Introduction

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components, such as collagen and fibronectin. This process leads to tissue stiffness, organ dysfunction, and eventually failure [1, 2]. Fibrosis contributes to nearly 45% of deaths in industrialized countries. It is not a disease itself, but rather a dysregulated tissue repair response that becomes chronic due to repetitive injury or inflammation [2].

Cardiac fibrosis is a hallmark of cardiac diseases such as myocardial infarction (MI), hypertensive heart disease, and chronic kidney disease-induced cardiorenal syndrome. Myocardial remodeling due to fibrosis results in reduced compliance, impaired systolic and diastolic function, and arrhythmias [3, 4]. The initial fibrotic response plays a reparative role. It helps maintain structural integrity and prevent ventricular wall rupture. However, the exaggerated fibrotic response can lead to ECM deposition and reactive fibrosis [5]. This maladaptive response leads to ventricular stiffening, impaired cardiac function, and the progression to heart failure (HF) [6].

The central role of fibroblasts and macrophages in cardiac fibrosis is increasingly recognized. Fibroblasts serve as the primary producers of ECM. Macrophages support their activation by secreting cytokines and growth factors, such as TGF-β [7–10]. RCMs are a critical component of the heart’s immune microenvironment. They make up approximately 5–10% of the non-myocyte population in the murine myocardium during steady-state conditions [11, 12]. These macrophages originate mainly from embryonic progenitors, such as yolk sac and fetal liver-derived cells. They maintain their population through local proliferation rather than monocyte recruitment [11, 12]. Both resident and monocyte-derived macrophages interact with fibroblasts directly or via paracrine mechanisms, facilitating the fibrotic process [7, 9]. These interactions highlight the importance of studying both macrophage subpopulations and their roles in fibrosis progression.

One emerging concept in cardiac fibrosis is macrophage-to-myofibroblast transformation (MMT). Recent studies have demonstrated that macrophages can transdifferentiate into myofibroblast-like cells [1, 13–15]. MMT is a crucial mechanism connecting chronic inflammation to tissue fibrosis. Inflammation is widely recognized as a key driver of fibrotic progression. Human studies and experimental models have consistently demonstrated the pivotal role of MMT in advancing fibrotic conditions [16–20]. Myofibroblasts represent the primary cell type responsible for producing collagen during the development of tissue fibrosis [21]. CD206⁺ M2 macrophages can be differentiated into myofibroblasts [22, 23]. MMT is characterized by the co-expression of F4/80 (CD68) and α-SMA. The trans-differentiation process is mediated by TGF-β/Smad3 signaling [21, 24].

In this review, we summarize the cellular dynamics of cardiac fibrosis. We focus on the interactions between macrophages and fibroblasts and their roles in ECM remodeling. Through current insights into macrophage plasticity and fibroblast activation, we aim to elucidate potential therapeutic strategies for mitigating cardiac fibrosis and improving cardiac function.

Cardiac cell composition

Changes in the number and function of various cardiac cells significantly impact myocardial structure and function. These cells include cardiomyocytes, fibroblasts, endothelial cells, and macrophages. Such cells contribute to the progression of HF [25]. Cardiomyocytes dominate the myocardial mass, providing contractile force, but their function relies on structural and immune support from other cell types. Fibroblasts regulate and remodel the ECM, and endothelial cells maintain vascular function [26]. Macrophages, another key component, are involved in immune modulation and tissue repair [27].

Single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) allow the identification of specific cell populations, including rare and disease-specific cell states, by analyzing transcriptional signatures [28]. Prior to the advent of these tools, histology and flow cytometry were commonly used but lacked the capacity to resolve cellular heterogeneity at the single-cell level [29]. In combination with spatial transcriptomics and multiplex imaging, they provide detailed insights into cellular interactions and their spatial organization, offering a comprehensive understanding of cardiac dynamics in both health and disease.

Resident and non-resident macrophages

RCMs are a specialized population of immune cells derived from embryonic progenitors. They are uniquely adapted to maintain cardiac homeostasis and facilitate tissue repair under both normal and pathological conditions [12, 43]. These macrophages self-renew locally within the myocardium and exhibit anti-inflammatory properties through the expression of CCR2^– markers, enabling them to suppress excessive inflammation and promote vascular remodeling following cardiac injury [44, 45]. Notably, RCMs play an active role in preserving cardiac structure and function by phagocytosing apoptotic cardiomyocytes, clearing cellular debris, and releasing cytokines that modulate fibroblast activation and ECM remodeling [43, 46, 47].

