Author + information
- Megan Monsanto, BS and
- Mark A. Sussman, PhD∗ ()
- Department of Biology, San Diego State University Heart Institute, San Diego State University, San Diego, California
- ↵∗Reprint requests and correspondence:
Dr. Mark A. Sussman, San Diego State University Heart Institute and Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, California 92182.
Activated cardiac fibroblasts (CFs) play a critical role in remodeling the heart following myocardial infarction (MI) but a clear consensus on the origin and biological properties of these dynamic cells remains elusive. In this issue of the Journal, Ruiz-Villalba et al. (1) endeavor to resolve the origin of CFs participating in tissue repair following MI by cell lineage tracing of 2 different suspected sources.
In general, the consensus is that CFs are recruited by endothelial cells via endothelial-mesenchymal transition (EndMT) or from the bone marrow; it also has been proposed that both resident CFs and migrating pericytes may be additional sources of fibroblast in the wake of cardiac damage (2–6). Indeed, the authors report that an undifferentiated epicardium-derived cell (EPDC) gives rise to CFs following injury in their model. Expansion and differentiation of EPDCs is secondary to an acute inflammatory response of infiltrating bone marrow–derived blood cells (BMCs) at the site of injury. Activated CFs mobilize and interact with BMCs, triggering cell polarization and matrix deposition contributing to scar formation. Ruiz-Villalba et al. (1) highlighted the clinical importance of elucidating mechanisms of post-MI ventricular remodeling with the hope of both providing a strategy to control cardiac fibrosis and modulating an environment favorable to cell-based therapies.
The adult mammalian heart has limited regenerative capacity, leading to loss of cardiomyocytes during acute infarction. Data from several studies (7–9) suggest that such factors as hypoxia, reactive oxygen species, and a cytokine-rich environment activate fibroblasts following MI. Characterization of CFs has been hampered by the lack of a robust CF-specific molecular marker and the cell population’s heterogeneity. Tracking of the CF origin in this study was accomplished by lineage tracing to examine the contribution of BMCs and EPDCs to CF formation after MI. However, the study stops short of examining the possibility that CFs may originate from endothelial cells via EndMT, an important point because both EndMT and bone marrow–derived fibroblasts within the myocardium have been associated with pathological remodeling. Additional studies with previously documented transgenic Tie2-Cre or VE-cadherin-Cre transgenic mouse lines would have helped resolve the possible endothelial origin of CFs.
Epicardial cells that line the surface of the heart were tracked using Cre/loxP transgenic mice to identify cells expressing Wilm's Tumor Gene 1 (Wt1), a transcriptional regulator that plays a key role in heart development and epicardial epithelial to mesenchymal transition (EMT) (10). The transgenic approach allows for Wt1+ cells to be permanently labeled with enhanced yellow fluorescent protein (eYFP). Immunofluorescence revealed eYFP/Wt1+ cells localized at the epicardium at embryonic day (E) 10, and through EMT gave rise to the EPDCs. At E11.5, eYFP+ EPDCs were the first cells to transition and occupy the myocardial interstitial space, completing the homing process between E15.5 and birth and remaining interstitial post-natally (Figure 1). Caveats when interpreting results from fate mapping studies using the murine Cre/loxP recombinase approach include variable sensitivity of the system, or lack thereof, depending on the reporter (10,11), but fate mapping remains the cutting-edge approach until real-time imaging to track individual cell movement is possible on live animals.
