The Role of Thrombospondin-2 in Myocardial Fibrosis

Abstract

Myocardial fibrosis is a common pathophysiological process observed in various cardiac diseases and cardiac aging, characterized by excessive accumulation of extracellular matrix proteins in the cardiac interstitium. This process is highly heterogeneous and dynamic, depending on the underlying cardiac pathology and disease stage, and represents a key pathological basis for the development and progression of heart failure. Thrombospondin-2 (TSP-2), a critical extracellular matrix regulatory protein, exhibits complex dual roles in different disease contexts and stages, exerting both cardioprotective and potentially pathological effects. Recent studies have demonstrated that TSP-2 is involved in the pathogenesis of heart failure and may regulate myocardial fibrosis through modulation of extracellular matrix remodeling and related signaling pathways. This review summarizes the regulatory roles of TSP-2 in myocardial fibrosis under various cardiac disease conditions and discusses its potential molecular mechanisms. By integrating current studies, this review provides new insights into the regulatory network of myocardial fibrosis and highlights the potential of TSP-2 as a therapeutic target. However, further mechanistic and translational studies are required to validate the precise role and clinical applicability of TSP-2 in cardiac fibrosis.

Graphical Abstract

Introduction

Myocardial fibrosis is a common feature of structural remodeling observed in various cardiac pathological conditions and during the aging process, characterized by the abnormal accumulation of extracellular matrix (ECM) in the cardiac stroma and stromal expansion.1,2 Upon activation, fibroblasts undergo transdifferentiation into myofibroblasts, which function as the principal cellular executors of the fibrotic response, driving aberrant overproduction and deposition of ECM components. Cardiac fibrosis typically occurs in conjunction with cardiac dysfunction, and its severity serves as a critical indicator for assessing patient prognosis. However, fibrosis possesses dual pathological and protective attributes: following myocardial infarction (MI), a moderate fibrotic response can replace necrotic myocardium by forming scar tissue, thereby maintaining ventricular geometry and preventing cardiac rupture, thus playing a necessary compensatory protective role during the acute phase.3 Myocardial fibrosis represents a common pathological feature of various cardiac diseases, including MI, pressure overload–induced hypertrophy, and end-stage heart failure, and plays a critical role in ventricular remodeling and functional deterioration.4 Under the stimulation of ischemia, mechanical stress, or an inflammatory microenvironment, quiescent cardiac fibroblasts are activated and transdifferentiate into myofibroblasts (marked by the expression of α-smooth muscle actin [α-SMA]). Activated myofibroblasts serve as the major effector cells driving cardiac fibrosis by acquiring enhanced ECM synthetic activity and promoting excessive deposition of ECM proteins, including type I and type III collagens and fibronectin.4–6 The progressive accumulation and remodeling of ECM within the myocardial interstitium and perivascular regions increase myocardial stiffness, reduce ventricular compliance, and contribute to diastolic dysfunction, with advanced fibrosis further impairing systolic performance.7 Furthermore, cardiac aging is also accompanied by interstitial and perivascular collagen deposition, leading to a progressive increase in ventricular stiffness and a decline in diastolic reserve; unlike pathological fibrosis, aging-related ECM accumulation primarily stems from the functional decline of matrix degradation pathways (such as the matrix metalloproteinase system), rather than enhanced collagen biosynthesis.1

However, myocardial fibrosis is not a single pathological process but rather encompasses multiple forms of ECM remodeling with distinct biological purposes and regulatory mechanisms. Depending on the underlying cause and disease stage, cardiac fibrosis can manifest as reparative scarring following a MI, reactive interstitial fibrosis under mechanical stress or chronic inflammatory stimulation, and age-related matrix remodeling. The former involves the replacement of necrotic myocardium with collagen deposits to maintain ventricular structural stability and prevent rupture; the latter two, however, are primarily characterized by persistent ECM accumulation, an imbalance in degradation, and reduced tissue compliance, ultimately leading to cardiac dysfunction. Although these processes are all accompanied by increased ECM deposition, they differ in their triggering mechanisms, cellular origins, and effects on cardiac function. Although myocardial fibrosis is a key pathological basis for the progression of various heart diseases to their end-stage, there are currently no approved specific therapeutic drugs targeting myocardial fibrosis itself.8 Current treatment strategies primarily aim to indirectly slow the progression of fibrosis by controlling the underlying cardiovascular disease, inhibiting pro-fibrotic signaling pathways, and improving cardiac remodeling. However, novel therapeutic strategies that directly target cardiac fibroblasts, ECM regulatory proteins, and key pro-fibrotic molecules remain in the exploratory phase. Therefore, further investigation of new fibrosis regulatory factors is of great significance for elucidating the molecular mechanisms underlying the onset and progression of myocardial fibrosis and for developing potential therapeutic strategies.

Thrombospondin-2 (TSP-2) belongs to the thrombospondin (TSP) family, a group of five structurally related secreted glycoproteins designated TSP-1 through TSP-5. With regard to their oligomerization status, TSP family members are sorted into two categories: TSP-1 and TSP-2 exist as homotrimers, whereas TSP-3, TSP-4, and TSP-5 exist as homopentamers.9 TSP-2 and TSP-1 are the most extensively studied TSPs. Although TSP-1 and TSP-2 share the same multi-domain architecture, their amino acid sequences differ, with the main differences concentrated in the N-terminal domains. This difference results in distinct ligand-binding capabilities between the two.10 Table 1 summarizes the main characteristics and differences between TSP-2 and TSP-1.

Table 1.