Under stress conditions such as hypertension or MI, RCMs undergo phenotypic changes, upregulating reparative programs that include the secretion of insulin-like growth factor-1 (IGF-1), a critical mediator for adaptive cardiac growth [45]. This function of RCMs is essential to counteract the fibrotic response, as it prevents excessive ECM deposition and maintains myocardial compliance [12, 45]. However, in conditions where RCM populations are depleted or impaired, their protective effects diminish, leading to exacerbated fibrosis and functional decline [46].

Non-resident macrophages (NRMs) are monocyte-derived and are actively recruited into the heart during injury or inflammation, where they adopt a predominantly pro-inflammatory phenotype characterized by CCR2⁺ markers [48, 49]. These macrophages amplify the inflammatory response by releasing high levels of inflammatory cytokines, including TNF-α, IL-6, IL-1β, and MCP-1, and pro-fibrotic factors, such as TGF-β, PDGF, and FGF, which stimulate fibroblast activation and drive the transition to pathological fibrosis [50, 51]. In acute MI, NRMs infiltrate the infarcted myocardium in large numbers, replacing a portion of the RCM population, and dominate the inflammatory phase by promoting ECM deposition and exacerbating cardiac remodeling [44]. Additionally, macrophages contribute to cardiac fibrosis through the actions of angiotensin II (Ang II) and aldosterone, which promote their polarization toward a fibrogenic phenotype [52].

The interaction between RCMs and NRMs is critical in shaping the progression and resolution of cardiac injury. RCMs are involved in orchestrating the transition from inflammation to repair by modulating NRM activity, thereby facilitating a controlled reparative response [43, 46]. However, when the recruitment of NRMs is excessive, their pro-inflammatory activity can overwhelm the reparative capacity of RCMs, resulting in maladaptive remodeling characterized by stiffened myocardium and impaired cardiac function [27, 44].

Emerging technologies, including single-cell transcriptomics and advanced imaging techniques, have provided deeper insights into the distinct roles and gene expression profiles of RCMs and NRMs [12]. These studies reveal that RCMs exhibit transcriptional programs geared toward cardiac repair and homeostasis, while NRMs display a more inflammatory signature associated with fibrosis and tissue damage [53, 54]. Understanding the precise mechanisms governing the behavior of these macrophage populations opens new avenues for targeted therapies aimed at reducing fibrosis and enhancing cardiac repair (Figure 1).

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Figure 1. The mechanism of resident macrophages and non-resident macrophages in cardiac fibrosis. Resident cardiac macrophages with a Ly6C^low F4/80^hi CCR2^– phenotype support cardiac function and attenuate excessive fibroblast activation by secreting growth factors that promote tissue repair. In contrast, infiltrating Ly6C^hi CCR2⁺ non-resident macrophages produce higher levels of inflammatory cytokines and pro-fibrotic mediators, such as TNF-α, IL-6, IL-1β, and MCP-1. These factors stimulate the differentiation of fibroblasts into myofibroblasts, which are characterized by elevated expression of α-SMA, Col I, and Col III

Interaction between macrophages and fibroblasts

Macrophages and fibroblasts are central to the processes of cardiac repair and fibrosis. They engage in dynamic interactions that modulate each other’s behavior. These interactions are influenced by various signaling pathways, cytokines, and growth factors, which can either promote or suppress fibrosis depending on the context. Macrophages recruit fibroblasts by releasing growth factors and cytokines [55]. In addition, macrophages express coagulation factor XIIIa and provide extracellular fibrin matrix to fibroblasts [56, 57]. Myofibroblasts, derived from fibroblast differentiation under the influence of cytokines such as TGF-β, are key players in ECM remodeling and scar formation during cardiac repair [58, 59]. TGF-β signaling also promotes the secretion of matrix proteins [60, 61]. Persistent activation of myofibroblasts, along with their interactions with macrophages, leads to excessive ECM deposition, contributing to pathological fibrosis [58, 62]. Myofibroblasts secrete factors such as IL-6 and COX-2-derived prostaglandins (PGE2 and PGD2), which enhance the anti-inflammatory phenotype of macrophages. In turn, alternatively activated macrophages downregulate myofibroblast activity, including cell migration and cytokine output, creating a feedback loop that can limit fibrosis [63]. After MI, macrophages infiltrate the infarcted myocardium and release cytokines and growth factors that recruit and activate fibroblasts. This recruitment is crucial for ECM deposition and scar formation but can also result in adverse remodeling if not properly regulated [64, 65]. Macrophages secrete MCP-1 and TGF-β, which are essential for initiating fibroblast activation but also drive fibrosis when overexpressed [60, 66].

