**3.1.2 Therapies using CPC for tissue engineering**

#### **3.1.2.1 Tissue engineered myocardial grafts**

Presently, many investigators are attempting to use cardiac stem cells to produce engineered myocardial sheet grafts for myoplasty of larger regions of infarct-damaged adult myocardium. Success has been limited to moderate. Major limiting factors to this approach remain. For example, integration of the graft into the existing myocardium so that it provides clinically significant augmented force development has been problematic. Additionally, achieving sufficient revascularization of the grafts to sustain viability has been difficult; see review by Sui (Sui et al., 2011). Although the potential utility of such grafts in congenital heart disease is not expectedly large, marked progress in this approach could conceivably provide alternative therapies for patients with failing Fontan circulations or possibly for those with cardiomyopathies.

#### **3.1.2.2 Tissue engineered myocardial vascular and valve grafts**

One area of potentially beneficial therapy that has previously received little attention in *congenital* heart disease patients is the engineering of vascular and valve grafts that are capable of meeting the rapid growth rate typical of the neonatal heart and great vessels during the first decade of life. The availability of graft materials with the ability to grow along with the young patient is highly desirable, but thus far not available. This is an area of recently increasing research interest, and one in which further research could provide enormous benefit to CHD patients.

#### **3.1.3 Mobilizing resident CPC**

#### **3.1.3.1 Alternatives to cell delivery: "Activation" of resident CPC**

Given the apparent difficulty in achieving clinically valuable augmentation of cardiac performance through the delivery of cardiomyocytes to a damaged heart, therapeutic

Myocardial Self-Repair and Congenital Heart Disease 283

from the secondary heart field) may participate in recovery from myocardial injury in a region-specific manner has also been shown. These cells apparently migrate and home to infarcted region of rat hearts, although they differentiate into neuronal, not myocardial cells (Beguin et al., 2011). Tamura (Tamura et al., 2011) used a mouse transgenic approach to tag neural crest cells and show that these cells migrated to the ischemic border zone of an infarct and transdifferentiated into cardiomyocytes. Although evidence for Nestin+ cells in the human heart is lacking, it is worth considering the different heart field lineages in the context of devising strategies for therapeutic targeting of human CPC. The targeting of neural crest-derived CPC to correct late ventricular arrhythmias occurring in patients after surgical repair operations for TOF is a potential therapeutic strategy(Di Felice &

In the setting of failing Fontan physiology the potential for boosting cardiac performance through manipulation of cell number represents a new horizon. Ventricular assist devices (VAD) are presently used as a bridge to transplant in pediatric Fontan patients (Fynn-Thompson & Almond, 2007). VAD are also used to reduce ventricular load with the objective of enhancing the ability of the ventricle to support a greater load, i.e., to "rest" or re-train the ventricle of patients with dilated cardiomyopathy (CMP), even restoring function to the point of enabling pump removal (Birks et al., 2011). It is conceivable that the use of VAD may increase cardiomyocyte number, and evidence of this comes from recent prospective studies of myocardial biopsies showing an increased number of diploid myocytes in end stage congestive heart failure patients supported as a bridge to transplant with LVAD (Wohlschlaeger et al., 2010). Manginas (Manginas et al., 2009) has shown that endothelial progenitor cells are also mobilized by VAD use in patients. EPC may be stimulated to home into myocardium supported by VAD and improve myocardial function. As reviewed by Tsiavou (Tsiavou & Manginas, 2010), rescue of myocardial function during cardiac support by VAD may be mediated by CD45+ EPC promoting neovascularization and transdifferentiating into or fusing with cardiomyocytes. However, the possible involvement of CPC fusion with existing myocytes as repair mechanism is not widely accepted. The implications of the above observations are that progenitor cells may at least be participating in, if not be a primary mechanism mediating these physiological changes

What physiological mechanisms might underlie the expansion of the myocardial cell population during VAD support? Current thinking is that paracrine mechanisms are involved. For example, mechanical stimulation by the VAD may induce the production of cytokines such as growth factors (see section 3.1.3.1), which then stimulate mitotic expansion of CPC. Additionally, the production of chemokines under the influence of the same mechanical stimulation may promote the homing of bone marrow-derived progenitor cells. Indeed, much of the success of cell delivery-based therapies, although still rather modest, are thought to be largely due to paracrine effects mediated through the injected cells, the carrier media or in some cases the physical-mechanical changes induced by

Zummo, 2009).

observed with VAD therapy.

injection of liquid boluses into the muscle wall.