The Main Characteristics and Differences Between TSP-2 and TSP-1

Feature	TSP-1	Ref	TSP-2	Ref
Family classification	A group A thrombospondin, encoded by the THBS1 gene, is a matricellular protein.		A group A thrombospondin, encoded by the THBS2 gene, is an important ECM regulatory protein.
Structural features	Its structure is that of a secreted glycoprotein with a homotrimeric arrangement, consisting of an N-terminal domain, oligomerization domain and procollagen-like domain, three type I repeats (TSRs), three type II repeats (EGF-like repeats), thirteen calcium-binding type III repeats, and a C-terminal domain.	[10]	It shares a highly similar modular structure with TSP-1. However, the two proteins exhibit amino acid sequence differences in specific regions, with the most significant differences found in the N-terminal heparin-binding region (sequence similarity of approximately 32%). These structural differences affect their ligand-binding properties and downstream biological functions	[10]
Major specific functional motifs and interacting molecules/receptors	The N-terminal domain can interact with heparin, heparan sulfate proteoglycans (HSPGs), integrins (α3β1, α4β1, and α6β1), versican, and low-density lipoprotein receptor-related protein (LRP). In addition, the KRFK (Lys-Arg-Phe-Lys) sequence present in the Type I repeat effectively activates transforming growth factor-β (TGF-β).	[11–18]	The N-terminal domain can interact with heparin, integrins (α4β1 and α6β1), and LRP. Because the KRFK sequence is absent from the Type I repeat of TSP-2, it cannot activate latent TGF-β.	[11,19–22]
Major cellular sources in the cardiovascular system	It is primarily produced by platelets, vascular smooth muscle cells, endothelial cells, fibroblasts, and myocytes exposed to stress stimuli.	[23–27]	It is mainly produced by fibroblasts and vascular smooth muscle cells; during pathological myocardial remodeling, the expression of TSP-2 in cardiac fibroblasts significantly increases.	[28–31]
Established cardiac functions	It contributes to cardiac remodeling by activating TGF-β-dependent fibrosis, modulating the inflammatory response, inhibiting angiogenesis, and regulating extracellular matrix deposition.	[27,32–35]	It maintains the homeostasis and structural integrity of the cardiac ECM and contributes to cardiac structural remodeling by regulating collagen fiber formation, cell–matrix interactions, and the levels of matrix metalloproteinases (particularly MMP-2).	[28,29,36–39]

TSP-2 is primarily synthesized by fibroblasts and smooth muscle cells, playing a key role in the late stages of tissue repair and during ECM remodeling;40 it is a crucial molecule for maintaining the structural integrity of the matrix. Cardiac spatial transcriptomics data further confirm that TSP-2 is primarily expressed by cardiac fibroblasts.41 In recent years, as our understanding of the mechanisms regulating ECM homeostasis has deepened, TSP-2 has been recognized as a factor associated with myocardial fibrosis.42 Current evidence suggests that TSP-2 exerts dual biological effects in myocardial fibrosis: it may play different or even opposing roles across various disease types, stages of injury, and microenvironmental conditions. On the one hand, it exerts a protective effect by stabilizing the ECM microenvironment; on the other hand, under conditions of persistent abnormal expression or in specific pathological environments, it may promote adverse ventricular remodeling.28,43–45 This article will provide an overview of the molecular characteristics and functional regulation of TSP-2, offer an in-depth analysis of its complex mechanisms of action in regulating myocardial fibrosis in the context of various heart diseases, and further explore its translational medical value as a potential therapeutic target.

Structure and Biological Functions of TSP-2

TSP-2 is a secreted, multimodular trimeric glycoprotein with a complex multi-domain molecular architecture. Its precursor polypeptide chain consists of 1172 amino acid residues, which undergo post-translational processing to form a mature trimer; the three subunits are covalently linked via interchain disulfide bonds to maintain structural integrity.9,46

The primary structure of TSP-2 consists of a series of conserved functional domains arranged sequentially from the N-terminus to the C-terminus:46 (1) N-terminal domain: The N-terminal domain contains the signal peptide responsible for protein secretion. Following maturation, this region exposes a heparin-binding site enriched in basic amino acid residues, which contributes to interactions with ECM components and cell surface molecules. (2) Oligomerization domain and procollagen-like domain: The oligomerization domain contains conserved cysteine residues involved in interchain disulfide bond formation, thereby stabilizing the homotrimeric structure of TSP-2. The adjacent procollagen-like domain is characterized by Gly-X-Y tripeptide repeats that form a collagen-like triple-helical structure and participate in ECM organization and protein–protein interactions. (3) Thrombospondin type I repeats (TSRs): TSP-2 contains three type I repeats, which harbor conserved motifs such as WxxW and CSVTCG. These repeats mediate interactions with extracellular receptors, including CD36, and contribute to the anti-angiogenic properties of thrombospondins through receptor-dependent signaling pathways. (4) Type II repeats (EGF-like repeats): Three epidermal growth factor-like repeats are located within the C-terminal signature domain. These modules contribute to the structural organization and conformational stability of TSP-2 and participate in calcium-dependent intramolecular interactions. (5) Calcium-binding type III repeats: The signature domain contains thirteen calcium-binding type III repeats (also referred to as aspartate-rich repeats) arranged in tandem. These repeats are enriched in acidic residues and coordinate Ca²⁺ ions, with individual repeats capable of binding one or two Ca²⁺ ions. Calcium coordination stabilizes the three-dimensional architecture of the signature domain and regulates ligand-binding properties. (6) C-terminal L-type lectin-like domain: The C-terminal lectin-like domain forms the terminal globular region of TSP-2 and, together with the adjacent EGF-like and calcium-binding type III repeats, constitutes the conserved signature domain involved in extracellular ligand recognition and molecular interactions. These structural domains are highly conserved among subgroup A thrombospondins. Comparative evolutionary analyses have demonstrated that the EGF-like repeat–calcium-binding type III repeat–lectin-like domain architecture is strongly conserved between Drosophila TSP and mammalian thrombospondins, highlighting its fundamental role in maintaining TSP family structure and function.46 Figure 1 presents a simple structural diagram of TSP-2.

Figure 1.

Simplified structure diagram of TSP-2. The TSP-2 precursor is processed to form a multi-domain trimeric glycoprotein. The figure shows its primary structure from the N-terminal to the C-terminal. WxxW, Tryptophan - Any amino acid - Any amino acid - Tryptophan; CSVTCG, Cysteine - Serine - Valine - Threonine - Cysteine - Glycine. The figure was constructed with BioRender (https://biorender.com).

