Scholarly article on topic 'Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts'

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts Academic research paper on "Biological sciences"

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{"Mesenchymal stem cell" / TGF-β / "Inflammatory fibroblast" / MCP-1 / Aging}

Abstract of research paper on Biological sciences, author of scientific article — Katarzyna A. Cieslik, JoAnn Trial, Jeffrey R. Crawford, George E. Taffet, Mark L. Entman

Abstract Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-β), due to reduced expression of TGF-β receptor I (TβRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-β promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-β signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TβRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ".

Academic research paper on topic "Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts"

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Journal of Molecular and Cellular Cardiology

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Review article

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts


Katarzyna A. Cieslik *, JoAnn Trial, Jeffrey R. Crawford, George E. Taffet, Mark L. Entman **

Division of Cardiovascular Sciences and the DeBakey Heart Center, Department of Medicine, Baylor College of Medicine, and Houston Methodist, Houston, TX 77030, USA


Article history:

Received 12 August 2013

Received in revised form 2 October 2013

Accepted 22 October 2013

Available online 31 October 2013


Mesenchymal stem cell TGF-ß

Inflammatory fibroblast MCP-1


Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-ß), due to reduced expression of TGF-ß receptor I (TßRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-ß promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-ß signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TßRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium".

© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.


1. Introduction...............................................................57

2. Fibrosis as a result of chemokine dysregulation; the myeloid (CD45+) fibroblast................................57

3. Aberrant MSC differentiation in aging; role of reduced TGF-p responsiveness..................................57

3.1. Consequences of loss of TGF-p responsiveness............................................58

3.1.1. Nanog reduction .....................................................58

3.1.2. Failure of MCP-1 suppression................................................58

4. TGF-p resistance and progressive fibrosis in the aging heart — an apparent paradox...............................58

4.1. MSC-derived fibroblast.......................................................58

4.2. Myeloid fibroblast.........................................................59

4.3. Inflammatory signaling.......................................................59

4.4. MSC-derived fibroblasts as a source of chronic MCP-1 generation....................................59

5. Chronic myocardial fibrosis arises from dysregulation of signaling between fibroblasts of two distinct origins — the inflammatory fibroblast hypothesis 59

6. Possible therapeutic strategy .......................................................60

7. Conclusions...............................................................61

Disclosure statement .............................................................61



☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Correspondence to: K.A. Cieslik, Baylor College of Medicine, Department of Medicine, Division of Cardiovascular Sciences, One Baylor Plaza, M.S. BCM620, Houston, Texas 77030. Tel.: +1 713 798 1952; fax: +1 713 796 0015.

** Correspondence to: M.L. Entman, Baylor College of Medicine, Department of Medicine, Division of Cardiovascular Sciences, One Baylor Plaza, M.S. BCM620, Houston, Texas 77030. Tel.: +1 713 7984188; fax: +1 713 796 0015.

E-mail addresses: (K.A. Cieslik), (M.L Entman).

0022-2828/$ - see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Secretion of extracellular matrix (ECM) proteins can be both adaptive and deleterious. Various pathological conditions trigger ECM (mostly collagens) deposition in the heart. A sudden loss of cardiomyocytes from a myocardial infarction (MI) causes activation of replace-ment/reparative fibrosis, where collagens substitute for necrotic cardiomyocytes and form a scar to preserve structural integrity of the heart [1]. In other pathophysiological conditions (in the absence of infarction) such as pressure overload [2], cardiomyopathy [3], and diabetes [4] adverse fibrosis has been observed as an interstitial or perivascular deposition of ECM. Interestingly, normal aging has been associated with progressive fibrosis [5], but the mechanism responsible for increased collagen content in the aging heart may be more complex than initially thought.

In the heart, both reparative scar formation [6] and adverse reactive fibrosis [7,8] are associated with pathophysiologic factors mediated by inflammation. Tissue inflammation is initiated by recruitment of leukocytes from blood through the venular endothelial barrier. This process can be modulated by endothelial and stromal cells. The endothelium regulates leukocyte recruitment by modulating expression of specific adhesion molecules that attract leukocytes and promote their adhesion to the endothelial cell border [9].

