Growth Factor Delivery Stimulates Endogenous Heart Repair After Heart Attacks in Pigs

Steven Chamulean and his colleagues at the University Medical Center Utrecht in Holland have examined the use of growth factors to induce healing in the heart after a heart attack. Because simply applying growth factors to the heart will cause them to simply be washed out, Chamulean and his coworkers embedded the growth factors in a material called hydrogel. They were able to measure how long the implanted growth factors lasted. As it turns out, when the growth factors were embedded in the hydrogel, they lasted for four days, and the hydrogel caused the growth factors to spread out into heart tissue with a gradient with the highest concentration at the site of injection (see Bastings, et al., Advanced Healthcare Materials 2013 doi: 10.1002/adhm.201300076).

In his new publication in the Journal of Cardiovascular Translational Research, Chamulean and his group used a new hydrogen called UPy to into which they embedded their growth factors. UPy stands for ureido-pyrimidinone end-capped poly(ethylene glycol) polymer. At the pH of our bodies, UPy hydrogels form a gel-like material made of fibers. When the pH changes, the gel becomes liquid. They embedded the growth factors insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF).

The experimental design of this paper used pigs that were given heart attacks and then reperfused 75 minutes later. One month later, the animals were broken into three groups: just hydrogel, hydrogel with growth factors embedded in it, and growth factors injected into other heart without hydrogel. One month later, the animals were examined for their heart function, and then the animals were sacrificed to examine their heart tissue.

In every case, the hearts treated with only the hydrogel did the poorest of the three groups. The animals injected with gel-less growth factors did better than the controls, but those animals treated with growth factors embedded in UPy hydrogel did the best. The physiological indicators of the hearts from the animals treated with UPy embedded with IGF-1 and HGF improved significantly more than the controls that were treated with only UPy hydrogel. The hearts from animals treated with IGF and HGF without hydrogels improved over controls, by not nearly as well as those treated with growth factor-embedded UPy hydrogels.

When the hearts were examined even more surprises were observed. The animals with hearts that had been treated with UPy + growth factors did not show the enlargement observed in the control hearts. This is significant, because enlargement of the heart is a side effect for a heart attack and is the sign of heart failure. The UPy + growth factor hearts also displayed many signs of dividing cells; far more than hearts from the other two groups. Since the heart has its own resident stem cell population, these growth factors stimulated these stem cells to divide and form new heart muscle, and new blood vessels. Blood vessel density was much higher in the UPy + growth factor group and the pressure against which blood flowed in these hearts was substantially less in this groups, demonstrating that not only was the blood vessel density higher, but blood flow through these vessel networks was much more efficient. There was also plentiful evidence of the formation of new muscle in the UPy + growth factor group. When these hearts were also stained for c-kit, which is a cell surface marker for cardiac stem cells, the UPy + growth factor hearts had lots of them – much more than the other two groups.

This paper reports significant findings because the resident stem cell population in the heart was actively mobilized without having to extract them by means of a biopsy. There is also evidence from Torella and others that IGF-1 and HGF can reactivate the sleeping cardiac stem cells of aged laboratory animals (Circulation Research 2004 94: 514-524). The UP{y hydrogels are well tolerated and are biodegradable. They provide a medium that stays in place and releases embedded growth factors in a sustained manner. The results in this paper provide the rationale to develop growth factor therapy for human patients.

When Is the Best Time to Treat Heart Attack Patients With Stem Cells?

Several preclinical trials in laboratory animals and clinical trials have definitively demonstrated the efficacy of stem cell treatments after a heart attack. However, these same studies have left several question largely unresolved. For example, when is the best time to treat acute heart attack patients? What is the appropriate stem cell dose? What is the best way to administer these stem cells? Is it better to use a patient’s own stem cells or stem cells from someone else?

A recent clinical trial from Soochow University in Suzhou, China has addressed the question of when to treat heart attack patients. Published in the Life Sciences section of the journal Science China, Yi Huan Chen and Xiao Mei Teng and their colleagues in the laboratory of Zen Ya Shen administered bone marrow-derived mesenchymal stromal cells at different times after a heart attack. Their study also examined the effects of mesenchymal stem cells transplants at different times after a heart attack in Taihu Meishan pigs. This combination of preclinical and clinical studies makes this paper a very powerful piece of research indeed.