The functional complexity of TSP-2 stems from its highly modular molecular architecture.10 At the molecular interaction level, the N-terminal heparin-binding domain recognizes and binds to cell surface heparan sulfate proteoglycans via a sequence rich in basic amino acid residues, thereby regulating cell adhesion and migration. The conserved CSVTCG motif within type I repeats act as an anti-angiogenic recognition site, binding to the CD36 receptor and activating downstream apoptotic signaling pathways, leading to programmed cell death in endothelial cells and thereby inhibiting pathological angiogenesis. In addition, specific functional peptide segments within calcium-binding type III repeats (such as the GVTDEKD sequence within the third repeat unit) can chelate basic fibroblast growth factor (FGF2) with high affinity, blocking its binding to tyrosine kinase receptors and thereby inhibiting FGF2-mediated pro-angiogenic effects.47 Notably, TSP-2 can also specifically interact with ECM proteins (such as matrix metalloproteinase-2 (MMP-2)48 and integrin receptors (including subtypes such as α4β1 and α6β1),11,19,49 thereby regulating ECM remodeling, cell migration, and intracellular signaling networks through multidimensional mechanisms.

The Role of TSP-2 in Myocardial Fibrosis Caused by Various Heart Diseases

The association between TSP-2 and myocardial fibrosis has been reported in various cardiac diseases. Notably, the biological effects of TSP-2 are significantly context-dependent: during the acute injury phase, it may exert a protective and reparative role by stabilizing the ECM; whereas during the chronic progression phase, it may promote pathological structural remodeling. This dynamic functional transition from adaptive repair to compensatory remodeling defines the dual role of TSP-2 in cardiac fibrogenesis. Figure 2 depicts the regulatory involvement of TSP-2 in fibrotic heart diseases arising from diverse etiologies. Table 2 summarizes the current research status and key findings regarding TSP-2 within this field.

Figure 2.

The regulatory role of TSP-2 in myocardial fibrosis caused by different etiologies. Under physiological conditions, TSP-2 mainly participates in maintaining ECM homeostasis and does not directly induce fibrosis. Its persistent high-level expression may reflect the progression of heart failure; however, in myocardial infarction, the pro-fibrotic effect of TSP-2 may contribute to structural remodeling of the infarcted area and prevent cardiac rupture. The role of TSP-2 in cardiac hypertrophy and aging remains to be further clarified. Solid lines indicate well-established or directly supported regulatory relationships, whereas dashed lines indicate potential or indirect associations based on limited evidence or findings from other tissues. The figure was constructed with BioRender (https://biorender.com).

Abbreviations: TSP-2, thrombospondin-2; ECM, extracellular matrix.

Table 2.

The Current Research Status and Main Findings of TSP-2 in the Field of Myocardial Fibrosis

Disease	Species	Research Type	Outcome	Ref
Coronary artery disease	Human	Prospective cohort study, Case-control study	An increase in TSP-2 levels is positively correlated with the risk of heart failure, death and re-hospitalization in patients with coronary artery disease; the TSP-2 gene polymorphism is significantly associated with early-onset coronary heart disease; the T→G point mutation in the 3’ untranslated region of the TSP-2 gene can reduce the risk of myocardial infarction.	[50–53]
Heart failure with preserved ejection fraction (HFpEF)	Human	Prospective cohort study, observational longitudinal study combined with machine learning	An increase in TSP-2 levels is associated with deterioration of cardiac function and increases the risk of death and adverse cardiovascular events; a decrease in TSP-2 levels is accompanied by a reduction in type I procollagen C-terminal propeptide.	[42,54,55]
Heart failure with reduced ejection fraction (HFrEF)	Human	Prospective cohort study, Cross-sectional study	An elevated level of TSP-2 is associated with the deterioration of heart failure and the development of new-onset heart failure, and it also increases the risk of death and cardiovascular events.	[41,56–59]
Heart hypertrophy	Rat/Mice	Animal experiments	TSP-2 mediates heart failure	[56]
Cardiac cell transplantation	Mice	Animal experiments	TSP-2 promotes fibrosis after cardiac cell transplantation and inhibits angiogenesis.	[28]
Pulmonary arterial hypertension	Human	Prospective cohort study	The increase in TSP-2 is associated with right ventricular systolic dysfunction and non-adaptive right ventricular remodeling.	[60]
General Population	Human	Cross-sectional observational study, prospective cohort study, case-control study	TSP-2 is positively correlated with new-onset heart failure and atrial fibrillation.	[41,61–63]
Older animals	Mice	Animal experiments	Knocking out the TSP-2 gene led to age-related dilated cardiomyopathy.	[64]
Aortic valve degeneration	Human	Observational experimental clinical research	TSP-2 is enriched in the bicuspid aortic valve and is involved in the formation of fibrosis.	[65]
Atrial fibrillation	Human	Prospective cohort combined with Mendelian randomization study	TSP-2 is involved in cardiac remodeling.	[66]

Cardiac Hypertrophy

Cardiac hypertrophy is an adaptive response induced by long-term hemodynamic pressure load (such as hypertension). Its pathological progression is accompanied by myocardial cell hypertrophy, fibroblast activation, and ECM remodeling and abnormal accumulation, which collectively promote the development of cardiac fibrosis and dysfunction.67–69 During the compensatory phase, the heart maintains ventricular structure and function through adaptive responses, including altered expression of matrix remodeling-related proteins such as TSP-2, which may contribute to ECM structural stability and regulation of MMP-mediated collagen turnover. However, clinical observations indicate that patients with cardiac hypertrophy accompanied by left ventricular systolic dysfunction exhibit significantly higher myocardial TSP-2 expression levels than those with preserved ejection fraction.56 These findings suggest that increased TSP-2 expression is associated with pathological remodeling and the progression toward cardiac dysfunction.

