Delivery of a Missing Protein Heals Damaged Hearts in Animals

Stanford University School of Medicine scientists have enabled the regeneration of damaged heart tissue in animals by delivering a protein to it by means of a bioengineered collagen patch.

“This finding opens the door to a completely revolutionary treatment,” said Pilar Ruiz-Lozano, PhD, associate professor of pediatrics at Stanford. “There is currently no effective treatment to reverse the scarring in the heart after heart attacks.”

Ruiz-Lozano and her colleagues published their data online in the journal Nature.

During a heart attack, cardiac muscle cells or cardiomyocytes die from a lack of blood flow. Replacing dead cells is vital for the organ to fully recover, but, unfortunately, the adult mammalian heart does not possess a great deal of regenerative ability. Therefore, scar tissue forms instead of heart muscle, and since scar tissue does not contract, it compromises the ability of the heart to function properly.

Heart attacks kill millions of people every year, and the number of heart attacks is predicted to rise precipitously in the next few decades. The number of heart attacks might even triple by 2030. Approximately, 735,000 Americans suffer a heart attack each year, and even though many victims survive the initial injury, the resulting loss of cardiomyocytes can lead to heart failure and even death. “Consequently, most survivors face a long and progressive course of heart failure, with poor quality of life and very high medical costs,” Ruiz-Lozano said. Transplanting healthy muscle cells and stem cells into a damaged heart have been tried, but these trials have mixed results, typically, and have yet to produce consistent success in promoting healing of the heart.

Previous heart regeneration studies in zebrafish have shown that the outer layers of the heart, known as the epicardium, is one of the driving tissues for healing a damaged heart. Ruiz-Lozano said, “We wanted to know what in the epicardium stimulates the myocardium, the muscle of the heart, to regenerate.” Since adult mammalian hearts do not regenerate effectively, Ruiz-Lozano and her co-workers wanted to know whether epicardial substances might stimulate regeneration in mammalian hearts and restore function after a heart attack.

She and her colleagues focused on Fstl1, which is a protein secreted by the epicardium, and acts as a growth factor for cardiomyocytes. Not only did this protein kick-start the proliferation of cardiomyocytes in petri dishes, but Ruiz-Lozano and others found that it was missing from damaged epicardial tissue following heart attacks in humans.

Next, Ruiz-Lozano and her colleagues reintroduced Fstl1 back into the damaged epicardial tissue of mice and pigs that had suffered a heart attack. They embedded a bioengineered patch on to the damaged heart tissue that was imbued with Fstl1. Then they sutured the patch, loaded with Fstl1, to the damaged tissue. These patches were made of natural material known as collagen that had been structurally modified to mimic certain mechanical properties of the epicardium.

Because the patches are made of collagen, they contain no cells, which mean that recipients do not need immunosuppressive drugs to avoid rejection. With time, the collagen material is absorbed into the heart. The elasticity of the material resembles that of the fetal heart, and seems to be one of the keys to providing a hospitable environment for muscle regrowth. New blood vessels regenerated there as well.

Within two to four weeks of receiving the patch, heart muscle cells began to proliferate and the animals progressively recovered heart function. “Many were so sick prior to getting the patch that they would have been candidates for heart transplantation,” Ruiz-Lozano said. The hope is that a similar procedure could eventually be used in human heart-attack patients who suffer severe heart damage.

The work integrated the efforts of multiple labs around the world, including labs at the Sanford-Burnham-Prebys Medical Discovery Institute in San Diego, UC-San Diego, Boston University School of Medicine, Imperial College London and Shanghai Institutes for Biological Sciences.

Stanford has a patent on the patch, and Ruiz-Lozano is chief scientific officer at Epikabio Inc., which has an exclusive option to license this technology.

Modified RNA Induces Vascular Regeneration After a Heart Attack

Regenerating the heart after a heart attack remains one of the Holy Grails of regenerative medicine. It is a daunting task. Even though text books may say, “the heart is just a pump,” this pump has a lot of tricks up its sleeve.

Stem cell treatments can certainly improve the structure and function of the heart after a heart attack, but getting the heart back to where it was before the heart attack is a whole different ball game. To truly regenerate, the heart, the organ or parts of it need to be reprogrammed to a time when the heart could regenerate itself. If that sounds difficult, it’s because it is. But some recent work suggests that it might at least partially possible.

Kenneth Chien and his colleagues from the Department of Stem Cell Biology and Regenerative Medicine at Harvard University have published a terrific paper in the journal Nature Biotechnology that tries to turn back to clock of the heart to augment its regenerative capabilities.

The outermost layer of the heart that surrounds the heart muscle is a layer called the “epicardium.”

epicardiumIn the epicardium are epicardial heart progenitors and these cells are activated within 48 hours after a heart attack in the mouse.  In the fetal heart, epicardial heart progenitors migrate into the heart and differentiate into heart muscle, blood vessels and smooth muscles.  In adults, these cells remain on the surface of the heart and differentiate largely into fibroblasts.  When it comes to regenerative medicine, can we take adult epicardial cells and reprogram them to act like fetal epicardial heart progenitors?

