Mesenchymal Stem Cells Engineered to Express Tissue Kallikrein Increase Recovery After a Heart Attack


Julie Chao is from the Department of Biochemistry and Molecular Biology, at the Medical University of South Carolina. Dr. Chao and her colleagues have published a paper in Circulation Journal about genetically modified mesenchymal stem cells and their ability to help heal a heart that has just experienced a heart attack.

Several laboratories have used mesenchymal stem cells (MSCs), particularly from bone marrow, to treat the hearts of laboratory animals that have recently experienced a heart attack. However, heart muscle after a heart attack is a very hostile place, and implanted MSCs tend to pack up and die soon after injection. Therefore, such injected cells do little good.

To fix this problem, researchers have tried preconditioning cells by growing them in a harsh environment or by genetically engineering them with genes that can increase their tolerance of harsh environments. Both procedures have worked rather well. In this paper, Chao and her group engineered bone marrow-derived MSCs to express the genes that encode “tissue kallikrein” (TK). TK circulates throughout our bloodstream but several different types of cells also secrete it. It is an enzyme that degrades the protein “kininogen” into small bits that have several benefits. Earlier studies from Chao’s own laboratory showed that genetically engineering TK into the heart improved heart function after a heart attack and increased the ability of MSCs to withstand harsh conditions (see Agata J, Chao L, Chao J. Hypertension 2002; 40: 653 – 659; Yin H, Chao L, Chao J. Journal of  Biol Chem 2005; 280: 8022 – 8030). Therefore, Chao reasoned that using MSCs engineered to express TK might also increase the ability of MSCs to survive in the post-heart attack heart and heal the damaged heart.

In this paper, Chao and others made adenoviruses that expressed the TK gene. Adenoviruses place genes inside cells, but they do not integrate those genes into the genome of the host cell. Therefore, they are safer to use than retroviruses. Chao and others used these TK-expressing adenoviruses to infect tissue and MSCs.

When TK-expressing MSCs were exposed to low-oxygen conditions, like what cells might experience in a post-heart attack heart, the TK-expressing cells were much heartier than their non-TK-expressing counterparts. When injected into rat hearts 20 minutes after a heart attack had been induced, the TK-expressing MSCs showed good survival and robust TK expression. Control hearts that had been injected with non-TK-expression MSCs or had not been given a heart attack showed no such elevation of TK expression.

There were also added bonuses to TK-expressing MSC injections. The amount of inflammation in the hearts was significantly less in the hearts injected with TK-expressing MSC injections compared to the controls. There were fewer immune cells in the heart 1 day after the heart attack and the genes normally expressed in a heart that is experiencing massive inflammation were expressed at lower levels relative to controls, if they were expressed at all.

Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E) neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups; **P<0.05 vs. MI/Control group. MSC, mesenchymal stem cell.
Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E)
neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows.
Original magnification, ×200. Data are mean ± SEM (n=5–8). *P

Another major bonus to the injection of TK-expressing MSCs into the hearts of rats was that these cells protected the heart muscle cells from programmed cell death. To make sure that this was not some kind of weird artifact, Chao and her team placed the TK-expressing MSCs in culture with heart muscle cells and then exposed them to low-oxygen tension conditions. Sure enough, the heart muscle cells co-cultured with the TK-expressing MSCs survived better than those co-cultured with non-TK-expressing MSCs.

TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups. Cultured cardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as evidenced by (D) Hoechst staining,
TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration
reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic
cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *Pcardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as
evidenced by (D) Hoechst staining,

Finally, when the hearts of the rats were examined 2 weeks after the heart attack, it was clear that the enlargement of the heart muscle (so-called “remodeling”) occurred in animals that had received non-TK-expressing MSCs or had received no MSCs at all, but did not occur in the hearts of rats that had received injections of TK-expressing MSCs. The heart scar was also significantly smaller in the hearts of rats that had received injections of TK-expressing MSCs, and had a greater concentration of new blood vessels. Apparently, the TK-expressing MSCs induced the growth of new blood vessels by recruiting EPCs to the heart to form new blood vessels.

In conclusion, the authors write that “MSCs genetically-modified with human TK are a potential therapeutic for ischemic heart diseases.”

Getting FDA approval for genetically engineered stem cells will not be easy, but TK engineering seems much safer than some of the other modifications that have been used. Also the vascular and cardiac benefits of this gene seem clear in this rodent model. Pre-clinical trials with larger animals whose cardiac physiology is more similar to humans is definitely warranted and should be done before any talk of human clinical trials ensues.

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