In Ren-2 transgenic hypertensive rats, cardiac expression of TSP-2 was markedly increased during the transition from compensated hypertrophy to heart failure and was associated with a failure-prone remodeling phenotype.56 In human cardiac samples obtained from patients with aortic stenosis undergoing valve replacement surgery, TSP-2 mRNA expression was also elevated in hypertrophied myocardium accompanied by reduced ejection fraction.56 The study further demonstrated that TSP-2 deficiency enhanced cardiac MMP-2 and MMP-9 activities, thereby accelerating ECM degradation and increasing susceptibility to cardiac rupture following angiotensin II (AngII) stimulation. Although TSP-2 deficiency resulted in reduced collagen deposition and attenuated histological fibrosis, these changes reflected impaired ECM stabilization rather than beneficial suppression of fibrotic remodeling. Markedly increased circulating TSP-2 concentrations have likewise been documented among individuals diagnosed with pulmonary arterial hypertension coexisting with left ventricular hypertrophy.60 In mice subjected to doxorubicin-induced cardiac injury, the absence of TSP-2 was associated with greater apoptotic loss of cardiomyocytes and disruption of the ECM.70 In rodent studies, the lack of TSP-2 has been shown to compromise cardiac interstitial matrix integrity and demonstrate a correlation with dilated cardiomyopathy.64,70 This may be related to reduced fibroblast contractility and decreased migration following TSP-2 deficiency.29

In experimental models featuring pulmonary arterial hypertension (PAH) or right-sided ventricular overload, cardiomyocyte-derived exosomes enriched in TSP-2 can be internalized by cardiac fibroblasts, triggering their transdifferentiation into myofibroblasts and enhancing TGF-β1 and collagen expression, thereby worsening interstitial fibrosis in the right ventricle.71 However, other studies have reported that specific overexpression of TSP-2 in mouse myocardial tissue does not induce a pathological cardiac phenotype.72 Conversely, in AngII-induced heart failure mice, although TSP-2 deficiency reduced total myocardial collagen, it increased the risk of myocardial rupture.56 These findings suggest that TSP-2 is critical for maintaining the integrity of the cardiac matrix, and that elevated levels of TSP-2 may represent a compensatory protective mechanism against pressure overload,73 whereas excessive inhibition or deletion of TSP-2 does not yield benefits beyond maintaining matrix integrity. Collectively, these findings indicate that the role of TSP-2 in the progression of cardiac hypertrophy and heart failure is clearly context-dependent. On the one hand, by regulating MMP activity, maintaining ECM structural stability, and promoting fibroblast-mediated matrix remodeling, TSP-2 may exert a protective effect during the early stages of pressure overload, preventing uncontrolled matrix degradation and ventricular structural damage; on the other hand, during persistent pressure stimulation or in the late stages of heart failure, elevated TSP-2 expression may reflect ongoing ECM remodeling and fibrotic responses; whether it directly promotes pathological fibrosis remains to be further clarified. Current evidence suggests that TSP-2 is not merely a pro-fibrotic factor but rather acts as a regulator of ECM homeostasis, exerting bidirectional regulatory effects under different pathological conditions. Therefore, future studies need to consider disease stage, type of cardiac load, cellular origin, and the TSP-2 regulatory network to further clarify whether its role in cardiac remodeling leans toward matrix protection or fibrosis promotion.

Heart Failure

Myocardial fibrosis, characterized by excessive collagen fiber deposition, is the core pathological basis for the onset and progression of heart failure. Regardless of whether the initial trigger is reparative or reactive fibrosis, a persistently activated fibrotic process exacerbates ventricular remodeling, promotes cardiac decompensation, and is closely associated with poor patient prognosis as an independent predictor.74 As an important regulatory molecule involved in the remodeling and fibrosis process of ECM, TSP-2 demonstrates marked elevation in the circulation of individuals with heart failure stemming from diverse etiologies, and its concentrations are linked to unfavorable cardiovascular prognosis across both preserved and reduced ejection fraction heart failure.54,57 However, current clinical evidence primarily supports an association between circulating TSP-2 levels and heart failure severity, disease progression, and prognosis, rather than directly demonstrating that elevated circulating TSP-2 drives myocardial collagen deposition or fibrosis. Crucially, circulating TSP-2 concentrations decline markedly among individuals diagnosed with terminal-stage cardiac failure after heart transplantation.41,58 This finding suggests that circulating TSP-2 reflects the advanced heart failure state and may partly originate from cardiac tissue; however, it does not by itself establish a causal role for TSP-2 in myocardial fibrosis.

In cardiac dysfunction precipitated by coronary artery disease, heightened circulating TSP-2 concentrations demonstrate a link to greater likelihood of mortality attributable to congestive heart failure throughout a 3-year follow-up, overall death from all causes, and recurrent hospital admissions.50 A proteomic cohort investigation conducted among individuals with ischemic heart disease likewise revealed that augmented TSP-2 expression exhibited an inverse relationship with left ventricular ejection fraction and a direct association with heart failure, a result subsequently corroborated in a murine model of cardiac failure precipitated by coronary artery occlusion and ensuing MI.51 The study further demonstrated that stimulation of human fibroblasts (CFs) with TGF-β and interleukin-1β (IL-1β) significantly increased the expression of the TSP-2-encoding gene in CFs, suggesting that TSP-2 can serve as a biomarker for heart failure following MI. These findings indicate that TSP-2 is closely associated with the inflammatory and fibrotic activation of cardiac fibroblasts; however, whether increased circulating TSP-2 directly mediates myocardial fibrosis remains to be further clarified. Moreover, TSP-2 has been linked to cardiac failure development among individuals with type 2 diabetes mellitus, older adults, hypertensive subjects, and obese persons.42,54,59,61

Interestingly, a large-scale community-based cohort study found that TSP-2 levels were already significantly elevated in peripheral blood samples collected 10 years prior to the diagnosis of heart failure.62 Proteomic analysis of plasma from human heart failure patients at different stages also revealed a strong association between TSP-2 and the progression of heart failure as well as end-stage heart failure, with levels decreasing significantly following heart transplantation.41 Together, these clinical observations suggest that circulating TSP-2 may serve as an early indicator of pathological remodeling and disease progression before overt heart failure develops. Nevertheless, these findings should be interpreted as evidence of association rather than direct proof that TSP-2 initiates or drives myocardial fibrosis. Animal studies have shown that during the transition from hypertension-related myocardial hypertrophy to heart failure, TSP-2 expression in the myocardium increases selectively, and this increase can predict the subsequent progression of heart failure.56 It is worth noting that while TSP-2 deficiency can reduce the fibrotic response under certain pathological conditions (such as in a cardiomyocyte transplantation model),28 it simultaneously leads to impaired ECM stability and increases the risk of cardiac rupture and dilated cardiomyopathy under pressure-loading stimulation,56 suggesting that TSP-2 plays a dual role in cardiac remodeling by both regulating fibrosis and maintaining matrix integrity.

