In this study we have demonstrated that (a) CPCs express functional anaphylatoxin receptors, (b) stimulation with C3a or C5a induces the expression of myofibroblast differentiation markers and EndMT-like gene expression, (c) C3a and C5a both increase CPC proliferation and migration and (d) C3a and C5a also induce telomerase activity (NFκB-dependent) and increase telomere maintenance, although not sufficiently to fully preserve telomere length. Taken together these data support a role for anaphylatoxins in CPC regulated cardiac healing and scar formation.
The discovery of endogenous c-kit–positive CPCs has driven a shift in cardiac biology. Work from multiple laboratories (revised in Barile et al. 2007; Laflamme and Murry 2011) have documented the heart’s ability to replace old and dying cells, and propose that this capacity depends on the persistence of a stem cell compartment. The initial interest generated by the identification and isolation of c-kit-positive CPCs was followed by a period of doubt, reflected in studies from different laboratories and accompanying editorials that questioned or neglected issues related to CPC function. These reservations regarding the role of CPCs in cardiac cell turnover are mainly related to the limited nature of myocyte renewal in the human heart (Bergmann et al. 2009). Although multiple studies in animal models have suggested that the adult heart is capable of some cellular turnover (reviewed in Choi et al. 2012), clinical evidence clearly shows that regeneration is inadequate after injury. This evidence supports the hypothesis that endogenous CPC populations probably have a role in cardiac homeostasis other than cardiomyocyte turnover, since the cardiac healing response is very poor. In adult mammals, cardiomyocyte regeneration is insufficient to functionally renew severely injured myocardium and consequently, scar tissue forms. Thus, the evolutionary adapted healing processes that initially benefited cardiac function finally results in a potentially maladaptive response in the long term.
After a myocardial infarction, massive cell infiltration into the myocardium results in fibrosis. MI injury is considered an inflammatory state, characterised by innate immune responses. Whereas it was initially thought that the immune system protects the body against foreign or non-self signals, there is now a body of evidence to suggest that the innate immune system is activated following tissue injury, triggering the release of endogenous ligands (Arslan et al. 2011). Participation of the complement system in myocardial ischemia was first demonstrated in 1971 in a rat MI model (Hill and Ward 1971). Administration of monoclonal antibodies against C5 and C5a as well as the C5a receptor reduced myocardial infarct size in pigs and rodents (Amsterdam et al. 1995; Vakeva et al. 1998; van der Pals et al. 2010; Zhang et al. 2007). However, a study concerning patients either undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) with pexelizumab (a single-chain fragment of a humanised monoclonal antibody against complement component C5) administration showed a significant reduction in mortality in the former case, but no benefit in patients with acute MI in the latter case (Testa et al. 2008). It is important to note that anaphylatoxin inhibition in these models has been performed in an acute MI phase but that the principal role of anaphylatoxins and their receptors here is to increase leukocyte recruitment to the reperfused myocardium following MI.
