Pressure-overloaded Myocardium Research Articles

Fibroblast Smad7 Induction Protects the Remodeling Pressure-Overloaded Heart.

Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β (transforming growth factor-β)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-β activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-β response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-β signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction. The effects of myofibroblast-specific Smad7 loss were studied in a mouse model of transverse aortic constriction, using echocardiography, histological analysis, and molecular analysis. Proteomic studies in S7KO (Smad7 knockout) and overexpressing cells were used to identify fibroblast-derived mediators modulated by Smad7. In vitro experiments using cultured cardiac fibroblasts, fibroblasts populating collagen lattices, and isolated macrophages were used to dissect the molecular signals responsible for the effects of Smad7. Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-β and angiotensin II stimulated fibroblast Smad7 upregulation via Smad3, whereas GDF15 (growth differentiation factor 15) induced Smad7 through GFRAL (glial cell line-derived neurotrophic factor family receptor α-like). MFS7KO (myofibroblast-specific S7KO) mice had increased mortality, accentuated systolic dysfunction and dilative remodeling, and accelerated diastolic dysfunction in response to transverse aortic constriction. Increased dysfunction in MFS7KO hearts was associated with accentuated fibrosis and increased MMP (matrix metalloproteinase)-2 activity and collagen denaturation. Secretomic analysis showed that Smad7 loss accentuates secretion of structural collagens and matricellular proteins and markedly increases MMP2 secretion. In contrast, Smad7 overexpression reduced MMP2 levels. In fibroblasts populating collagen lattices, the effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Surprisingly, MFS7KO mice also exhibited significant macrophage expansion caused by paracrine actions of Smad7 null fibroblasts that stimulate macrophage proliferation and fibrogenic activation. Macrophage activation involved the combined effects of the fibroblast-derived matricellular proteins CD5L (CD5 antigen-like), SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), ECM1 (extracellular matrix protein 1), and TGFBI (TGFB induced). The antifibrotic effects of Smad7 in the pressure-overloaded heart protect from dysfunction and involve not only reduction in collagen deposition but also suppression of MMP2-mediated matrix denaturation and paracrine effects that suppress macrophage activation through inhibition of matricellular proteins.

Bone marrow-fibroblast progenitor cell-derived small extracellular vesicles promote cardiac fibrosis via miR-21-5p and integrin subunit αV signalling.

Cardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin-10 (IL10) reduces pressure overload (PO)-induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro-fibrotic miRNAs enriched in sEV-derived from IL10 KO FPCs promote cardiac fibrosis in pressure-overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV-2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis-associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFβ-treated IL10-KO-FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR-21a-5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR-21a-5p binds and stabilizes ITGAV (integrin av) mRNA. Finally, miR-21a-5p-silenced in sEV reduced PO-induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC-derived sEV exacerbate cardiac fibrosis through the miR-21a-5p/ITGAV/Col1α signalling pathway, suggesting miR-21a-5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.

Systemic proinflammatory-profibrotic response in aortic stenosis patients with diabetes and its relationship with myocardial remodeling and clinical outcome

