Fibrotic Signaling in Cardiac Fibroblasts and Vascular Smooth Muscle Cells: The Dual Roles of Fibrosis in HFpEF and CAD.

Patients with heart failure with preserved ejection fraction (HFpEF) and atherosclerosis-driven coronary artery disease (CAD) will have ongoing fibrotic remodeling both in the myocardium and in atherosclerotic plaques. However, the functional consequences of fibrosis differ for each location. Thus, cardiac fibrosis leads to myocardial stiffening, thereby compromising cardiac function, while fibrotic remodeling stabilizes the atherosclerotic plaque, thereby reducing the risk of plaque rupture. Although there are currently no drugs targeting cardiac fibrosis, it is a field under intense investigation, and future drugs must take these considerations into account. To explore similarities and differences of fibrotic remodeling at these two locations of the heart, we review the signaling pathways that are activated in the main extracellular matrix (ECM)-producing cells, namely human cardiac fibroblasts (CFs) and vascular smooth muscle cells (VSMCs). Although these signaling pathways are highly overlapping and context-dependent, effects on ECM remodeling mainly act through two core signaling cascades: TGF-ß and Angiotensin II. We complete this by summarizing the knowledge gained from clinical trials targeting these two central fibrotic pathways.

Integrin α11ß1 and syndecan-4 dual receptor ablation attenuate cardiac hypertrophy in the pressure overloaded heart.

Pathological myocardial hypertrophy in response to an increase in left ventricular (LV) afterload may ultimately lead to heart failure. Cell surface receptors bridge the interface between the cell and the extracellular matrix (ECM) in cardiac myocytes and cardiac fibroblasts and have been suggested to be important mediators of pathological myocardial hypertrophy. We identify for the first time that integrin α11 (α11) is preferentially upregulated among integrin ß1 heterodimer-forming α-subunits in response to increased afterload induced by aortic banding (AB) in wild-type (WT) mice. Mice were anesthetized in a chamber with 4% isoflurane and 95% oxygen before being intubated and ventilated with 2.5% isoflurane and 97% oxygen. For pre- and postoperative analgesia, animals were administered 0.02-mL buprenorphine (0.3 mg/mL) subcutaneously. Surprisingly, mice lacking α11 develop myocardial hypertrophy following AB comparable to WT. In the mice lacking α11, we further show a compensatory increase in the expression of another mechanoreceptor, syndecan-4, following AB compared with WT AB mice, indicating that syndecan-4 compensated for lack of α11. Intriguingly, mice lacking mechanoreceptors α11 and syndecan-4 show ablated myocardial hypertrophy following AB compared with WT mice. Expression of the main cardiac collagen isoforms col1a2 and col3a1 was significantly reduced in AB mice lacking mechanoreceptors α11 and syndecan-4 compared with WT AB.NEW & NOTEWORTHY Despite their putative importance in stress sensing, the specific integrin α-subunit(s) involved in cardiac hypertrophy has not been identified. Here, we show that α11 and syndecan-4 are critical and interdependent mediators of the hypertrophic response to increased LV afterload. We demonstrate in cells lacking both receptors an interdependent reduction in cell attachment to the major cardiac extracellular matrix components, suggesting that their interplay represents an important mechanism for stress sensing in cardiac cells.

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction.

Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor ß receptor I (TGFßRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation.

Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin.

