Background: Endothelial cells(ECs) serve as an instructive platform to support hematopoietic stem cells(HSCs) for homeostasis, however the underlying mechanism by which ECs regulate HSCs remains unclear. In humans, poor graft function(PGF) is characterized by pancytopenia post allogeneic hematopoietic stem cell transplantation(allo-HSCT) and serves as an appropriate model to study hematopoiesis in real world. Our series of studies reported that the impaired BM ECs are responsible for defective hematopoiesis post allo-HSCT(2013 BBMT, 2014 BBMT, 2015 BMT, 2016 Blood, 2018 AJH, 2019 Blood Advances), whereas prophylactic strategies to improve BM ECs promote hematopoietic reconstitution post allo-HSCT(2019 Blood Advances), further indicating the critical role of BM ECs in regulating HSCs. Autophagy, an essential homeostatic process responsible for nutrient deprivation, can be activated as cytoprotective response. Conversely, over-activated autophagy contributes to cancer progression. Beclin-1, an autophagy-related protein, plays a critical role in the formation of autophagosomes. Accumulating evidence has demonstrated the essential roles of ECs in supporting HSCs and of autophagy in benefiting ECs, raising the question of whether EC autophagy plays a critical role in supporting HSCs. Moreover, the effect of EC autophagy on HSC support needs to be elucidated and validated in a human pancytopenia disease model.
Aims: The current study was performed to investigate whether the autophagy status in ECs regulates their ability to support hematopoiesis. Moreover, we evaluated the effect of EC autophagy on HSC support in PGF patients, and the results may provide a promising therapeutic target for PGF patients post allo-HSCT.
Methods: Human Umbilical Vein Endothelial Cells(HUVECs) were genetically knockdown or overexpressed of Beclin-1. RNA-seq analyses were performed to elucidate the regulatory mechanism underlying hematopoietic supporting ability of HUVECs, which were further confirmed by qRT-PCR. In vitro pharmacologic regulations of autophagy(Rapamycin, an autophagy activator, and Hydroxychloroquine, an autophagy inhibitor) were administrated to HUVECs. Subsequently, a prospective case-control study was conducted. The levels of autophagy-related markers(LC3, Beclin1, and p62), and intracellular autophagosomes in BM ECs from PGF patients and their matched good graft function(GGF) patients were evaluated by monodansylcadaverine(MDC) staining, flow cytometry, western blot and transmission electron microscopy. HUVECs or BM ECs from PGF patients were cocultured with normal CD34+ cells. The quantity and function, especially the HSCs supporting ability of HUVECs or BM ECs from PGF patients were evaluated by colony-forming unit assay after Rapamycin treatment.
Results: Inhibiting autophagy by Beclin-1 knockdown significantly reduced the hematopoiesis-supporting ability of HUVECs, which could be restored by activating autophagy through up-regulating Beclin-1. During the above process, autophagy positively regulated hematopoiesis-regulating genes in HUVECs. In addition, in vitro pharmacologic regulations of autophagy by Rapamycin or Hydroxychloroquine could modulate the hematopoietic supporting ability of HUVECs through regulating autophagy. Moreover, genetic and pharmacologic down-regulating autophagy in HUVECs impaired their quantity and function, which could be restored by up-regulating autophagy in HUVECs. Subsequently, the prospective case-control study demonstrated that defective autophagy, reduced Beclin-1 expression and deficient CFU plating efficiency in BM ECs from PGF patients when compared to their matched GGF patients. Importantly, Rapamycin quantitatively and functionally improved the impaired BM ECs from PGF patients in vitro, and especially enhanced their ability to support HSCs by activating the defective autophagy.
Summary / Conclusions: Our results suggest that the autophagy status of ECs modulates their ability to support hematopoiesis by regulating the Beclin-1 pathway. Defective autophagy in BM ECs may be involved in the pathogenesis of PGF post allo-HSCT. Although further validation is required, our data suggest that Rapamycin could improve the impaired HSC-supporting ability of ECs by activating autophagy, thus providing a promising therapeutic approach for PGF patients.
Disclosures
No relevant conflicts of interest to declare.
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