How Cardiospheres Heal the Heart

In 2007, Eduardo Marbán and his colleagues have discovered a stem cell population from the hearts of mice and humans that grow as small balls of cells in culture (see RR Smith, et al., Circulation. 2007 Feb 20;115(7):896-908). He called these cells “cardiospheres” and in a follow-up study showed that these cells have the ability to differentiate into heart muscle cells, blood vessel cells, or other types of heart-specific cells (PV Johnson, et al., Circulation. 2009 Sep 22;120(12):1075-83). Other animal experiments by Marban’s group showed that not only were cardiospheres easily obtained by means of heart biopsies, but injection of these cells directly into the heart after a heart attack augmented healing of the heart and accelerated the recovery of heart function and while preserving heart structure (ST Lee, et al., J Am Coll Cardiol. 2011 Jan 25;57(4):455-65; CA Carr, et al., PLoS One. 2011;6(10):e25669; Shen D, Cheng K, Marbán E. J Cell Mol Med. 2012 Sep;16(9):2112-6).

All of these very hopeful results in culture and in animal studies eventually gave way to a small human clinical trial in which a heart patient’s own cardiospheres were transplanted into their own hearts.  This clinical trial, the CADUCEUS trial (which stands for cardiosphere-derived autologous stem cells to reverse ventricular dysfunction), prevent patient’s hearts from worsening, but more remarkably, the heart scars of these patients were partially erased 6 months after treatment.  A one-year follow-up showed that patients had improved global heart function that directly correlated to the shrinkage of their heart scars.

These results are very encouraging and Marbán made it clear that he wants to conduct larger clinical trials.  However, he still had a gaggle of unanswered questions about his cardiospheres.  Do these cells affect blood vessel formation?  Can they prevent the enlargement of the heart that occurs after a heart attack (known as cardiac remodeling)?  Can the benefits of these cells be solely linked to their effects on the heart scar?  Do cardiospheres prevent the formation of the heart scar?  Do they only help heal the area of the heart where they are administered or do they also help more far-flung regions of the heart?  These are all good questions, and answers to them are necessary if Marbán and his group is to conduct larger and more intense clinical trials with human heart patients.  Therefore, he turned to an animal model system to address these questions in detail.  In particular, he chose Wistar Kyoto rats.

Readers of this blog will recognize the experimental strategy; break the rats into three groups, induce experimental heart attacks in two groups, give one group cultured cardiospheres and leave the other one alone.  Thus you have a sham group that underwent surgery but was not given a heart attack, a heart attack group that did not receive cardiospheres, and a heart attack group into which 2 million rat cardiospheres were injected at four different sites near the site of the infarct.

This experiment, did far more than simply monitor the heart function of the animals for several weeks.  Instead, some of these animals were sacrificed and their hearts were subjected to extensive biochemical and molecular biological tests.   The goal of these experiments was to determine not just if the cardiospheres helped heal the heart.  Marbán and his group already knew that they do.  They wanted to know how they heal the heart.

The cardiosphere-treated animals showed substantial improvements in their heart function as opposed to their non-treated counterparts.  The treated animals had heart that did not undergo remodeling and also pumped better.  Hearts from the cardiosphere-treated animals had less dead heart tissue and more live tissue.  They had smaller heart scars, and better preservation of cellular structure in the heart.  When biochemical markers of proliferating cells were measured in these hearts, the cardiosphere-treated hearts showed robust increases in cell proliferation far above those hearts that were not treated with cardiospheres.  Thus cardiospheres seem to induce resident heart cells to divide and replace dead and dying heart cells.

A common response to a heart attack is that the surviving heart cells enlarge (hypertrophy).  The cardiosphere-treated hearts showed no such response.  Also, when the blood vessel density of the heart tissue was determined, the cardiosphere-treated hearts had close the twice the vessel density of the non-treated hearts.  This was the case near the site of cardiosphere injection, but it also held, albeit not as robustly, in areas far from the site of cardiosphere injection.  This suggests that blood vessel formation is due to secreted molecules.

To test this possibility, Marbán and his crew rigged a culture assay in which rings of tissue from the aorta (the largest blood vessel in the body), were embedded in collagen and treated with culture media from cardiospheres, standard culture cell culture media, or cell culture medium from endothelial cells.  The cardiosphere culture medium, which contains a cocktail of molecules secreted by growing cardiospheres as they have grown in culture, induced far more blood vessels in this system than the other two.  This confirms the notion that cardiospheres secrete blood vessels-inducing molecules that this increases the vascularization of the heart muscle, this aiding its survival.