The possibility of a circulating BMC origin for CFs as previously suggested (4) was addressed by Ruiz-Villalba et al. (1) by tracing BMC cardiac homing after monomeric red fluorescent protein (mRFP) bone marrow transplantation into irradiated recipient mice. BMC contribution to the cardiac interstitium was found to be very low (<2% of nonmyocardial cells) and therefore does not seem to be the major source of CFs in the healthy adult heart. Subsequently, after showing negligible BMC infiltration into the normal undamaged myocardium, mRFP+ BMCs were transplanted into Wt1Cre-eYFP+ mice subjected to ligation of the left anterior descending coronary artery to induce MI. Contribution of EPDCs and BMCs in the context of ventricular remodeling was assessed at multiple time points after injury. As expected, extensive myocardial cell death was observed concurrent with an increase in circulating mRFP+ BMCs in the infarct zone (IZ) following left anterior descending coronary artery ligation. Expansion of resident epicardium-derived CFs follows BMC infiltration into the IZ, with the number of EPDCs steadily increasing to match that of the BMCs at the 1-week time point (mRFP/eYFP cell ratio, 0.97 ± 0.18). The authors demonstrated that migratory homing of EPDCs occurs in response to the myocardially secreted chemokine stromal cell-derived factor 1α. Interestingly, they found that EPDC and BMC compose 90.2 ± 2.1% of total cells present in the IZ. Spatially, both cell types are tightly packed and polarized with the long axes of CFs and BMCs running parallel to one another.
Characterization of the interstitial EPDC revealed a population of CD90+/CD31−/smooth muscle actin, type αlow cells that when sorted from infarcted hearts, showed at least a partial population with CF-like qualities including high levels of collagen I transcript. In contrast, CD45+ sorted BMCs showed nearly undetectable levels of collagen I messenger ribonucleic acid, reinforcing the conclusion that fibroblast origin is not from the bone marrow.
To verify that the CFs differentiate from resident interstitial EPDCs and not from adult epicardium EMT reactivation, the authors checked for expression of classic epicardial markers, such as Wt1 and Raldh2, in the eYFP+ subepicardial cell population. Significant expression of epicardial markers was not seen in the interstitial space of the subepicardium at any stage examined in this study, confirming that the adult epicardium is not the source of newly formed CFs.
In summary, Ruiz-Villalba et al. (1) conclude interstitial EPDCs are the main source of CFs in the ischemic heart and that patterned collagen deposition in the post-MI fibrotic scar depends on interaction of BMCs with the epicardium-derived CFs. Speculatively, the authors assert that saturation of BMCs and EPDCs at the site of injury prevents efficient engraftment of injected stem/progenitor cells, but this remains a hypothetical possibility without basis and requires longitudinal time course studies following injury to truly assess the question of donated cell engraftment. Indeed, many studies with adoptive transfer of stem cells following injury report significantly reduced scar formation and collagen deposition. Thus, although the study by Ruiz-Villalba et al. brings the field a step closer to understanding the cellular origins of cardiac fibrosis, the authors do not explore potential routes of minimizing malignant scarring to improve cell-based therapies. Drugs that target fibrotic proteins, such as transforming growth factor-β and platelet-derived growth factor, are currently being considered as antifibrotic treatments (12).
However, it remains to be seen if identifying EPDCs as the primary origin of CFs in the ischemic context will be of notable clinical relevance for future development of antifibrotic drugs. Although targeting the cellular source of EPDC-derived CFs is one possibility, it would need to be determined whether other cell populations would compensate for the loss of a select cell type in the heart and, equally important, whether short-term scar reduction in the wake of MI has long-term deleterious consequences for repair, remodeling, and functional competency of the heart. In the end, although the contribution of EPDC is appreciable in the context of this study, the debate about how to best deal with fibroblasts in the injured myocardium will undoubtedly continue for years to come.
↵∗ Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology.
Ms. Monsanto is supported by the American Heart Association Cardiovascular Disease Student Scholarship and Rees-Stealy Research Foundation. Dr. Sussman is supported by National Institutes of Health grants R01HL067245, R37HL091102, R01HL105759, R01HL113656, R01HL113647, and R01HL122525; has received an award from the Foundation Leducq Transatlantic Network; and is a founder and co-owner of CardioCreate Inc.
- 2015 American College of Cardiology Foundation
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