Antioxidants As A Novel Treatment To Revert Impaired Angiogenic Potential Driven By Metabolic Alterations Observed In Shwachman-Diamond Syndrome Derived Mesenchymal Stromal Cells

Introduction and aims: MSCs are crucial elements in the BM niche homeostasis. Importantly, our group previously demonstrated that semi-cartilaginous pellets derived from SDS-MSCs was impaired to generate in vivo heterotopic ossicles compared to HDs, due to severe vascularization defect. Moreover, SDS-MSCs showed a defective in vitro ability to form capillary-like networks compared to HD-MSCs (1). Angiogenesis required an adjustment of metabolic activities and altered cellular metabolism adversely affects angiogenic sprouting (2). In this context, antioxidants represent a novel treatment to revert aberrant angiogenesis (3). Therefore, we aimed to characterize the SDS-MSC metabolic profile as contributing factor of their angiogenic impairment and we investigated antioxidants as new therapeutic strategies to revert SDS angiogenic defect. Methods and materials: BM derived HD- and SDS-MSCs were stimulated with DMEM±NAC (1mM) or DMSO (0.05%v/v) for 48h. The in vitro angiogenic capability of MSCs was evaluated by performing Matrigel angiogenesis assay, by analysing viability (AnnexinV) and the expression of angiogenic molecules (RT-PCRs/ELISA). Heterotopic angiopoietic assay was performed in NSG mice to investigate in vivo MSCs angiogenic potential. The plugs were explanted after 10/21 days and stained by hematoxylin-eosin. Concerning the metabolic analyses, oxidative phosphorylation (OxPhos) was assayed by oximetry and bioluminescent ATP synthesis assay. Spectrophotometric analyses were performed to evaluate ATP/AMP ratio, lactate fermentation yield, malondialdehyde (MDA) level, iron content and enzymatic activity measurements. Instead, reactive oxygen species (ROS) was quantified by flow cytometry. SDS BM biopsies were collected during routinely diagnostic procedures and stained by hematoxylin-eosin. Results: We confirmed the impaired angiogenic potential in a cohort of 13 SDS- compared to 14 HD-MSCs. SDS-MSCs showed a defective capability to recreate a defined capillary-like network compared to HDs. Accordingly, ImageJ analyses showed that several angiogenic elements, including branches and meshes, were significantly reduced in SDS-MSCs. Furthermore, the angiogenic defect of SDS-MSCs was correlated neither to low cells viability, nor to a differential expression of angiogenic molecules (i.e. VEGF). To assess angiogenesis in SDS, we analysed BM biopsies of 2 SDS patients. Importantly, vasculature appeared aberrant with the present of dilated vessels, supporting our hypothesis of compromised angiogenic potential. Therefore, we investigated SDS-MSCs metabolism (n=6). Concerning the I-III-IV mitochondrial complexes pathway, SDS-MSCs consumed 57% less oxygen (p=0.004) and produced 64% less ATP compared to HD-MSCs (p=0.002). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). Therefore, Complex IV activity was 61% lower in SDS- vs HD-MSCs (p=0.002), highlighting its role in the SDS OxPhos defect. As for the energetic status, SDS-MSCs showed low intracellular ATP/AMP ratio (mean=1.1,range=0.8-1.6 vs mean=3.6,range=3.1-4.0,in HDs; p=0.002). Importantly, SDS-MSCs compensated for their mitochondrial defect increasing anaerobic glycolysis. Indeed, the SDS-MSCs lactate dehydrogenase (LDH) activity was increased by 30% (p=0.002) and the SDS lactate fermentation yield resulted 40% higher in SDS than HD-MSCs (p=0.002). The amount of ROS in SDS-MSCs was increased by 27% compared to HD-MSCs (n=5) and also the SDS-MSCs lipid peroxidation level was significantly higher over HDs (mean=12.7µMofMDA/mg,range=10.5-14.1µM/mg vs mean=6.6µM/mg,range=5.9-7.4µM/mg; p=0.002). Importantly, SDS oxidative damage was associated to 55% increase of total intracellular iron. Particularly, Fe3+/Fe2+ ratio in SDS-MSCs was increased by 58% compared to HDs. Then, we treated SDS-MSCs for 48h with NAC, a broad-range antioxidant (n=5), or with DMSO, which acts at very low concentrations as scavenger, specifically on lipid peroxidation products (n=6). Interestingly, NAC/DMSO stimulated SDS-MSCs increased by 75% the oxygen consumption and by 70% the ATP synthesis in both the electron transport chain pathways, thus resulting in levels comparable to HD-MSCs. The SDS OxPhos restoration was respectively associated to a 53% and 60% increase of Complex IV activity after NAC/DMSO stimulation (p<0.0001 for both). Moreover, antioxidants corrected the SDS energetic defect restoring ATP/AMP ratio to the levels of HDs and reducing by 30% the lactate fermentation yield. Importantly, SDS-MSCs lipid peroxidation level was respectively reduced by 60% and 51% after NAC/DMSO treatments (p<0.0001 for both). Moreover, antioxidants decreased SDS iron content (p<0.0001 for both), and completely restored the Fe3+/Fe2+ ratio to HDs levels. Finally, we investigated the effect of antioxidants on the SDS-MSCs angiogenic potential. We showed that SDS-MSCs defective capability to form capillary-like network was completely restored after antioxidant stimulations (n≥5). Several angiogenic elements, including branches and segments, were significantly increased in NAC/DMSO stimulated SDS-MSCs to HD levels. As in vitro results, we demonstrated that SDS-MSCs in vivo angiogenic potential was severely impaired (n=3). Notably, analyses at different time points of harvested plugs contained SDS-MSCs highlighted absence of vessels in contrast to plugs with HD-MSCs, that displayed perfused vascular structures. Interestingly, preliminary results suggested that antioxidants increased SDS-MSCs in vivo ability to sustain angiogenesis. Indeed, SDS plugs derived from antioxidant treated mice showed functional vessels within just 10 days (Figure1). Discussion: Our results demonstrated that SDS-MSCs showed impaired in vitro and in vivo angiogenic potential. Severe metabolic alterations with high oxidative damage in SDS-MSCs contributed to their angiogenic defect. Importantly, NAC and DMSO treatments induced SDS metabolic remodelling and restored the angiogenic impairment. Conclusion: Our study demonstrated for the first time to our knowledge that SDS angiogenic impairment was driven by metabolic alterations, providing a more comprehensive understanding of the BM microenvironment. Therefore, antioxidants restored mutually metabolic and angiogenic impairments representing a promising therapeutic approach in the management of SDS.