Enzyme Helps Stem Cells Improve Recovery From Limb Injury


Ischemia refers to the absence of oxygen in a tissue or organ. Ischemia can cause cells to die and organs to fail and protecting cells, tissues and organs from ischemia-based damaged is an important research topic.

Perfusion refers to the restoration of the blood flow to an organ or tissue that had experienced a cessation of blood flow for a period of time. Even though the restoration of circulation is far preferable to ischemia, perfusion has its own share of side effects. For example, perfusion heightens cells death and inflammation and this can greatly exacerbate the physical condition of the patient after a heart attack, traumatic limb injury, or organ donation.

“Think about trying to hold onto a nuclear power plant after you unplug the electricity and cannot pump water to cool it down,” said Jack Yu, Chief of Medical College of Georgia’s Section of Plastic and Reconstructive Surgery. “All kinds of bad things start happening,”

Earlier studies in the laboratory of Babak Baban have shown that stem cells can improve new blood vessel growth and turn down the severe inflammation after perfusion (see Baban, et al., Am J Physiol Regul Integr Comp Physiol. 2012 Dec;303(11):R1136-46 and Mozaffari MS, Am J Cardiovasc Dis. 2013 Nov 1;3(4):180-96). Baban is an immunologist in the Medical College of Georgia and College of Dental Medicine at Georgia Regents University.

The new study from the Baban laboratory shows that an enzyme called indolamine 2,3,-dioxygenase or IDO can regulate inflammation during perfusion. IDO is widely known to generate immune tolerance and dampen the immune response in the developing embryo and fetus, but it turns out that stem cells also make this enzyme.

In their study, Including IDO with bone marrow-derived stem cells increased the healing efficiency of injected stem cells.

 Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment). The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present. doi:10.1371/journal.pone.0095720.g001
Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment).
The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present.
doi:10.1371/journal.pone.0095720.g001

Also indicators of inflammation, swelling, and cell death decreased in animals that received bone marrow-derived stem cell injections and had IDO.  Baban’s group also showed that the injected stem cells increased endogenous expression of IDO in the perfused tissues.

BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury. Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification
BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury.
Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification

Baban thinks that even though these experiments were performed in mice, because mice tend to be a rather good model system for limb perfusion/ischemia, these results might be applicable in the clinic.  “We don’t want to turn off the immune system, we want to turn it back to normal,” said Baban

According to Baban’s collaborator, Jack Yu, even a short period of inadequate blood supply and nutrients results in the rapid accumulation of destructive acidic metabolites, reactive oxygen species (also known as free radicals), and cellular damage.  The power plant of the cell, small structures called the mitochondria, tend to be one of the earliest casualties of ischemia/perfusion.  Since mitochondria require oxygen to make a chemical called ATP, which is the energy currency in cells, a lack of oxygen makes the mitochondria leaky, swollen and dysfunctional.

“The mitochondria are very sick,” said Yu. ” When blood flow is restored, it can put huge additional stress on sick powerhouses.  “They start to leak things that should not be outside the mitochondria.”

Without adequate energy production and a cellular power plant that leaks, the cells fill with toxic byproducts that cause the cells to commit a kind of cellular hari-kari.  Inflammation is a response to dying cells, since the role of inflammation is to remove dead or dying cells, but inflammation can give the coup de grace to cells that are already on the edge.  Therefore, inflammation can worsen the problem of cell death.

Even though these results were applied to limb ischemia perfusion, Baban and his colleagues think that their results are applicable to other types of ischemia perfusion events, such as heart attacks and deep burns.  Yu, for example, has noticed that in the case of burn patients, the transplantation of new tissue into areas that have undergone ischemia perfusion can die off even while the patient is still in the operating room.

“It cuts across many individual disease conditions,”  said Yu.  Transplant centers already are experimenting with pulsing donor organs to prevent reperfusion trauma.

The next experiments will include determining if more is better.  That is, if giving more stem cells will improve the condition of the injured animal.  In these experiments, which were published in the journal PLoS One, only one stem cell dose was given.  Also, IDO-enhancing drugs will be examined for their ability to prevent reperfusion damage.

Even though stem cells are not given to patients in hospitals after reperfusion, stem cell-based treatments are being tested for their ability to augment healing after reperfusion.  Presently, physicians reestablish blood flow and then give broad-spectrum antibiotics.  The results are inconsistent.  Hopefully, this work by Baban and others will pave the road for future work that leads to human clinical trials.

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mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).