Loss of Adrenergic Nerves in the Bone Marrow Microenvironment Drives an Aging HSC Niche Phenotype

Hematopoietic stem cell (HSC) aging is accompanied by an expansion of myeloid-biased HSCs with declined long-term self-renewal functions, contributing to the development of hematologic malignancies. Recent studies have suggested a role for the bone marrow microenvironment in HSC aging but the extrinsic molecular mechanisms driving their aging remain largely unknown. Mesenchymal stem cells (MSCs) form a critical component of the HSC niche, promoting HSC quiescence and balanced differentiation. Some MSC activity is found associated with arterioles, which are densely innervated by the sympathetic nervous system (SNS). We used tridimensional whole-mount bone marrow confocal immunofluorescence imaging of HSCs and vascular structures to investigate the effects of aging on the HSC microenvironment. Comparative bone marrow analyses of aged (20-24 months) C57BL/6 and Nestin-GFP transgenic mice (where GFP marks MSCs) with young (2 month-old) C57BL/6 mice, revealed significant reductions in sympathetic innervation (3-fold, p<0.01) as determined by staining for tyrosine hydroxylase expressing nerve fibers. The SNS neuropathy of aged mice was accompanied with vascular remodeling characterized by an increase in total vascular density marked by CD31⁺/CD144⁺ (2-fold, p<0.01) and shortening of arterioles marked by CD31⁺/CD144⁺ and Nestin-GFP⁺ (5-fold reduction, p<0.001), reduction in α-smooth muscle actin (αSMA) pericytes (12-fold, p<0.01) and expansion of Nestin⁺ MSCs (4 fold, p<0.05). Whereas a subset of HSCs (~35%) are associated with arterioles in young mice, analyses of HSC localization in aged bone marrow revealed that HSCs were redistributed and expanded away from arterioles (p=0.0047). Functional analysis revealed a diminished in vitro clonal capacity of aged MSCs (determined by CFU-F and mesensphere assays; 3-fold, p<0.0001), reduced HSC maintenance activity determined by the ability to produce HSC maintenance factors (Cxcl12, Scf and Angpt1, 2-fold, p<0.01), and increased proliferative state (2-fold, p<0.0001), all indicative of reduced activity. Since SNS nerves regulate circadian egress of HSCs through regulation of MSC activity, we evaluated the circadian oscillations of HSCs in blood and niche factors in Nestin-GFP⁺ stromal cells. Aged mice exhibited ablated oscillations of progenitor release determined by peripheral blood colony-forming units in culture (CFU-C/ml blood) and phenotypic progenitors (LSK/ml blood). Furthermore, unlike the robust circadian oscillations of Cxcl12 levels of young MSCs, no circadian expression pattern was detected in aged mice, suggesting that the SNS neuropathy associated with aging has functional consequences by leading to disrupted circadian rhythms and expanded MSC populations with reduced HSC maintenance activity. To determine whether the loss of SNS nerves drives bone marrow vascular remodeling and HSC aging, we surgically sympathectomized young Nestin-GFP mice by unilateral microsection of both the sciatic and femoral nerves, while the sham-operated contralateral side served as control. Strikingly, four months following surgery, phenotypic HSCs selectively expanded in sympathectomized femurs (2-fold, p<0.05) and exhibited myeloid-biased differentiation upon competitive bone marrow transplantation compared to control (3-fold, p<0.01). Furthermore, similar to our observations in aged mice, chronic surgical sympathectomy (16 weeks) induced dramatic "aging-like" alterations of bone marrow arteriolar structures characterized with arteriolar shortening and loss of αSMA⁺ and Nestin-GFP⁺ arterioles (4-fold, p<0.01; 3 fold, p<0.001). These results thus suggest that aging-associated sympathetic neuropathy and the loss of β-adrenergic signals in the bone marrow microenvironment drive aging of HSCs by remodeling vascular niches and thus represent a potent therapeutic target for stem cell rejuvenation.