Abstract Body (Do not enter title and authors here): Background: We previously reported that Exendin-4 administration reduced hematopoietic stem/progenitor cells (HSPCs) proliferation and plaque inflammation and size in LDLr^-/- mice on HFD, accompanied by decreased alpha-ketoglutarate (α-KG) levels in HSPCs.
Hypothesis: α-KG could orchestrate HFD in plaque inflammation and progression.
Methods: LDLR^-/- mice were placed on HFD for six weeks. From the third week onwards, they were given 2% α-KG in the drinking water for a further four weeks. At the end of the experiment, the heart was dissected for H&E analysis. Fresh bone marrow cells (BMC) were stained with surface markers of HSPCs and GMPs for FACS analysis. The FACS-sorted GMPs were subjected to targeted metabolomics and single-cell RNA sequencing (scRNA-seq). The results of scRNA-seq were validated by qPCR and western blot using BMCs of LDLr^-/- mice. LDLR^-/- mice were lethally irradiated and transplanted with wild-type (WT) or OXGR1^-/- BMCs. After four weeks, the mice were fed an HFD with or without α-KG, as described above. Ribose-5-phosphate expression and IMPDH2 activity were quantified in lineage^-/low cells treated with PBS or PNP by ELISA.
Results: HFD induced greater plasma levels of α-KG in LDLr^-/- mice than chow diet group. Compared to the effects of HFD alone, the addition of α-KG further increased GMP frequency in BM, myeloid cells in blood and plaque size (p<0.05 for all). Following BMT and HFD, α-KG increased plaque progression in LDLr^-/- mice transplanted with WT BMC, but not with OXGR1^-/- BMC. Compared with the chow diet group, the glycolysis and pentose phosphate pathways were promoted in the GMPs of the HFD mice with or without α-KG treatment. scRNA-seq analysis identified purine nucleoside phosphorylase (PNP) as being higher in GMPs of LDLr^-/- mice on HFD plus α-KG than on HFD alone. Western blot analysis confirmed higher PNP expression in BMC of LDLr^-/- mice received WT BMC and HFD plus α-KG, compared to those on HFD alone. However, no difference was observed in mice transplanted with OXGR1^-/- BMC. In vitro, adding PNP to BMCs induced neutrophil production compared with controls. Exposure to PNP triggered ribose-5-phosphate expression and IMPDH2 activity in lineage^-/low cells. PNP-induced GMP proliferation and neutrophil production were abrogated by IMPDH2 inhibitor.
Conclusions: On top of HFD, the α-KG/OXGR1 led to pronounced inflammatory myeloid cell production and plaque progression in LDLr^-/- mice via PNP-mediated GMP proliferation.