Study of Angiogenic Potential and Oxidative Metabolism in Mesenchymal Stromal Cells Derived from Shwachman-Diamond Syndrome Patients

Introduction: Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive bone marrow (BM) failure disorder. SDS BM biopsies showed tortuous vessels with collapsed lumens, which might promote progression of malignant clones.

Mesenchymal stromal cells (MSCs) are crucial elements in the BM niche homeostasis. Our group previously demonstrated that the in vivo ability of semi-cartilaginous pellets derived from SDS-MSCs to generate complete heterotopic ossicles was severely impaired in comparison with healthy donors (HDs). In addition, after specific angiogenic stimuli, SDS-MSCs showed an in vitro defective capability to form a well-defined tubular network. The aim of this study was to deeply investigate the cellular and molecular mechanisms underlying the SDS-MSCs impaired angiogenic potential.

Methods: The angiogenic capability of BM derived SDS- and HD-MSCs was evaluated by performing in vitro Matrigel angiogenesis assay, by analysing cell viability and the expression of key angiogenic molecules before and after the angiogenic assay (RT-PCRs and/or ELISA). Concerning energy metabolism, oxidative phosphorylation (OxPhos) has been assayed by oximetry and bioluminescent ATP synthesis assay, and ATP/AMP ratio, malondialdehyde (MDA) level, and complex IV and lactate dehydrogenase (LDH) activity have been evaluated by spectrophotometric analyses.

Results: We confirmed the impaired angiogenic potential of SDS-MSCs, previously observed in a restricted cohort of patients (n=6), in 13 SDS- compared to 14 HD-MSCs. In detail, after 3h of angiogenic stimuli, SDS-MSCs showed a defective capability to recreate a defined capillary-like network compared to HD-MSCs in a Matrigel-based assay. Accordingly, ImageJ Angiogenesis Analyzer showed that several angiogenic elements, including branches and meshes, were significantly reduced in SDS-MSCs. Furthermore, we demonstrated that the angiogenic defect of SDS-MSCs was correlated neither to a lower cellular viability than HD-MSCs, nor to a different expression of the main angiogenic molecules.

Different reports showed a close relationship between aberrant angiogenesis and oxidative stress. Based on previous studies that demonstrated a defective energy metabolism and an increased oxidative stress in SDS patient-derived lymphoblastoid cells, we performed metabolic analyses of 6 SDS- and 6 HD-MSCs from the cohort used for angiogenic assay. In detail, we assayed OxPhos metabolism, evaluating oxygen consumption and ATP production. Concerning the I, III, and IV complexes pathway, we demonstrated that SDS-MSCs consumed 54% less oxygen (p=0.009) and they produced 60% less ATP compared to HD-MSCs (p=0.004). Accordingly, the analysis of the II, III, and IV complexes pathway demonstrated that the oxygen consumption was reduced by 61% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). In addition, the P/O ratio, an index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both electron transport chain pathways (p=0.004 for both).

Moreover, OxPhos defect of SDS-MSCs was associated to the decreased enzymatic activity of complex IV compared to HD-MSCs (mean=9.5mU of oxidated cytochrome c/mg protein, range=7.8-11.5mU/mg vs mean=25.6mU/mg, range=21.6-31.9mU/mg; p=0.002).

Consequently, SDS-MSCs showed a 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). This decrease was accompanied by an increased LDH activity (mean=0.4mU/mg protein, range=0.4-0.6mU/mg vs mean=0.3mU/mg, range=0.27-0.32mU/mg, in HDs; p=0.0022) in SDS-MSCs, showing an attempt to compensate the mitochondrial defect by the anaerobic glycolysis enhancement.

Finally, we demonstrated that the level of lipid peroxidation of SDS-MSCs was significantly increased compared to HDs (mean=11.8µM of MDA/mg protein, range=10.4-15.2µM/mg vs mean=6.5µM/mg, range=5.0-7.7µM/mg; p=0.0022), suggesting an oxidative stress production increment.

Conclusions: We demonstrated that the angiogenic defect in SDS-MSCs coexists with metabolic alterations. In addition, we demonstrated for the first time to our knowledge, that SDS-MSCs showed a defective oxidative metabolism, characterized by a decreased energy production and a high level of oxidative damage.

These results underline the key role of MSCs in SDS BM niche and provide new insights into the pathogenesis of this rare disease.

Biondi:Incyte: Consultancy, Other: Advisory Board; Bluebird: Other: Advisory Board; Novartis: Honoraria; Amgen: Honoraria. Dufour:Gilhead: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Biocryst: Consultancy.