Furthermore, in the transaortic constriction (TAC) rat model, the TSP-2 gene was similarly persistently upregulated, indicating that it plays a continuous role in chronic fibrotic remodeling, which may support the view that TSP-2 acts as a marker of long-term mechanical transduction activation.29 These experimental findings suggest that TSP-2 participates in the regulation of myocardial fibrotic remodeling under pathological stress conditions. However, given that complete TSP-2 deficiency disrupts ECM stability and increases the risk of cardiac rupture, the biological effects of TSP-2 appear to be context- and stage-dependent rather than representing a simple pro-fibrotic function. Therefore, although elevated TSP-2 expression is consistently observed in heart failure and is associated with myocardial remodeling, disease progression, and fibrosis-related phenotypes, current evidence does not support the conclusion that circulating TSP-2 itself directly promotes myocardial collagen deposition. Given the risk that TSP-2 deficiency may disrupt the maintenance of matrix homeostasis, traditional systemic or long-term strategies for inhibiting TSP-2 may pose potential safety concerns. Therefore, future research should avoid simply treating TSP-2 as a single pro-fibrotic target; instead, strategies such as inducible conditional gene regulation, cell-specific interventions, or stage-specific modulation could be adopted to elucidate its pathological role based on a clear understanding of the disease stage and cellular origin. At the same time, potential therapeutic approaches targeting TSP-2 activity—such as antibodies, siRNA, or small molecules—require systematic evaluation of their effects on scar stability, ECM integrity, angiogenesis, and non-specific off-target effects. Although systematic studies on the molecular networks, cell-specific functions, and disease-stage dependence of TSP-2 in regulating heart failure-associated myocardial fibrosis are currently lacking, an in-depth analysis of its bidirectional regulatory mechanisms may help develop more precise ECM modulation strategies and provide a new theoretical basis for the treatment of heart failure.

MI

MI is ischemic necrosis of the myocardium caused by a sudden interruption or reduction in coronary blood flow. As a severe form of ischemic injury, myocardial necrosis triggers reparative scarring characterized by fibrosis; this protective mechanism is designed to prevent serious mechanical complications, such as post-infarction cardiac rupture.2 Decades of research have shown that post-infarction inflammation and fibrosis require strict regulation to protect the ventricle from catastrophic structural failure while limiting the progression of heart failure.75

The deposition of an organized ECM (primarily a network of collagen fibers) is critical for maintaining cardiac structural integrity, preventing cardiac rupture, and avoiding adverse ventricular remodeling. However, long-term excessive activation of CFs promotes pathological accumulation of the ECM, leading to reduced myocardial compliance and ultimately contributing to the onset and progression of heart failure following MI.76,77 Proteomic analyses have demonstrated that TSP-2 protein levels are markedly upregulated in the sera of patients after MI, and show a robust correlation with heart failure developing in the post-infarction period. Furthermore, a marked increase in TSP-2 gene expression in the heart was also observed in mice following coronary artery ligation.51 The absence of TSP-2 not only causes defects in CF adhesion and disrupted fiber formation but also promotes increased angiogenesis.78 Given the critical role of TSP-2 in maintaining ECM integrity, the TSP-2 gene is rapidly activated after MI promoting collagen deposition and thereby maintaining cardiac structural stability. In vitro experiments indicate that subjecting human cardiac myoblasts (hCMPCs) to hypoxia can mimic the infarct microenvironment; following hypoxia, TSP-2 expression in hCMPCs is significantly upregulated; if TSP-2 is knocked out, cell proliferation, migration capacity, and MMP activity are all markedly enhanced.79 Single-cell sequencing of a rat MI model revealed a marked increase in TSP-2 mRNA expression in CFs; silencing the TSP-2 gene resulted in impaired CF migration and compromised cellular function, indicating that TSP-2 promotes post-infarction scar formation.29

Interestingly, clinical studies have found that the T→G single-nucleotide polymorphism in the 3′-untranslated region of the TSP-2 gene is associated with a reduced risk of early MI; it is speculated that the underlying mechanism may involve the inhibition of MMP-2 activity and the downregulation of angiogenesis.80 However, some studies have failed to replicate this association.81,82 Furthermore, in patients with ST-segment elevation MI, peripheral blood TSP-2 levels did not show a significant increase during the acute phase or 3 months post-infarction.83 Consequently, consistent data supporting the impact of TSP-2 genetic polymorphisms on MI vulnerability are currently insufficient, precluding definitive conclusions. Taken together, the exact contribution of TSP-2 in the context of MI has yet to be fully elucidated. Existing animal studies suggest that it may exert a potential protective effect during the early repair phase by inhibiting MMP activity and reducing excessive ECM degradation.84 Overall, the role of TSP-2 following MI remains somewhat controversial. Current evidence suggests that TSP-2 may exert a protective effect during the early repair phase of infarction by regulating MMP activity, maintaining ECM integrity, and promoting stable scar formation; however, its sustained elevation may also reflect—or even contribute to—late-stage ECM remodeling and fibrosis. Therefore, its specific role may depend on the different time points following MI, the cellular origin, and the local microenvironment. Future studies using multi-phase animal models are needed to further clarify the regulatory boundaries of TSP-2 between MI repair and adverse remodeling.

Cardiac Aging

With advancing age, the heart undergoes progressive aging, the core characteristic of which is functional decline resulting from morphological and structural remodeling. Progressive collagen deposition in aging myocardium leads to interstitial fibrosis, resulting in reduced ventricular compliance and impaired diastolic function. Heart failure has emerged as the predominant reason for hospital admissions within the population aged 65 years and above. Simultaneously, the occurrence of coronary artery disease, elevated blood pressure, and diabetes mellitus increases markedly with advancing age, and senescence per se can expedite the advancement of ischemic, hypertensive, and diabetic cardiomyopathies. Animal models suggest a marked increase in collagen content in the aging heart, a finding confirmed in mice, rats, and aged rabbits.85–87 Furthermore, the relative ratio of Type I to Type III collagen shifts, with an increase in the proportion of Type III collagen.88 Autopsy specimens from elderly individuals without organic heart disease also exhibit similar patterns of collagen remodeling,89 suggesting that aging itself can induce cardiac interstitial fibrosis.