Background Pressure overload of the heart occurs in patients with hypertension or valvular stenosis and induces cardiac fibrosis because of excessive production of extracellular matrix by activated cardiac fibroblasts. This initially provides essential mechanical support to the heart, but eventually compromises function. Osteopontin is associated with fibrosis; however, the underlying signaling mechanisms are not well understood. Herein, we examine the effect of thrombin-cleaved osteopontin on fibrosis in the heart and explore the role of syndecan-4 in regulating cleavage of osteopontin. Methods and Results Osteopontin was upregulated and cleaved by thrombin in the pressure-overloaded heart of mice subjected to aortic banding. Cleaved osteopontin was higher in plasma from patients with aortic stenosis receiving crystalloid compared with blood cardioplegia, likely because of less heparin-induced inhibition of thrombin. Cleaved osteopontin and the specific osteopontin peptide sequence RGDSLAYGLR that is exposed after thrombin cleavage both induced collagen production in cardiac fibroblasts. Like osteopontin, the heparan sulfate proteoglycan syndecan-4 was upregulated after aortic banding. Consistent with a heparan sulfate binding domain in the osteopontin cleavage site, syndecan-4 was found to bind to osteopontin in left ventricles and cardiac fibroblasts and protected osteopontin from cleavage by thrombin. Shedding of the extracellular part of syndecan-4 was more prominent at later remodeling phases, at which time levels of cleaved osteopontin were increased. Conclusions Thrombin-cleaved osteopontin induces collagen production by cardiac fibroblasts. Syndecan-4 protects osteopontin from cleavage by thrombin, but this protection is lost when syndecan-4 is shed in later phases of remodeling, contributing to progression of cardiac fibrosis.

Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks.

Cancer-associated fibroblasts: how do they contribute to metastasis?

Cancer-associated fibroblasts (CAFs) are activated fibroblasts in the tumor microenvironment. They are one of the most prominent cell types in the stroma and produce large amounts of extracellular matrix molecules, chemokines, cytokines and growth factors. Importantly, CAFs promote cancer progression and metastasis by multiple pathways. This, together with their genetic stability, makes them an interesting target for cancer therapy. However, CAF heterogeneity and limited knowledge about the function of the different CAF subpopulations in vivo, are currently major obstacles for identifying specific molecular targets that are of value for cancer treatment. In this review, we discuss recent major findings on CAF development and their metastasis-promoting functions, as well as open questions to be addressed in order to establish successful cancer therapies targeting CAFs.

Sweet, yet underappreciated: Proteoglycans and extracellular matrix remodeling in heart disease.

Extracellular matrix remodeling is extensive in several heart diseases and hampers cardiac filling, often leading to heart failure. Proteoglycans have over the last two decades emerged as molecules with important roles in matrix remodeling and fibrosis in the heart. Here we discuss and review current literature on proteoglycans that have been studied in cardiac remodeling. The small leucine rich proteoglycans (SLRPs) are located within the extracellular matrix and are organizers of the matrix structure. Membrane-bound proteoglycans, such as syndecans and glypicans, act as receptors and direct cardiac fibroblast signaling. Recent studies indicate that proteoglycans are promising as diagnostic biomarkers for cardiac fibrosis, and that they may provide new therapeutic strategies for cardiac disease.

Mechanical regulation of cardiac fibroblast profibrotic phenotypes.

Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs.

The Soft- and Hard-Heartedness of Cardiac Fibroblasts: Mechanotransduction Signaling Pathways in Fibrosis of the Heart.

Cardiac fibrosis, the excessive accumulation of extracellular matrix (ECM), remains an unresolved problem in most forms of heart disease. In order to be successful in preventing, attenuating or reversing cardiac fibrosis, it is essential to understand the processes leading to ECM production and accumulation. Cardiac fibroblasts are the main producers of cardiac ECM, and harbor great phenotypic plasticity. They are activated by the disease-associated changes in mechanical properties of the heart, including stretch and increased tissue stiffness. Despite much remaining unknown, an interesting body of evidence exists on how mechanical forces are translated into transcriptional responses important for determination of fibroblast phenotype and production of ECM constituents. Such mechanotransduction can occur at multiple cellular locations including the plasma membrane, cytoskeleton and nucleus. Moreover, the ECM functions as a reservoir of pro-fibrotic signaling molecules that can be released upon mechanical stress. We here review the current status of knowledge of mechanotransduction signaling pathways in cardiac fibroblasts that culminate in pro-fibrotic gene expression.

The Heparan Sulfate Proteoglycan Glypican-6 Is Upregulated in the Failing Heart, and Regulates Cardiomyocyte Growth through ERK1/2 Signaling.

