Beneficial effects of exercise initiated before development of hypertrophic cardiomyopathy in genotype-positive mice.

The effect of exercise on disease development in hypertrophic cardiomyopathy (HCM) genotype-positive individuals is unresolved. Our objective was to test the effect of exercise training initiated before phenotype development on cardiac fibrosis, morphology, and function in a mouse model of HCM. Genotype-positive Myh6 R403Q mice exposed to cyclosporine A (CsA) for induction of HCM (HCM mice) were allocated to high-intensity interval treadmill running or sedentary behavior for 6 wk. CsA was initiated from week 4 of the protocol. Cardiac imaging and exercise testing were performed at weeks 0, 3, and 6. After protocol completion, arrhythmia provocation was performed in isolated hearts, and left ventricles (LVs) were harvested for molecular biology and histology. Exercised HCM mice ran farther and faster and exhibited attenuated left atrial (LA) dilatation compared with sedentary mice. Exercised HCM mice had no difference in fibrosis compared with sedentary HCM mice despite lower expression of key extracellular matrix (ECM) genes collagen 1 and 3, fibronectin, and lysyl oxidase, accompanied by increased activation of Akt, GSK3b, and p38. Exercise did not have negative effects on LV function in HCM mice. Our findings indicate mild beneficial effects of exercise initiated before HCM phenotype development, specifically lower ECM gene expression and LA dilatation, and importantly, no detrimental effects.NEW & NOTEWORTHY Genotype-positive hypertrophic cardiomyopathy (HCM) mice had beneficial effects of exercise initiated before phenotype development. Exercised HCM mice had increased exercise capacity, smaller left atria, no increase in hypertrophy, or reduction of function, and a similar degree of fibrosis despite reduction of central extracellular matrix (ECM) genes, including collagens, compared with sedentary HCM mice.

Cancer-associated fibroblasts in desmoplastic tumors: emerging role of integrins.

The tumor microenvironment (TME) is a complex meshwork of extracellular matrix (ECM) macromolecules filled with a collection of cells including cancer-associated fibroblasts (CAFs), blood vessel associated smooth muscle cells, pericytes, endothelial cells, mesenchymal stem cells and a variety of immune cells. In tumors the homeostasis governing ECM synthesis and turnover is disturbed resulting in abnormal blood vessel formation and excessive fibrillar collagen accumulations of varying stiffness and organization. The disturbed ECM homeostasis opens up for new types of paracrine, cell-cell and cell-ECM interactions with large consequences for tumor growth, angiogenesis, metastasis, immune suppression and resistance to treatments. As a main producer of ECM and paracrine signals the CAF is a central cell type in these events. Whereas the paracrine signaling has been extensively studied in the context of tumor-stroma interactions, the nature of the numerous integrin-mediated cell-ECM interactions occurring in the TME remains understudied. In this review we will discuss and dissect the role of known and potential CAF interactions in the TME, during both tumorigenesis and chemoresistance-induced events, with a special focus on the "interaction landscape" in desmoplastic breast, lung and pancreatic cancers. As an example of the multifaceted mode of action of the stromal collagen receptor integrin α11ß1, we will summarize our current understanding on the role of this CAF-expressed integrin in these three tumor types.

Fibroblast integrin α11ß1 is a collagen assembly receptor in mechanoregulated fibrillar adhesions.