Since the complement system is activated in the milieu of tissue injury, the effect of C3a and C5a on CSC migration and proliferation is of specific interest, because these anaphylatoxins may contribute to the mobilisation, recruitment, and proliferation of cells at an injury site, thus facilitating wound healing. Our data support the role of anaphylatoxins in recruiting cells involved in tissue healing: C3a and C5a are able to induce CPC proliferation and migration, which supports the role of anaphylatoxins in the control of tissue healing. Indeed, apart from the known chemotactic role that C3a and C5a play with regard to leukocytes, anaphylatoxins are also chemotactic factors for other cells like mesenchymal stem cells (Schraufstatter et al. 2009) or neural stem cells (Shinjyo et al. 2009). Our data also shows that in addition to this enhancement in proliferation and migration, anaphylatoxins also induce telomerase mRNA and activity. Although addition of C5a significantly increases telomere length compared to controls, and to a lesser degree C3a, telomerase activity is not enough to maintain telomere length compared to the initial state. Interestingly a large body of data has accumulated regarding the capacity of telomerase to support roles other than telomere preservation (reviewed in Martinez and Blasco 2011). One of these roles is the facilitation of cell growth and proliferation. For instance, hTERT overexpression lengthens the proliferative lifespan of human bone marrow stromal cells (Simonsen et al. 2002) being also able to induce hyperplasia and hypertrophy in murine cardiac myocytes (Kohl 2001). Moreover, ectopically expressed hTERT confers resistance to the anti-proliferative effect of transforming growth factor β (TGF-β) in p16-null human mammary epithelial cells (Stampfer et al. 2001). In addition, telomerase activation in human mammary epithelial cells coincides with the stimulation of a cellular mitogenic program (Smith et al. 2003), indicating that telomerase may affect epithelial cell proliferation not only by stabilising telomeres, but also by affecting the expression of growth-promoting genes. There is also data that supports an extra-telomeric role for telomerase in protection against oxidative stress; in this regard Schraufstatter et al. (2009) demonstrated that C3a and C5a protect MSCs from oxidative damage. This can be translated into an in vivo scenario, where one would expect that the anaphylatoxins would recruit healing cells to areas of tissue injury, where these cells would encounter the production of oxidants due to neutrophil recruitment and reperfusion injury. Therefore all these data support a role for anaphylatoxins in promoting the recruitment of healing cells to areas of injury in order to restore tissue homeostasis. Telomerase activity would not only improve cell proliferation and survival but also protect cells against the hostile environment – providing protection from apoptosis, oxidative stress, and DNA damage.
Injured ischemic myocardium progresses to necrosis and subsequent healing; the local response to irreversible injury is the formation of granulation tissue, the accumulation of collagen, and resultant replacement fibrous scar (Sun et al. 2002). Although clinical and biological data shows that the heart has a very low endogenous capacity for regeneration when severe damage is inflicted, multiple endogenous putative cardiac stem cell or progenitor cell populations have been identified and isolated. Markers, traditionally associated with blood, bone marrow or pluripotent stem cells, have been used by several independent groups to identify these cells in adult or postnatal hearts in humans and other mammalian species (Smith et al. 2008). We have worked with a population (CPCs), as described by Messina et al. (Messina et al. 2004), this population expresses the cardiac markers, Nkx2.5, Gata4, Cx43 and Mef2c (Figure 1B) and therefore had a putative cardiogenic potential. Thus our first task was to understand why, if these cells have the potential to differentiate in cardiomyocytes, physiological cardiac healing usually results as a fibrotic scar and shows very low levels of cardiomyocyte replacement. We investigated the potential of these cells to differentiate to all three lineages described, and facilitated differentiation by using 10% FBS to improve myofibroblast differentiation, and EGM on Matrigel to increase endothelial lineage differentiation. Interestingly anaphylatoxins repress Gata4 expression and increase the expression of some early cardiac markers (Nkx2.5, Tbx3, Tbx5, Actc1) in CPCs. Endothelial cell (EC) lineage differentiation was clearly improved by culture on Matrigel using EGM media and in these conditions, stimulation with C3a and C5a anaphylatoxins reduced endothelial gene expression. In agreement with this data, immunofluorescence also showed the absence of ECs in the presence of anaphylatoxins. Inhibition of EC differentiation is paralleled by induction of Twist and Snail genes together with Fsp1 expression. Fsp1 is one of the markers of fibroblast formation and is a cytoskeletal protein belonging to the calmodulin-S100-troponin C superfamily of intracellular calcium binding proteins associated with cytoskeletal fibres, cell motility, and mesenchymal phenotype (Zimmer et al. 1995). Therefore the expression of Snail1 and Twist1 genes, together with the significant increase in Fsp1 expression, indicated that an endMT-like differentiation event might be occurring in CPCs. In addition, anaphylatoxins induce the expression of an array of genes associated with myofibroblasts that probably abolishes their role in regeneration but hints at their relevance in the cardiac healing process that follows MI. In this scenario, complement anaphylatoxins would activate migration of CPCs to the damaged area, induce their proliferation and finally, upon persistence of local signalling, would promote their differentiation into myofibroblast cells that would be able to fill and quickly repair the damaged heart area, preventing further cardiac complications.