Pressure overload is a frequent cause of heart failure. Heart failure affects millions of patients worldwide and is a major cause of morbidity and mortality. Cell surface proteoglycans are emerging as molecular players in cardiac remodeling, and increased knowledge about their regulation and function is needed for improved understanding of cardiac pathogenesis. Here we investigated glypicans (GPC1-6), a family of evolutionary conserved heparan sulfate proteoglycans anchored to the extracellular leaflet of the cell membrane, in experimental and clinical heart failure, and explored the function of glypican-6 in cardiac cells in vitro. In mice subjected to pressure overload by aortic banding (AB), we observed elevated glypican-6 levels during hypertrophic remodeling and dilated, end-stage heart failure. Consistently, glypican-6 mRNA was elevated in left ventricular myocardium from explanted hearts of patients with end-stage, dilated heart failure with reduced ejection fraction. Glypican-6 levels correlated negatively with left ventricular ejection fraction in patients, and positively with lung weight after AB in mice. Glypican-6 mRNA was expressed in both cardiac fibroblasts and cardiomyocytes, and the corresponding protein displayed different sizes in the two cell types due to tissue-specific glycanation. Importantly, adenoviral overexpression of glypican-6 in cultured cardiomyocytes increased protein synthesis and induced mRNA levels of the pro-hypertrophic signature gene ACTA1 and the hypertrophy and heart failure signature genes encoding natriuretic peptides, NPPA and NPPB. Overexpression of GPC6 induced ERK1/2 phosphorylation, and co-treatment with the ERK inhibitor U0126 attenuated the GPC6-induced increase in NPPA, NPPB and protein synthesis. In conclusion, our data suggests that glypican-6 plays a role in clinical and experimental heart failure progression by regulating cardiomyocyte growth through ERK signaling.

Syndecans in heart fibrosis.

Heart disease is a deadly syndrome affecting millions worldwide. It reflects an unmet clinical need, and the disease mechanisms are poorly understood. Cardiac fibrosis is central to heart disease. The four-membered family of transmembrane proteoglycans, syndecan-1 to -4, is believed to regulate fibrosis. We review the current literature concerning syndecans in cardiac fibrosis. Syndecan expression is up-regulated in response to pro-inflammatory stimuli in various forms of heart disease with fibrosis. Mice lacking syndecan-1 and -4 show reduced activation of pro-fibrotic signaling and increased cardiac rupture upon infarction indicating an important role for these molecules. Whereas the short cytoplasmic tail of syndecans regulates signaling, their extracellular part, substituted with heparan sulfate glycosaminoglycan chains, binds a plethora of extracellular matrix (ECM) molecules involved in fibrosis, e.g., collagens, growth factors, cytokines, and immune cell adhesion proteins. Full-length syndecans induce pro-fibrotic signaling, increasing the expression of collagens, myofibroblast differentiation factors, ECM enzymes, growth factors, and immune cell adhesion molecules, thereby also increasing cardiac stiffness and preventing cardiac rupture. Upon pro-inflammatory stimuli, syndecan ectodomains are enzymatically released from heart cells (syndecan shedding). Shed ectodomains affect the expression of ECM molecules, promoting ECM degradation and cardiac rupture upon myocardial infarction. Blood levels of shed syndecan-1 and -4 ectodomains are associated with hospitalization, mortality, and heart remodeling in patients with heart failure. Improved understanding of syndecans and their modifying enzymes in cardiac fibrosis might contribute to the development of compounds with therapeutic potential, and enzymatically shed syndecan ectodomains might constitute a future prognostic tool for heart diseases with fibrosis. Graphical Abstract Graphical abstract summarizing the contents of the current review on syndecans in cardiac fibrosis. The heart is subjected to various forms of pathological stimuli, e.g., myocardial infarction, hypertension, valvular stenosis, infection, or an inherited genetic mutation, triggering responses in cells resident in the heart. Here, we focus on the responses of cardiac fibroblasts directing changes in the extracellular matrix resulting in cardiac fibrosis. A family of four transmembrane proteoglycans, syndecan-1 to -4, is expressed in the cell membrane of cardiac fibroblasts and is generally up-regulated in response to the above-mentioned pathological stimuli. Syndecans carry glycosaminoglycan chains on their extracellular domain, binding a plethora of molecules involved in fibrosis, e.g., growth factors, cytokines, immune cell adhesion proteins, and pathogens. Syndecans have a short cytoplasmic tail involved in pro-fibrotic signaling. The signaling and cellular processes governed by syndecans in the heart in response to pathological stimuli regulate important aspects of extracellular matrix remodeling and fibrosis and have mainly been studied in cardiac remodeling in response to cardiac infarction and pressure overload. In general, adequate timing and the quantity and quality of fibrosis are absolutely crucial for heart function and survival, determining cardiac stiffness, contractility, compliance, probability of rupture, dilation, and diastolic and systolic function. Syndecan-1 and -4 have mainly been studied in the heart and are discussed in this review (LV left ventricle).

Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice.

AIMS: In pressure overload, left ventricular (LV) dilatation is a key step in transition to heart failure (HF). We recently found that collagen VIII (colVIII), a non-fibrillar collagen and extracellular matrix constituent, was reduced in hearts of mice with HF and correlated to degree of dilatation. A reduction in colVIII might be involved in LV dilatation, and we here examined the role of reduced colVIII in pressure overload-induced remodelling using colVIII knock-out (col8KO) mice. METHODS AND RESULTS: Col8KO mice exhibited increased mortality 3-9 days after aortic banding (AB) and increased LV dilatation from day one after AB, compared with wild type (WT). LV dilatation remained increased over 56 days. Forty-eight hours after AB, LV expression of main structural collagens (I and III) was three-fold increased in WT mice, but these collagens were unaltered in the LV of col8KO mice together with reduced expression of the pro-fibrotic cytokine TGF-ß, SMAD2 signalling, and the myofibroblast markers Pxn, α-SMA, and SM22. Six weeks after AB, LV collagen mRNA expression and protein were increased in col8KO mice, although less pronounced than in WT. In vitro, neonatal cardiac fibroblasts from col8KO mice showed lower expression of TGF-ß, Pxn, α-SMA, and SM22 and reduced migratory ability possibly due to increased RhoA activity and reduced MMP2 expression. Stimulation with recombinant colVIIIα1 increased TGF-ß expression and fibroblast migration. CONCLUSION: Lack of colVIII reduces myofibroblast differentiation and fibrosis and promotes early mortality and LV dilatation in response to pressure overload in mice.

Syndecan-4 is a key determinant of collagen cross-linking and passive myocardial stiffness in the pressure-overloaded heart.

AIMS: Diastolic dysfunction is central to the development of heart failure. To date, there is no effective treatment and only limited understanding of its molecular basis. Recently, we showed that the transmembrane proteoglycan syndecan-4 increases in the left ventricle after pressure overload in mice and man, and that syndecan-4 via calcineurin/nuclear factor of activated T-cells (NFAT) promotes myofibroblast differentiation and collagen production upon mechanical stress. The aim of this study was to investigate whether syndecan-4 affects collagen cross-linking and myocardial stiffening in the pressure-overloaded heart. METHODS AND RESULTS: Aortic banding (AB) caused concentric hypertrophy and increased passive tension of left ventricular muscle strips, responses that were blunted in syndecan-4(-/-) mice. Disruption of titin anchoring by salt extraction of actin and myosin filaments revealed that the effect of syndecan-4 on passive tension was due to extracellular matrix remodelling. Expression and activity of the cross-linking enzyme lysyl oxidase (LOX) increased with mechanical stress and was lower in left ventricles and cardiac fibroblasts from syndecan-4(-/-) mice, which exhibited less collagen cross-linking after AB. Expression of osteopontin (OPN), a matricellular protein able to induce LOX in cardiac fibroblasts, was up-regulated in hearts after AB, in mechanically stressed fibroblasts and in fibroblasts overexpressing syndecan-4, calcineurin, or NFAT, but down-regulated in fibroblasts lacking syndecan-4 or after NFAT inhibition. Interestingly, the extracellular domain of syndecan-4 facilitated LOX-mediated collagen cross-linking. CONCLUSIONS: Syndecan-4 exerts a dual role in collagen cross-linking, one involving its cytosolic domain and NFAT signalling leading to collagen, OPN, and LOX induction in cardiac fibroblasts; the other involving the extracellular domain promoting LOX-dependent cross-linking.

Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart.

Sustained pressure overload induces heart failure, the main cause of mortality in the Western world. Increased understanding of the underlying molecular mechanisms is essential to improve heart failure treatment. Despite important functions in other tissues, cardiac proteoglycans have received little attention. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is essential for pathological remodeling, and we here investigated its expression and shedding during heart failure. Pressure overload induced by aortic banding for 24 h and 1 week in mice increased syndecan-4 mRNA, which correlated with mRNA of inflammatory cytokines. In cardiac myocytes and fibroblasts, tumor necrosis factor-α, interleukin-1ß and lipopolysaccharide through the toll-like receptor-4, induced syndecan-4 mRNA. Bioinformatical and mutational analyses in HEK293 cells identified a functional site for the proinflammatory nuclear factor-κB transcription factor in the syndecan-4 promoter, and nuclear factor-κB regulated syndecan-4 mRNA in cardiac cells. Interestingly, tumor necrosis factor-α, interleukin-1ß and lipopolysaccharide induced nuclear factor-κB-dependent shedding of the syndecan-4 ectodomain from cardiac cells. Overexpression of syndecan-4 with mutated enzyme-interacting domains suggested enzyme-dependent heparan sulfate chains to regulate shedding. In cardiac fibroblasts, lipopolysaccharide reduced focal adhesion assembly, shown by immunohistochemistry, suggesting that inflammation-induced shedding affects function. After aortic banding, a time-dependent cardiac recruitment of T lymphocytes was observed by measuring CD3, CD4 and CD8 mRNA, which was reduced in syndecan-4 knockout hearts. Finally, syndecan-4 mRNA and shedding were upregulated in failing human hearts. Conclusively, our data suggest that syndecan-4 plays an important role in the immune response of the heart to increased pressure, influencing cardiac remodeling and failure progression.

Syndecan-4 signaling via NFAT regulates extracellular matrix production and cardiac myofibroblast differentiation in response to mechanical stress.

Pressure overload activates cardiac fibroblasts leading to excessive production of extracellular matrix which may contribute to compromised heart function. The activated fibroblast acquires smooth muscle-like features such as expression of smooth muscle α-actin (SMA) and SM22 and is therefore referred to as myofibroblast. The molecular mechanisms underlying mechanical stress-induced myofibroblast differentiation are poorly defined. The objective of this study was to examine the potential roles of the transmembrane proteoglycan syndecan-4 and the calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) in myofibroblast differentiation. Aortic banding resulted in elevated collagen I and III, fibronectin, SMA and SM22 mRNA in the left ventricles of wild-type mice, whereas this response was markedly reduced in syndecan-4(-/-) mice. Myofibroblast differentiation in vitro was associated with increased SMA, collagen I and III expression and NFAT-luciferase activity, all of which were reduced in fibroblasts from syndecan-4(-/-) mice or after treatment with calcineurin/NFAT blockers. Following cyclic stretch, NFATc4 was activated in cardiac fibroblasts in a syndecan-4- and calcineurin-dependent manner. Syndecan-4 and calcineurin co-localized and mechanical stress resulted in dephosphorylation of serine179 of syndecan-4, an intracellular residue critical for calcineurin interaction. Over-expression of NFATc4 up-regulated collagen III, MRTF-A (a transcriptional regulator of SMA) and the NFAT-target regulator of calcineurin 1.4 (RCAN1.4). Our data demonstrate that syndecan-4 is important for the differentiation of cardiac fibroblasts into myofibroblasts in the pressure-overloaded heart and that the calcineurin/NFAT pathway is engaged upon mechanical stress in a syndecan-4-dependent manner, playing an active role in myofibroblast differentiation and extracellular matrix production. This article is part of a Special Issue entitled 'Possible Editorial'.