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Mechanism by Which Injured Tissue “Tells” Stem Cells to Leave Bone Marrow

Urao et al. Stem Cells. 2012 Jan 30.

In addition to the established role of bone marrow derived stem cells in producing blood cells, an interesting aspect of these stem cells is to assist/accelerate tissue healing after injury. Perhaps the most studied example of this is in the situation of myocardial infarction (heart attack), in which damaged heart muscle sends out signals to the bone marrow, which cause selective homing of bone marrow stem cells into the damaged heart tissue. This is believed to occur via activation of the transcription factor HIF-1 alpha due to lack of oxygen in the tissue. HIF-1 alpha binds to DNA and induces activation of a variety of genes that are involved in angiogenesis such as VEGF, FGF-2, and IL-20. Additionally, HIF-1 alpha stimulates production of the chemokine stromal derived factor (SDF)-1, which attracts bone marrow stem cells by binding to the CXCR4 receptor. The importance of SDF-1 in terms of bone marrow stem cell migration is exemplified in the situation of bone marrow transplantation. When a transplant is performed the bone marrow recipient is administered the donor stem cells intravenously and not intraosteolly (inside the bone). The reason for this is because the bone marrow itself constantly produces SDF-1 which attracts injected stem cells that express CXCR4.

During infarction, the concentration of SDF-1 produced by the damaged heart muscle is higher than the concentration of SDF-1 in the bone marrow, and as a result, stem cells from the bone marrow leave the bones, enter circulation, and home to the heart. Similar examples are found in the situation of stroke. In stroke patients, not only do bone marrow stem cells enter circulation after the stroke, but it has been reported that patients with higher number of stem cells in circulation actually have better outcomes.

The possibility of chemically “mobilizing” bone marrow stem cells into circulation is very attractive. On the one hand, it would be conceptually possible to augment the extent of regeneration by increasing the number of circulating stem cells, and on the other hand, it may be possible to perform “bone marrow transplantation” without the painful procedure of drilling holes through the bones of the donor. In fact, the second possibility is actually part of clinical practice. Doctors use the drug G-CSF, otherwise known as Neupogen, to cause donor migration of bone marrow stem cells into circulation, which are then harvested by leukopheresis, so that bone marrow puncture is not needed. The first possibility, the therapeutic use of bone marrow mobilization has resulted in mixed data. Some groups have demonstrated significant improvement in heart attack patients treated with G-CSF, whereas others have reported no benefit. Recently a new way of mobilizing stem cells has been approved by the FDA: a small molecule drug called Mozobil which blocks the interaction between SDF-1 and CXCR4. This drug was developed by the company Anormed and sold to Genzyme, a major Biopharmaceutical company.

In a recent paper, the role of oxidative stress was investigated in the animal model of critical limb ischemia. Critical limb ischemia is a condition in which patients experience poor circulation in the lower extremities, usually as a result of advanced peripheral artery disease. To replicate this condition in animals, the femoral artery which feeds the leg is ligated, and perfusion of the leg is measured, usually with Doppler ultrasound. In the mouse model there is a gradual recovery of blood flow as a result of spontaneous angiogenesis (new blood vessel formation). It is believed that bone marrow stem cells are involved in the formation of these new blood vessels.

While it is known that ischemia in the leg muscle is associated with recruitment of stem cells by production of SDF-1, little is known involving the changes that occur in the bone marrow as a result of ischemia in the leg.

Scientists demonstrated that after mice are subjected to hindlimb ischemia, there is a major increase in the production of free radicals in the bone marrow, specifically in the endosteal and central region of the bone marrow. Interestingly, these free radicals appear to be made by the enzyme Nox2 because mice lacking this enzyme do not have free radicals produced in the bone marrow as a result of leg ischemia. The enzyme appears to be expressed mainly in the Gr-1(+) myeloid suppressor cells that are found in the bone marrow. Free radicals were found to be associated with expression of HIF-1 alpha, implying occurrence of localized hypoxia. As can be expected, HIF-1 alpha expression was also found to associate with production of the angiogenic cytokine VEGF. It appeared that bone marrow VEGF expression was associated with expansion of bone marrow Lin(-) progenitor cell survival and expansion, leading to their mobilization into systemic circulation. It was furthermore demonstrated that ischemia of the leg increased expression of the proteolytic enzymes MT1-MMP and MMP-9 activity in the bone marrow, which did not occur in mice lacking Nox2.

The identification of NOX2 as being critical in the mobilization of bone marrow stem cells in response to ischemia suggests that antioxidants may actually modulate the extent of bone marrow stem cell mobilization. Conversely, if one believes the concept proposed, then oxidative stress (at least in a short term setting) would be beneficial towards mobilization. This is supported by studies showing that hyperbaric oxygen induces transient mobilization of bone marrow stem cells. For example Dhar et al. published (Equine peripheral blood-derived mesenchymal stem cells: Isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment. Equine Vet J. 2012 Feb 15) that hyperbaric oxygen treatment in horses increased yield of mesenchymal stem cells collected from peripheral blood. Thom et al (Vasculogenic stem cell mobilization and wound recruitment in diabetic patients: increased cell number and intracellular regulatory protein content associated with hyperbaric oxygen therapy. Wound Repair Regen. 2011 Mar-Apr;19(2):149-61) reported 2-fold increases in hematopoietic stem cells (identified by CD34 expression) in diabetic patients who received hyperbaric oxygen. This study also demonstrated that the CD34 cells that were found in circulation contained high expression of HIF-1 alpha, implying that they may possess angiogenic activity. An interesting experiment would have been if they removed the cells and assessed in vitro angiogenic activity. Indeed it is known that in patients with diabetes the CD34 cells possess a reduced angiogenic activity. If hyperbaric oxygen stimulates this angiogenic activity, it may be a relatively non-invasive method of augmenting the “rejuvenation” potential of the patient’s own stem cells. Another interesting finding of the study was that hyperbaric oxygen was associated with an increase in nitric oxide production by platelets. Since nitric oxide can act as an anticoagulant, this may be another benefit of using hyperbaric oxygen.

One important question is the potency of the stem cell mobilization induced by hyperbaric oxygen. Specifically, while it is nice that an increase in CD34 cells is observed, what activity do these cells actually have ? An earlier study by Thom et al (Stem Cell Mobilization by Hyperbaric Oxygen. Am J Physiol Heart Circ Physiol. 2006 Apr;290(4):H1378-86) demonstrated that the colony-forming ability of the mobilized cells was actually 16-20 fold higher compared to controls. Colony-forming ability is an assessment of the stem cells to generate new cells in vitro.

Thus the paper we discussed sheds some interesting light on the connection between “oxidative medicine” and stem cell biology. Obviously more studies are needed before specific medical recommendations can be made, however, given the large number of patients being treated with alternative medicine techniques such as hyperbaric oxygen, one must ask whether other treatments of this nature also affect stem cells. For example, what about ozone therapy? Or intravenous ascorbic acid?

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