CPC culture and characterisation
Small biopsies of murine adult hearts were placed on gelatin/fibronectin plates (Figure 1A (1)). Following an initial outgrowth of fibroblast-like cells, within 5–7 days of explant plating, small, round and poorly adherent cells appeared and expanded (Figure 1A (2)). These cells, called explant-derived cells (EDCs) could be detached by gently pipetting, and were harvested and cultured to form cardiospheres (Figure 1A (3)). Using immunofluorescence, EDCs were found to express the cardiac markers, Nkx2.5, Gata4, Cx43, and Mef2c (Figure 1B) and also cell surface makers c-kit and Sca-1 (Figure 1B). During cardiosphere culture, proliferative cells (Ki67 positive) were found primarily in the external part of the sphere, in contrast to the CPC marker c-kit predominately found in the core. Sca-1 was more homogenously expressed throughout the whole culture. EDCs showed a very low expression for the vascular markers CD31 and aSMA.
Cardiospheres were then expanded as a monolayer culture to passage 2 (p2); these cells, present within 3–4 weeks of biopsy, were termed multipotent cardiac progenitor cells (CPCs; Figure 2A). Flow cytometry on EDCs revealed that 84 ± 0.3% of these cells expressed the stem cell marker c-kit, 71 ± 9% expressed Sca-1 and 11 ± 2% expressed CD45 (Figure 2B) Gene expression was analysed using immunofluorescence and RT-PCR. CPCs expressed the pluripotent genes Bmi1, Nestin, Rex1, Tert, and lacked Oct4 and Sox2 expression (Figure 2B and some early cardiac transcription factors Tbx3, and Gata4. In order to evaluate their differentiation potential, CPCs were cultured with differentiation medium (Figure 2D and 2E) or co-cultured with neonatal rat cardiomyocytes (NRCMs; Figure 2F and 2G). After 7 days in differentiation medium culture, CPCs showed upregulation of cardiac genes such as Troponin T and α-Actinin (Figure 2D), which was confirmed by western-blot (Figure 2E). GFP+ CPCs were co-cultured with NRCMs and after 7 days isolated by FACS for molecular analysis. Gene expression studies showed upregulation of Gata4, α-Actinin, Troponin T, β-MyHC and α-SMA (Figure 2F) and immunofluorescence analysis revealed the presence of GFP+ CPCs derived cells expressing Tropomyosin (Figure 2G).
CPCs express functional anaphylatoxin receptors
Functional expression of the anaphylatoxin receptors C3aR and C5aR on non-immune cells has been previously reported for other cells like neurons and astrocytes (Van Beek et al. 2000) or mesenchymal stem cells (Schraufstatter et al. 2009), between others. To evaluate the potential of complement anaphylatoxins to activate CPCs, we checked the expression of C3a and C5a receptors on CPCs. We detected cell surface expression of C3aR and C5aR determined by immunofluorescence (Figure 3A), which was confirmed by Western blotting for their respective proteins (Figure 3B). The entire cell population expressed cell surface C3aR and C5aR in moderate to high levels. Interestingly, densitometry analysis of western blot membrane and normalized the expression versus C5aR the expression of C3aR in CPCs was 7,9 fold higher than in endothelial cells (ECs) or 2,5 fold higher compared to mouse embryonic fibroblasts (MEFs). To probe the functionality of the C3a and C5a receptors, we explored the activation of signalling pathways known to be triggered by anaphylatoxins in other cell types. These pathways are the G-protein calcium-release dependent phosphorylation of PKC, and also the phosphorylation-dependent activation of ERK1/2 (Monsinjon et al. 2003), AKT/PKB (Schraufstatter et al. 2009), and NFkB (Pan 1998). We found substantial activation of both the ERK and PKC pathways, but we did not detect the phosphorylated forms of Akt (Figure 3C). In addition C3a, and to a larger extent C5a, were able to induce NFκB activation, measured as the phosphorylation of IKKa (Figure 3C) and the nuclear translocation of p65 (Figure 3D).
