TGF-β Pathway Inhibition Improves Hematopoietic Deficiency and Immune Imbalance By Repairing Endothelial Progenitor Cell Function in Aplastic Anemia

Background: Aplastic anemia(AA), one of bone marrow(BM) failure syndromes, is a rare and life-threatening disease. However, the pathogenesis of AA remains to be elucidated and therapeutic strategies need to be further established. Our recent studies reported the dysfunction of BM endothelial progenitor cells(EPCs), one of critical components in BM microenvironment, are involved in hematopoiesis defect and immune imbalance in hematopietic dysfunction-related diseases, including AA(2016Blood, 2022Haematologica, 2022BMC Med, 2023Sci China Life Sci). Therefore, unveiling the mechanism underlying BM EPC dysfunction may provide a potential target for alleviating AA. Hyperactive TGF-β signaling is reported to inhibit the proliferation of hematopoietic stem cells(HSCs) and lead to BM failure in mice, while TGF-β inhibition promotes hematopoietic recovery. Latest studies indicate one of TGF-β signaling inhibitors, luspatercept, promotes erythroid maturation in patients with myelodysplastic neoplasms and β-thalassaemia. However, whether TGF-β inhibition could restore hematopoiesis and immune status via repairing BM EPCs, and therefore provide a potential therapeutic option for AA patients remain to be elucidated.

Aims: To investigate whether TGF-β signaling is activated in BM EPCs of AA patients compared with healthy controls(HCs). Moreover, to explore whether activation of TGF-β signaling in EPCs impairs their biological functions in regulating homeostasis of HSC and T cell differentiation. Finally, to determine whether TGF-β inhibition could restore their multi-lineage hematopoiesis and immune balance via repairing BM EPCs in AA.

Methods: A prospective case-control study enrolled AA patients and their age-matched HCs to compare the expression levels of TGF-β1 and TGF-βRI using flow cytometry. BM EPC functions were evaluated by proliferation, apoptosis, migration and tube formation assays. Direct-coculture systems of EPCs and CD34⁺ or CD3⁺ cells were used to examine the hematopoiesis-supporting and immune regulation capacity of EPCs, respectively. An impaired EPC model derived from HCs induced by TGF-β1 and a classical AA mice model were established to further validate the role of TGF-β signaling in regulating hematopoiesis and immunological status in vitro and in vivo. Finally, RNA-seq analysis and qRT-PCR were performed to validate previous findings.

Results: Compared to HCs, decreased and dysfunctional EPCs with hyperactive TGF-β signaling, which are characterized as decreased double stained cell numbers, reduced angiogenesis and migration capacities, higher apoptosis ratio, impaired HSC-supporting ability and imbalanced T cell differentiation-supporting ability, were observed in AA patients, which could be improved by TGF-β inhibition. Consistently, the same findings were observed in an impaired EPC model triggered by TGF-β1 in vitro. Furthermore, AA mice presented the damage in BM endothelial cells with activated TGF-β signaling, whereas TGF-β inhibition restored hematopoietic defect and rebalanced T cell subsets via repairing endothelial cells, thus contributing to a better performance in peripheral blood including increased erythroid and megakaryocytic lineages. Mechanismly, RNA-seq and qRT-PCR analysis further validated TGF-β signaling is involved in BM EPC dysfunction in AA patients.

Summary/Conclusion: We found for the first time that dysfunctional BM EPCs with hyperactive TGF-β signaling are involved in AA, whereas inhibition of TGF-β could promote multi-lineage hematopoiesis recovery and immune balance in AA via repairing dysfunctional EPCs. Our study suggests TGF-β inhibitors, including luspatercept, might be a potential therapeutic strategy for AA patients, which provides a rationale for further clinical trials in AA patients to validate our preliminary findings in the future.