Endothelial mTOR Preserves Hematopoietic Stem Cell Fitness By Suppressing Thrombospondin1

Abstract

Hematopoietic stem cells (HSC) reside in close proximity to blood vessels within the bone marrow (BM) termed 'perivascular niches' which provide instructive cues for maintaining HSC self-renewal potential. The perivascular niche is comprised of endothelial cells (ECs) lining the BM blood vessels (BMECs), and an array of perivascular stromal cells that collectively serve to regulate balanced HSC self-renewal and differentiation. While young HSCs are endowed with robust self-renewal potential, aging is associated with a significant decline in HSC fitness including a loss of their self-renewal ability and a biased differentiation towards the myeloid lineages at the expense of lymphopoiesis. These aging related HSC defects result in anemias, impaired response to immunizations, and an increased risk for developing clonal disorders including acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). However, mechanisms that drive HSC defects during aging remain poorly understood, and represents a critical barrier towards developing strategies for preservation of hematopoietic fitness during aging. While HSC aging was generally assumed as a predominantly cell-intrinsic process, recent studies have identified that aging of the BM niche plays an instructive role in promoting age-related HSC defects. Supporting this, we recently discovered that aging is associated with a significant decline in mTOR activity within BMECs that impairs their capacity to serve as a HSC-supportive niche. Our findings showed that deletion of mTOR specifically within ECs of young (4 month old) mice (Endothelial Cell specific mTOR Knock Out; mTORECKO) is sufficient to recapitulate HSC aging phenotypes including loss of HSC self-renewal, and a myeloid-biased differentiation. Here, we sought to identify the mechanisms by which endothelial mTOR preserves HSC fitness. To identify candidate factors that promote HSC aging in mTORECKO mice, we performed a plasma proteomic analysis that revealed a putative role for Thrombospondin1 (Thbs1) wherein mTORECKO mice displayed elevated levels of Thbs1. Analysis of cultured BMECs revealed that inhibition of mTOR signaling within BMECs was sufficient to increase Thbs1 expression. Thbs1 is a well-defined antiangiogenic factor that has recently been identified to play a critical role in promoting a multitude of age-related cardiovascular and inflammatory disorders. However, whether Thbs1 regulates HSC activity is not known. To this end, we performed ex vivo HSC expansion transplantation assays which revealed that Thbs1 causes a significant decline in long term HSC engraftment. RNA Seq analysis of HSC expansions revealed that Thbs1 activates chronic inflammatory responses within HSCs that have been suggested to promote aging related HSC defects. To determine whether Thbs1 promotes HSC aging in mTORECKO mice, we crossed them with Thbs1 global knockout mice (Thbs1KO) to generate mTORECKOThbs1KO mice. Analysis of mTORECKOThbs1KO mice revealed that deletion of Thbs1 was sufficient to restore hematopoietic defects in mTORECKO mice including restoration of peripheral blood myeloid bias, BM HSC frequency and hematopoietic progenitor activity. Furthermore, serial HSC transplantation analysis confirmed that deletion of Thbs1 was sufficient to restore HSC self-renewal and lymphoid output in mTORECKO mice, identifying a putative role for Thbs1 as a pro-geronic factor for HSCs. To determine whether Thbs1 promotes HSC dysfunction during physiological aging, we aged Thbs1KO mice for 18 months and analyzed their hematopoietic parameters. Our analysis revealed that deletion of Thbs1 was able to prevent the age-associated increase in peripheral blood myeloid bias and BM HSC frequency. Notably, HSCs derived from aged (18 month old) Thbs1KO mice demonstrated a suppression of inflammatory responses by RNA Seq analysis, and a restoration of self-renewal ability and balanced lineage output in serial HSC transplantation assays, that are indistinguishable from HSCs derived from young (3 month old) mice. Our findings identify Thbs1 as a novel pro-geronic factor for HSC aging that can be targeted to preserve hematopoietic healthspan.