Evidence also suggests that other cells such as fibroblasts [10,11] and macrophages [12] may play important roles in leukocyte recruitment as well. It has been demonstrated in in vitro studies that fibroblasts derived from chronically inflamed tissue supported leukocyte diapedesis [13], in contrast to fibroblasts derived from healthy donors [14]. The described effect was attributed to the presence of MCP-1 [13]. MCP-1 is a strong monocyte and T lymphocyte chemoattractant [15]. Elevated expression levels of MCP-1 and CCR2 (an MCP-1 receptor) has been reported in various diseases characterized by inflammation such as rheumatoid arthritis, multiple sclerosis and asthma [16]. In this communication, we focus on the role of inflammatory signaling between different fibroblast precursors as the source of chronic progressive cardiac fibrosis.

2. Fibrosis as a result of chemokine dysregulation; the myeloid (CD45+) fibroblast

CD45+ fibroblasts of myeloid origin have been identified in various models of adverse fibrosis studied in our laboratory. When we examine cardiac fibrosis as a consequence of non infarctive brief daily coronary occlusion (ischemia/reperfusion cardiomyopathy, I/RC) [7] or angiotensin II infusion [17] and in a chronic condition such as aging [5], all models were characterized by the presence of CD45+ myeloid fibroblasts in the myocardium. Fibroblasts of myeloid origin are spindle-shaped and smallerthan structural (mesenchymal) fibroblasts; they express collagen type I and alpha-smooth muscle actin (a-SMA), and are highly proliferative [7]. The presence of these cells in the heart relies on expression of MCP-1 since genetic deletion of MCP-1 [17,18] or its receptor [19] prevents development of cardiac fibrosis. Our in vitro studies using human monocytes and a cardiac endothelial layer confirmed that in response to MCP-1, monocytes and T cells migrate through the endothelial barrier [20,21] and after transmigration the monocytes transition into myeloid fibroblasts. This transition is dependent on Th1 and Th2 lymphocyte induction and secretion of specific lymphokines and requires transendothelial migration [5]. The role of MCP-1 in fibrosis has been studied by other laboratories as well. Its importance in the promotion of fibrosis in various models and organs has been confirmed in kidney [22], liver [23] and lung [24].

In the acute models of fibrosis following daily ischemia and reperfusion [25] or angiotensin II infusion [17], expression of MCP-1 was elevated for up to 14 days and then its level was reduced to baseline even in the presence of continuing stimulus [25], suggesting that an MCP-1 repressing mechanism was activated. Despite the continued insult, the number of myeloid fibroblasts was also gradually reduced

[17] and fibrosis did not progress [25]. Suppression of MCP-1 production correlates with induction of de novo TGF-p synthesis [6]; moreover, TGF-p-dependent activation of Smad 3 attenuates MCP-1 transcriptional activation [26]. TGF- null mice, on the other hand, are characterized by an excessive inflammation and massive and unattenuated infiltration of leukocytes [27,28]. Genetic deletion of thrombospondin-1, a protein that determines TGF-p biological activity, resulted in prolonged post-infarction inflammation [29], implying that TGF-p plays a crucial role in resolution of inflammation.

In contrast to acute models, in a chronic model, aging, we have demonstrated a progressively elevated MCP-1 expression starting in middle age (14 months of age) accompanied by an increasing number of myeloid fibroblasts [5]. This indicates that in the aging heart the factor that suppresses MCP-1 expression is not operant. As a result, progressive fibrosis was observed with aging accompanied by a progressive increase in T lymphocytes (CD3) and the Th2 lymphokine (1L-13) [5]. All of these markers are compatible with persistence of MCP-1 induced chemotaxis.

Interestingly, elevated recruitment of CD45+ cells in aged mice was observed not only in the heart [5] but also in the liver [30], kidney [31] and lung [32] suggesting perhaps a common mechanism behind this phenomenon.