Animal studies suggest that TSP-2 expression is significantly increased in the blood vessels of aging mice, while neovascularization is suppressed, capillary density is reduced, and type I collagen expression is decreased.90 Diminished type I collagen abundance alongside elevated type III collagen levels may contribute to vascular rigidity and impaired elasticity, thereby elevating susceptibility to cardiovascular conditions exemplified by atherosclerosis. Clinical studies further reveal that elevated circulating TSP-2 levels are significantly associated with loss of functional capacity in the elderly,91 the onset of heart failure,61 and the frailty phenotype.92 This evidence suggests that elevated circulating TSP-2 may be associated with adverse cardiovascular outcomes in elderly individuals. However, experimental studies have revealed a more complex role for TSP-2 in cardiac aging. Mortality rates in TSP-2 knockout mice were significantly higher than those in wild-type mice between 24 and 60 weeks of age. In aged wild-type mice, myocardial TSP-2 expression increased compared with young mice, accompanied by age-related collagen accumulation. Notably, TSP-2 deficiency resulted in severe age-associated dilated cardiomyopathy, systolic dysfunction, and myocardial fibrosis, whereas cardiac capillary density remained unchanged. Restoration of TSP-2 expression in knockout mice improved survival and prevented the development of dilated cardiomyopathy,64 indicating that endogenous TSP-2 is essential for maintaining cardiac ECM integrity during aging. These findings suggest that age-related collagen accumulation may partly result from impaired matrix turnover and defective collagen organization rather than solely from increased collagen synthesis.

However, rescue of a TSP-2-deficient phenotype and therapeutic augmentation of TSP-2 in wild-type aging hearts represent distinct biological questions that require separate experimental validation. Furthermore, because most available studies rely on global TSP-2 knockout models, the observed cardiac phenotypes may also involve systemic alterations in vascular or connective tissue homeostasis. Therefore, the precise contribution of cardiomyocyte- or cardiac fibroblast-derived TSP-2 to age-related myocardial remodeling remains to be determined. Overall, current evidence supports a critical role for TSP-2 in preserving cardiac matrix stability during aging, but whether TSP-2 augmentation represents a therapeutic strategy for age-associated cardiac fibrosis requires further investigation.

Molecular Mechanisms of TSP-2 in Cardiac Fibrosis

TSP-2 serves as a critical modulator of ECM assembly and exhibits potent anti-angiogenic activity. Excessive TSP-2 may contribute to the pathological advancement of myocardial fibrosis through multiple mechanisms, including augmented ECM deposition, suppression of neovascularization, and initiation of TGF-β-mediated pro-fibrotic signaling pathways. Figure 3 illustrates the pathways and signaling networks related to myocardial fibrosis involving TSP-2.

Figure 3.

TSP-2 is involved in pathways and signaling networks associated with myocardial fibrosis. Currently, there is no direct evidence demonstrating that TSP-2 directly drives cardiac fibrosis. Based on available cardiac studies and cross-tissue evidence, TSP-2 may indirectly contribute to myocardial fibrosis through mechanisms including ECM deposition, inhibition of angiogenesis, and regulation of pro-fibrotic signaling pathways such as TGF-β signaling. Solid lines indicate well-established or directly supported regulatory relationships, whereas dashed lines indicate potential or indirect associations based on limited evidence or extrapolation from findings in other tissues. The figure was constructed with BioRender (https://biorender.com).

Abbreviations: MMP2, matrix metalloproteinase 2; MMP9, matrix metalloproteinase 9; miR29, microRNA-29; ECM, extracellular matrix; Wnt/β-catenin, wingless-related integration site/beta-catenin; VEGF, vascular endothelial growth factor; IGF-1, insulin-like growth factor-1; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; TGF-β1, transforming growth factor-beta 1; CD36, cluster of differentiation 36; FGF2, fibroblast growth factor 2; Smad2/3, mothers against decapentaplegic homolog 2/3; PI3K/Akt, phosphatidylinositol 3-kinase/protein kinase B; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha.

Regulation of ECM Metabolism and CF Activation

Characterized as an adaptive complex meshwork, the ECM consists of structural protein constituents (represented by collagen and laminin), adhesive glycoprotein molecules (such as fibronectin), and regulatory-type matricellular proteins. It maintains tissue homeostasis and structural integrity by providing mechanical support and regulating cell-matrix interactions and the bioavailability of growth factors.93,94 The spatiotemporal dynamic remodeling of the ECM is precisely and synergistically regulated by proteolytic systems (such as MMPs) and post-translational modification systems (such as lysyl oxidase (LOX)).95,96 The pathological essence of myocardial fibrosis lies in an imbalance in ECM remodeling, specifically manifested as pathological deposition of interstitial type I and type III collagen or dysfunction of matrix degradation pathways. Crucially, the biosynthesis, secretion, assembly, and cross-linking maturation of collagen fibers are all precisely regulated by physicochemical factors in the ECM microenvironment—such as mechanical stress, cytokine concentrations, and matrix stiffness—forming complex self-regulatory loops.