C3a and C5a promote CPC proliferation and migration
Proliferation and migration to damaged tissues are two important characteristics of progenitor cells that enable them to perform their physiological role in maintaining tissue homeostasis. In the context of cardiac homeostasis, the release of complement anaphylatoxins is a signal of tissue damage, which in turn may have an effect on any cardiac progenitors close to the damaged area. To probe this hypothesis we assessed the proliferative and migration ability of CPCs upon C3a and C5a stimulation (Figure 4A). CPCs were cultured in the presence of C3a and C5a, and their proliferation rate was measured by 3H-thymidine incorporation into their DNA. CPCs cultured in the presence of C3a and C5a proliferated faster than the control cells. This proliferation enhancement was dose dependent and peaked at C3a = 50 nM (3-fold vs. control), and C5a = 15 nM (4-fold vs. control; Figure 4A). Moreover effect of C5a is quite more stronger to activate migration and proliferation than C3a. The addition of both anaphylatoxins at the described peak concentration together the effect of C5a overlaps C3a with out any cooperation.
In some models cell proliferation is linked to cell migration (Diez-Juan and Andres 2003). Interestingly, in addition to the modulation of the chemotactic activity of immune cells, C3a and C5a has also been shown to induce migration of non-immune cells such as neural stem cells (Shinjyo et al. 2009) or mesenchymal stem cells (Schraufstatter et al. 2009), between others. Therefore we next explored the potential of C3a and C5a as chemotactic factors in CPCs. Using a Boyden chamber, CPCs were placed in the upper transwell compartment and stimulated with C3a or C5a in the lower compartment (Figure 4B). Both anaphylatoxins were able to induce cell migration when present at the same concentration that induced cell proliferation in the previous assay. C5a was more efficient at inducing cell migration at lower doses; at higher concentrations the stimulation was reduced, resulting in a bell-shaped response curve (Figure 4B), which may be explained by the fact that this agent primarily stimulates chemotaxis. In fact, at higher concentrations, diffusion of C5a from the lower to the upper compartment of the Boyden chamber could disrupt the C5a gradient and thus prevent chemotactic migration. In contrast, the use of a chemoattractant that stimulates chemokinesis would not result in a bell-shaped dose–response, since such a reaction is independent of chemical gradients.
Anaphylatoxins C3a and C5a induce telomerase activity and telomere maintenance in CPCs
It is well known that cell proliferation is dependent on telomere maintenance, which is mainly achieved through the action of telomerase (Tert) (Choudhary et al. 2012). Moreover, multiple experimental data show that telomere length maintenance is crucial to the preservation of the regenerative potential of adult endogenous stem cells (reviewed in (Choudhary et al. 2012)). Thus, we assessed if C3a and C5a could be considered as a pro-healing stimuli by activating CPC proliferation in combination with telomerase induction.
To test this idea, we measured telomerase expression and activity in CPCs cultured in the presence of C3a or C5a at the optimal doses of 50 nM and 15 nM respectively (Figure 5A). We found, by qPCR analysis, that CPCs cultured with C3a and C5a showed more than a 2-fold increase in Tert mRNA (Figure 5A). This result was confirmed by measuring telomerase activity using a telomeric amplification protocol (TRAP). CPCs cultured in the presence of C3a and C5a showed an incremental increase in telomerase activity of 3-fold and 5-fold respectively, with respect to the control cells (Figure 5A). We next examined whether the pro-telomeric role of C3a and C5a anaphylatoxins is correlated to the presence of longer telomeres than in controls. We used a telomeric probe, normalised with a centromeric probe, to quantify the telomere length of CPCs grown in presence of C3a or C5a for 20 population doublings. As shown in Figure 5B and C, C3a-CPCs and C5a-CPCs display longer telomeres than their control counterparts. However, this telomere maintenance effect is not capable of completely preventing telomere shortening, since C5a-CPCs at p20 have still significantly shorter telomeres than their counterparts at p0. In addition, this increment was only statistically significant (P = 0.001) in the C5a treated CPCs (Figure 5B). Taken together, these results suggest that C3a and C5a have a ‘pro-telomeric’ effect on CPCs, although this is not sufficient to retain telomere length at their initial length.
