Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α.

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.

Hypoxia decreases sclerostin expression and increases Wnt signaling in osteoblasts.

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF-alpha isoforms and thereby enables HIF-alpha/beta-driven gene transcription, increases bone mineral content and cross-sectional area compared to wild-type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated beta-catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased beta-catenin gene reporter activity. Hypoxia and its mimetics increased expression of the BMP antagonists gremlin and noggin and decreased Smad-1/5/8 phosphorylation. As a partial explanation for the mechanism of regulation of sclerostin by oxygen, MEF2 reporter assays revealed decreased activity. Modulation of VEGF signaling under normoxia or hypoxia revealed no influence upon Sost transcription. These data suggest that hypoxia inhibits sclerostin expression, through enhanced antagonism of BMP signaling independent of VEGF.

The effect of oxygen tension on the long-term osteogenic differentiation and MMP/TIMP expression of human mesenchymal stem cells.

The use of mesenchymal stem cells in tissue engineering to augment the repair of a variety of tissues including bone is a rapidly growing and exciting field. Although oxygen tension is a powerful stimulus for cells both in vitro and in vivo, the oxygen environment in which such cells would undergo differentiation is commonly overlooked. We examined the effect of long-term (21-days) low oxygen tension (1, 2 and 5%) on the osteogenic differentiation and matrix metalloproteinase (MMP)/tissue inhibitor of MMP (TIMP) expression of human mesenchymal stem cells (MSCs). Our data suggest that MSCs undergo osteoblastic differentiation most rapidly under 21% oxygen while oxygen tensions below 5% have an inhibitory effect. Interestingly, there was not a statistically significant difference in osteogenic markers between 5 and 21% oxygen. In addition, our data suggest that oxygen tension affects the expression of individual MMP and TIMPs differently. Low oxygen tension has an inhibitory effect on MMP-13 and TIMP-1 expression, which are involved in extracellular matrix remodeling and potentially vascular invasion. In contrast, MMP-2, a metalloproteinase involved in cell migration was not affected by oxygen tension. This data suggests that 21% oxygen may be beneficial for rapid osteogenic differentiation as would be required for the production of individual patient ex vivo constructs. In addition, this has important in vivo implications relating to the importance of early vascularization of sites of orthopedic injury. By augmenting the neovascularization process, it may be possible to facilitate more rapid differentiation of progenitors and thus the repair process.