Abstract Background It is unclear whether and how diabetes mellitus may aggravate myocardial fibrosis and remodeling in the pressure-overloaded heart. We investigated the impact of diabetes on the prognosis of aortic stenosis (AS) patients and its underlying mechanisms using comprehensive noninvasive imaging studies and plasma proteomics. Methods Severe AS patients undergoing both echocardiography and cardiovascular magnetic resonance (CMR) (n=253 of which 66 had diabetes) comprised the imaging cohort. The degree of replacement and diffuse interstitial fibrosis by late gadolinium enhancement (LGE) and extracellular volume fraction (ECV) was quantified using CMR. Plasma samples were analyzed with the multiplex proximity extension assay for 92 proteomic biomarkers in a separate biomarker cohort of severe AS patients (n=100 of which 27 had diabetes). Results In the imaging cohort, diabetic patients were older (70.4±6.8 vs. 66.7±10.1 years) and had a higher prevalence of ischemic heart disease (28.8% vs. 9.1%), with more advanced ventricular diastolic dysfunction. On CMR, diabetic patients had increased replacement and diffuse interstitial fibrosis (LGE% 0.3 [0.0–1.6] versus 0.0 [0.0–0.5], p=0.009; ECV% 27.9 [25.7–30.1] versus 26.7 [24.9–28.5], p=0.025) (Figure 1). Plasma proteomics analysis of the biomarker cohort revealed that 9 proteins (E-selectin, interleukin-1 receptor type 1, interleukin-1 receptor type 2, galectin-4, intercellular adhesion molecule 2, integrin beta-2, galectin-3, growth differentiation factor 15, and cathepsin D) are significantly elevated in diabetic AS patients (Figure 2). Pathway over-representation analyses of the plasma proteomics with Gene Ontology terms indicated that pathways related to inflammatory response and extracellular matrix components were enriched, suggesting that diabetes is associated with systemic effects that evoke proinflammatory and profibrotic response to the pressure-overloaded myocardium. During follow-up (median 6.3 years [IQR 5.2–7.2]) of the imaging cohort, 232 patients received aortic valve replacement (AVR) with 53 unexpected heart failure admissions or death. Diabetes was a significant predictor of heart failure and death, independent of clinical covariates and AVR (hazard ratio 1.88, 95% confidence interval 1.06–3.31, p=0.030). Conclusion Plasma proteomic analyses indicate that diabetes potentiates the systemic proinflammatory and profibrotic milieu in AS patients. These systemic biological changes underlie the increase of myocardial fibrosis, diastolic dysfunction, and worse clinical outcomes in severe AS patients with concomitant diabetes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Research Foundation of Korea

Abstract P1018: Bone Marrow Progenitor Cell-Derived Exosomes Activate Cardiac Fibroblasts Via Mir-21a-5p Mediated Integrin Alpha V Stabilization

Abstract: Heart failure is leading cause of death worldwide, and cardiac fibrosis is the hallmark of heart failure. We previously showed that interleukin-10 (IL10) treatment significantly reduces pressure overload-induced cardiac fibrosis. We also have demonstrated that fibroblast progenitor cells (bone marrow-prominin positive cells; FPCs) play a prominent role in developing cardiac fibrosis, yet the mechanism is largely unknown. Here, we hypothesized that pro-fibrotic miRNAs enriched in exosomes derived from IL10 KO FPCs promote fibroblast activation and cardiac fibrosis in pressure-overloaded myocardium. Methods: Exosomes were isolated from WT and IL-10KO FPCs. Exosomal miRNAs profiling was performed using fibrosis-associated miRNA profiler kit from Qiagen. To determine the contribution of exosomal miR21 on TAC-induced fibrosis, miR21 level was reduced in FPCs-derived exosomes using miR21 silencer. Modified exosomes were injected into heart immediately after TAC surgery. Results: FPCs were identified as CD45+/prominin-1 cells using FACS. TGFβ treatment exacerbated fibrotic genes (Col1α, fibronectin, periostin, and αSMA) expression in IL10KO FPCs compared to WT-FPCs. Pathway-based miRNA array revealed that IL10KO FPCs exosomes are enriched with miR-21a-5p and facilitated fibroblasts activation as observed by qPCR (αSMA and Col1α), western (Col1α and TGFβ levels), wound healing, and immunostaining assay (Col1α/DAPI expression) as compared to WT control. Interestingly, miR21 inhibition in IL10KO FPCs exosome using antimiR 21, reduced TGFβ-induced cardiac fibroblast activation. Target prediction database analysis revealed that Integrin Subunit Alpha V (ITGAV) is a strong regulatory target for miRNA21. At molecular level, we found that exosomal miR-21a-5p stabilizes ITGAV, which ultimately enhances fibroblast activation and cardiac fibrosis both in vitro and in vivo. Conclusions: In conclusion, we report that FPCs derived miR21 packaged in exosomes activates cardiac fibroblasts in IL10-KO mice via miR-21a-5p/ITGAV/Col1α signaling axis and increased cardiac fibrosis. IL10 treatment significantly reduced miR21 exosomal packaging, inhibited fibroblasts activation, and reduced cardiac fibrosis following TAC.

