Fibroblast Smad7 Induction Protects the Remodeling Pressure-Overloaded Heart.

BACKGROUND: Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-ß (transforming growth factor-ß)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-ß activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-ß response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-ß signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction. METHODS: 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. RESULTS: Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-ß 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). CONCLUSIONS: 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.

Macrophages in the infarcted heart acquire a fibrogenic phenotype, expressing matricellular proteins, but do not undergo fibroblast conversion.

Although some studies have suggested that macrophages may secrete structural collagens, and convert to fibroblast-like cells, macrophage to fibroblast transdifferentiation in infarcted and remodeling hearts remains controversial. Our study uses linage tracing approaches and single cell transcriptomics to examine whether macrophages undergo fibroblast conversion, and to characterize the extracellular matrix expression profile of myeloid cells in myocardial infarction. To examine whether infarct macrophages undergo fibroblast conversion, we identified macrophage-derived progeny using the inducible CX3CR1CreER mice crossed with the PDGFRαEGFP reporter line for reliable fibroblast identification. The abundant fibroblasts that infiltrated the infarcted myocardium after 7 and 28 days of coronary occlusion were not derived from CX3CR1+ macrophages. Infarct macrophages retained myeloid cell characteristics and did not undergo conversion to myofibroblasts, endothelial or vascular mural cells. Single cell RNA-seq of CSF1R+ myeloid cells harvested from control and infarcted hearts showed no significant expression of fibroblast identity genes by myeloid cell clusters. Moreover, infarct macrophages did not express significant levels of genes encoding structural collagens. However, infarct macrophage and monocyte clusters were the predominant source of the fibrogenic growth factors Tgfb1 and Pdgfb, and of the matricellular proteins Spp1/Osteopontin, Thbs1/Thrombospondin-1, Emilin2, and Fn1/fibronectin, while expressing significant amounts of several other matrix genes, including Vcan/versican, Ecm1 and Sparc. ScRNA-seq data suggested similar patterns of matrix gene expression in human myocardial infarction. In conclusion, infarct macrophages do not undergo fibroblast or myofibroblast conversion and do not exhibit upregulation of structural collagens but may contribute to fibrotic remodeling by producing several fibrogenic matricellular proteins.

Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation.

RATIONALE: TGF (transforming growth factor)-ß is critically involved in myocardial injury, repair, and fibrosis, activating both Smad (small mothers against decapentaplegic)-dependent and non-Smad pathways. The in vivo role of TGF-ß signaling in regulation of macrophage function is poorly understood. We hypothesized that in the infarcted myocardium, activation of TGF-ß/Smad signaling in macrophages may regulate repair and remodeling. OBJECTIVE: To investigate the role of macrophage-specific TGF-ß Smad3 signaling in a mouse model of myocardial infarction and to dissect the mechanisms mediating Smad-dependent modulation of macrophage function. METHODS AND RESULTS: TGF-ßs markedly activated Smad3 in macrophages, without affecting Smad-independent pathways. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-ß release. MyS3KO (myeloid cell-specific Smad3 knockout) mice had no baseline defects but exhibited increased late mortality and accentuated dilative postmyocardial infarction remodeling. Adverse outcome in infarcted MyS3KO mice was associated with perturbations in phagocytic activity, defective transition of macrophages to an anti-inflammatory phenotype, scar expansion, and accentuated apoptosis of border zone cardiomyocytes. In vitro, Smad3 null macrophages exhibited reduced expression of genes associated with eat-me signals, such as Mfge8 (milk fat globule-epidermal growth factor factor 8), and reduced capacity to produce the anti-inflammatory mediators IL (interleukin)-10 and TGF-ß1, and the angiogenic growth factor VEGF (vascular endothelial growth factor). Mfge8 partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Impaired anti-inflammatory actions of Smad3 null macrophages were associated with marked attenuation of phagocytosis-induced PPAR (peroxisome proliferator-activated receptor) expression. MyS3KO mice had no significant alterations in microvascular density and interstitial fibrosis in remodeling myocardial segments. CONCLUSIONS: We demonstrate that Smad3 critically regulates function of infarct macrophages, by mediating acquisition of a phagocytic phenotype and by contributing to anti-inflammatory transition. Smad3-dependent actions in macrophages protect the infarcted heart from adverse remodeling.

