NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation

Mesenchymal stromal cells (MSCs) are multipotent and can be derived from different adult tissues including fat. Our repeated attempts to produce long-term proliferative cultures of rat abdominal adipose stem cells (aASCs) under normal oxygen concentration (21%) were unsuccessful. We set to examine the events controlling this cytostasis of aASCs and found that it resulted from overproduction of reactive oxygen species (ROS) that led to apoptosis. ROS overproduction in aASCs was accompanied by increased expression of NOX1 but not of NOX2 or NOX4. NOX family members are an important source of intracellular ROS pointing to NOX1 involvement in ROS accumulation. This was verified when aASCs that were grown under 3% oxygen conditions expanded long term, displaying reduced NOX1 expression and decreased ROS accumulation. NOX1 involvement in aASC cytostasis was reaffirmed when cells that were expanded under normoxic conditions in the presence of a specific NOX1 inhibitor, ML171, demonstrated reduced ROS accumulation, reduced apoptosis and long-term expansion. aASC expansion arrest was accompanied also by a weak fat differentiation and migratory potential, which was enhanced by NOX1 inhibition. This suggests an inhibitory role for NOX1-induced ROS overproduction on aASCs, their fat differentiation and migratory potential. In contrast to aASCs, similar cells produced from subcutaneous fat were easily expanded in normoxic cultures, exhibiting low ROS concentrations, a low number of apoptotic cells and improved fat differentiation and migration. Taken together, our results show, for the first time, that NOX1-induced ROS accumulation halts ASC expansion and reduces their differentiation and migratory potential under normoxic conditions. Importantly, this phenotype comprises a tissue-specific signature as it was evident in aASCs but not in subcutaneous ASCs. NOX-induced ROS accumulation and cytokine production by fat are part of the metabolic syndrome. The similarity of this phenomenon to aASC phenotype may indicate that they arise from similar molecular mechanisms.Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are produced and propagated from a wide range of adult tissues.¹ MSCs were suggested to originate from a perivascular source in various adult tissues.² Although they were originally derived mainly from bone marrow, adipose-derived MSCs (ASCs) were recently demonstrated to harbor properties similar to bone marrow-derived MSCs.³ MSCs that were expanded under ‘hypoxic'' conditions (1–5% oxygen) demonstrated improved culture expansion, differentiation and genomic stability compared with MSCs that were grown under ‘normoxic'' conditions (atmospheric oxygen level).^{4, 5, 6} Reduced reactive oxygen species (ROS) accumulation was suggested as a possible explanation to the improved expansion of MSCs under low oxygen conditions.⁷ROS are produced in cells mostly by the mitochondrial oxidative phosphorylation process or as cellular signaling molecules mainly by the family of NOX NADPH oxidases.⁸ NOX family members generate superoxides and other downstream ROS products.^{8, 9} NOX1 the first NOX2 homolog to be described^{10, 11} is highly expressed in colon epithelium, and expressed in many other tissues and cells, including fibroblasts.^{9, 12, 13} ROS overproduction leads to many destructive cellular processes, such as aging, DNA damage and apoptosis.^{14, 15} Importantly, NOX-induced ROS accumulation in fat tissue during obesity was shown to be the cause of the deregulated production of adipocytokines and the induction of the metabolic syndrome.^{16, 17, 18} Development of the metabolic syndrome was correlated with the accumulation of abdominal/visceral fat rather than the accumulation of total body fat, indicating the importance of abdominal fat in the development of this syndrome.¹⁹Here we show that the inability of abdominal rat ASCs to reach long-term culture expansion (i.e expansion arrest), their weak fat differentiation and migratory potential and their increased cytokine expression results from NOX1-induced ROS accumulation that leads to their apoptotic death. Specific inhibition of NOX1 enabled long-term propagation of abdominal ASCs (aASCs) and their improved fat differentiation and migration. The role of the tissue origin in the aASC phenotype was demonstrated as ASCs from subcutaneous fat displayed reduced ROS accumulation, long-term culture propagation, improved fat differentiation and reduced cytokine expression compared with aASCs.