Endothelial cell-derived thrombospondin-1 negatively affects hematopoietic function during aging Free

The pool of candidates for treatment of hematological malignancies via bone marrow (BM) transplants has greatly expanded due to advancements in conditioning strategies, but these carry an increased risk of failure and complication as patients age. Age-related defects to the hematopoietic stem cell (HSC) microenvironment, the BM niche, have emerged as a significant driving force for hematopoietic aging. However, insights into how this occurs are lacking. Using our previously defined premature vascular aging model, we identified thrombospondin-1 (Thbs1) as a pro-gerontic factor of HSCs. Thbs1 is a secreted, matrix-bound glycoprotein that plays major roles in regulating cellular interactions between cells and the surrounding matrix. Notably, Thbs1 is an anti-angiogenic factor and a regulator of platelet aggregation. Although Thbs1 is detectable at low levels within the plasma of healthy individuals, Thbs1 can be elevated in age-related diseases, including those within the cardiovascular and metabolic systems, leading to it being detectable in multiple fluid compartments, such as blood.

Despite being well-studied within age-related diseases, Thbs1 has not been thoroughly studied within hematopoiesis. We found that competitive HSC transplantation assays utilizing aged HSCs isolated from global knockout of Thbs1 (Thbs1^gKO) mice demonstrate a preservation of stem cell activity including increased engraftment and balanced, multi-lineage potential. This data suggests that Thbs1 may influence HSC function through intrinsic and/or extrinsic mechanisms. In a follow-up reciprocal transplantation study, wild type HSCs were transplanted into pre-conditioned Thbs1^gKO recipients or vice versa, or donor HSCs were transplanted into recipients of the same genotype. Results from this experiment showed that only when Thbs1gKO HSCs were transplanted into Thbs1^gKO recipients, would it lead to both increased engraftment and multi-lineage potential, illustrating the importance of both intrinsic and extrinsic Thbs1. To identify which niche cells produce Thbs1, we created a Thbs1-GFP reporter mouse and used flow cytometry and confocal imaging to unveil endothelial cells (ECs), megakaryocytes (MKs), osteoblasts, and myeloid- and LepR+ stromal-cells to be Thbs1-producing cells in the BM niche. Cell-specific deletion of Thbs1 from these cells followed by competitive stem cell transplantations in young mice revealed that only KO of EC- and MK- derived Thbs1 showed increased HSC engraftment mimicking results utilizing young HSCs from Thbs1^gKO. However, when aging these models and performing competitive transplants, only HSCs derived from EC-KO of Thbs1 were able to recapitulate the global KO phenotype of young-like engraftment and lineage potential.

Myelosuppressive treatment can cause irreversible damage to the BM niche, hindering the recovery of the hematopoietic system. Both MKs and ECs have been shown to play important roles in this; MKs aid in re-establishing HSC quiescence and ECs are known to re-establish hematopoiesis post chemotherapeutic treatment. Due to the anti-angiogenic nature of Thbs1, there is a likelihood that Thbs1 plays a role in vascular recovery post insult. To this end, possible roles of Thbs1 during myelosuppressive recovery are being assessed. 5-fluorouracil (5-FU) sub-lethal myelosuppression on our Thbs1-GFP reporter showed dynamic Thbs1 expression throughout recovery within BM cells, indicating a possible cell-specific role Thbs1 plays during recovery. Young Thbs1^gKO mice exhibited earlier and more robust complete blood count recovery post myelosuppressive treatment, notably in the clinical marker of neutrophil recovery. Competitive transplantation following this treatment also shows increased engraftment in the KO group as compared to the control. When performing the same treatment on our cell-specific Thbs1 KO models in myeloid cells, osteoblasts, and ECs, only ECs showed similar improvements seen in the global KO model. Other cell-specific models are currently being tested, and transplantation experiments are ongoing. Collectively, we show that EC-derived Thbs1 plays a crucial role in the aging of the BM niche and recovery post myelosuppressive treatment, validating it as a potential therapeutic target to support the regeneration of the hematopoietic system following myelosuppression in aged patients facing malignancy.