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

RATIONALE: 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)-ß 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. OBJECTIVES: To examine the role of fibroblast-specific TGF-ß/Smad3 signaling in the remodeling pressure-overloaded myocardium. METHODS AND RESULTS: 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-ß1, TGF-ß2, and TGF-ß3 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. CONCLUSIONS: In the pressure-overloaded myocardium, TGF-ß/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.

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

TGF-ßs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-ß1, -ß2 and -ß3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Protective effects of macrophage-specific integrin α5 in myocardial infarction are associated with accentuated angiogenesis.

Macrophages sense changes in the extracellular matrix environment through the integrins and play a central role in regulation of the reparative response after myocardial infarction. Here we show that macrophage integrin α5 protects the infarcted heart from adverse remodeling and that the protective actions are associated with acquisition of an angiogenic macrophage phenotype. We demonstrate that myeloid cell- and macrophage-specific integrin α5 knockout mice have accentuated adverse post-infarction remodeling, accompanied by reduced angiogenesis in the infarct and border zone. Single cell RNA-sequencing identifies an angiogenic infarct macrophage population with high Itga5 expression. The angiogenic effects of integrin α5 in macrophages involve upregulation of Vascular Endothelial Growth Factor A. RNA-sequencing of the macrophage transcriptome in vivo and in vitro followed by bioinformatic analysis identifies several intracellular kinases as potential downstream targets of integrin α5. Neutralization assays demonstrate that the angiogenic actions of integrin α5-stimulated macrophages involve activation of Focal Adhesion Kinase and Phosphoinositide 3 Kinase cascades.

The role of Smad2 and Smad3 in regulating homeostatic functions of fibroblasts in vitro and in adult mice.

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-ß-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.

Anti-inflammatory therapies in myocardial infarction: failures, hopes and challenges.

In the infarcted heart, the damage-associated molecular pattern proteins released by necrotic cells trigger both myocardial and systemic inflammatory responses. Induction of chemokines and cytokines and up-regulation of endothelial adhesion molecules mediate leukocyte recruitment in the infarcted myocardium. Inflammatory cells clear the infarct of dead cells and matrix debris and activate repair by myofibroblasts and vascular cells, but may also contribute to adverse fibrotic remodelling of viable segments, accentuate cardiomyocyte apoptosis and exert arrhythmogenic actions. Excessive, prolonged and dysregulated inflammation has been implicated in the pathogenesis of complications and may be involved in the development of heart failure following infarction. Studies in animal models of myocardial infarction (MI) have suggested the effectiveness of pharmacological interventions targeting the inflammatory response. This article provides a brief overview of the cell biology of the post-infarction inflammatory response and discusses the use of pharmacological interventions targeting inflammation following infarction. Therapy with broad anti-inflammatory and immunomodulatory agents may also inhibit important repair pathways, thus exerting detrimental actions in patients with MI. Extensive experimental evidence suggests that targeting specific inflammatory signals, such as the complement cascade, chemokines, cytokines, proteases, selectins and leukocyte integrins, may hold promise. However, clinical translation has proved challenging. Targeting IL-1 may benefit patients with exaggerated post-MI inflammatory responses following infarction, not only by attenuating adverse remodelling but also by stabilizing the atherosclerotic plaque and by inhibiting arrhythmia generation. Identification of the therapeutic window for specific interventions and pathophysiological stratification of MI patients using inflammatory biomarkers and imaging strategies are critical for optimal therapeutic design.

Reduced Vitamin D Receptor on Circulating Endothelial Progenitor Cells: A New Risk Factor of Coronary Artery Diseases.

AIM: Endothelial progenitor cells (EPCs) are shown to participate in the pathological processes of atherosclerosis. While Vitamin D and its receptor axis might exert some effects on EPCs' function. But their exact relationship with clinical patients is still elusive, which inspired us to explore the potential association of vitamin D receptor (VDR) expression on circulating EPCs and serum vitamin D levels among patients with coronary artery disease (CAD). METHODS: Two hundred patients with CAD after their admission to hospital and one hundred healthy controls were enrolled. Medical history data were retrieved and fresh blood samples were collected for flow cytometry analysis. VDR expressions on EPCs were evaluated according to the standardized protocol. Logistic regression analysis was used to investigate the potential risk factor of CAD. RESULTS: CAD patients were found to have lower log10VDR-MFIs than those of control group, especially for patients with diabetes (p＜0.001). Log10VDR-MFIs were inversely correlated with glycated hemoglobin (R=－0.472, p＜0.001), and while EPCs challenged with high glucose had lower VDR expression. Multivariate logistic regression analysis revealed that lower log10VDR-MFIs were independently associated with the risk of CAD (OR=0.055, p=0.008). CONCLUSION: A significant decrease of VDR expression on circulating EPCs was observed among CAD patients, particularly among those also with diabetes. VDR expression on EPCs was independently negatively correlated with HbA1c and high glucose decreased EPCs' VDR expression. Low levels of VDR expression on circulating EPCs might serve as a potential risk factor of CAD.

