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Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Targeting cardiac fibroblasts: The pressure is on a
a
bcd
Thomas Moore-Morris , Michel Pucéat & Sylvia M Evans a
INSERM UMRS_910; Aix-Marseille Université; Marseille, France
b
Skaggs School of Pharmacy; University of California San Diego; La Jolla, CA USA
c
Department of Medicine; University of California San Diego; La Jolla, CA USA
d
Department of Pharmacology; University of California San Diego; La Jolla, CA USA Published online: 30 Oct 2014.
Click for updates To cite this article: Thomas Moore-Morris, Michel Pucéat & Sylvia M Evans (2014) Targeting cardiac fibroblasts: The pressure is on, Cell Cycle, 13:17, 2647-2648, DOI: 10.4161/15384101.2014.954212 To link to this article: http://dx.doi.org/10.4161/15384101.2014.954212
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
FEATURE Cell Cycle 13:17, 2647--2648; September 1, 2014; © 2014 Taylor & Francis Group, LLC
Targeting cardiac fibroblasts: The pressure is on Thomas Moore-Morris1, Michel Puceat1, and Sylvia M Evans2,3,4,* INSERM UMRS_910; Aix-Marseille Universite; Marseille, France; 2Skaggs School of Pharmacy; University of California San Diego; La Jolla, CA USA; 3Department of Medicine; University of California San Diego; La Jolla, CA USA; 4Department of Pharmacology; University of California San Diego; La Jolla, CA USA
Downloaded by [Georgetown University] at 06:51 02 March 2015
1
Heart failure is a major health issue, with its incidence on the rise. It is well established that fibroblast accumulation in stressed myocardium causes arrhythmias and drives adverse remodeling that underlies heart failure. Hence, defining mechanisms responsible for this surge in cardiac fibroblast numbers represents a key clinical issue. Recent studies have proposed that endothelial-to-mesenchymal transition (EndoMT) and recruitment of circulating fibroblast progenitors generate significant numbers of fibroblasts and could be targeted to alleviate fibrosis.1,2 Here, we discuss limitations of these studies arising from markers utilized for cardiac fibroblasts, as well as our recently reported findings on cardiac fibroblast markers and origins3 and their clinical impact. Fibroblasts are abundant interstitial cells principally known for secreting extracellular matrix, particularly collagen type I. Markers used to identify fibroblasts include CD90 (Thy1), DDR2, and fibroblast specific protein 1 (FSP1), although none are fibroblast-specific.2 To label cardiac fibroblasts in heart at baseline, we used a Collagen1a1-GFP reporter line4 that we found was not expressed in nonfibroblast lineages including endothelium, pericytes, and haematopoietic lineages. The mesenchymal marker PDGFRa, expressed by fibroblasts,5 was coincident with Collagen1a1-GFP. In contrast, a marker commonly used to label fibroblasts, Fibroblast Specific Protein 1 (FSP1), was expressed in leukocytes and was absent from Collagen1a1-GFPC fibroblasts. Following pressure overload, Collagen1a1-GFPC fibroblasts proliferated, and constituted the major cell population in fibrotic lesions as identified by excess
collagen deposition. Interestingly, a smooth muscle actin (aSMA), used to identify activated fibroblasts, termed “myofibroblasts,” was expressed in only approximately 15% of fibroblasts in interstitial lesions, and not in perivascular lesions, suggesting that many fibroblasts would be overlooked when using this marker, at least in the context of pressure overload. Following pressure overload, FSP1 marked a significant proportion of CD45C immune cells, and labeled only 50% of fibroblasts in perivascular lesions, but did not label fibroblasts in interstitial lesions, demonstrating the limitations of this marker for identifying fibrotic fibroblasts (Fig. 1). Observed heterogeneity of fibroblast gene expression in interstitial and perivascular lesions is consistent with work of others6 and could be further analyzed by approaches such as single cell transcriptomics and clonal analysis. EndoMT of subsets of endocardial cells of the atrioventricular canal and outflow tract during midgestation generates mesenchymal valve progenitor cells. It has been suggested that an analogous process is reactivated following pressure overload, generating fibroblasts from the microvasculature.1 However, our lineage tracing of adult endothelium with an inducible endothelial specific Cre, VEcadherin-CreERT2 did not reveal evidence that EndoMT had occurred.3 A previous study performed to assess the occurrence of EndoMT utilized an endothelial Cre, Tie1-Cre, which labels both endothelial and haematopoietic lineages.1 It has emerged from work of others,7 and our work, that a majority of FSP1 expressing cells in fibrotic lesions are immune cells, not fibroblasts. These FSP1 expressing