A few experiments have suggested that we can.  In 2011, Smart and others used a small peptide called thymosin β4 to reprogram epicardial cells in mice to form heart muscle and other heart-specific tissues.  Even though the reprogramming was not terribly robust, Smart and others convincingly showed that it was real (Nature 474,640–644).

The Chien group used modified RNA molecules made with unusual nucleotides that encoded the protein vascular endothelial growth factor-A (VEGF-A) to reprogram the epicardium of mice.  VEGF-A is very good and reprogramming the epicardium, and this modified RNA technique does not induce and immune response the way injecting DNA does and the RNA causes bursts of VEGF-A activity that efficiently reprograms the epicardium.

After giving mice heart attacks, Chien and others injected the VEGF-A modified RNAs into the border of the infarcted area of the heart. The modified RNAs induced new gene expression that is normally seen during the establishment of blood vessels.  VEGF-A expression was elevated for up to 6 days after the injections, and animals that had their hearts injected with modified VEGF-A RNA had smaller scars in their hearts, less cell death, and greater tissue volume in their hearts than animals that received either injections of VEGF-A DNA, buffer, or modified RNA that expressed a glowing protein.  Also, the effects of the modified VEGF-A RNA could be abrogated with co-administrating the drug Avastin, which is an antagonist of VEGF-A

Tests with cultured heart cells showed that VEGF-A modified RNA induced blood vessel-specific genes.  These inductions were sensitive to drugs that blocked the VEGF-A receptor, which shows that it is indeed the VEGF-A protein that is inducing these trends.  Finally, a heart muscle gene, Tnnt2 is also induced by the modified VEGF-A RNA.  When the efficacy of the modified VEGF-A RNA was tested in living animals, if was clear that the most numerous cells induced by the modified VEGF-A RNA was endothelial cells, which line blood vessels, followed by smooth muscle cells, and then by heart muscle cells.

Thus, the growth factor VEGF-A can signal to epicardial heart progenitor cells to heal the heart after a heart attack in mice.  It works through the VEGF-A receptor (KDR), and it induces epicardial derived cells (EPDCs) to differentiate into blood vessels, heart muscle cells, and smooth muscle cells, all of which are required to heal the heart.  If VEGF-A signaling can be used to augment heart healing after a heart attack, it might provide a new strategy for healing the heart after a heart attack in a manner that helps the heart heal itself from the inside rather than placing something from the outside into it.

Stem Cells in the Epicardium of the Heart

Congestive heart failure is the leading cause of morbidity and mortality worldwide. Implanting stem cells into the damaged heart to regenerate the dead heart cells is a potentially exciting prospect for regenerative medicine. Finding the right cell for the job is the greatest challenge, and to this end the heart itself may provide an interesting source of stem cells for regenerative medicine. This source of cells resides on the outside of the heart, a layer known as the epicardium.

Epicardial cells

After a heart attack, the cells of the epicardium differentiate into smooth muscle cells and heart-specific fibroblasts. They do not form heart muscle cells or blood vessels, but they do secrete a whole cadre of growth factors that encourage the heart to form blood vessels. In mice, preconditioning the heart with a protein called “thymosin beta4” induces the epicardial cells to migrate into the heart and form new heart muscle cells (Smart et al., Nature 2001 474: 640-4). Unfortunately, using thymosin beta4 in human patients who have had heart attacks fails to elicit appropriate changes in the myocardium (Zhou et al., J Mol Cell Cardiol 2012 52: 43-7).

Chong and his colleagues have discovered a new stem cell in the epicardium of mice that can grow for long periods of time in culture and are found near blood vessels. Chong and others call this new epicardial stem cell a “cardiac colony-forming-unit fibroblast” or cCFU-F for short (Chong et al., Cell Stem Cell 2011 9: 527-40).

These cCFU-Fs form from the epicardium, but they do not express heart-specific genes (e.g., c-Kit, CD31, Flk1, CD45, Nkx2-5, NG2). When the gene expression profile of cCFU-Fs was examined in some detail, they expressed the same clusters of genes as bone marrow stem cells (Pelekanos et al., Stem Cell Research 2012 8: 58-73). Are these two cell populations the same? Apparently not. Chong and his crew tried to reconstitute the cCFU-Fs in mice that had had their bone marrow completely replaced with green-glowing bone marrow stem cells. The glowing bone marrow stem cells never contributed to the cCFU-F population.

Therefore, can cCFU-Fs contribute to heart regeneration after a heart attack? Can they be primed to form heart muscle with thymosin beta4? Many questions abound, but these cCFU-Fs seem to represent an easily accessed and robust population for regenerative medicine for the heart after a heart attack.