Dysfunction of the thymus in mice with hypertension.

The aim of this study was to evaluate thymus function in mice with hypertension. A total of 60 C57BL/6J mice were randomized into control, sham surgery and two-kidney, one-clip groups (n=20 in each). At 4 or 8 weeks after surgery, mice were sacrificed, and blood, spleens, kidneys and thymuses were harvested. The results of reverse transcription-quantitative polymerase chain reaction analysis revealed that the mRNA levels of Forkhead box protein N1 (Foxn1) and autoimmune regulator (AIRE) in the thymus tissue of mice from the HTN group were significantly lower than those from the control group at 4 and 8 weeks (P<0.05). Foxn1 and AIRE expression was also reduced in the sham surgery group at 4 weeks after surgery, but had recovered 4 weeks later. Similar results were observed for the expression of signal-joint T cell receptor excision circles and the percentages of T cell subsets. The present study indicates that impaired thymus function is associated with hypertension in mice, which suggests that thymus function may be a novel target for the treatment of patients with hypertension.

Reduced T-Cell Thymic Export Reflected by sj-TREC in Patients with Coronary Artery Disease.

AIM: Immunologic dysfunction was recently found to be one of the most important mechanisms underlying the initiation and development of atherosclerosis. Thymus involution can contribute to immune disturbance and disequilibrium of T-cell subsets. This study aimed to explore whether recent thymic emigration (RTE) is impaired in patients with coronary artery disease (CAD). METHODS: Content of signal-joint T cell receptor excision circles (sj-TREC) in T lymphocytes, a molecular marker of RTE, was assessed among CAD patients and age-matched controls. Monochrome multiplex quantitative PCR method was used to assess the samples' telomere length in order to exclude the potential influence of T cell proliferation on the dilution of sj-TREC. Patients were grouped according to Gensini score (GS) (low, GS ＜18; intermediate, GS 18-41; high, GS ＞41). Ordinary logistic regression models were used to determine potential risk factors for CAD and GS tertiles. RESULTS: Average copy numbers of sj-TREC per 10(6) T lymphocytes among patients with unstable angina, stable angina, and controls were 726±429, 1213±465, and 1795±838, respectively (P＜0.001). However, there was no significant difference in telomere length among groups. Moreover, the content of sj-TREC in the high GS group was most significantly reduced than the low GS group (P＜0.001). Multivariate logistic regression analysis revealed that lower sj-TREC was independently associated with the progression of CAD (OR=0.44, P＜0.001) and higher GS (OR=0.4, P＜0.001). CONCLUSION: Impaired RTE could be partly responsible for CAD development. Mechanisms may be involved in the disturbance of T lymphocyte compartment and interruption of maintained immune tolerance resulting from thymus involution.

Danhong Promotes Angiogenesis in Diabetic Mice after Critical Limb Ischemia by Activation of CSE-H 2 S-VEGF Axis.

The aim of this paper is to investigate effect and mechanism of Danhong injection (DH) on angiogenesis in the diabetic hind limb ischemia mouse model. Thirty diabetic hind limb ischemic model mice and ten normal mice, established by intraperitoneal (i.p.) injection of streptozotocin (STZ) or PBS and ligation/excision of femoral artery, and then twenty diabetic hind limb ischemic model mice of all were evenly randomized to saline (control, n = 10) and DH i.p. injection (2 mL/kg weight for 7 days, n = 10) groups. Limb perfusion recovery and femoral blood hydrogen sulfide (H2S) and vessel regeneration and lower limb vascular endothelial growth factor (VEGF)/cystathionine γ-lyase (CSE) expression were evaluated during intervention and after euthanasia, respectively. DH i.p. increased ischemic limb perfusion and promoted collateral circulation generation without decreasing blood glucose level. Increased local CSE-H2S-VEGF expression contributed to beneficial effects of DH injection. In conclusion, activation of local CSE-H2S-VEGF axis might participate in proangiogenesis effects of DH injection in diabetic hind limb ischemia model mice, suggesting a potential therapy for diabetic patients with critical limb ischemia.