Long-term Dexamethasone Treatment Increases Cardiac Galectin-3 Levels.

Glucocorticoids, which are widely prescribed around the world, cause cardiac remodeling in long-term treatment by triggering insulin resistance and increasing blood pressure. However, its role in cardiac remodeling remains unclear. Galectin-3 (gal-3) is a member of a beta-galactoside-binding animal lectins, upregulated as a result of insulin resistance and in the pressure-overloaded myocardium and regulate cardiac remodeling. We hypothesized that gal-3 may be upregulated in the myocardium with prolonged use of glucocorticoids and associated with cardiac hypertrophy. To examine the involvement of glucocorticoids in gal-3 levels in rat myocardium, sixteen female Wistar Albino rats were assigned to control (C; n = 8) and dexamethasone (Dex; n = 8) groups. Daily dexamethasone was injected subcutaneously for 28days at a dose of 1mg.kg-1. Control animals were injected with the same volume of saline. The body weight and heart weights were determined. Gal-3 levels in myocardium were determined by Western blot. Our data shows that dexamethasone administration resulted in significant increase in heart weight (p < 0.05) and HW/BW ratios (p < 0.001) and 28days of dexamethasone administration with the dose of 1mg.kg-1 caused a twofold increase in the gal-3 expression in the left ventricle (p < 0.001). The finding of the current study is the first to show that dexamethasone causes an increase in gal-3 levels in myocardium. Our study provides an important step in the development of possible therapeutics by determining that dexamethasone causes an increase in gal-3 levels in the myocardium and raises awareness about the follow-up of patients receiving long-term glucocorticoid therapy.

Abstract P374: Functional Impairment Of Cardiac Endothelial Cells In Diabetic Failing Heart Via Dysregulated Exosomal Mirnas

Introduction: Cardiac endothelial cells (ECs) and fibroblasts (FBs) together maintain cardiac homeostasis. Their functional impairment aggravates complications in the heart. In diabetes, acute inflammation leads to cardiac FBs activation, which predisposes the diabetic myocardium to severe fibrosis. Further, inflammation-related vascular dysfunction is a major end-organ complication in diabetics. However, it is not known, whether myofibroblast regulates ECs function in a diseased diabetic heart. Therefore, we hypothesized that “myofibroblast in diabetic heart secretes exosomes packed with antiangiogenic/profibrotic factors, which impede EC function and exaggerate pathological remodelling in pressure-overloaded (PO) myocardium. Methods: Exosomes were isolated from diabetic mice plasma and FB condition media by ultracentrifugation and characterized by nanosight &amp; electron microscopy. We cultured mouse primary heart ECs in growth media and treated with exosomes derived from FBs (treated with 25mM glucose or 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Results: Ang II and glucose significantly activate FBs as shown by qPCR (fibronectin, collagenase1α1) and western blot (pSmad2, p-p38). Exosomes derived from diabetic Ang II treated FBs significantly impaired ECs function as shown by Matrigel tube formation and Boyden chamber migration assays. Interestingly, ECs markers (eNOS, VEGF, CD31) genes and proteins expression were significantly inhibited in ECs treated with exosomes-derived from glucose and Ang II treated FBs. We, further, checked the effect of diabetic mice plasma exosomes on ECs function and found significantly impaired as shown by tube formation and migration data. Finally, microRNA (miR) array and qPCR analysis revealed that miR-216a-5p, miR-26a-5p and miR7a-5p were highly upregulated in exosomes derived from FBs co-treated with glucose and AngII. Conclusions: Taken together, this study demonstrates that glucose and AngII co-treated FBs-derived exosomes are enriched in pro-fibrotic factors and can lead to EC dysfunction and promotes cardiac fibrosis in PO myocardium. In future studies, we will modulate the target miRs in diabetic FBs to see whether it rescue reparative function of ECs and inhibits fibrosis in failing heart.