immune cells are labeled by most endothelial Cres. Thus, the assumption that FSP1 is a specific marker for fibroblasts would lead to the mistaken conclusion that fibroblasts were derived from endothelial or haematopoietic lineages.1 Utilizing a pan-haematopoietic Cre, Vav-Cre, in conjunction with Collagen1a1-GFP and PDGFRa, we could not find evidence for any fibroblasts of haematopoietic origin in fibrotic lesions following pressure overload.3 Fibroblasts have been shown to derive from epicardial EMT during midgestation, although other developmental origins have not been excluded.2 Using epicardial-specific Cre drivers, we observed that most, but not all, cardiac fibroblasts were of epicardial origin. We identified a second cardiac fibroblast lineage residing mainly in the septum that we show likely derives from endocardial EndoMT associated with endocardial cushion formation. In this manner, our study demonstrated that resident cardiac fibroblasts develop from 2 distinct lineages that are differentially distributed within myocardium (Fig. 1). Combined epicardial and endothelial lineage tracing accounted for approximately 95% of fibroblasts in fibrotic hearts. Although we could detect no EndoMT, it remains a possibility that other, non-fibroblast lineages labeled by epicardial Cres may also contribute to fibroblasts during fibrosis. In this regard, pericytes, derived from epicardial lineages in heart, become fibrogenic in various other organs, notably liver and kidney.4,5 In the pressure overload mouse model, both interstitial and perivascular fibrotic lesions develop. The 2 developmentallyderived fibroblast lineages we identified
*Correspondence to: Sylvia M Evans; Email: [email protected] Submitted: 07/29/2014; Accepted: 08/04/2014 http://dx.doi.org/10.4161/15384101.2014.954212 Comment on: Moore-Morris T, et al. J Clin Invest 2014; 124(7):2921-34; PMID:24937432; http://dx.doi.org/10.1172/JCI74783
www.landesbioscience.com
Cell Cycle
2647
Downloaded by [Georgetown University] at 06:51 02 March 2015
Figure 1. Cardiac fibroblast markers and lineages following pressure-overload. At baseline and following pressure overload, all fibroblasts are marked by Collagen1a1-GFP and PDGFRa. FSP1 labels most haematopoietic cells and a subset of fibroblasts (50%) in perivascular areas. aSMA labels a small subset of fibroblasts (15%) in interstitial lesions. Spatially complementary endocardial- and epicardial-derived lineages behave similarly following pressure overload.
contributed to both perivascular and interstitial fibrotic lesions, located within myocardial regions reflecting their original developmental distribution. Intriguingly, References 1. 2.
3.
Zeisberg EM, et al. Nat Med 2007; 13(8):952-61; PMID:17660828 Zeisberg EM, et al. Circ Res 2010; 107(11): 1304-12; PMID:21106947; http://dx.doi.org/10.1161/ CIRCRESAHA.110.231910 Moore-Morris T, et al. J Clin Invest 2014; 124(7): 2921-34; PMID:24937432; http://dx.doi.org/10.1172/ JCI74783
2648
our analyses demonstrated that both lineages responded very similarly to pressure overload in terms of proliferation and transcriptional regulation, despite their 4.
5.
Kisseleva T, et al. Proc Natl Acad Sci U S A 2012; 109 (24):9448-53; PMID:22566629; http://dx.doi.org/ 10.1073/pnas.1201840109 Smith CL, et al. Circ Res 2011; 108(12):e15-26; PMID: 21512159; http://dx.doi.org/10.1161/CIRCRESAHA. 110.235531
Cell Cycle
distinct developmental origins and highlight the possibility of identifying common therapeutic targets. Hence, our study predicts that targeting proliferation of resident cardiac fibroblasts will be critical for mitigation of fibrosis. This hypothesis is in agreement with previous clinical observations on the anti-fibrotic action of Losartan, an Angiotensin II receptor blocker, likely mediated by its action on fibroblast proliferation. However, as fibrosis occurs rapidly, developing approaches to resolve established scarring will be important. In this context, much may be learned from models of fibrosis in the liver, where fibroblasts undergo apoptosis and quiescence during scar resolution.4 Interestingly, we identified upregulated genes associated with “negative regulation of cell death” in cardiac fibroblasts from hypertrophic hearts, suggesting cardiac fibroblasts could be particularly resistant to apoptosis. 6.
7.