The induction of TERT expression by anaphylatoxins C3a and C5a is dependent of NFκB activation
To gain insight into the molecular mechanisms responsible for C3a and C5a anaphylatoxin mediated Tert induction we examined the Tert promoter and found two NFκB binding sites. Since C3a and C5a are able to induce NFκB signalling in CPCs, this molecular pathway appeared to be a good candidate for the mediation of C3a and C5a dependent Tert mRNA expression.
To test this possibility, we used two complementary approaches. First, we performed a chromatin immunoprecipitation assay for the NFκB subunit, p65; we used primer pairs that cover the NFκB site at −326-316 in the murine TERT promoter so that we could determine if p65 is recruited to the endogenous TERT promoter upon anaphylatoxin stimulation. These CHiP assays confirmed that C3a/C5a stimulation induces the recruitment of p65 to the consensus site in the murine TERT promoter (Figure 6B). Second, to further confirm that NFκB was responsible, at least in part, for inducing TERT mRNA expression in anaphylatoxin-stimulated CPCs, we used a mutant of IκBα protein that is refractory to IKK phosphorylation. This mutant IκBα S32/S36-A behaves as a potent dominant negative IκBα protein that attenuates NFκB transactivation (Brown et al. 1995). CPCs were transduced with a retroviral vector coding for IκBα to block NFκB activation or with a control vector, and Tert mRNA expression was assessed by RT-qPCR. As shown in Figure 6A, attenuation of NFκB activation abolishes anaphylatoxin dependent Tert induction in CPCs. Taken together these experiments demonstrate that NFκB is the main signalling pathway involved in anaphylatoxin dependent Tert expression.
Anaphylatoxins C3a and C5a abolish the cardiac and endothelial potential of CPCs and promote myofibroblastic differentiation mediated by an endothelial to mesenchymal transition-like process
Although some experimental results have shown a limited regenerative response after heart injury, cardiac wound healing in mammals is hampered by the fact that the regeneration of heart muscle is virtually absent, and that damaged myocardium is replaced by scar tissue. The above results point towards pro-regenerative activity for complement anaphylatoxins: both of them promote CPC migration, proliferation, telomerase activity, and telomere maintenance. All of these anaphylatoxin-mediated processes can be envisioned to activate and expand the resident CPC pool within the heart. However, to be effective in regenerating damaged tissue, a stem cell pool must preserve its differentiation potential when it proliferates. A pro-regenerative event would mean that CPCs maintain their ability to differentiate into the main cardiogenic lineages (smooth muscle/myofibroblast, endothelium and cardiomyocytes), or are even able to enhance their ability to differentiate towards a cardiomyocyte fate, the main cell type that is not replenished after MI. Therefore we examined the effect of CPC exposure to complement anaphylatoxins.
CPCs treated with either C3a or C5a (for 72 h) were assessed, by RT-qPCR, for the expression of some well-known endothelial, myofibroblast, and cardiomyocyte lineage markers. We chose Gata4, Nkx2.5, Tbx5, and Tbx3 as cardiogenic markers, Actc1 as an early cardiomyocyte marker, CD31, vWF, and E-Cadherin as endothelial markers, Myocardin, SM22α, and TCF21 as myofibroblast markers, and SM22α, α-SMA, Vimentin, Desmin, and Collagen 1a (Col1a) as mature myofibroblast markers. We found that upon anaphylatoxin stimulation, CPCs appeared to initiate a myofibroblastic transcription program, with a more than 5-fold increase in the pro-myofibroblastic transcription factors Myocardin and TCF21. In addition, there was a clear increase in the expression of the myofibroblast markers Desmin, α-SMA, Col1a, and Vimentin, which clearly indicates differentiation towards a mature myofibroblast fate (Figure 7A). The induction of myofibroblast genes was accompanied by a reduction of endothelial markers with more than a 3-fold decrease in the quantity of CD31 and E-Cadherin mRNA detected, and a 2-fold decrease in the case of vWF. It therefore seemed clear that anaphylatoxin stimulation compromises the endothelial fate of CPCs and promotes a clear bias towards the myofibroblastic fate. This was further confirmed by immunofluorescence analysis of the myofibroblast marker α-SMA, and of two endothelial markers CD31, and the surface protein lectin. As shown in Figure 6B, CPCs treated with C3a or C5a showed increased levels of α-SMA positive cells, typical of myofibroblastic lineages. Conversely, CPCs stimulated with C3a or C5a lost CD31expression and showed a reduction in tomato-lectin binding on their membranes. As a positive control, we cultured CPCs in endothelial medium (EGM2) to direct endothelial differentiation. At the mRNA level, this resulted in a 2.5-3-fold increase in endothelial marker levels and the abolition of myofibroblast markers (Figure 7A). Immunofluorescence studies revealed that, with respect to the control, EGM2 cultured CPCs presented higher levels of lectin and slightly increased levels of CD31 (Figure 7B); this mirrors the small increase in CD31 mRNA expression in EGM2 cultured CPCs with respect to control CPCs.