4-Methylumbelliferone Attenuates Macrophage Invasion and Myocardial Remodeling in Pressure-Overloaded Mouse Hearts.

[Figure: see text].

Cardiovascular Outcomes with Sacubitril-Valsartan in Heart Failure: Emerging Clinical Data.

One of the defining features of heart failure (HF) is neurohormonal activation. The renin-angiotensin-aldosterone-system (RAAS) and sympathetic nervous system (SNS) cause vasoconstriction and fluid retention and, in response, the secretion of natriuretic peptides (NPs) from volume and pressure-overloaded myocardium promotes vasodilation and diuresis. Inhibition of the RAAS with either angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) has been the cornerstone of medical treatment for HF with a reduced ejection fraction (HFrEF) but, until recently, it was unclear how the beneficial effects of NPs may be augmented in patients with HF. Neprilysin, a metalloproteinase widely distributed throughout the body, plays a role in degrading the gross excess of circulating NPs in patients with HF. Early studies of neprilysin inhibition suggested possible physiological benefits. In 2014, the PARADIGM-HF trial found that sacubitril-valsartan, a combination of the ARB valsartan, and the neprilysin inhibitor sacubitril, was superior to enalapril in patients with HFrEF, reducing the relative risk of cardiovascular (CV) death or first hospitalisation with HF by 20%. Almost half of the patients with HF symptoms have a “preserved” ejection fraction (HFpEF); however, the PARAGON-HF study found that sacubitril-valsartan in patients with LVEF ≥45% had no effect on CV death or first and recurrent hospitalisations with HF compared to valsartan. Guidelines across the world have changed to include sacubitril-valsartan for patients with HFrEF yet, nearly 6 years after PARADIGM-HF, there is still uncertainty as to when and in whom sacubitril-valsartan should be started. Furthermore, there may yet be subsets of patients with HFpEF who might benefit from treatment with sacubitril-valsartan. This review will describe the mechanisms behind the outcome benefit of sacubitril-valsartan in patients with HFrEF and to consider its future role in the management of patients with HF.

Abstract 362: Role of Dysregulated Exosomal MiRNAs in Functional Impairment of Cardiac Endothelial Cells

Objective: Endothelial cells (ECs) play critical role to maintain the normal heart function. It is shown that fibroblast-derived exosomes have the ability to enhance cardiac myocytes hypertrophy in pressure-overloaded myocardium. However, their effect on endothelial cell function has not been studied. Others and we have previously shown that stress-induced chronic inflammation induces cardiac fibroblasts and mediates endothelial cells dysfunction. Here we hypothesized that activated cardiac fibroblasts-derived exosomes (FB-Exo) mediates cardiac ECs dysfunction and leads for cardiac pathology and restoring the altered FB-Exo contents will improve endothelial cells function and biology. Methods: We cultured mouse primary endothelial cells in EC growth media. Cells were treated with fibroblasts-derived-Exo. Exosomes were isolated from fibroblast condition media by ultracentrifugation and characterized by nanosight &amp; electron microscopy. Results: Fibroblasts were significantly activated by TGFβ treatment as shown by qPCR and western blot (smad2/3, p-Smad2/3, p38, p-p38) data. Endothelial cells dysfunction as shown by Matrigel assay, real-time Q-PCR and western data (eNOS and Hif1α) data was observed in TGFβ-FB-Exo treatment endothelial cell. MTT, TUNEL and migration assay also followed the same trend as TGFβ-FB-Exo treatment significantly induced endothelial cell death and inhibits its proliferation and migration. Furthermore, microRNA array and PCR analysis revealed dysregulation of miR-132-3p, miR-2001-3p and miR-125b-5p in TGFβ-FB-Exo treated endothelial cells. Conclusions: Taken together, this study demonstrates that TGFβ treated fibroblasts-derived exosomes are enriched in pro-fibrotic factors and can lead to endothelial dysfunction and promotes cardiac fibrosis in PO myocardium. In future study, we will modulate the target miRs in fibroblasts to see whether it rescue reparative function of endothelial cell and inhibits cardiac fibrosis in failing heart.