**3.2 Potential roles of CPC in therapy for CHD 3.2.1 CPC silently contribute to therapy** 

**3.2.1.1 (Pre-) Failing Fontan rescue via activation-RV strengthening** 

approaches that are designed to mobilize resident CPC to expand the population of cardiomyocytes in situ are being given much more consideration. Therapeutic exploitation of the paracrine environments of the CPC niche and enhancing homing to sites of repair is a very attractive alternative approach to cell-based therapies. In concept, it is a matter of using biomolecules to mimic or enhance endogenous CPC "awakening" mechanisms to obtain greater quantities of cells to differentiate into functional cardiomyocytes. Recent reports reveal that this general approach has a high potential for success and is quite worthy of further investigation. High mobility group box protein 1 (HMGB1), an endogenous chromatin-associated protein, and Thymosine Beta4, a G-actin monomer binding protein, have been identified as paracrine factors potentially able to promote regeneration of myocardium. HMGB1 is released from necrotic cells and has been shown to stimulate the homing of fibroblasts and smooth muscle cells. It has been identified as a potential mediator of resident stem cell activation/mobilization (Palumbo & Bianchi, 2004). Limana (Limana et al., 2005) demonstrated that injection of HMGB1 into the infarcted region of mice induced the appearance of new myocytes and an increase in ventricular performance, leading these investigators to conclude that HMGB1 is a "potent inducer of myocardial regeneration." They demonstrated that c-kit+ CPC express the receptor for HMGB1 and that treatment increased the number of c-kit+ cells in mouse heart. Thymosine Beta4 has been shown to stimulate epicardial-derived cells (EPDC) to migrate and potentially promote neovascularization in the infarcted mouse heart (Smart et al., 2010); see review by (Bollini et al., 2011). Although there was no proof that EPDC were a source of the new cardiomyocytes, they may facilitate collateral vessel growth and thereby support the cardiomyocyte regeneration process. Damaged myocardium has a different paracrine environment, which if properly understood and exploited, may provide unique approaches for therapy via resident CPC activation.

Growth factor treatment has also been used to activate resident CPC populations for myocardial repair. Linke (Linke et al., 2005) used a canine MI model to show that IGF-1 and HGF treatment (intra-myocardial injection) could increase the density of proliferating (Ki67+) CPC following MI. These same investigators recently extended their observations, showing that CPC aging is related to a decline in signaling through IGF-1 and HGF, which in turn reduces their effect to antagonize the aging effect that the local renin-angiotensin system induces on CPC. They found that IGF-1 and HGF were able to partially reverse agerelated decline in cardiac function in rats (Gonzalez et al., 2008). Consistent with these reports of IGF-1 stimulation of CPC proliferation, D'Amario (D'Amario et al., 2011) has proposed that CPC senescence is regulated by paracrine and autocrine signaling: positive through IGF-1 & -2, and balanced by an opposing signal mediated primarily by angiotensin II, all acting via their cognate receptors. Accordingly, they propose that IGF-1 promotes CPC proliferation and survival via IGF-1 receptor signaling, CPC differentiation via action on both IGF-1 and -2 receptors, and increasing angiotensin signaling and reduced IGF-1 receptor signaling with aging promotes CPC and cardiomyocyte apoptosis.

CPC from the secondary/anterior heart field (i.e., Nestin+) are resident in the secondary heart field regions of the rat heart (Drapeau et al., 2005). The recent review by Di Felice (Di Felice & Zummo, 2009) surveyed the many mutations of secondary heart field cells that are associated with human Tetralogy of Fallot. They concluded that potentially improved approaches to therapy would be better informed by a clearer understanding of the behavior and patterns of migration of CPC of the secondary heart field. That neural stem cells (CPC