Fibroblasts and macrophages reciprocally regulate each other through the colony-stimulating factor 1 (CSF-1) and its receptor (CSF-1R). Fibroblasts secrete CSF-1 to sustain macrophage populations, while macrophages, in turn, influence fibroblast survival and activity through the secretion of cytokines, such as PDGFs [62]. This signaling axis is critical in fibrotic conditions, as CSF-1R blockade has been shown to attenuate fibrosis by limiting macrophage recruitment and reducing fibroblast activation [67, 68]. Matrix metalloproteinases (MMPs), secreted by both macrophages and fibroblasts, play a pivotal role in ECM degradation and remodeling. Dysregulation in MMP activity contributes to persistent ECM deposition, reinforcing tissue stiffness and fibrotic progression [62, 69]. This feedback loop highlights the need for strategies targeting MMP regulation to mitigate fibrotic remodeling. Studies on macrophage-derived factors, such as low-density lipoprotein receptor-related protein 1 (LRP1), have revealed their role in rejuvenating fibroblast functions. Young macrophages secrete factors that suppress fibrotic signaling, while aged macrophages show impaired ability to regulate fibroblasts, leading to exacerbated fibrosis [70].

Cardiac injury triggers the release of damage-associated molecular patterns (DAMPs) from necrotic myocardial cells, which act through toll-like receptor 4 (TLR4) to activate fibroblasts. This activation increases fibroblast proliferation, motility, and expression of ECM components such as collagen. DAMPs, including HMGB1 and S100 proteins, are pivotal in this process, as demonstrated by their ability to induce myocardial inflammation and fibrosis when injected into the heart [71]. In inflammatory dilated cardiomyopathy (DCM), fibroblasts respond to IL-17A by producing granulocyte/macrophage colony-stimulating factor (GM-CSF). This cytokine attracts pro-inflammatory macrophages, which further exacerbate cardiac fibrosis through a feed-forward loop. The IL-17A-GM-CSF axis demonstrates how cytokine-mediated interactions between fibroblasts and macrophages contribute to the progression of cardiac inflammation and fibrosis [72, 73]. Macrophage migration inhibitory factor (MIF), secreted by macrophages, exhibits antifibrotic effects by inhibiting fibroblast proliferation and matrix deposition via CD74/AMPK signaling. While primarily studied in liver fibrosis, similar mechanisms are likely applicable to the heart, where MIF may regulate macrophage-fibroblast interactions to suppress excessive ECM production [74].

Macrophage-to-myofibroblast transformation

MMT and cardiac fibrosis

MMT significantly contributes to myocardial fibrosis (MF). In scRNA-seq and lineage-tracing experiments, macrophages derived from circulating monocytes were shown to preferentially undergo MMT. In a myocardial ischemia-reperfusion (MIR) injury model, macrophages producing high levels of TGF-β were identified as key contributors to fibrotic remodeling. Notably, S100a9^hi macrophages, a subset identified through scRNA-seq, activated the Myd88/NFκB/NLRP3 and TGF-β/Smad3 signaling pathways. This dual activation facilitated both macrophage-to-myofibroblast transition and the transformation of cardiac fibroblasts into myofibroblasts [87]. A study [85] showed that this transition was driven by an increased ALKBH5 expression, which mediated m6A demethylation of IL-11 mRNA, enhancing its stability and protein expression. Mice with macrophage-specific deletion of ALKBH5 exhibited reduced MMT, attenuated cardiac fibrosis, and improved cardiac function. Notably, targeted siRNA delivery against ALKBH5 or IL-11 receptor α in macrophages effectively mitigated fibrosis in mice [85]. In cardiac fibrosis post-MI, macrophages were observed to acquire fibrotic markers such as fibroblast activation protein-α (FAP) and collagen type I during the reparative phase. The IKKε-p38 signaling axis plays a pivotal role in regulating macrophage responses to inflammation and tissue repair. In MI models using IKKε-deficient mice, reduced phosphorylation of p38 in macrophages lacking IKKε has been associated with impaired resolution of inflammation and an increased transition of MMT, which exacerbates fibrosis. This highlights the significance of p38 signaling in macrophage plasticity and its potential impact on fibrotic progression [88].

Integrated single-cell transcriptomics revealed that macrophages release profibrotic signals, including FN1, SPP1, and THBS1. In MI and DCM, macrophages expressing elevated levels of M1 polarization markers and myofibroblast markers directly facilitate MF progression. Transcription factors such as FOSB in DCM and MI and KLF6 in HF were identified as key regulators of this transformation. Moreover, the study highlights that macrophages undergoing MMT exhibit significant activation of toll-like receptors, ERK signaling, and the HIF-1 pathway, which are critical for promoting fibrosis in response to myocardial injury [89]. Treatment with eplerenone, an MR antagonist, was shown to inhibit MMT transformation by reducing the expression of intercellular adhesion molecule-1 (ICAM-1) and MCP-1, thereby limiting macrophage-driven inflammatory injury. The findings reveal the MR/SGK-1 pathway as a key regulator of MMT and underscore its role in the pathogenesis of unilateral ureteral obstruction (UUO)-induced cardiac fibrosis [90]. In another study using a rat model of UUO, significant cardiac fibrosis and MMT were observed, processes that were effectively suppressed by the MR antagonist, eplerenone. In vitro studies further demonstrated that aldosterone-activated MR promoted MMT and increased the secretion of connective tissue growth factor (CTGF) from macrophages. Blocking MR or CTGF inhibited these effects, suggesting that the MR/CTGF pathway plays a pivotal role in MMT-driven cardiac fibrosis [91, 92].