3. Aberrant MSC differentiation in aging; role of reduced TGF-p responsiveness

Our studies of fibrosis in aging, however, also uncovered another distinct class of fibroblasts. We identified these fibroblasts as CD44+CD45neg cells arising from mesenchymal stem cells (MSC) of non-myeloid origin [33]. We have found that the number of these cells progressively increases in the aging myocardium and concomitantly with age progression a higher number of these cells differentiate into collagen producing fibroblasts but not myofibroblasts [33]. These fibroblasts were larger than the myeloid fibroblasts and were CD45neg. In contrast to the myeloid fibroblasts, they did not become myofibroblasts (i.e. they were a-SMA negative). These fibroblasts appeared to emanate from the endogenous MSC found in the heart that we have previously implicated as the major source of fibroblasts for myocardial scar formation after MI [34]. In the heart, cardiac MSC are biased toward a fibroblast lineage [34] although they maintain their plasticity and ability to differentiate into other lineages as well (in vitro) [35]. In the aging heart, MSC are dysregulated and demonstrate abnormalities in lineage choice as well as a defect in conversion of fibro-blasts to myofibroblasts [36]. This latter abnormality results in the poorer quality scars found after MI in aging mice [37-39].

We have examined the mechanism by which resistance to TGF-p might influence the aberrant function of MSC in the aging heart. Multipotency of these stem cells is maintained mostly by Nanog [40], a transcription factor that is associated with various proteins in repression complexes, which controls other genes' transcription and prevents differentiation [41]. Nanog expression is directly activated by various factors, including TGF-p [42,43]. TGF-(3 signals via two serine/thereonine kinase receptors: type I receptor (T(3RI) and type II receptor (TpRll) [44]. Three types of T(3RI have been identified: activin like kinase (ALK) 1, 2 and 5 [45-47]. The T(3RI in MSC and mesenchymal fibroblasts described below has been identified as ALK5 [36].

Interestingly, MSC isolated from 30-month old hearts display a reduced expression of T(3RI (Fig. 1A) and therefore their TGF-p responsiveness is diminished. Concomitantly with reduced T(3RI expression the Nanog level was reduced by ~80% in aged cardiac MSC compared to young controls (Fig. 1B, C left panel). Since TGF-p downregulates expression of MCP-1 [26] by controlling the activity of AP-1, a transcription factor involved in MCP-1 transcriptional induction, we decided to examine MCP-1 mRNA levels in MSC derived from young and aged hearts. We found that as MCP-1 mRNA expression increases with age, the Nanog transcript level proportionally declined (Fig. 1B, C). Thus, defective TGF-p signaling appeared to result in increased MSC to fibroblast

Fig. 1. The inflammatory phenotype of MSC derived from aged hearts concurs with their reduced expression of TpRI and Nanog. A. TpRI mRNA expression in MSC derived from young and aged hearts. n = 3, 3 for young and aged mouse hearts from C57BL/6. p = 0.053 evaluated by Student's t test. B. Expression of Nanog and MCP-1 transcripts in MSC derived from 3-, 14- and 24-30-month old mice. n = 5,4, 5 indicate number of donor mice per age group starting from the youngest. C. Tables show Nanog (left) and MCP-1 (right) expression with corresponding statistical analysis. mRNA levels were examined by qPCR performed on a CFX 96 Real Time PCR Detection System using SYBR green technology. Primers were validated according to MIQE quidelines [103]. Gene expression was normalized to the level of an endogenous reference (Hprt). B and C are two different representations of the same experiments. * denotes p < 0.05 compared to control sample as analyzed by ANOVA. The data for Nanog expression is modified with permission from Cieslik K.A., Trial J., Entman M.L. Defective myofibroblast formation from mesenchymal stem cells in the aging murine heart: Rescue by activation of the AMPK pathway. The American Journal of Pathology (2011), 179,1792-1806.

transition and augmented production of MCP-1. We will discuss this in the next section.

31. Consequences of loss ofTGF-fi responsiveness

3.1.1. Nanog reduction

A reduced level of Nanog may allow MSC escape from an undifferentiated state since it has been demonstrated that a low level of Nanog correlates with increased cell differentiation [48] and we observed an elevated number of CD44+CD45neg fibroblast precursors in the aged myocardium [33]. To examine further the role of reduced TGF-p responsiveness, a potential culprit in aberrant MSC conduct, we subjected MSC derived from young hearts to chronic inhibition of TpRI by supplementing their culture medium with a TpRI kinase inhibitor (LY364947). TpRI inhibition in young cells resembled the scenario in aging presented in Fig. 1B and C: Nanog expression was reduced (Fig. 2).