Braitsch CM, et al. J Mol Cell Cardiol 2013; 65:10819; PMID:24140724; http://dx.doi.org/10.1016/j. yjmcc.2013.10.005 Kong P, et al. Am J Physiol Heart Circ Physiol 2013; 305(9):H1363-72; PMID:23997102; http://dx.doi. org/10.1152/ajpheart.00395.2013
Volume 13 Issue 17
Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Targeting cardiac fibroblasts: The pressure is on a
a
bcd
Thomas Moore-Morris , Michel Pucéat & Sylvia M Evans a
INSERM UMRS_910; Aix-Marseille Université; Marseille, France
b
Skaggs School of Pharmacy; University of California San Diego; La Jolla, CA USA
c
Department of Medicine; University of California San Diego; La Jolla, CA USA
d
Department of Pharmacology; University of California San Diego; La Jolla, CA USA Published online: 30 Oct 2014.
Click for updates To cite this article: Thomas Moore-Morris, Michel Pucéat & Sylvia M Evans (2014) Targeting cardiac fibroblasts: The pressure is on, Cell Cycle, 13:17, 2647-2648, DOI: 10.4161/15384101.2014.954212 To link to this article: http://dx.doi.org/10.4161/15384101.2014.954212
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
FEATURE Cell Cycle 13:17, 2647--2648; September 1, 2014; © 2014 Taylor & Francis Group, LLC
Targeting cardiac fibroblasts: The pressure is on Thomas Moore-Morris1, Michel Puceat1, and Sylvia M Evans2,3,4,* INSERM UMRS_910; Aix-Marseille Universite; Marseille, France; 2Skaggs School of Pharmacy; University of California San Diego; La Jolla, CA USA; 3Department of Medicine; University of California San Diego; La Jolla, CA USA; 4Department of Pharmacology; University of California San Diego; La Jolla, CA USA
Downloaded by [Georgetown University] at 06:51 02 March 2015
1
Heart failure is a major health issue, with its incidence on the rise. It is well established that fibroblast accumulation in stressed myocardium causes arrhythmias and drives adverse remodeling that underlies heart failure. Hence, defining mechanisms responsible for this surge in cardiac fibroblast numbers represents a key clinical issue. Recent studies have proposed that endothelial-to-mesenchymal transition (EndoMT) and recruitment of circulating fibroblast progenitors generate significant numbers of fibroblasts and could be targeted to alleviate fibrosis.1,2 Here, we discuss limitations of these studies arising from markers utilized for cardiac fibroblasts, as well as our recently reported findings on cardiac fibroblast markers and origins3 and their clinical impact. Fibroblasts are abundant interstitial cells principally known for secreting extracellular matrix, particularly collagen type I. Markers used to identify fibroblasts include CD90 (Thy1), DDR2, and fibroblast specific protein 1 (FSP1), although none are fibroblast-specific.2 To label cardiac fibroblasts in heart at baseline, we used a Collagen1a1-GFP reporter line4 that we found was not expressed in nonfibroblast lineages including endothelium, pericytes, and haematopoietic lineages. The mesenchymal marker PDGFRa, expressed by fibroblasts,5 was coincident with Collagen1a1-GFP. In contrast, a marker commonly used to label fibroblasts, Fibroblast Specific Protein 1 (FSP1), was expressed in leukocytes and was absent from Collagen1a1-GFPC fibroblasts. Following pressure overload, Collagen1a1-GFPC fibroblasts proliferated, and constituted the major cell population in fibrotic lesions as identified by excess
collagen deposition. Interestingly, a smooth muscle actin (aSMA), used to identify activated fibroblasts, termed “myofibroblasts,” was expressed in only approximately 15% of fibroblasts in interstitial lesions, and not in perivascular lesions, suggesting that many fibroblasts would be overlooked when using this marker, at least in the context of pressure overload. Following pressure overload, FSP1 marked a significant proportion of CD45C immune cells, and labeled only 50% of fibroblasts in perivascular lesions, but did not label fibroblasts in interstitial lesions, demonstrating the limitations of this marker for identifying fibrotic fibroblasts (Fig. 1). Observed heterogeneity of fibroblast gene expression in interstitial and perivascular lesions is consistent with work of others6 and could be further analyzed by approaches such as single cell transcriptomics and clonal analysis. EndoMT of subsets of endocardial cells of the atrioventricular canal and outflow tract during midgestation generates mesenchymal valve progenitor cells. It has been suggested that an analogous process is reactivated following pressure overload, generating fibroblasts from the microvasculature.1 However, our lineage tracing of adult endothelium with an inducible endothelial specific Cre, VEcadherin-CreERT2 did not reveal evidence that EndoMT had occurred.3 A previous study performed to assess the occurrence of EndoMT utilized an endothelial Cre, Tie1-Cre, which labels both endothelial and haematopoietic lineages.1 It has emerged from work of others,7 and our work, that a majority of FSP1 expressing cells in fibrotic lesions are immune cells, not fibroblasts. These FSP1 expressing