Immunofluorescence analysis showed that when CPCs are in their multipotential state, they express moderate levels of both myofibroblast and endothelial markers (Figure 7) and so they can be considered as being in a state of balance between these two fates. Complement anaphylatoxin stimulation seems to break this balance, pushing CPCs towards the myofibroblast lineage. We then hypothesized about the mechanism underlying this C3a/C5a-dependent balance shift and noticed that the decrease in the endothelial markers could point to an endothelial to mesenchymal like transition (EndTM), which would be capable of altering this equilibrium. To test this hypothesis, we treated CPCs with the optimal C3a and C5a doses and measured the mRNA levels of two key transcription factors involved in EndMT, Snail1 and Twist1 (Huber et al. 2005). Figure 7A shows that cells treated with C3a and C5a presented higher levels of Twist1 (almost 2-fold) and Snail (> 5-fold) mRNAs. We also assessed the expression of the known EndMT marker Fsp1 and confirmed the C3a/C5a-dependent induction of an EndMT-like process; CPCs treated with these anaphylatoxins showed a 5-fold increase in the Fsp1 mRNA levels (Figure 7A).
Therefore a likely explanation for the observed results is that C3a and C5a promote an EndMT-like process in CPCs that restricts their endothelial potential and pushes them towards the mesenchymal fate inducing the expression of the myofibroblastic lineage promoters and thus committing them to differentiate towards myofibroblasts that express the typical markers Desmin, α-SMA and, SM22α. In addition, the cardiogenic potential of CPCs is abolished upon C3a and C5a stimulation, since this negates C3a and C5a dependent commitment of CPCs into myofibroblasts. Expression of the key pioneer transcription factor in cardiogenesis, Gata4, was clearly repressed upon anaphylatoxin treatment (Figure 7A); however the expression of other cardiogenic factors did not follow this pattern. When CPCs were treated with C3a and C5a, we found that while Gata4 mRNA decreased 5-fold, the mRNA levels of its downstream factors Nkx2.5, Tbx5 and Tbx3 markedly increased (2.6 to 17-fold), along with an increase in mRNA from the early cardiac marker Actc1, which rose by 3 - 4-fold.
Gata4 is known to be crucial for cardiomyocyte differentiation; therefore, the repression of Gata4 expression in CPCs stimulated by C3a and C5a clearly points toward a role for these factors in the compromise of cardiogenic differentiation potential in these CPCs. A role for C3a and C5a in the compromise of cardiogenic differentiation is further supported by the appearance α-SMA positive cells and by the induction of myofibroblast markers in C3a/C5a treated CPCs, since this process is obstructing cardiomyocyte differentiation (Wu et al. 2006). Therefore the C3a and C5a mediated transcriptional activation of Tbx3, Tbx5 and Nkx2.5 might suggest an alternative role for these transcription factors in the maturation of and/or functional properties of myofibroblasts. Another interesting possibility is the implication of these mesodermal factors in the C3a/C5a mediated proliferation of CPCs.
Thus, we conclude that C3a and C5a promote the differentiation and commitment of CPCs towards myofibroblast lineage, blocking their cardiac and endothelial potential.