Cyanotic Congenital Heart Diseases Alter Metholome In Bone Marrow Niche And Induce Mesenchymal Stem Cell Premature Senescene Through Nothch1 Promoter Hypermethylation

Background and ObjectiveCongenital heart disease (CHD) is the leading birth defects worldwide, About 10% of CHD are cyanotic congenital heart diseases (CCHD). Bone marrow-derived mesenchymal stem cells (BMSCs) are the most popular cell type for regenerative therapy. BMSCs reside in a relatively hypoxic bone marrow niche. Severe hypoxia in CCHD patients will probably aggravated the hypoxic status in bone marrow niche and induce a critical metabolic change which may affect biology behavior of BMSCs. This study was designed to investigate the profound hypoxia induced metabolome alteration in bone marrow niche and their effects on BMSCs biology as well as the molecular mechanisms.MethodsThe oxygen tension in bone marrow niche was assayed by bone marrow blood gas analysis and the expression hypoxia inducible factor-la (HIF-la) was also analyz-ed. The ability of proliferation, multilineage potential and anti-apoptosis in BMSCs from NCCHD group and CCHD group was analyzed by normoxic experiment in vitro. Metabolic profiling in bone marrow niche of CCHD patients was investigated through GC-TOF/MS based metabolomics approach and high concentration of D-galactose was found in bone marrow niche of CCHD patients. Then high dose of D-galactose was added in the media to estimate its effect on BMSCs biology behavior. Notchl pathway and senescence-related pathway among NCCHD, CCHD and D-Gal group were assayed by Western Blot. Time-of-flight mass spectrometer was applied to analyze the methylation level in Notchl gene promoter. Jagged-1 peptide which is a agonist of Notch1 receptor was applied in the experiment in vitro to estimate its effect on BMSCs from CCHD patients.ResultsWe found remarkably low oxygen tension(CCHD 3.3% VS NCCHD 6.5%, P<0.01) and overexpression of HIF-la in the bone marrow niche of CCHD patients. The ability of proliferation, multilineage potential and anti-apoptosis in BMSCs of CCHD group were severely impaired. The results of metabonomics demonstrated that severe hypoxia enhanced the anaerobic glycolysis and gluconeogenesis, inhibited the TCA cycle, and caused plenty of D-galactose accumulated in bone marrow niche of CCHD patients. D-galactose could arrest cell cycle; impair the potential of proliferation, multilineage differentiation and anti-apoptosis in BMSCs; upregulate the level of P-galactosidase, indicating D-galactose can induce BMSCs premature senescence. Western Blot assay showed that the expression of Notchl, Id1 and pRb were inhibited and P16 was significantly upregulated in CCHD and D-Gal group, while there was no difference in the expression of P53 and P21. We found the methylation level of CpGll.12.13, CpG24 and CpG33 dinucleotides were significantly higher in BMSCs of passage 3 to 5 from CCHD and D-Gal group, demonstrating that D-galactose can induce hypermethylation in Notchl gene promoter and that can be maintained in different cell generations. Jagged-1 peptide could upregulate the expression of Nothl, Idl, pRb and down-regulate the level of P16. What’s more, Jagged-1 peptide can observably the ability of proliferation, multilineage potential and anti-apoptosis in BMSCs of CCHD group.ConclusionProfound hypoxia caused by CCHD induced energy metabolism alteration and D-galactose accumulation in bone marrow niche. High dose of D-galactose induced BMSCs premature senescence and impair its ability of proliferation, differentiation and anti-apoptosis by causing hypermethyation in Notchl gene promoter which could inhibit the activity of Notch pathway and upregulate P16 pathway. And the hypermethyation in Notchl gene promoter can be inherited through successive cell generations. Jagged-1 could reverse the senescence status of BMSCs from CCHD group by activating Notch pathway.