P4997Raf kinase inhibitor protein of the bone marrow contributes to cardiac fibrogenesis in pressure-overloaded myocardium

Abstract Background Raf Kinase Inhibitor Protein (RKIP) regulates myocardial remodeling under conditions of enhanced myocardial oxidative stress in pressure-overloaded left ventricle (LV) modulating myocardial production of reactive oxygen species (ROS). A second mode of action may be the mobilization of circulating fibroblasts (fibrocytes) from the bone-marrow (BM). However the underlying mechanisms are incompletely understood. Methods and results To further characterize the role of RKIP in BM cells for myocardial remodeling 10-week-old wild-type (WT) C57BL/6N mice were subjected to transplantation of bone marrow (BMT) from 10-week-old C57BL/6-RKIP-deficient (RKIP−/−) N or WT C57BL/6N mice expressing green fluorescent protein (GFP)+ ubiquitously. 28 days later, transverse aortic constriction (TAC, 360 μm) or SHAM-operation was performed. 5 weeks post TAC, LV systolic pressure (LVSP) and heart weight to tibia length ratio were significantly increased in both types of BMT, compared with corresponding SHAM. Increased afterload elicited myocardial fibrosis as assessed by picrosirius red staining (WT/WT SHAM 15±2.5%, WT/WT TAC 21.3±1.4%, p&lt;0.05; RKIP−/−/WT SHAM 17±2%, RKIP−/−/WT TAC 18±3%, p=ns) and significantly increased the number of LV fibroblasts per mm2 estimated by immunostaining for intracellular fibronectin, which were further reduced by transplantation of RKIP−/−N BM (WT/WT SHAM 5499±313, WT/WT TAC 7493±741 per mm2, p&lt;0.05; RKIP−/−/WT SHAM 5737±259, RKIP−/−/WT TAC 5282±551, per mm2, p=ns). Moreover, transplantation of RKIP−/−N BM significantly diminished the number of circulating BM-derived GFP+ fibroblasts in the peripheral blood and LV myocardium during pressure overload (WT/WT SHAM 961±129, WT/WT TAC 2326±273 per mm2, p&lt;0.05; RKIP−/−/WT SHAM 1041±209, RKIP−/−/WT TAC 1518±107, per mm2, p=ns). The myocardial redox status was assessed by the co-immunostaining for ROS production marker 8-hydroxyguanosin (8-dOHG), cardiomyocyte marker α-sarcomeric actin and fibroblast marker intracellular fibronectin. Pressure overload during 5 weeks significantly increased the percentages of 8-dOHG+cardiomyocytes (WT/WT SHAM 34±9%, WT/WT TAC 63±6%, p&lt;0.05; RKIP−/−/WT SHAM 29±6%, RKIP−/−/WT TAC 31±8%, p=ns) and 8-dOHG+fibroblasts (WT/WT SHAM 57±6%, WT/WT TAC 73±4%, p&lt;0.05; RKIP−/−/WT SHAM 58±2%, RKIP−/−/WT TAC 58±7%, p=ns) in mice transplanted with WT BM but not with RKIP−/−N BM. Conclusions In pressure-overload induced enhanced myocardial ROS production, deficiency of RKIP-expression in the bone marrow abrogates left ventricular fibrosis by reduction of myocardial ROS production and mobilization of BM-derived fibroblasts. These findings suggest that the function of RKIP in the bone marrow may be important for maladaptive myocardial remodelling. Acknowledgement/Funding Deutsche Forschungsgemeinschaft: KA4024/3-1, SFB TRR219; Saarland University HOMFOR, Dr. Marija Orlovich foundation, Corona foundation s199/10060/2014

Abstract 250: Smad3-Mediated Induction of Smad7 in Activated Myofibroblasts Protects the Remodeling Myocardium