Erythropoietin receptor (EPOR) signaling has emerged as a significant regulator of MMT in fibrosis. Under TGF-β1 stimulation, EPOR activation in BMDMs enhances the expression of both F4/80 and α-SMA, indicating an increased MMT process [93]. In an MI model, macrophages undergoing MMT can express fibroblast markers, such as type I collagen (COL1A1), FSP1, and FAP, contributing to cardiac fibrosis (Figure 2). Depletion of macrophages reduces these collagen-producing cells, indicating that macrophages are key drivers of fibrotic remodeling in the heart [13, 94, 95]. Research on the MMT’s role in cardiac fibrosis remains limited. However, the emerging concept of MMT offers valuable insights into the mechanisms driving cardiac fibrosis. It also highlights MMT’s potential as a therapeutic target. Targeting MMT pathways could pave the way for novel strategies to treat cardiovascular diseases associated with cardiac fibrosis, providing hope for more effective interventions and improved clinical outcomes.

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Figure 2. Diagram of MMT in myocardial infarction. Upon MI, circulating monocytes infiltrate the damaged site and primarily differentiate into an M2 pro-fibrotic phenotype rather than an M1 pro-inflammatory phenotype. The TGF-β/Smad3 pathway mediates MMT, leading these cells to express fibroblast-associated markers such as α-SMA, COL1A1, FSP1, and FAP. MI: myocardial infarction; MMT: macrophage-to-myofibroblast transformation

Conclusion

Cardiac fibrosis is a critical factor in the progression of MI, HF, and hypertensive cardiomyopathy. This pathological process is driven by excessive deposition of ECM components, such as collagen type I and type III. These components are primarily produced by activated fibroblasts and myofibroblasts. The interplay between fibroblasts and macrophages plays a pivotal role in this process. Macrophages regulate fibroblast activation through cytokines and growth factors, including TGF-β, PDGF, and IL-6.

A significant breakthrough in understanding fibrosis is the recognition of MMT. During MMT, macrophages express dual markers such as CD68, F4/80 (macrophage markers), and α-SMA or collagen type I (myofibroblast markers). This process is primarily regulated by the TGF-β/Smad3 signaling pathway and influenced by factors like CTGF and MR activation. Inhibition of pathways like MR/CTGF with agents such as eplerenone has shown potential in suppressing MMT and reducing fibrosis.

Other regulatory mechanisms of MMT include epigenetic modifications like ALKBH5-mediated m6A RNA demethylation, which impacts macrophage plasticity and differentiation. Furthermore, macrophage subsets such as CD206⁺ M2 macrophages are more prone to MMT, contributing significantly to collagen deposition and scar formation. Depleting macrophages or inhibiting specific pathways can reduce MMT-derived myofibroblasts, highlighting therapeutic potential.

Despite these insights, research on MMT in cardiac fibrosis remains incomplete. The specific contributions of RCMs versus NRMs during different stages of fibrosis need further clarification. Additionally, the environmental cues such as hypoxia, mechanical stress, and inflammatory signals that drive MMT require more detailed investigation. Understanding how these factors interact with signaling pathways like TGF-β/Smad3, MR/CTGF, and ALKBH5 could uncover new intervention points. However, potential effects on macrophage-mediated tissue repair and immune regulation should be considered.

This review underscores the importance of MMT in cardiac fibrosis and its potential as a therapeutic target. By identifying key markers and pathways involved in macrophage-fibroblast interactions, this work lays the foundation for developing targeted therapies. Future studies integrating advanced techniques like scRNA-seq, spatial transcriptomics, and lineage tracing will be crucial for mapping the precise roles of macrophage subsets in fibrosis. Translating these insights into clinical applications could lead to innovative treatments for fibrotic heart diseases. This progress offers new hope for patients with MI, HF, and related conditions.

In conclusion, targeting MMT and its regulatory pathways holds promise for mitigating cardiac fibrosis. Addressing current knowledge gaps and pursuing these research avenues may transform the treatment landscape for cardiovascular diseases associated with fibrosis.