Study of angiogenic potential and metabolism in mesenchymal stromal cells derived from Shwachman-Diamond Syndrome patients and new therapeutic strategies

Shwachman-Diamond syndrome (SDS) bone marrow (BM) biopsies showed tortuous and collapsed vessels, highlighting angiogenic abnormalities. Importantly, our group demonstrated that SDS BM mesenchymal stromal cells (MSCs) showed in vitro and in vivo impaired angiogenic potential compared to healthy donors (HD)-MSCs. Recent evidence underlined that pathological angiogenesis was accompanied by altered metabolism and that the use of antioxidants represented a novel strategy to target angiogenic defects. Thus, the aims of this study were to characterize the metabolic status of SDS-MSCs and to investigate if the treatment with antioxidants may impact on the SDS impaired angiogenic potential. Firstly, we assayed oxidative phosphorylation (OxPhos), evaluating oxygen consumption and ATP production. Concerning the I-III-IV mitochondrial complexes pathway, we demonstrated that SDS-MSCs consumed 57% less oxygen (p=0.004,n=6) and they produced 64% less ATP compared to HD-MSCs (p=0.002,n=6). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002,n=6) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002,n=6). In addition, the P/O ratio, index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both electron transport chain pathways (p=0.002 for both, n=6). Therefore, we demonstrated that complexIV activity was 61% lower in SDS-MSCs vs HD-MSCs (p=0.002,n=6), highlighting its role in the observed SDS OxPhos defect. Evaluating the energetic status, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1,range=0.8-1.6vsmean=3.6,range=3.1-4.0, in HDs; p=0.002,n=6). This decrease was accompanied by an increased lactate dehydrogenase (LDH) activity (mean=0.4mU/mg protein,range=0.4-0.6mU/mgvsmean=0.3mU/mg,range=0.27-0.32mU/mg, in HDs; p=0.002,n=6) in SDS-MSCs, showing an attempt to compensate the mitochondrial defect by the anaerobic glycolysis enhancement. Furthermore, we demonstrated that the amount of ROS in SDS-MSCs was basally increased by 27% compared to HDs (n=5) and the SDS lipid peroxidation level was significantly higher compared to HDs (mean=12.7µMofMDA/mg,range=10.5- 14.14µM/mgvsmean=6.6µM/mg,range=5.9-7.4µM/mg;p=0.002,n=6). In this contest, dimethylsulfoxide (DMSO), used at low concentration, represents an innovative antioxidant molecule, acting particularly on lipid peroxidation. Thus, we treated MSCs with 0.05% DMSO for 48h. Surprisingly, we demonstrated that stimulated SDS-MSCs completely restored the ability to form tubular-like structures in Matrigel-based assay (n=7). Moreover, the SDS angiogenic defect correction was accompanied with a metabolic remodelling (n=6). Concerning oxidative phosphorylation metabolism, DMSO treatment increased the oxygen consumption and the ATP synthesis in both the electron transport chain pathways to HDs level and it also restored the activity of complexIV. Moreover, DMSO corrected the SDS energetic defect by restoring ATM/AMP ratio and LDH activity to the levels of HDs. Importantly, also oxidative stress in DMSO stimulated SDS-MSCs resulted comparable to HD controls. To confirm if the effect of DMSO was due properly to its antioxidant properties, we treated SDSMSCs for 48h with N-Acetylcysteine (NAC) (n=5), one of the most used antioxidants. The SDS angiogenic potential and the metabolic alterations were completely restored and there were not differences between DMSO and NAC treatment. In conclusion, we demonstrated that the altered metabolism of SDS-MSCs could be responsible of the observed angiogenic defect. In addition, we showed that antioxidants restored the metabolic alterations and the angiogenic potential in SDS-MSCs, paving the way for new therapeutic strategies.