TGFβs are induced in the infarcted and pressure-overloaded myocardium and play an essential role in repair and remodeling by activating Smad-dependent and Smad-independent cascades. Fibroblasts are major targets of TGFβs. Activation of Smad3 signaling in cardiac fibroblasts is crucial in post-infarction repair, mediating formation of aligned myofibroblast arrays. TGFβ responses are tightly regulated through induction of endogenous suppressive signals, such as the inhibitory Smads, that prevent excessive TGFβ signaling. We hypothesized that the inhibitory Smad7 may be induced in activated myofibroblasts infiltrating infarcted and pressure-overloaded hearts, protecting the myocardium from adverse remodeling and fibrosis by restraining TGFβ actions. Smad7 is markedly induced in infarcted and pressure-overloaded mouse hearts, and is localized in cardiomyocytes, myofibroblasts and activated macrophages. Induction of Smad7 in myofibroblasts is mediated through TGFβ/Smad3 signaling in vitro and in vivo. Mice lacking Smad7 in activated myofibroblasts (MFS7KO) and Smad7 fl/fl controls underwent non-reperfused myocardial infarction and transverse aortic constriction protocols. Following infarction, MFS7KO mice had higher mortality, worse systolic dysfunction and accentuated diastolic dysfunction when compared to Smad7 fl/fl. Following pressure overload, MFS7KO mice had accentuated interstitial and perivascular fibrosis and increased cardiomyocyte hypertrophy. In vitro, Smad7 overexpression attenuated myofibroblast conversion and reduced expression of profibrotic genes. Conversely, Smad7 knockdown promoted a matrix-synthetic fibroblast phenotype. Smad7 overexpression abrogated TGF-β-stimulated Smad2/3 and Erk activation without affecting TβRI and TβRII phosphorylation. Akt and p38 activation were unaffected by Smad7 overexpression. In conclusion, in remodeling hearts, Smad7 induction in activated myofibroblasts restrains fibrosis, negatively regulating Smad2/3 and Erk signaling through actions downstream of the TβRs. The protective effects of fibroblast Smad7 in cardiac remodeling may have important therapeutic implications for heart failure patients.

Protective Effects of Activated Myofibroblasts in the Pressure-Overloaded Myocardium Are Mediated Through Smad-Dependent Activation of a Matrix-Preserving Program.

The heart contains abundant interstitial and perivascular fibroblasts. Traditional views suggest that, under conditions of mechanical stress, cytokines, growth factors, and neurohumoral mediators stimulate fibroblast activation, inducing ECM (extracellular matrix) protein synthesis and promoting fibrosis and diastolic dysfunction. Members of the TGF (transforming growth factor)-β family are upregulated and activated in the remodeling myocardium and modulate phenotype and function of all myocardial cell types through activation of intracellular effector molecules, the Smads (small mothers against decapentaplegic), and through Smad-independent pathways. To examine the role of fibroblast-specific TGF-β/Smad3 signaling in the remodeling pressure-overloaded myocardium. We examined the effects of cell-specific Smad3 loss in activated periostin-expressing myofibroblasts using a mouse model of cardiac pressure overload, induced through transverse aortic constriction. Surprisingly, FS3KO (myofibroblast-specific Smad3 knockout) mice exhibited accelerated systolic dysfunction after pressure overload, evidenced by an early 40% reduction in ejection fraction after 7 days of transverse aortic constriction. Accelerated systolic dysfunction in pressure-overloaded FS3KO mice was associated with accentuated matrix degradation and generation of collagen-derived matrikines, accompanied by cardiomyocyte myofibrillar loss and apoptosis, and by enhanced macrophage-driven inflammation. In vitro, TGF-β1, TGF-β2, and TGF-β3 stimulated a Smad3-dependent matrix-preserving phenotype in cardiac fibroblasts, suppressing MMP (matrix metalloproteinase)-3 and MMP-8 synthesis and inducing TIMP (tissue inhibitor of metalloproteinases)-1. In vivo, administration of an MMP-8 inhibitor attenuated early systolic dysfunction in pressure-overloaded FS3KO mice, suggesting that the protective effects of activated cardiac myofibroblasts in the pressure-overloaded myocardium are, at least in part, because of suppression of MMPs and activation of a matrix-preserving program. MMP-8 stimulation induces a proinflammatory phenotype in isolated macrophages. In the pressure-overloaded myocardium, TGF-β/Smad3-activated cardiac fibroblasts play an important protective role, preserving the ECM network, suppressing macrophage-driven inflammation, and attenuating cardiomyocyte injury. The protective actions of the myofibroblasts are mediated, at least in part, through Smad-dependent suppression of matrix-degrading proteases.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Pharmacologic inhibition of the enzymatic effects of tissue transglutaminase reduces cardiac fibrosis and attenuates cardiomyocyte hypertrophy following pressure overload