In our quest to understand the biology of cardiac MSC in aging, we challenged the multipotentiality of these cells by subjecting them to adipocytic, chondrocytic and osteogenic differentiation programs. As TGF-p, and more specifically activated Smad3, negatively controls adi-pogenesis [49] it seems logical that reduced TGF-p responsiveness

Fig. 2. Inhibition of TpRI activity in MSC derived from young mouse hearts resulted in a proinflammatory phenotype and reduction of Nanog levels. mRNA levels of Nanog and MCP-1 expression in MSC treated with or without 500 nM LY364947 (inhibitor) for 21 days. Cells derived from 4 different animals were used in 4 independent experiments. Gene expression was normalized to the level of an endogenous reference (Hprt). * denotes p < 0.05 compared to control sample as analyzed by Student's t test.

would increase MSC "sensitivity" to adipogenic signals. These abnormalities are associated in vivo with elevated insulin levels [33]. Thus TGF-p plays a crucial role in maintaining the MSC primitive state and prevents skewing into lineages other than the "default" fibroblast. Abnormal stem cell differentiation with aging has been reported in satellite cells, mouse hematopoietic stem cells and bone marrow MSC [50,51].

3.1.2. Failure of MCP-1 suppression

As discussed above, TGF-p exerts an important anti-inflammatory role which features suppression of chemokines [6,26,29]. This property also appears to be pertinent to MSC and their progeny. The aging MSC fail to suppress MCP-1 production despite ample supplies of cardiac TGF-p (Figs. 1B, C), and similarly, inhibition of TpRI in MSC derived from young hearts also results in elevated expression of MCP-1 (Fig. 2).

4. TGF-p resistance and progressive fibrosis in the aging heart — an apparent paradox

TGF-p is a canonical growth factor that promotes extracellular protein synthesis [52]. Its role in fibrosis has been well established in the heart, lung, kidney, and liver [53-56], and inhibition of TGF-p signaling has been shown to reduce adverse fibrosis in many models [55,56]. The finding that TGF-p resistance actually enhances fibrosis by several mechanisms in the aging heart involving fibroblast precursors of two separate origins has afforded us important insights into the signaling mechanisms promoting this critical paradox and a better understanding of the emergence of chronic ongoing fibrosis.

Changes of TGF-p signaling have been associated with age-related osteoarthritis and skin photodamage. The former has been linked to a switch from ALK5 to preferential ALK1 mediated signaling [57], and the latter showed reduced expression ofTpRIl [58]. Both mechanisms link reduced Smad2/3 mediated signaling with aging similar to our aging heart model [36].

4.1. MSC-derived fibroblast

The first signaling relates to the species of fibroblasts found in the aging heart that arise from TGF-p resistant MSC described above [36]. Because they retain the TGF-p resistance [36], these fibroblasts do not become myofibroblasts and appear to be associated with the interstitial fibrosis seen in aging [33]. One potential mechanism relates to the fact that loss of TGF-p induced Nanog suppression invites an increased

differentiation of MSC into fibroblasts, thus increasing the fibroblast population. Our studies also suggest a role for signaling via farnesyl transferase and ERK in the TGF-p resistant fibroblasts [33]. Others have also reported that a-SMA deficiency has been previously noted as a factor associated with enhanced fibrosis in the lung [59] and kidney [60].

4.2. Myeloid fibroblast

The second signaling event relates to the myeloid fibroblasts responding to a persistence of MCP-1 expression that normally abates in other more acute models of adverse fibrosis [8,20]. In the aging model, the myeloid fibroblast expressed a-SMA but a-SMA expression was not evident in the mesenchymal fibroblast [33]. Therefore TGF-p resistance was confined to the endogenous MSC and their fibroblast progeny [36]. It suggested the possibility that MSC, or cells arising from them, might be an important factor in the persistence of MCP-1 production, and promote formation of myeloid fibroblasts.

4.3. Inflammatory signaling

Both of these fibrosis mechanisms depend on dysregulation of inflammatory signaling very commonly associated with MSC differentiation. Myeloid fibroblasts and MSC differentiation are both potently regulated by TGF-p, which also influences monocyte and T lymphocyte signaling [61,62]. Fibroblasts emanating from MSC may increase in number because of the increased MSC differentiation resulting from Nanog alterations but are also resistant to TGF-p suppression of a variety of inflammatory cytokines and chemokines. The remainder of this section begins an investigation of potential mechanisms linking the mechanisms by which fibroblasts of two different origins combine to induce fibrosis in aging.