Solid epithelial cancers with significant desmoplasia are characterized by an excessive deposition of collagen-based matrix, which often supports tumor progression. However, the mechanism of how collagen receptors mediate collagen fibrillogenesis still remains mostly unclear. We show that the collagen-binding integrin α11ß1 can co-localize with tensin-1 and deposit collagen I in human pancreatic ductal adenocarcinoma (PDAC) stroma. In addition to the canonical fibrillar adhesion integrin α5ß1 expressed by human PDAC cancer-associated fibroblasts (CAFs), tensin-1-positive fibrillar adhesions contained α11ß1 but lacked α1ß1 and α2ß1. CAFs lacking α5ß1 expression displayed mechanoregulated and tensin-1 dependent α11ß1 fibrillar adhesions, suggesting independent roles of the two integrins with regards to fibrillar adhesions-based de novo fibrillogenesis. Further, we demonstrate that cell surface-associated collagen I assembly necessitated α11ß1, but not α5ß1 expression. In summary, α11ß1 integrin is a novel component of fibrillar adhesions, which is strategically positioned to mediate de novo collagen fibrillogenesis at the cell surface under pro-fibrotic conditions.

Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development.

Cardiac fibrosis is a central pathological feature in several cardiac diseases, but the underlying molecular players are insufficiently understood. The extracellular matrix proteoglycan versican is elevated in heart failure and suggested to be a target for treatment. However, the temporal expression and spatial distribution of versican and the versican cleavage fragment containing the neoepitope DPEAAE in cardiac fibrosis remains to be elucidated. In this study, we have examined versican during cardiac fibrosis development in a murine pressure overload model and in patients with cardiomyopathies. We found that versican, mainly the V1 isoform, was expressed immediately after induction of pressure overload, preceding collagen accumulation, and versican protein levels extended from the perivascular region into the cardiac interstitium. In addition, we found increased production of versican by collagen expressing fibroblasts, and that it was deposited extensively in the fibrotic extracellular matrix during pressure overload. In cardiac cell cultures, the expression of versican was induced by the pro-fibrotic transforming growth factor beta and mechanical stretch. Furthermore, we observed that the proteolytic cleavage of versican (DPEAAE fragment) increased in the late phase of fibrosis development during pressure overload. In patients with hypertrophic and dilated cardiomyopathies, we found elevated levels of versican and a positive correlation between versican and collagen mRNA in the heart, as well as increased cleavage of full-length protein. Taken together, the temporal expression profile and the spatial distribution of both the full-length versican and the DPEAAE fragment observed in this study indicates a role for versican in development of cardiac fibrosis.

Lumican accumulates with fibrillar collagen in fibrosis in hypertrophic cardiomyopathy.

AIMS: Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM. METHODS: Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n = 15), and controls from hearts deemed unsuitable for transplantation (n = 8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n = 8-10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n = 3) and examined using confocal microscopy. RESULTS: Lumican mRNA was increased threefold in HCM patients (P < 0.05) and correlated strongly with expression of collagen I (R2  = 0.60, P < 0.01) and III (R2  = 0.58, P < 0.01). Lumican protein was increased by 40% in patients with HCM (P < 0.01) and correlated with total (R2  = 0.28, P = 0.05) and interstitial (R2  = 0.30, P < 0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P < 0.001), and lumican protein increased 20-fold (P < 0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P < 0.01; mice: +13%, P < 0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (-15 nm, P < 0.05), overlapped more frequently with (+7.3%, P < 0.05) and to a larger degree with (+5.6%, P < 0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8 nm, P < 0.001), longer (+345.9 nm, P < 0.001), and fewer (-8.9%, P < 0.001) collagen fibres. CONCLUSIONS: The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM.

Inhibition of the extracellular enzyme A disintegrin and metalloprotease with thrombospondin motif 4 prevents cardiac fibrosis and dysfunction.

AIMS: Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. METHODS AND RESULTS: The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e' and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-ß target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-ß levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-ß-binding proteins, i.e. latent-binding protein of TGF-ß and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. CONCLUSION: Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-ß availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

The extracellular matrix glycoprotein ADAMTSL2 is increased in heart failure and inhibits TGFß signalling in cardiac fibroblasts.