Tissue transglutaminase (tTG) is a multifunctional protein with a wide range of enzymatic and non-enzymatic functions. We have recently demonstrated that tTG expression is upregulated in the pressure-overloaded myocardium and exerts fibrogenic actions promoting diastolic dysfunction, while preventing chamber dilation. Our current investigation dissects the in vivo and in vitro roles of the enzymatic effects of tTG on fibrotic remodeling in pressure-overloaded myocardium. Using a mouse model of transverse aortic constriction, we demonstrated perivascular and interstitial tTG activation in the remodeling pressure-overloaded heart. tTG inhibition through administration of the selective small molecule tTG inhibitor ERW1041E attenuated left ventricular diastolic dysfunction and reduced cardiomyocyte hypertrophy and interstitial fibrosis in the pressure-overloaded heart, without affecting chamber dimensions and ejection fraction. In vivo, tTG inhibition markedly reduced myocardial collagen mRNA and protein levels and attenuated transcription of fibrosis-associated genes. In contrast, addition of exogenous recombinant tTG to fibroblast-populated collagen pads had no significant effects on collagen transcription, and instead increased synthesis of matrix metalloproteinase (MMP)3 and tissue inhibitor of metalloproteinases (TIMP)1 through transamidase-independent actions. However, enzymatic effects of matrix-bound tTG increased the thickness of pericellular collagen in fibroblast-populated pads. tTG exerts distinct enzymatic and non-enzymatic functions in the remodeling pressure-overloaded heart. The enzymatic effects of tTG are fibrogenic and promote diastolic dysfunction, but do not directly modulate the pro-fibrotic transcriptional program of fibroblasts. Targeting transamidase-dependent actions of tTG may be a promising therapeutic strategy in patients with heart failure and fibrosis-associated diastolic dysfunction.

Interleukin-10 Inhibits Bone Marrow Fibroblast Progenitor Cell-Mediated Cardiac Fibrosis in Pressure-Overloaded Myocardium.

Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload-induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload-induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC-mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload-induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis. Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow. Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow-depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction-induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with α-smooth muscle actin or collagen 1α in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction-induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-β-induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction-based selective miRNA analysis revealed that transforming growth factor-β-induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-β-induced fibrotic signaling in BM-FPCs. Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad-miRNA-21-mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

Tissue transglutaminase induction in the pressure-overloaded myocardium regulates matrix remodelling.

Tissue transglutaminase (tTG) is induced in injured and remodelling tissues, and modulates cellular phenotype, while contributing to matrix cross-linking. Our study tested the hypothesis that tTG may be expressed in the pressure-overloaded myocardium, and may regulate cardiac function, myocardial fibrosis and chamber remodelling. In order to test the hypothesis, wild-type and tTG null mice were subjected to pressure overload induced through transverse aortic constriction. Moreover, we used isolated cardiac fibroblasts and macrophages to dissect the mechanisms of tTG-mediated actions. tTG expression was upregulated in the pressure-overloaded mouse heart and was localized in cardiomyocytes, interstitial cells, and in the extracellular matrix. In contrast, expression of transglutaminases 1, 3, 4, 5, 6, 7 and FXIII was not induced in the remodelling myocardium. In vitro, transforming growth factor (TGF)-β1 stimulated tTG synthesis in cardiac fibroblasts and in macrophages through distinct signalling pathways. tTG null mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross-linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure-overloaded myocardium. In vitro, tTG did not modulate TGF-β-mediated responses in cardiac fibroblasts; however, tTG loss was associated with accentuated proliferative activity. Moreover, when bound to the matrix, recombinant tTG induced synthesis of tissue inhibitor of metalloproteinases (TIMP)-1 through transamidase-independent actions. Following pressure overload, endogenous tTG mediates matrix cross-linking, while protecting the remodelling myocardium from dilation by exerting matrix-preserving actions.