Gata4 has been proved to be a critical early factor in cardiogenesis, it lays upstream of Tbx5, Nkx2.5, and Act1 in the cardiogenic transcription network, and acts together with Baf60c to generate a chromatin state competent for cardiogenic differentiation (Takeuchi and Bruneau 2009). Therefore C3a and C5a may block cardiomyocyte differentiation by repressing Gata4 expression. The cardiogenic factors Nkx2.5, Actc1 and specially Tbx3 and Tbx5 may have roles other than their cardiogenic ones, including modulating the response of CPCs to complement anaphylatoxins, for instance they might be involved in the C3a/C5a dependent myofibroblast differentiation process. It is known that several mesodermal cells (cardiomyocytes and myofibroblasts) share parts of their transcriptional differentiation networks, supporting the existence of a common myocardial and smooth muscle cell precursor in the developing embryo. This is consistent with in vivo studies, which show the co-expression of numerous smooth muscle genes in myocardial progenitor cells (Wu et al. 2006). Therefore Tbx3 and Tbx5 would be common to both cell types, and Gata4 levels would be critical in modulating the fate decision of progenitor cells. Interestingly it has been proposed that Gata4 expression can play a role in the developmental regulation of cardiac fibroblasts and has a function in the maintenance of cardiac-resident progenitors (Jankowski 2009). This raises the hypothesis that C3a and C5a are factors that affect CPC fate by switching the balance of different lineage specific factors: they shut down the expression of endothelial and cardiac factors and induce the expression of myofibroblast factors (TCF21, SM22α and Myocardin), irreversibly pushing CPCs towards the myofibroblast fate.
Taken together, these findings shed some light onto what have been described as endogenous cardiac stem cells and some mechanistic insight about the limited potential of CPCs to generate new cardiomyocytes after cardiac injury. We can hypothesize that anaphylatoxin release at early time after cardiac injury is able to increase CPC numbers and to promote migration towards the site of injury. This signal also increases CPC potential to differentiate into myofibroblast lineages that would participate in scar formation. This situation gives an advantage to cardiac tissue promoting a fast healing in MI. Differentiation toward myofibroblast in CPCs would make available a higher number of myofibroblasts to contribute to other sources of myofribroblasts (Krenning et al. 2010) during the resolution stage of MI. This situation is different from physiological cardiac cell turnover. During life possibly other signals in absence of inflammatory signals will permit cardiomyocyte differentiation to replace exhausted cells. Interestingly recent evidence reported by Jianqin Ye et al. (2012) have shown that there is a significant increase in the proliferative capacity of CS-forming cells isolated from the “middle aged” heart following acute MI resulting in a significant rise in the number of CSs in vitro and this increase is most pronounced within the first week post-MI. In addition they show that show for the first time that the CS cells obtained from 1-week post-MI hearts engraft in ischemic myocardium and restore cardiac function at 25 days post-injection in vivo. However, we did not find evidence for differentiation of these cells into mature cardiomyocytes or new vessels althouhgt promoted angiogenesis in vivo. Their data suggest that early signals that happens after MI would commit these cells to a more healing phenotype instead of regenerative fate.
To heal the heart adding new cardiomyocyte probably is a more delicate and time-consuming situation that mammalian wounded heart cannot go through in a wild environment. Thus the signals involved in cardiac healing have been selected to permit a fast healing situation increasing myofibroblast differentiation but in other hand this impairs cardiomyocyte renewal. Deeper understanding of these mechanisms would help to improve wounded heart regeneration.