term regenerating capacity compared with young wt mice (engraftment at 20 weeks: 41% vs 62%, P=0.038). Conversely, aged EC-miR-126 overexpressing mice (Spred1^f/fTie2-cre+) had higher density of arterioles and lower frequency of HSCs that exhibited enhanced self-renewal capacity compared with aged wt controls (engraftment at 20 weeks: 42% vs 9%, P=0.0016). Finally, aged HSCs co-cultured with aged ECs had lower levels of miR-126 and reduced long-term engraftment in congenic recipient mice compared with aged HSCs co-cultured with young ECs (engraftment at 16 weeks: 46.2% vs 65.7%, P=0.0003); this reduction could be rescued by co-treatment with miR-126 mimic (16 weeks: 46.2% vs 57.2%, P=0.03).In summary, we uncover a crucial role of inflammaging-mediated loss of EC miR-126 that lead to aging BM vascular remodeling with decreased density of arterioles and EC-miR-126 supply to HSCs, that expand, but lose regenerative capacity. Targeting miR-126 loss with miR-126 mimic restores miR-126 levels and reconstitution capacities in aged HSCs, therefore representing a potential therapeutic approach to mitigate aging-related HSC dysfunction and rejuvenate hematopoiesis.