You are hereDecember 10, 2012 | Mesenchymal Stem Cells
Signalling behind MSC Mobilisation Uncovered
Original article from STEM CELLS
Adult stem/progenitor cells are able to differentiate into many cell types and can also be recruited to a site of injury where they either repair the injured tissue or contribute to tissue remodeling (Ferrari et al, Takahashi et al, Lagasse et al, Orlic et al and Kale et al). Mesenchymal stem cells (MSCs) in peripheral blood are one such stem cell known to act in this way, and it is believed that promigratory factors released from injured tissue or surrounding inflammatory cells create a signal for their recruitment (Caplan and Correa, Krankel et al and Wojakowski et al). However the primary endogenous factors activated or released in response to injury to stimulate the mobilization of MSCs are largely unknown. Transforming growth factor beta proteins (TGFβs) are synthesized in a latent form sequestered in extracellular matrix (ECM) (Kanzaki et al and Munger et al) and perturbations in the ECM associated with phenomena such as angiogenesis, wound repair, inflammation, and cell growth (Annes et al) release active TGFβs. TGFβ1 can be released from the bone matrix to induce MSCs migration for bone remodeling (Tang et al), but less is understood about a potential role in the vasculature where shear stress and arterial injury can induce activation of TGFβ1 (Ahamed et al and Qi et al). Now, in a study in Stem Cells, researchers from the group of Xu Cao at the Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, using two separate models of arterial damage, have found that MSCs become mobilized into peripheral blood and migrate to injured sites to participate in vascular repair and remodeling by a mechanism controlled by active TGFβ1 (Wan et al).
A mouse model of femoral artery injury (Sata et al) was initially utilised to assess if Sca1+CD29+CD11b−CD45− MSCs or Nestin+ MSCs are mobilized in response to injury. The group found that overall, the number of MSCs was elevated compared to a control group at 3 days post-injury and this was maintained for 2 weeks. This model and an additional rat model (balloon injury of the carotid artery (Xing et al)) were then used to study whether these MSCs are specifically recruited to an injury site. Neointimal tissue, a thickened layer of cells in an artery caused by migration and proliferation of cells from outwith the wounded area, was observed at the intraluminal side of the injury site after 1 week, becoming thicker at two weeks and growing till complete re-endothelialization at 6 weeks. At 1 week and 2 weeks Nestin+ cells were detected in a single layer of the intraluminal side of the neointima which were also Sca1+ indicative of these cells being MSCs. Approximately 20% of the Nestin+ cells were also CD31+ at two weeks suggesting that some Nestin+ MSCs may transform to endothelial cells for endothelium repair. Next using a novel genetic mouse model generated by crossing mice carrying a nestin promoter/enhancer-driven cre-recombinase (Nestin-cre) with C57BL/6J-Gtrosa26 tm1EYFP (R26-stop-EYFP) mice in which both Nestin+ cells and their descendants at the neointimal tissue can be marked, myofibroblast differentiation was analysed. At 1 and 2 weeks, approximately 57% and 45% of cells in all layers of the neointimal tissue were EYFP+, where only the cells in the outer layer remained Nestin+, suggesting that the EYFP+ cells were myofibroblastic cells.
Mechanisms behind MSCs migration were then analysed, focusing on the role of TGFβ1. This investigation found that active TGFβ1 expression increased in the intimal and medial layers of the injured arteries at 3 days, 1 week, and 2 weeks post-injury in both models, while no active TGFβ1 was found in uninjured arteries. A cell migration assay using aorta-conditioned medium (CM) was next used to further study the role of TGFβ1, finding that injury-associated CM greatly increased migration of MSCs compared to uninjured CM. This increased migration was inhibited after the addition of neutralizing antibodies against TGFβ1 or TGFβ3, but not TGFβ2, which correlated to the discovery of high levels of both TGFβ1 and TGFβ3 in the injury-associated CM. Downstream analysis of TGFβ signalling was studied through the use of several small molecule inhibitors; addition of a TβRI/Smad inhibitor (SB505124) or a c-Jun N-terminal kinases (JNK) inhibitor (SP600125) reduced the increase in migration mediated by the injury-associated CM but inhibitors of extracellular signal-regulated kinases (U0126), p38 (SB202190) or RhoA (Y27632) had little or no effect.
In vivo, active TGFβ1 level in the blood was found to be elevated at 3 days and reached 8-fold at 1 week and 10-fold at 2 weeks post-injury. This level of TGFβ1 was also found to be able to increase MSC numbers in peripheral blood by 2.5–5-fold at 24 hours, while haematopoietic stem cell (HSC) and endothelial progenitor cell (EPC) numbers remained unchanged. Biotin labelling of TGFβ1 and later analysis of streptavidin-FITC-bound cells demonstrated that TGFβ1 bound almost exclusively to Nestin+Sca1+CD29+CD11b− cells. Additionally almost all sorted Sca1+CD29+CD11b−CD45− cells from bone marrow and peripheral blood were Nestin+ after TGFβ1 injection suggesting that the cells in peripheral blood are MSCs and had mobilized from the bone marrow. These cells were capable of osteogenesis, adipogenesis, and chondrogenesis and could differentiate into smooth muscle- or myofibroblast-like cells. However, injection of a TβRI inhibitor SB-505124 (SB) blocked the increase in Nestin+ cells in peripheral blood at 3 days and 2 weeks post-arterial injury, and neointima formation was reduced to very low levels, with no Nestin+ cells on the endoluminal side of the neointima. Further signalling mechanisms were investigated through analyses of MCP1 and Chemokine (C-X-C motif) ligand 12 (CXCL12)/SDF1α have both been reported to be involved in the recruitment of stem/progenitor cells to the vasculature (see paper for extended references). A neutralizing antibody against MCP1, but not SDF1α, inhibited the increase in MSC migration after exposure to injury-associated CM, while high levels of MCP1 were observed mostly in the smooth muscle cells and neointimal cells. TGFβ has been previously shown to stimulate MCP1 expression in the vascular smooth muscle cells (Zhang et al) and indeed MCP1 production in the injury models was inhibited by selective TβRI kinase inhibitor SB505124 or neutralizing antibodies against TGFβ1 or TGFβ3, indicating that TGFβs were primary factors that regulate MCP1 expression via a TβRI-mediated pathway. Additionally a selective MCP-1 receptor antagonist (RS 504393) significantly inhibited neointima formation and the recruitment of the Nestin+ cells to neointima after injury.
Overall, this report suggests that MSCs are mobilized into peripheral blood in response to injury, whereafter they become recruited to the injury site to mediate repair. This recruitment is mediated by Smad and JNK signaling and TGFb1 is required for the mobilization and recruitment of MSCs from bone marrow to the peripheral blood. Additionally, MCP1, a downstream target of TGFb, acts as a chemoattractant allowing or the homing of MSCs to injury sites. Together, these findings may provide a basis for the study into the improvement of cell mobilization and homing in MSC-based therapy.
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