approaches that are designed to mobilize resident CPC to expand the population of cardiomyocytes in situ are being given much more consideration. Therapeutic exploitation of the paracrine environments of the CPC niche and enhancing homing to sites of repair is a very attractive alternative approach to cell-based therapies. In concept, it is a matter of using biomolecules to mimic or enhance endogenous CPC "awakening" mechanisms to obtain greater quantities of cells to differentiate into functional cardiomyocytes. Recent reports reveal that this general approach has a high potential for success and is quite worthy of further investigation. High mobility group box protein 1 (HMGB1), an endogenous chromatin-associated protein, and Thymosine Beta4, a G-actin monomer binding protein, have been identified as paracrine factors potentially able to promote regeneration of myocardium. HMGB1 is released from necrotic cells and has been shown to stimulate the homing of fibroblasts and smooth muscle cells. It has been identified as a potential mediator of resident stem cell activation/mobilization (Palumbo & Bianchi, 2004). Limana (Limana et al., 2005) demonstrated that injection of HMGB1 into the infarcted region of mice induced the appearance of new myocytes and an increase in ventricular performance, leading these investigators to conclude that HMGB1 is a "potent inducer of myocardial regeneration." They demonstrated that c-kit+ CPC express the receptor for HMGB1 and that treatment increased the number of c-kit+ cells in mouse heart. Thymosine Beta4 has been shown to stimulate epicardial-derived cells (EPDC) to migrate and potentially promote neovascularization in the infarcted mouse heart (Smart et al., 2010); see review by (Bollini et al., 2011). Although there was no proof that EPDC were a source of the new cardiomyocytes, they may facilitate collateral vessel growth and thereby support the cardiomyocyte regeneration process. Damaged myocardium has a different paracrine environment, which if properly understood and exploited, may provide unique approaches for therapy via

Growth factor treatment has also been used to activate resident CPC populations for myocardial repair. Linke (Linke et al., 2005) used a canine MI model to show that IGF-1 and HGF treatment (intra-myocardial injection) could increase the density of proliferating (Ki67+) CPC following MI. These same investigators recently extended their observations, showing that CPC aging is related to a decline in signaling through IGF-1 and HGF, which in turn reduces their effect to antagonize the aging effect that the local renin-angiotensin system induces on CPC. They found that IGF-1 and HGF were able to partially reverse agerelated decline in cardiac function in rats (Gonzalez et al., 2008). Consistent with these reports of IGF-1 stimulation of CPC proliferation, D'Amario (D'Amario et al., 2011) has proposed that CPC senescence is regulated by paracrine and autocrine signaling: positive through IGF-1 & -2, and balanced by an opposing signal mediated primarily by angiotensin II, all acting via their cognate receptors. Accordingly, they propose that IGF-1 promotes CPC proliferation and survival via IGF-1 receptor signaling, CPC differentiation via action on both IGF-1 and -2 receptors, and increasing angiotensin signaling and reduced IGF-1

CPC from the secondary/anterior heart field (i.e., Nestin+) are resident in the secondary heart field regions of the rat heart (Drapeau et al., 2005). The recent review by Di Felice (Di Felice & Zummo, 2009) surveyed the many mutations of secondary heart field cells that are associated with human Tetralogy of Fallot. They concluded that potentially improved approaches to therapy would be better informed by a clearer understanding of the behavior and patterns of migration of CPC of the secondary heart field. That neural stem cells (CPC

receptor signaling with aging promotes CPC and cardiomyocyte apoptosis.

resident CPC activation.

from the secondary heart field) may participate in recovery from myocardial injury in a region-specific manner has also been shown. These cells apparently migrate and home to infarcted region of rat hearts, although they differentiate into neuronal, not myocardial cells (Beguin et al., 2011). Tamura (Tamura et al., 2011) used a mouse transgenic approach to tag neural crest cells and show that these cells migrated to the ischemic border zone of an infarct and transdifferentiated into cardiomyocytes. Although evidence for Nestin+ cells in the human heart is lacking, it is worth considering the different heart field lineages in the context of devising strategies for therapeutic targeting of human CPC. The targeting of neural crest-derived CPC to correct late ventricular arrhythmias occurring in patients after surgical repair operations for TOF is a potential therapeutic strategy(Di Felice & Zummo, 2009).