As previously mentioned, TSP-2 plays a broad role in the homeostasis of the ECM: it not only regulates collagen synthesis and MMP expression,97 but also maintains matrix structural integrity by inhibiting active MMPs in the ECM, promotes the ordered assembly of collagen fibers, and suppresses pathological angiogenesis.98 Deletion of the TSP-2 gene leads to impaired MMP recycling and accelerates ECM degradation.40 Notably, TSP-2 deficiency induces an ECM remodeling phenotype characterized by impaired collagen cross-linking, altered structural organization of fibrotic collagen, and disruption of the matrix architecture. The underlying mechanism is that TSP-2 deficiency lifts the repression of miR-29, leading to elevated miR-29 levels, which in turn suppress LOX expression, reduce collagen cross-linking efficiency, and ultimately compromise ECM structural stability.99 In degenerative valvular heart diseases (including aortic, mitral, and tricuspid valve lesions), upregulated TSP-2 expression is closely associated with abnormal ECM remodeling in the valvular stroma.65,100 Current research indicates that, although TSP-1 and TSP-2 share a high degree of structural homology, their interaction patterns with cell receptors are not entirely consistent. TSP-2 can mediate cellular responses through specific integrins. For example, TSP-2 can bind to the α4β1 integrin and regulate endothelial cell adhesion, migration, and angiogenesis-related responses to TSP-2. In vitro and in vivo studies have shown that the α4β1 integrin is involved in regulating TSP-2-mediated angiogenesis.19 Furthermore, the N-terminal domain of TSP-2 can be recognized by the α6β1 integrin, suggesting that α6β1 may be involved in mediating the interaction between TSP-2 and the ECM.11 However, compared with TSP-1, the receptor profile of TSP-2 and its downstream signaling pathways have not yet been fully elucidated. Therefore, whether some of the receptors identified for TSP-1 (such as αvβ3 integrin and CD47) also mediate TSP-2 function remains to be further verified.

TSP-2 is an important regulatory molecule that maintains the integrity of the ECM ultrastructure. In dermal fibroblasts (DFs), TSP-2 gene knockout significantly reduces the binding capacity of von Willebrand factor (vWF) to the ECM and leads to abnormal collagen fiber alignment, but does not significantly alter collagen expression levels.101 Interestingly, TSP-2 deficiency in DFs activates the TGF-β3 and Wnt/β-catenin signaling pathways, promoting fibroblast proliferation and directed migration.102 In contrast, In PAH-related myocardial fibrosis, TSP-2 drives the transdifferentiation of cardiac fibroblasts into myofibroblasts, enhances collagen deposition, and gives rise to disorganized ECM architecture; conversely, miR-29a-3p can mitigate ECM remodeling and fibrosis through direct suppression of TSP-2.71 These results suggest that TSP-2’s regulation of ECM homeostasis is significantly dependent on cell type and disease context. In the heart, TSP-2 may contribute to the onset and progression of cardiac fibrosis by modulating ECM structural integrity and the activation status of fibroblasts. However, current research is insufficient regarding how TSP-2 coordinates ECM remodeling and fibrosis in cardiac fibroblasts; further studies are needed to elucidate its specific molecular mechanisms and therapeutic potential.

Inhibit Angiogenesis

Inhibited angiogenesis can promote the formation of a pro-fibrotic microenvironment through microcirculatory dysfunction. Insufficient vascular endothelial growth factor (VEGF) signaling can impair the maintenance and repair capabilities of capillaries, leading to capillary sparsity, reduced tissue perfusion, and disruption of ECM homeostasis, thereby promoting fibrotic remodeling.103 In pathological conditions such as ischemic heart disease and diabetic cardiomyopathy, enhancing angiogenesis may serve as a key strategy for improving tissue repair. Previous studies have shown that therapeutic VEGF delivery can promote angiogenesis in ischemic regions, improve local perfusion, and mitigate post-ischemic ventricular remodeling.104,105 However, the efficacy of angiogenesis therapy is influenced by factors such as vascular maturity, inflammatory responses, and the fibrotic microenvironment. Conversely, persistent angiogenesis deficiency may lead to endothelial dysfunction, myocardial hypoxia, and fibroblast activation, further promoting abnormal ECM deposition. As an important endogenous anti-angiogenic factor, TSP-2 may be involved in the regulatory processes of angiogenesis and ECM remodeling described above. In TSP-2 knockout mice, both the scar tissue and vascular density formed following intramyocardial injection of fetal mouse cardiomyocytes were significantly reduced compared to wild-type mice,28 suggesting that TSP-2 not only participates in ECM remodeling following myocardial injury but also plays a crucial regulatory role in angiogenesis during the repair process.

By restraining endothelial cell proliferation and inducing apoptosis, TSP-2 markedly attenuates gelatinase activity, consequently suppressing angiogenesis and preventing ECM degradation, which contributes to the promotion of myocardial fibrosis. Studies have confirmed that TSP-2 inhibits the proliferation of human microvascular endothelial cells mediated by various growth factors, including VEGF, insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF); this may be one of the key mechanisms by which it regulates microvascular formation.30 As important regulators of angiogenesis, MMPs play a role that extends far beyond simple ECM degradation; they promote angiogenesis through multiple mechanisms, including cleaving VEGF, modulating receptor signaling, and releasing various growth factors.106,107 Among these MMPs, MMP-2 and MMP-9 interact particularly closely with TSP-2. Study has shown that TSP-2 can inhibit MMP-9 activity and thereby suppress angiogenesis; upon TSP-2 knockout, MMP-9 activity significantly increases, and the inhibitory effect on angiogenesis is markedly reversed.40 TSP-2 is overexpressed in mouse heart failure, and following TSP-2 knockout, the activities of both MMP-2 and MMP-9 are significantly elevated.56 These findings indicate that TSP-2 regulates angiogenesis at least in part by directly modulating the activity of MMPs. Mechanistically, the thrombospondin type I repeat domains of TSP-2 have been shown to interact with MMP-2 and inhibit the activation of pro-MMP-2, whereas the precise mechanisms by which TSP-2 modulates MMP-9 activity remain to be further clarified.108 Additionally, TSP-2 inhibits angiogenesis through its interaction with CD36 and is regulated by histidine-rich glycoprotein.109 TSP-2 can also bind to FGF2 via its calcium-binding III-repeat domain, thereby blocking the interaction between FGF2 and its receptor and exerting an anti-angiogenic effect.47 Therefore, TSP-2 may influence angiogenesis and extracellular matrix degradation by regulating MMP activity. Given the important roles of microvascular homeostasis and ECM remodeling in cardiac fibrosis, this mechanism may be involved in TSP-2-mediated regulation of myocardial fibrosis; however, its specific role and molecular mechanisms in cardiac tissue require further research and validation.