4.4. MSC-derived fibroblasts as a source of chronic MCP-1 generation

Fig. 3. Fibroblasts derived from aged MSC present an inflammatory phenotype.

A. Increased synthesis of MCP-1 mRNA and protein in fibroblasts derived from 30 month old MSC mice (aged) compared with young controls from 3 month old mice (young). Gene expression was normalized to the level of an endogenous reference (Hprt). mRNA was measured in quiescent fibroblasts. Expression of protein secreted into the culture medium within 24 h was measured on RayBio Mouse Cytokine Antibody Array and normalized to the positive control. * denotes p < 0.05 (Student's t test).

B. Elevated level of superoxide in fibroblasts derived from 30 month old cardiac MSC visualized via MitoSox. n = 3,3 for young and aged donors.

To further assess the role of TGF-p in cardiac fibrosis in the aging heart, we analyzed mouse hearts from 3 and 30 month old C57BL/6 mice for TGF-(31 transcript levels by quantitative PCR (qPCR). We found a modest increase of TGF-(31 in aged hearts (1.19 ± 0.075 in 3 month old versus 2.23 ± 0.43 in 30 month old) that was statistically significant (p = 0.037). TGF-p is present in the uninjured heart mostly in a latent, inactive form. This latent TGF-p is bound to extracellular matrix (ECM) and its release from matrix is triggered by various enzymes [63]. For this potential signaling, TGF-(31 is plentiful in the aged heart and its release in the bioactive form [64] from the matrix is via enzymes whose expression is enhanced by IL-13 [65] or MMP9 [64] (which are also elevated in the aged heart [5,66]).

Although TGF-p seems to be essential for development of fibrosis via induction of ECM protein synthesis [67] and promotion of fibroblast maturation [68] its role in aging dependent fibrosis may be different and related to signal alteration via downregulation of the receptor [58] or switch of preferential effector downstream of the receptor [57].

Next, we examined the expression of MCP-1 transcript and protein levels in MSC-derived fibroblasts (Fig. 3A). The defect we observed in aged MSC (Fig. 1B, C right panel) was passed on to their progeny fibroblasts; their expression of MCP-1 was elevated by 7 and 4 fold in mRNA and secreted protein levels respectively when compared to young controls.

In addition to the failure of TGF-p suppression, several mechanisms may play roles in the upregulated expression of MCP-1. We hypothesized that reactive oxygen species (ROS) are important in the chronic induction of MCP-1 in fibroblasts derived from aged MSC based on our previously demonstrated association between ROS levels and MCP-1 expression [25,69,70]. Analysis of quiescent cultured fibroblasts revealed an elevated production of superoxide in aged fibroblasts when compared to young controls (Fig. 3B) suggesting that an altered mitochondrial

function may be responsible for the defect. However we have not excluded other pathways that might be involved in this exaggerated MCP-1 synthesis. Others have shown that the angiotensin II type I receptor 1 (AT-1) may be involved in MCP-1 induction in a model of pressure overloaded hearts [71]. Increased local expression of ACE and AT-1 in the aging heart has been reported by our laboratory before [5].

5. Chronic myocardial fibrosis arises from dysregulation of signaling between fibroblasts of two distinct origins — the inflammatory fibroblast hypothesis

Our studies demonstrated a major role for the age dependent increase in myeloid fibroblasts in cardiac fibrosis of the aging mouse, which was likewise accompanied by increases in MCP-1 as well as myeloid fibroblasts [5]. As opposed to the acute models described above, this chronic fibrosis was attended by an age-dependent increase in the presence of myeloid fibroblasts as well as chemokines and lym-phokines that characterized the signal cascade driving their formation [5]. The aging model, therefore, represented an opportunity to gain potential insight into ongoing chronic fibrosis in the heart but not present in the acute models.

We postulated that the chronic models involved important dysregulation in a signaling process that ordinarily suppresses inflammatory signaling related to myeloid fibrosis [5]. As described above, studies had suggested that the fibroblasts mediating myocardial scar formation after ischemia did not arise from myeloid fibroblasts but appeared to arise from MSC [34]. Subsequent studies demonstrated that these MSC-derived fibroblasts were progressively abnormal in the aging mouse; as described above, there was a marked reduction in TGF-(3 responsiveness

and the fibroblasts did not convert to myofibroblasts [36]. This abnormality resulted in defects in myofibroblast function manifested both in vivo and in vitro [36-39]. The pathologic consequence of this with respect to scar formation is discussed in detail elsewhere [39]. In this report, we will address the significance of TGF-p resistance in MSCs as it affects inflammatory signaling.

