HIF-1α Agonist Ameliorates Acute Graft-Versus-Host Disease Via Restraining SIRT1/ PARP1 in Bone Marrow Endothelial Progenitor Cells

Background：Acute Graft-Versus-Host Disease(aGVHD) is a prevalent complication after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Bone marrow Endothelial Progenitor Cells(BMEPCs) are crucial in hematopoietic-supporting after allo-HSCT. Our former study has showed the reduced HIF-1α expression and impaired BMEPC function in aGVHD patients. HIF-1α plays an important role in endothelial cells’ survival. Roxadustat is a HIF-1α agonist that has been widely used recently for the clinical treatment of renal hypertension. However, its role in allo-HSCT requires further investigation.

Aims:Our study aims to investigate the role of HIF-1α in the development of aGVHD after allo-HSCT. Furthermore, our study aims to verify the efficacy of HIF-1α agonists in prophylaxis of aGVHD in animal models.

Methods:30 patients with aGVHD and 30 patients without GVHD were included in our study. SiRNA transfection knocked down HIF-1α in human umbilical vein endothelial cells(HUVECs) as a low HIF-1α expression endothelial cell model. Endothelial cell-specific HIF-1α knockout mice were generated using the Cre-LoxP system. The mouse aGVHD model was established by irradiating C57 mice followed by infusion of allogeneic mouse(BALB/C) bone marrow hematopoietic stem cells(HSCs) and splenocytes.

Results:The expression of HIF-1α and autophagy-related proteins Beclin-1 and LC3 in EPCs from aGVHD patients was downregulated compared to non-GVHD patients. Moreover, EPCs from patients with aGVHD exhibited impaired cellular function, including cell proliferation, migration, and angiogenesis. HSCs co-culturing with EPCs from aGVHD patients performed elevated levels of reactive oxygen species(ROS), apoptosis and impaired colony formation ability. Furthermore, the knockdown of HIF-1α in HUVECs led to the activation of NAD+ consuming proteins SIRT1 and PARP1, which caused a decrease in NAD+ levels and abnormal mitochondrial morphology. Intervention with HIF-1α activators Roxadustat, EPCs from aGVHD patients showed restored cellular function and hematopoiesis-supporting ability. Likewise, the supplementation of NAD+ precursors, β-nicotinamide mononucleotide(NMN), effectively increased NAD+ levels in endothelial cells and restored cellular function. After inducing aGVHD by infusion of bone marrow HSCs and splenocytes from BALB/C mice, HIF-1α knockout C57 mice showed significantly higher aGVHD clinical scores and reduced survival compared to controls, and histopathology of the gut, colon and skin showed a more extensive inflammatory infiltration. Intraperitoneal injection of Roxadustat and NMN after the induction of aGVHD, restored HIF-1α expression and decreased ROS levels in BMEPCs of HIF-1α knockout C57 mice,with less weight loss and higher survival rates.

Conclusion:The results revealed that the autophagy and mitochondrial dysfunction was mediated by low expression of HIF-1α in EPCs of patients with aGVHD. The deficiency of HIF-1α led to abnormal activation of SIRT1 and PARP1, which in turn affected the levels of NAD+ and mitochondrial status, ultimately impairing cellular function and hematopoiesis-supporting ability in BMEPCs and HIF-1α knockout mice models. Moreover，We found that Roxadustat and NMN enhance the autophagy and improved mitochondrial function of BMEPCs in vitro. In vivo, treatment with Roxadustat and NMN reduced the incidence of aGVHD and improved survival of HIF-1α knockout mice. This therapeutic approach holds great promise as a novel strategy for the prevention and treatment of aGVHD in patients following allo-HSCT.