The results of the clinical trial came from 42 heart attack patients who were treated 3 hours after suffering a heart attack, or 1 day, 3 days, 2 weeks or 4 weeks after a heart attack. The patients were evaluated with echocardiogram to ascertain heart function and magnetic resonance imaging of the heart to determine the size of the heart scar, the thickness of the heart wall, and the amount of blood pumped per heart beat (stroke volume).

When the data were complied and analyzed, patients who received their stem cell transplants 2-4 weeks after their heart attacks fared better than the other groups. The heart function improved substantially and the size of the infarct shrank the most. 4 weeks was better than 2 weeks,

The animal studies showed very similar results.

Eight patients were selected to receive additional stem cell transplants. These patients showed even greater improvements in heart function (ejection fraction improved to an average of 51.9% s opposed to 39.3% for the controls).

These results show that 2-4 weeks constitutes the optimal window for stem cell transplantation. If the transplant is given too early, then the environment of he heart is simply too hostile to support the survival of the stem cells. However, if the transplant is performed too late, the heart has already experiences a large amount of cell death, and a stem cell treatment might be superfluous. Instead 2-4 weeks appears to be the “sweet spot” when the heart is hospitable enough to support the survival of the transplanted stem cells and benefit from their healing properties. Also, this paper shows that multiple stem cell transplants a two different times to convey additional benefits, and should be considered under certain conditions.

Grafted Stem Cell Derivatives Restore Normal Heart Rhythms in Mice

American researchers, in collaboration with technicians from Fujifilm VisualSonics, Inc., have used advanced ultrasonic software to document microscopic, regenerative improvements to heart muscle that has suffered from previous damage.

High-frequency ultrasound and special cardiac-assessment software was developed by FujiFilm VisualSonics, Inc of Toronto, Canada. Scientists from Mayo Clinic implanted engineered cells into the damaged hearts of mice and then used the special software and ultrasound imaging to observe the regeneration of the heart so that it began to contract with normal cardiac rhythms.

After a heart attack, dead heart tissue is replaced with a cardiac scar that consists of scar tissue that neither contracts nor conducts the signals to contract. Depending of the size of the heart scar, the heart can beat abnormally. An abnormal heart beat is known as arrhythmia. Arrhthymias come in three different categories: a heart that beats too fast (tachycardia), a heart that beats too slowly (bradycardia), and a heart that beats erratically. Arrhythmias after a heart attack can be life-threatening, and restoring normal heart rhythm to the heart after a heart attack is very important.

In this experiment, mice were given heart attacks, and then undifferentiated induced pluripotent stem cells (iPSCs) were implanted into these hearts. Those mice that received induced pluripotent stem cells gradually normalized, their heart beat. The resynchronization of the heart beat of these mice was imaged with high-resolution ultrasound.

Satsuki Yamada, first author of this paper, said, “A high-resolution ultrasound revealed harmonized pumping [of the heart] where iPS cells were introduced to be the previously damaged heart tissue.” Yamada also noted that Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, and achieves resynchronization of the heart beat after a heart attack.

This experiment shows, for the first time that undifferentiated iPSCs have the potential to stabilize a patient’s heart after a heart attack. The healing of the heart was documented by ultrasound imaging and by “speckle-tracking echocardiogram.,” Speckle-tracking echocardiography was designed by VevoStrain Advanced Cardiac Analysis Software, which was manufactured by VisualSonics.

This software package provides advanced imaging and quantification capabilities for studying sensitive movements in heart muscles and it is also the only commercial cardiac-strain package optimized for assessing cardiovascular function preclinical rodent studies.

Yamada and her co-researchers utilized this software during the implantation and observation of the iPSCs within the hearts of mice. This software package the motion of the heart wall both at the regional and global levels and from several different perspectives, measurements of these movements, the changes in dimension in the left ventricle during the heart cycle.

The software definitely showed that homogeneous wall movement was restored in those mice that had received implants of iPSCs.