Based on these observations we postulate that MCP-1 secreted by MSC (that are in a perivascular location [72]) attracts the migration of myeloid leukocytes through an endothelial barrier and promotes leukocyte influx into the tissue via an MCP-1 gradient. A number of experiments indicate an anti-inflammatory or immunosuppressive role for MSC. It has been demonstrated that bone marrow MSC can suppress T cell [73], B cell [74] and NK cell [75] proliferation. In vitro studies have demonstrated that human MSC can induce the generation of regulatory T cells, a T cell subtype that prevents autoimmunity and maintains immune homeostasis [76]. It is thought that MSC exert their immunosuppressive property via secretion of various mediators (for an extensive review see [77], and that this paracrine effect may have a potential therapeutic use in inflammatory dysregulations such as multiple sclerosis [78], inflammatory bowel disease [79] and diabetes [80]. In fact, several clinical trials are already completed or ongoing, that study systemic use of MSC in immunological disorders ( In parallel, there are reports describing MSC inhibition of inflammatory-dependent fibrosis in skin [81] and kidney [82] models. From a study by Waterman et al. it is clear that MSC function can be modulated by external signals [83], suggesting that the environment of the aging heart may alter the function of resident MSC.

We hypothesize that MCP-1 released by mesenchymal fibroblasts promotes the migration of monocytes and T lymphocytes away from sub-endothelial compartments, as described by McGettrick at el [84], and their subsequent transition to fibroblasts. Monocytes that migrate through endothelium differentiate early after a chemoattractive stimulus to M1 (classically) and later to M2 (alternatively) activated macrophages [85]. As the M1 response transitions towards the M2 response the cells start expressing collagen type I (MCP-1 increases expression of TGF-p and collagen type I in human monocytes [86]) and effectively become myeloid derived fibroblasts. This process is supported by T cells that migrate together with monocytes in response to the MCP-1 gradient [5]. Once the T cells are in the tissue, they polarize to Th1 (proinflammatory) and Th2 (profibrotic) subtypes. Th2 lymphocytes promote the transition from monocytes to M2 alternatively activated macrophages and finally to myeloid fibroblasts [87], and Th1 lymphocytes activate lysyl oxidase (LOX) 1 and 3, enzymes that crosslink fibrillar collagen [88]. Thus, it is clear that MCP-1 affects various cells by orchestrating several processes leading to myeloid dependent fibrosis [5].

The functional relationship between the roles of fibroblasts of mesenchymal and myeloid origins in the progression of chronic cardiac fibrosis has been delineated in Fig. 4. The role of the inflammatory fibroblast and its chemokine(s) expression has been suggested in other models such as myocardial infarction (MI) [10], rheumatoid arthritis [11] and skin fibrosis [89].

In this review we extensively discuss two types of fibroblasts that are of mesenchymal and myeloid origins, both of which participate in adverse fibrosis in aging. Several sources of mesenchymal fibroblasts other than from resident cardiac MSC have been implicated in the development of fibrosis. Epithelial cells that acquire a mesenchymal phe-notype (EMT) have been shown to contribute to a pool of fibroblasts, especially under inflammatory conditions [90]. Likewise, endothelial to mesenchymal transition (EndoMT) has been linked to fibrosis [91] as well as bone marrow MSC [92] and pericytes [93]. Input of these cells (especially originating from EMT and EndoMT) into fibrosis has been recently challenged by other scientists based on lack of specificity of one of the marker used to characterized these cells, FSP-1 [94,95].Ad-ditionally it was demonstrated that these mesenchymal fibroblasts that originated from various sources mature into myofibroblasts expressing a-SMA. We have demonstrated that in the aging heart the majority of

endothelial cells

myeloid fibroblast

Fig. 4. Proposed mechanism of fibrosis in the aging heart. In the aged heart MSC differentiate into mesenchymal fibroblasts that have reduced TGF-ß responsiveness but secrete elevated levels of MCP-1. MCP-1 enhances monocyte transendothelial migration contributing to an increased number of myeloid fibroblasts in the aged heart. Fibroblasts of mesenchymal and myeloid origin participate in development of fibrosis in the aging hearts by producing and depositing collagens. Mo and M^ denote monocyte and macrophage respectively.