Abstract 99: IL10-inhibits Fibroblast Progenitor Cell-mediated Cardiac Fibrosis in Pressure-overloaded Myocardium

Background: Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Recent studies suggest the contribution of bone marrow fibroblasts progenitor cells (BM-FPC) in pressure overload (PO)-induced cardiac fibrosis. Previously we have shown that interleukin-10 suppress PO-induced cardiac fibrosis, however, its role on inhibition of BM-FPC-mediated fibrosis is not known. Thus, we hypothesized that IL-10 inhibits PO-induced homing and transition of BM-FPC to myoFBs and therefore, attenuates cardiac fibrosis. Methods and Results: Cardiac fibrosis was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Systemic recombinant IL-10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO-enhanced FPC (Prominin1 + cells) mobilization and homing in IL-10 KO mice compared to WT mice. Furthermore, bone marrow transplantation (BMT) experiment was performed wherein WT marrow from GFP mice was repopulated in IL-10 KO mice. FPC mobilization was significantly reduced in BMT-IL10 KO mice compared to IL-10 KO mice after TAC. Furthermore, immunofluorescence result in BMT mice showed that subsets of myoFBs are derived from BM after TAC. To identify the molecular mechanism, wild type BM-FPC were treated with TGFβ 2 with or without IL10. IL10 treatment significantly inhibits TGFβ 2 -induced FPC to myoFBs transition. As miRNAs are key players in cardiac fibrosis, next we performed fibrosis-associated miRNA profiling using miRNA array kit. TGFβ 2 -induced miR-208, 155, 21 and 145 expression was markedly inhibited by IL-10. Conclusion: Taken together, our findings suggest that both reduced homing to heart and transition of FPC to myofibroblasts mediate anti-fibrotic effect of IL10 during PO-induced heart failure. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.

Myocardial Galectin-3 Expression Is Associated with Remodeling of the Pressure-Overloaded Heart and May Delay the Hypertrophic Response without Affecting Survival, Dysfunction, and Cardiac Fibrosis

The β-galactoside-binding animal lectin galectin-3 is predominantly expressed by activated macrophages and is a promising biomarker for patients with heart failure. Galectin-3 regulates inflammatory and fibrotic responses; however, its role in cardiac remodeling remains unclear. We hypothesized that galectin-3 may be up-regulated in the pressure-overloaded myocardium and regulate hypertrophy and fibrosis. In normal mouse myocardium, galectin-3 was constitutively expressed in macrophages and was localized in atrial but not ventricular cardiomyocytes. In a mouse model of transverse aortic constriction, galectin-3 expression was markedly up-regulated in the pressure-overloaded myocardium. Early up-regulation of galectin-3 was localized in subpopulations of macrophages and myofibroblasts; however, after 7 to 28 days of transverse aortic constriction, a subset of cardiomyocytes in fibrotic areas contained large amounts of galectin-3. Invitro, cytokine stimulation suppressed galectin-3 synthesis by macrophages and cardiac fibroblasts. Correlation studies revealed that cardiomyocyte- but not macrophage-specific galectin-3 localization was associated with adverse remodeling and dysfunction. Galectin-3 knockout mice exhibited accelerated cardiac hypertrophy after 7 days of pressure overload, whereas female galectin-3 knockouts had delayed dilation after 28 days of transverse aortic constriction. However, galectin-3 loss did not affect survival, systolic and diastolic dysfunction, cardiac fibrosis, and cardiomyocyte hypertrophy in the pressure-overloaded heart. Despite its potential role as a prognostic biomarker, galectin-3 is not a critical modulator of cardiac fibrosis but may delay the hypertrophic response.

Differential protein expression and basal lamina remodeling in human heart failure.

A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts. Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion. Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls. Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease.

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model.

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.