Fibrosis accompanies most heart diseases and is associated with adverse patient outcomes. Transforming growth factor (TGF)ß drives extracellular matrix remodelling and fibrosis in the failing heart. Some members of the ADAMTSL (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs-like) family of secreted glycoproteins bind to matrix microfibrils, and although their function in the heart remains largely unknown, they are suggested to regulate TGFß activity. The aims of this study were to determine ADAMTSL2 levels in failing hearts, and to elucidate the role of ADAMTSL2 in fibrosis using cultured human cardiac fibroblasts (CFBs). Cardiac ADAMTSL2 mRNA was robustly increased in human and experimental heart failure, and mainly expressed by fibroblasts. Over-expression and treatment with extracellular ADAMTSL2 in human CFBs led to reduced TGFß production and signalling. Increased ADAMTSL2 attenuated myofibroblast differentiation, with reduced expression of the signature molecules α-smooth muscle actin and osteopontin. Finally, ADAMTSL2 mitigated the pro-fibrotic CFB phenotypes, proliferation, migration and contractility. In conclusion, the extracellular matrix-localized glycoprotein ADAMTSL2 was upregulated in fibrotic and failing hearts of patients and mice. We identified ADAMTSL2 as a negative regulator of TGFß in human cardiac fibroblasts, inhibiting myofibroblast differentiation and pro-fibrotic properties.

Integrin α11 cytoplasmic tail is required for FAK activation to initiate 3D cell invasion and ERK-mediated cell proliferation.

Integrin α11ß1 is a collagen-binding integrin, which is receiving increasing attention in the context of wound healing and fibrosis. Although α11ß1 integrin displays similar collagen specificity to α2ß1 integrin, both integrins have distinct in vivo functions. In this context, the contribution of α11 subunit cytoplasmic tail interactions to diverse molecular signals and biological functions is largely unknown. In the current study, we have deleted the α11 cytoplasmic tail and studied the effect of this deletion on α11 integrin function. Compared to wild-type cells, C2C12 cells expressing tail-less α11 attached normally to collagen I, but formed fewer focal contacts. α11-tail-less cells furthermore displayed a reduced capacity to invade and reorganize a 3D collagen matrix and to proliferate. Analysis of cell signaling showed that FAK and ERK phosphorylation was reduced in cells expressing tail-less α11. Inhibition of ERK and FAK activation decreased α11-mediated cell proliferation, whereas α11-mediated cell invasion was FAK-dependent and occurred independently of ERK signaling. In summary, our data demonstrate that the integrin α11 cytoplasmic tail plays a central role in α11 integrin-specific functions, including FAK-dependent ERK activation to promote cell proliferation.

α11ß1 Integrin is Induced in a Subset of Cancer-Associated Fibroblasts in Desmoplastic Tumor Stroma and Mediates In Vitro Cell Migration.

Integrin α11ß1 is a collagen receptor that has been reported to be overexpressed in the stroma of non-small cell lung cancer (NSCLC) and of head and neck squamous cell carcinoma (HNSCC). In the current study, we further analyzed integrin α11 expression in 14 tumor types by screening a tumor tissue array while using mAb 203E3, a newly developed monoclonal antibody to human α11. Different degrees of expression of integrin α11 were observed in the stroma of breast, ovary, skin, lung, uterus, stomach, and pancreatic ductal adenocarcinoma (PDAC) tumors. Co-expression queries with the myofibroblastic cancer-associated fibroblast (myCAF) marker, alpha smooth muscle actin (αSMA), demonstrated a moderate level of α11+ in myCAFs associated with PDAC and HNSCC tumors, and a lack of α11 expression in additional stromal cells (i.e., cells positive for fibroblast-specific protein 1 (FSP1) and NG2). The new function-blocking α11 antibody, mAb 203E1, inhibited cell adhesion to collagen I, partially hindered fibroblast-mediated collagen remodeling and obstructed the three-dimensional (3D) migration rates of PDAC myCAFs. Our data demonstrate that integrin α11 is expressed in a subset of non-pericyte-derived CAFs in a range of cancers and suggest that α11ß1 constitutes an important receptor for collagen remodeling and CAF migration in the tumor microenvironment (TME).