mesenchymal fibroblasts are a-SMAneg [33]. Although that does not exclude the above described cells from contributing to the fibroblast pool (because the defect in TGF-ß signaling may not be restricted only to resident MSC), our studies demonstrated that the changes seen in resident MSC recapitulate the abnormalities see in vivo [34,39].

6. Possible therapeutic strategy

Whether advanced fibrosis can be reversed is controversial. It has been demonstrated that the collagen content of the left ventricle in aged animals increases from 5.5% to 12% [96] and it has been postulated that fibrosis reversibility depends on the degree of cross-linked ECM [97]. From our acute model of fibrosis we found that once the ischemia/ reperfusion ceased, the collagen content in the heart was reduced within 30 days [98]. As we reported before that elevated collagen deposition is related to a CD45+ cell influx and their transition into fibroblasts [5,7,17], a possible strategy would be to reduce the ability of these cells to transmigrate or differentiate. On the other hand, in the aging heart dysfunctional mesenchymal fibroblasts display upregulated collagen synthesis [33]. Collagen synthesis originating from both these sources of fibroblasts could be modulated via reduction of prenylation. We discovered that small G proteins (Rho and Ras) are involved in myeloid and mesenchymal fibroblast-dependent fibrosis respectively. Two enzymes, geranylgeranyltransferase (GGTase) and farnesyltransferase (FTase) transfer the isoprenyl chain formed during cholesterol biosynthesis onto a variety of proteins, which then affects their membrane targeting, cellular localization and proteinprotein interactions [99]. GGTase targets proteins such as Rho, whereas FTase attaches farnesyl groups to Ras proteins [100]. We found that genetic inhibition of ROCK-1, a Rho effector, resulted in attenuated fibrosis and reduced number of myeloid fibroblasts in the heart [101], suggesting that the use of a specific GGTase inhibitor could shut down myeloid cell-dependent fibrosis. Also, the use of an FTase inhibitor, as we have established before, reduced collagen expression

in mesenchymal fibroblasts via targeting the Ras/Erk pathway 33]. Taken together we postulate that aged dependent cardiac fibrosis could be reduced by targeting prenyl transferase pathways (via GGTase and FTase inhibitors) or prenyl chain biosynthesis (via statins). Use of statins in treatment of fibrosis has been shown to be beneficial in experimental models [ 102]. Finally, from the prevention standpoint, treatments or behavioral modifications that reduce insulin resistance and lower arterial insulin levels may prevent these changes in MSC that we have suggested to arise from high insulin levels 33].

7. Conclusions

We present here a new concept of biological cross-talk between fibroblasts of myeloid and mesenchymal origin that supports the development of chronic fibrosis in the aging uninjured heart. The evidence suggests that MCP-1 and TGF-p are major players in this scenario. In the aging model chemokine synthesis is persistent 5] suggesting a dysregulation of immune suppression. We demonstrate that unresponsiveness of mes-enchymal cells to TGF-p due to their defect in T(3RI expression results in insufficient suppression of chemokine synthesis in these cells (MSC and their fibroblast progeny). This increases the rate of differentiation of MSC (due to reduction of Nanog) and results in an elevated number of collagen expressing mesenchymal fibroblasts as observed in our studies [ 33]. In addition, resistance to TGF-p in MSC-derived fibroblasts prevents normal suppression of MCP-1. The resultant chronic expression of MCP-1 derived from stromal cells activates an influx of monocytes and their transition to myeloid fibroblasts 5]. Therefore, fibroblasts from two different origins contribute to fibrosis in the aging heart.

Disclosure statement

This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This work was supported by the NIH grant R01HL089792 (MLE), the Medallion Foundation grants (KC andJT) and the Hankamer Foundation.


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MSC: mesenchymal stem cells TGF-ß: transforming growth factor-ß TßRI: TGF-ß receptor I

MCP-1: monocyte chemoattractant protein-1 a-SMA: alpha smooth muscle actin