When iPSCs were implanted into mice that had dysfunctional immune systems, they produced tumors, but in mice with normal immune systems, the implanted iPSCs did not produce tumors. What became of those cells is uncertain, but they clearly helped heal the heart and did not cause tumors.

Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.
Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.

This paper is interesting and suggests that undifferentiated cells can also exert healing effects on the heart.

Inhibition of a Heart-Specific Enzyme After a Heart Attack Decreases Heart Damage and Prevents Remodeling

Cardiac Troponin I-interacting Kinase or TNNI3K is an enzyme that was initially identified in fetal and adult heart tissue, but was undetectable in other tissues. The function of this enzyme remains unknown, but Chinese scientists showed that overexpression of TNNI3K in cultured heart muscle cells causes them to blow up and get large (hypertrophy). Earlier this year, a research team from Peking Union Medical College showed that overexpression of TNNI3K in mice caused enlargement of the heart (Tang H., et al., J Mol Cell Cardiol 54 (2013): 101-111). These results suggested that TNNI3K is a potential therapeutic target for heart attack patients.

To that end, Ronald Vagnozzi and his colleagues in the laboratory of Thomas Force at Temple University School of Medicine and their collaborators designed small molecules that can inhibit TNNI3K activity, and these small molecules decrease cardiac remodeling after a heart attack in rodents. Large animal trials are planned next.

In the first experiments of this paper, Vagnozzi and others showed that the levels of TNNI3K in the heart increase after a heart attack. Measurements of TNNI3K protein levels failed to detect it in all tissue other than the heart. Furthermore, it was present throughout the heart, and mainly in heart muscle and not in blood vessels, fibroblasts, and other types of non-muscle heart tissues.

Next, Vagnozzi and others measured TNNI3K protein levels in heart transplant patients. The heart tissues of these patients, who had badly dysfunctional hearts showed higher than usual levels of TNNI3K protein. Thus, TNNI3K is associated with heart tissue and is up-regulated in response to heart dysfunction.

The next experiment examined the effects of overexpressing the human TNNI3K gene in mice. While the overexpression of TNNI3K did not affect heart function of structure under normal circumstances, under pathological conditions, however, this is not he case. If mice that overexpressed TNNI3K where given heart attacks and then “reperfused,” means that the blood vessel that was tied off to cause the heart attack was opened and blood flowed back into the infarcted area. In these cases, mice that overexpressed TNNI3K had a larger area of cell death in their hearts than their counterparts that did not overexpress TNNI3K. The reason for this increased cell death had to do with the compartment in the cell that generated most of the energy – the mitochondrion. TNNI3K causes the mitochondria in heart muscle cells to go haywire and kick out all kinds of reactive oxygen-containing molecules that damage cells.

Cell damage as a result of reactive oxygen-containing molecules (known as reactive oxygen species or ROS) activates a pathway in heart cells called the “p38” pathway, which leads to programmed cell death.

p38 signaling

Once Vagnozzi and his colleagues nailed down the function of TNNI3K in heart muscle cells after a heart attack, they deleted the gene that encodes TNNI3K and gave those TNNI3K-deficient mice heart attacks. Interestingly enough, after a heart attack, TNNI3K-deficient mice showed much small dead areas than normal mice. Also, the levels of the other mediators of TNNI3K-induced cell death (e.g., oxygen-containing molecules, p38, ect.) were quite low. This confirms the earlier observations that TNNI3K mediates the death of heart muscle cells after a heart attack, and inhibiting TNNI3K activity decreases the deleterious effects of a heart attack.

And now for the pièce de résistance – Vagnozzi and his crew synthesized small molecules that inhibited TNNI3K in the test tube. Then they gave mice heart attacks and injected these molecules into the bellies of the mice. Not only were the infarcts, or areas of dead heart muscle cells small in the mice injected with these TNNI3K inhibitors, but the heart of these same mice did not undergo remodeling and did not enlarge, showed reduced scarring, and better ventricular function. This is a proof-of-principle that inhibiting TNNI3K can reduce the pathological effects of a heart attack.

This strategy must be tested in large animals before it can move to human trials, but the strategy seems sound at this point, and it may revolutionize the treatment of heart attack patients.