TGF-β Signaling

In mammals, the TGF-β superfamily encompasses three distinct isoforms, namely TGF-β1, TGF-β2, and TGF-β3, which profoundly govern cellular proliferation, lineage commitment, motility, and apoptosis via autocrine and paracrine signaling.110 The TGF-β signaling pathway is a central molecular mechanism underlying the development of cardiac fibrosis. It activates quiescent CFs through both canonical (Smad-dependent) and non-canonical (Smad-independent) signaling routes, driving their conversion into α-SMA-positive myofibroblasts and fostering excessive ECM deposition.111,112 In cardiac remodeling, TGF-β activates the Smad2/3-MMP2/9 signaling axis to promote oxidative stress, cardiomyocyte apoptosis, and interstitial fibrosis.113 As a member of the stromal protein family, TSP-2 is significantly upregulated in cardiac hypertrophy and heart failure; given that TGF-β also regulates the balance between tissue repair and fibrosis, a pathophysiological association between the two may exist. However, whether TSP-2 directly participates in TGF-β signaling regulation remains controversial. Structural and functional studies indicate that although TSP-2 shares approximately 60% sequence homology with TSP-1, TSP-2 cannot directly activate latent TGF-β due to the absence of the conserved KRFK (Lys-Arg-Phe-Lys) motif.20 This stands in stark contrast to the function of TSP-1, which activates TGF-β via the KRFK sequence and promotes fibrosis.12,114

Previous studies have shown that TSP-2 can inhibit TGF-β activation,115 possibly by suppressing TSP-1-mediated TGF-β activation.47 However, recent tissue-specific studies have revealed the complexity of TSP-2 function: in an idiopathic pulmonary fibrosis model, TSP-2 overexpression significantly activated the classical TGF-β1/Smad2/3 signaling pathway, inducing a cascade of pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and abnormal collagen network deposition.116 In colorectal cancer, TSP-2 similarly promotes tumor progression by activating the TGF-β/Smad axis.117 In contrast, in liver fibrosis, TSP-2 activates the FAK/TGF-β signaling pathway by binding to Toll-like receptor 4; specific knockdown of TSP-2, however, leads to inactivation of the TGF-β1/Smad2/3 pathway and reduced collagen deposition, suggesting that TSP-2 may regulate fibrosis through distinct mechanisms in different tissues.118 The aforementioned cross-tissue evidence indicates that TSP-2’s regulation of TGF-β signaling exhibits significant microenvironment-dependence and tissue specificity. Although existing studies support a pathological association between TSP-2 and cardiac fibrosis, there remains insufficient evidence to confirm whether it directly regulates myocardial ECM remodeling via the TGF-β1/Smad signaling axis. Therefore, elucidating the signaling networks of TSP-2 in cardiac fibrosis represents an important direction for future research.

Other Methods

As a member of the matricellular protein family, TSP-2 possesses dual biological functions: regulating ECM assembly and modulating innate immunity. In terms of inflammatory signal transduction, TSP-2 can promote NF-κB nuclear translocation and transcriptional activity by activating the PI3K/Akt signaling cascade, thereby inducing the expression of the pro-inflammatory cytokine IL-6.119 Importantly, TSP-2 lacks intrinsic kinase activity and therefore cannot directly phosphorylate the NF-κB p65 subunit. Instead, as an ECM glycoprotein, TSP-2 may regulate p65 phosphorylation indirectly through receptor-mediated intracellular signaling pathways, such as PI3K/Akt activation or other upstream kinase cascades, ultimately reinforcing NF-κB-dependent inflammatory responses and promoting the expression of cytokines including IL-6 and tumor necrosis factor-α (TNF-α).120 Therefore, the inflammatory regulatory role mediated by TSP-2 may constitute one of the key potential mechanisms underlying its effects on cardiac fibrosis. However, direct evidence for this regulatory axis in cardiac tissue remains limited and requires further validation.

Conclusion

This Review integrates current evidence to provide an overview of the molecular structure, biological functions and multifaceted roles of TSP-2 in cardiac fibrosis. Clinical and experimental studies have demonstrated that increased TSP-2 expression is associated with heart failure and cardiac fibrosis; however, elevated TSP-2 levels may represent either a marker of ongoing ECM remodelling and disease progression or an adaptive response aimed at preserving ECM integrity in response to cardiac injury or haemodynamic stress. Emerging evidence suggests that TSP-2 is not simply a profibrotic mediator, but rather a context-dependent regulator of ECM homeostasis, whose effects are shaped by disease aetiology, disease stage, cellular origin and the surrounding microenvironment.

However, direct causal evidence defining the role of TSP-2 in cardiac fibrosis remains limited. Several key questions remain unresolved: (1) genetic or pharmacological intervention studies are still needed to directly establish the contribution of TSP-2 to fibrotic remodelling; (2) the precise molecular pathways through which TSP-2 regulates cardiac fibrosis remain incompletely understood; and (3) whether the function of TSP-2 differs among heart failure subtypes arising from distinct aetiologies, including ischaemic, pressure overload-induced and metabolic cardiomyopathies, remains to be determined. Future studies integrating longitudinal analyses, diverse disease models and single-cell approaches will be essential to define the spatiotemporal expression patterns, cell-specific functions and regulatory mechanisms of TSP-2, thereby determining its potential utility as a biomarker or therapeutic target for cardiac fibrosis. However, therapeutic strategies targeting TSP-2 should be approached cautiously, given the potential risk of disrupting its essential role in maintaining cardiac ECM integrity.

Funding Statement

This work was supported by the Heilongjiang Provincial Natural Science Foundation of China (Grant NO. PL2026H322) and Youth Research Project on Traditional Chinese Medicine from the Heilongjiang Provincial Administration of Traditional Chinese Medicine (Grant NO. ZHY2026-314).

Data Availability

Data sharing is not applicable to this article as no data were created or analysed in this study.

Ethics Approval and Consent to Participate

Ethical approval and consent were deemed not applicable.

Author Contributions

WG: Conceptualization, Methodology, Visualization, Writing – original draft. JP: Methodology, Visualization, Writing – review & editing. H-xL: Formal Analysis, Writing – original draft. J-hL: Formal Analysis, Writing – original draft. JC: Conceptualization, Writing – original draft. Lf-OY: Visualization, Writing – original draft. QT: Conceptualization, Writing – review & editing. H-yL: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition. All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest that pertain to this work.