Marbán and his team also examined the molecule that forms the heart scar; collagen and how cardiospheres affect the synthesis and deposition of collagen.  They discovered that cardiospheres actually degrade the collagen at the heart scar.  They showed that cardiosphere secrete enzymes that have been documented to degrade collagen (Matrix Metalloproteases 2 and 13 for those who are interested).  Marbán and others also discovered that cardiospheres put the kibosh on collagen synthesis.  When they measured biochemical markers of collagen synthesis (hydroxyproline), they were present at rather low levels.  Thus cardiospheres prevent the deposition of the heart scar and also actively degrade it.

Thus, Marbán and his colleagues showed that cardiospheres: 1) prevent the tissue-level changes associated with cardiac remodeling; 2) preserve heart function locally and globally; 3) increase the proliferation of heart muscle cells at the site of the infarct, and to a lesser effect, throughout the heart; 4) induce the formation of new blood vessels at the site of injection, and, to a lesser extend, further from the site of cardiosphere injection; and 5) actively prevent the formation of the heart scar by inhibiting its formation and degrading whatever collagen has been deposited.

Thus cardiospheres decrease the formation of collagen and therefore, decrease the stiffness of the wall of the heart.  They also product new blood vessels and provide a supportive environment for the formation of new heart muscle cells.

This paper was published in PLoS One (2014) 9(2):e88590.

May Marbán’s clinical trials increase!!

Transplanted Human Umbilical Cord Blood Cells Improved Long-Term Heart Muscle Structure and Function in Rats After a Heart Attack

Jianyi Zhang, from the University of Minnesota Health Science Center, in Minneapolis, Minnesota and his co-workers have shown that the transplantation of human umbilical cord blood cells into the rat hearts after a heart attack experience long-term effects that are not observed in the control animals that did not receive the stem cells. Furthermore, none of these laboratory animals required immunosuppressive therapy. The study is scheduled to be published in the journal Cell Transplantation.

“Myocardial infarction induced by coronary artery disease is one of the major causes of heart attack,” said Dr. Zhang. “Because of the loss of viable myocardium after an MI, the heart works under elevated wall stress, which results in progressive myocardial hypertrophy and left ventricular dilation that leads to heart failure. We investigated the long-term effects of stem cell therapy using human non-hematopoietic umbilical cord blood stem cells (nh-UCBCs). These cells have previously exhibited neuro-restorative effects in a rodent model of ischemic brain injury in terms of improved LV function and myocardial fiber structure, the three-dimensional architecture of which make the heart an efficient pump.”

According to Zhang and his co-authors, stem cell researchers have intently examined the ability of stem cells to regenerate and heal damaged heart tissue. Many laboratories all over the world have employed different types of stem cells, different animal models, and distinct modes of stem cell delivery into the heart tissue, and different stem cell doses. All of these studies have produced varying levels of improvement of left ventricular function. Zhang and others also note that, for the most part, the underlying mechanisms by which implanted stem cells improve heart function are “poorly understood and that the overall regeneration of heart muscle cells is modest at best.

In order to investigate the heart’s remodeling processes and to characterize the alterations in cardiac fiber architecture, Zhang’s team used diffusion tensor MRI (DTMRI), which has been previously used to study heart muscle fiber structure in both humans and animals. Most previous studies have concentrated on the short-term effects of umbilical cord blood cells (UCBCs) on damaged heart muscles. Fortunately, this study, which examined the long-term effects of UCBCs, not only demonstrated evidence of significantly improved heart function in treated rats, but also showed evidence of delay and prevention of myocardial fiber structural remodeling. Keep in mind that such alterations in heart muscle fiber structure could have resulted in heart failure.

When compared to the age-matched but untreated rat hearts that had suffered a heart attack, the regional heart muscle function of non-hematopoietic UCBC-treated hearts was significantly improved and the preserved myocardial fiber structure seems to have served as an “underlying mechanism for the observed function improvements.”

“Our data demonstrate that nh-UCBC treatment preserves myocardial fiber structure that supports the improved LV regional and chamber function,” concluded the researchers.

“This study provides evidence that UCBCs could be a potential therapy with long-term benefits for MI” said Dr. Amit N. Patel, director of cardiovascular regenerative medicine at the University of Utah and section editor for Cell Transplantation. “Preservation of the myocardial fiber structure is an important step towards finding an effective therapy for MIs”

See: Chen, Y.; Ye, L.; Zhong, J.; Li, X.; Yan, C.; Chandler, M. P.; Calvin, S.; Xiao, F.; Negia, M.; Low, W. C.; Zhang, J.; Yu, X. The Structural Basis of Functional Improvement in Response to Human Umbilical Cord Blood Stem Cell Transplantation . Cell Transplant. Appeared or available online: December 10, 2013.