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A4624 - Hypoxia- and SU5416-Induced Downregulation of Nestin Causes Cell Cycle Arrest and Promotes Apoptosis in Bone Marrow Mesenchymal Stem Cells
Author Block: D. Farkas, S. Hultman, K. Dandamudi, A. R. Bhagwani, L. Farkas; Virginia Commonwealth University, Richmond, VA, United States.
Rationale: Recent studies indicate that bone marrow cells may be impacted in Pulmonary Hypertension (PH), yet current studies have largely focused on the hematopoietic system. Mesenchymal stem cells (MSCs) are important for maintenance and function of the bone marrow (BM) hematopoietic stem cell niche, our study evaluated the change in function and gene expression of nestin, an intermediate filament protein implicated in cell growth of different progenitor cell populations, as well as angiogenesis.
Methods: BM-MSCs were isolated from the BM of rats with chronic hypoxia and SU5416 (H/S) induced PH. Control BM-MSCs were derived from rats housed under normoxic and chronic hypoxic conditions. Naive BM-MSCs were further exposed to hypoxia and SU5416 for 7 days in vitro. Nestin expression was manipulated by siRNA-mediated gene knockdown and adenovirus-mediated overexpression. Proliferation was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation and flow cytometry, apoptosis by Annexin V assay.
Results: BM-MSCs from rats with H/S-induced PH grew smaller colonies with larger cell size. These changes were mirrored by reduced proliferation, cell cycle arrest and increased apoptosis in H/S BM-MSCs. At the same time, we found reduced expression of TNF-stimulated gene 6 protein (TSG-6) and nestin in H/S BM-MSCs. Then we studied the direct influence of hypoxia and SU5416 on BM-MSC function, and found that 7 days of hypoxia and SU5416 induced cell cycle arrest associated with slower cell growth, and decreased Nestin expression. Nestin overexpression partially rescued BM-MSCs from H/S-induced growth inhibition. Then, we identified by siRNA-mediated knockdown of Nestin expression that reduced level of Nestin is sufficient to block cell growth and cause cell cycle arrest in BM-MSCs.
Conclusions: Hypoxia and SU5416 impair growth of BM-MSCs and reduce expression of Nestin both in vivo and in vitro. Our data further suggest a causal relationship between Nestin expression and cell growth in BM-MSCs. Because mutations in bone morphogenic protein receptor 2 are important for a relevant portion of PH patients, further experiments are underway to determine the role of bone morphogenic protein signaling for Nestin expression in BM-MSCs.