#### **3.2 Potential roles of CPC in therapy for CHD 3.2.1 CPC silently contribute to therapy**

#### **3.2.1.1 (Pre-) Failing Fontan rescue via activation-RV strengthening**

In the setting of failing Fontan physiology the potential for boosting cardiac performance through manipulation of cell number represents a new horizon. Ventricular assist devices (VAD) are presently used as a bridge to transplant in pediatric Fontan patients (Fynn-Thompson & Almond, 2007). VAD are also used to reduce ventricular load with the objective of enhancing the ability of the ventricle to support a greater load, i.e., to "rest" or re-train the ventricle of patients with dilated cardiomyopathy (CMP), even restoring function to the point of enabling pump removal (Birks et al., 2011). It is conceivable that the use of VAD may increase cardiomyocyte number, and evidence of this comes from recent prospective studies of myocardial biopsies showing an increased number of diploid myocytes in end stage congestive heart failure patients supported as a bridge to transplant with LVAD (Wohlschlaeger et al., 2010). Manginas (Manginas et al., 2009) has shown that endothelial progenitor cells are also mobilized by VAD use in patients. EPC may be stimulated to home into myocardium supported by VAD and improve myocardial function. As reviewed by Tsiavou (Tsiavou & Manginas, 2010), rescue of myocardial function during cardiac support by VAD may be mediated by CD45+ EPC promoting neovascularization and transdifferentiating into or fusing with cardiomyocytes. However, the possible involvement of CPC fusion with existing myocytes as repair mechanism is not widely accepted. The implications of the above observations are that progenitor cells may at least be participating in, if not be a primary mechanism mediating these physiological changes observed with VAD therapy.

What physiological mechanisms might underlie the expansion of the myocardial cell population during VAD support? Current thinking is that paracrine mechanisms are involved. For example, mechanical stimulation by the VAD may induce the production of cytokines such as growth factors (see section 3.1.3.1), which then stimulate mitotic expansion of CPC. Additionally, the production of chemokines under the influence of the same mechanical stimulation may promote the homing of bone marrow-derived progenitor cells. Indeed, much of the success of cell delivery-based therapies, although still rather modest, are thought to be largely due to paracrine effects mediated through the injected cells, the carrier media or in some cases the physical-mechanical changes induced by injection of liquid boluses into the muscle wall.

Myocardial Self-Repair and Congenital Heart Disease 285

with expansion of the myocardial cell population, conceivably the benefits of such therapy

The presence of resident CPC in myocardium is well supported through multiple studies. There is still much independent confirmation to be completed to clarify the promise of cardiac progenitor cell-mediated repair. Importantly, the major novel discoveries in the field of CPC biology have been made by only a small group of investigators and interpretation of some data is impaired by the lack of independent corroborating studies. Controversies also continue regarding the origin of CPC: during cardiac development, e.g., Isl1+ CPC, or from bone marrow, e.g., c-Kit+ CPC. Most likely, both sources are important but their therapeutic utilization may need to be approached with different strategies. Methods for activating resident CPC to realize their potential for effecting endogenous cardiac repair are still in the early discovery period. The fundamental question of CPC role in homeostatic maintenance of the myocardium throughout life has yet to be fully clarified, although an understanding of this highly significant role appears to be limited only by a lack of detection. Nonetheless, the potential applications of CPC-focused therapies in congenital heart disease treatments

We thank Olaf Reinhartz, MD for reviewing this manuscript, and Laura Piechura for

Abu-Issa, R. & Kirby, M. L. (2007). Heart Field: From Mesoderm to Heart Tube. *Annual* 

Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. & Meyers, E. N. (2002). Fgf8 Is Required

Amir, G., Ma, X., Reddy, V. M., Hanley, F. L., Reinhartz, O., Ramamoorthy, C. & Riemer, R.

Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D.,

2007) pp. S40-45, ISBN 1743-4300 (Electronic) 1743-4297 (Linking)

*review of cell and developmental biology* Vol.23, 2007) pp. 45-68, ISBN 1081-0706 (Print)

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K. (2008). Dynamics of Human Myocardial Progenitor Cell Populations in the Neonatal Period. *The Annals of thoracic surgery* Vol.86, No.4, 2008) pp. 1311-1319,

Kroumpouzou, E., Traill, T. A., Leblanc-Straceski, J., Renault, B., Kucherlapati, R., Seidman, J. G. & Seidman, C. E. (1997). Mutations in Human Tbx5 [Corrected] Cause Limb and Cardiac Malformation in Holt-Oram Syndrome. *Nature genetics* Vol.15, No.1, (Jan, 1997) pp. 30-35, ISBN 1061-4036 (Print) 1061-4036 (Linking) Bayes-Genis, A., Roura, S., Prat-Vidal, C., Farre, J., Soler-Botija, C., de Luna, A. B. & Cinca, J.

(2007). Chimerism and Microchimerism of the Human Heart: Evidence for Cardiac Regeneration. *Nature clinical practice. Cardiovascular medicine* Vol.4 Suppl 1, (Feb,

are likely manifold, awaiting only further investigation and implementation.

may be far greater for the pediatric population than the adult.

**4. Conclusions** 

**5. Acknowledgement** 

1081-0706 (Linking)

(Linking)

ISBN 1552-6259

editorial assistance.

**6. References** 