immune cells are labeled by most endothelial Cres. Thus, the assumption that FSP1 is a specific marker for fibroblasts would lead to the mistaken conclusion that fibroblasts were derived from endothelial or haematopoietic lineages.1 Utilizing a pan-haematopoietic Cre, Vav-Cre, in conjunction with Collagen1a1-GFP and PDGFRa, we could not find evidence for any fibroblasts of haematopoietic origin in fibrotic lesions following pressure overload.3 Fibroblasts have been shown to derive from epicardial EMT during midgestation, although other developmental origins have not been excluded.2 Using epicardial-specific Cre drivers, we observed that most, but not all, cardiac fibroblasts were of epicardial origin. We identified a second cardiac fibroblast lineage residing mainly in the septum that we show likely derives from endocardial EndoMT associated with endocardial cushion formation. In this manner, our study demonstrated that resident cardiac fibroblasts develop from 2 distinct lineages that are differentially distributed within myocardium (Fig. 1). Combined epicardial and endothelial lineage tracing accounted for approximately 95% of fibroblasts in fibrotic hearts. Although we could detect no EndoMT, it remains a possibility that other, non-fibroblast lineages labeled by epicardial Cres may also contribute to fibroblasts during fibrosis. In this regard, pericytes, derived from epicardial lineages in heart, become fibrogenic in various other organs, notably liver and kidney.4,5 In the pressure overload mouse model, both interstitial and perivascular fibrotic lesions develop. The 2 developmentallyderived fibroblast lineages we identified
*Correspondence to: Sylvia M Evans; Email: [email protected] Submitted: 07/29/2014; Accepted: 08/04/2014 http://dx.doi.org/10.4161/15384101.2014.954212 Comment on: Moore-Morris T, et al. J Clin Invest 2014; 124(7):2921-34; PMID:24937432; http://dx.doi.org/10.1172/JCI74783
www.landesbioscience.com
Cell Cycle
2647
Downloaded by [Georgetown University] at 06:51 02 March 2015
Figure 1. Cardiac fibroblast markers and lineages following pressure-overload. At baseline and following pressure overload, all fibroblasts are marked by Collagen1a1-GFP and PDGFRa. FSP1 labels most haematopoietic cells and a subset of fibroblasts (50%) in perivascular areas. aSMA labels a small subset of fibroblasts (15%) in interstitial lesions. Spatially complementary endocardial- and epicardial-derived lineages behave similarly following pressure overload.
contributed to both perivascular and interstitial fibrotic lesions, located within myocardial regions reflecting their original developmental distribution. Intriguingly, References 1. 2.
3.
Zeisberg EM, et al. Nat Med 2007; 13(8):952-61; PMID:17660828 Zeisberg EM, et al. Circ Res 2010; 107(11): 1304-12; PMID:21106947; http://dx.doi.org/10.1161/ CIRCRESAHA.110.231910 Moore-Morris T, et al. J Clin Invest 2014; 124(7): 2921-34; PMID:24937432; http://dx.doi.org/10.1172/ JCI74783
2648
our analyses demonstrated that both lineages responded very similarly to pressure overload in terms of proliferation and transcriptional regulation, despite their 4.
5.
Kisseleva T, et al. Proc Natl Acad Sci U S A 2012; 109 (24):9448-53; PMID:22566629; http://dx.doi.org/ 10.1073/pnas.1201840109 Smith CL, et al. Circ Res 2011; 108(12):e15-26; PMID: 21512159; http://dx.doi.org/10.1161/CIRCRESAHA. 110.235531
Cell Cycle
distinct developmental origins and highlight the possibility of identifying common therapeutic targets. Hence, our study predicts that targeting proliferation of resident cardiac fibroblasts will be critical for mitigation of fibrosis. This hypothesis is in agreement with previous clinical observations on the anti-fibrotic action of Losartan, an Angiotensin II receptor blocker, likely mediated by its action on fibroblast proliferation. However, as fibrosis occurs rapidly, developing approaches to resolve established scarring will be important. In this context, much may be learned from models of fibrosis in the liver, where fibroblasts undergo apoptosis and quiescence during scar resolution.4 Interestingly, we identified upregulated genes associated with “negative regulation of cell death” in cardiac fibroblasts from hypertrophic hearts, suggesting cardiac fibroblasts could be particularly resistant to apoptosis. 6.
7.
Braitsch CM, et al. J Mol Cell Cardiol 2013; 65:10819; PMID:24140724; http://dx.doi.org/10.1016/j. yjmcc.2013.10.005 Kong P, et al. Am J Physiol Heart Circ Physiol 2013; 305(9):H1363-72; PMID:23997102; http://dx.doi. org/10.1152/ajpheart.00395.2013
Volume 13 Issue 17
