Cardiac Myofibroblast Differentiation Research Articles

DLK1 overexpression improves sepsis-induced cardiac dysfunction and fibrosis in mice through the TGF-β1/Smad3 signaling pathway and MMPs.

Sepsis is a serious inflammatory disease caused by bacterial infection. Cardiovascular dysfunction and remodeling are serious complications of sepsis, which can significantly affect sepsis patients' mortality. Delta-like homologue 1 (DLK1) has been reported could inhibit cardiac myofibroblast differentiation. However, the function of DLK1 in sepsis is unknown. In the present study, the DLK1 expression was first identified based on the online dataset GSE79962 analysis and cecal ligation and puncture (CLP)-induced sepsis mouse model. DLK1 expression was significantly reduced in septic heart tissues. In septic mouse heart, CLP operation decreased the fractional shortening (EF) (%) and ejection fraction (FS) (%) and caused significant edema, disordered myofilament arrangement, and degradation and necrosis in myocardial cells; CLP operation also increased collagen deposition and elevated the protein levels of fibrotic markers (α-SMA and F-actin). DLK1 overexpression in septic mice could effectively increase EF (%) and FS (%), attenuate CLP-caused ECM degradation and deposition and partially inhibit the CLP-induced TGF-β1/Smad signaling activation. In conclusion, DLK1 expression was poorly expressed in the CLP-induced septic mouse heart. DLK1 overexpression partially alleviated sepsis-induced cardiac dysfunction and fibrosis, with the involvement of the TGF-β1/Smad3 signaling pathway and MMPs.

PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction

After myocardial infarction (MI), fibroblasts progress from proliferative to myofibroblast states, resulting in fibrosis. Platelet-derived growth factors (PDGFs) are reported to induce fibroblast proliferation, myofibroblast differentiation, and fibrosis. However, we have previously shown that PDGFs improve heart function post-MI without increasing fibrosis. We treated human cardiac fibroblasts with PDGF isoforms then performed RNA sequencing to show that PDGFs reduced cardiac fibroblasts myofibroblast differentiation and downregulated cell cycle pathways. Using mouse/pig MI models, we reveal that PDGF-AB infusion increases cell-cell interactions, reduces myofibroblast differentiation, does not affect proliferation, and accelerates scar formation. RNA sequencing of pig hearts after MI showed that PDGF-AB reduces inflammatory cytokines and alters both transcript variants and long noncoding RNA expression in cell cycle pathways. We propose that PDGF-AB could be used therapeutically to manipulate post-MI scar maturation with subsequent beneficial effects on cardiac function.

The Novel AT2 Receptor Agonist β-Pro7-AngIII Exerts Cardiac and Renal Anti-Fibrotic and Anti-Inflammatory Effects in High Salt-Fed Mice.

A high salt (HS) diet is associated with an increased risk for cardiovascular diseases (CVDs) and fibrosis is a key contributor to the organ dysfunction involved in CVDs. The activation of the renin angiotensin type 2 receptor (AT2R) has been considered as organ protective in many CVDs. However, there are limited AT2R-selective agonists available. Our first reported β-substituted angiotensin III peptide, β-Pro7-AngIII, showed high selectivity for the AT2R. In the current study, we examine the potential anti-fibrotic and anti-inflammatory effects of this novel AT2R-selective peptide on HS-induced organ damage. FVB/N mice fed with a 5% HS diet for 8 weeks developed cardiac and renal fibrosis and inflammation, which were associated with increased TGF-β1 levels in heart, kidney and plasma. Four weeks' treatment (from weeks 5-8) with β-Pro7-AngIII inhibited the HS-induced cardiac and renal fibrosis and inflammation. These protective effects were accompanied by reduced local and systemic TGF-β1 as well as reduced cardiac myofibroblast differentiation. Importantly, the anti-fibrotic and anti-inflammatory effects caused by β-Pro7-AngIII were attenuated by the AT2R antagonist PD123319. These results demonstrate, for the first time, the cardio- and reno-protective roles of the AT2R-selective β-Pro7-AngIII, highlighting it as an important therapeutic that can target the AT2R to treat end-organ damage.

The landscape of accessible chromatin in quiescent cardiac fibroblasts and cardiac fibroblasts activated after myocardial infarction

ABSTRACT After myocardial infarction, the massive death of cardiomyocytes leads to cardiac fibroblast proliferation and myofibroblast differentiation, which contributes to the extracellular matrix remodelling of the infarcted myocardium. We recently found that myofibroblasts further differentiate into matrifibrocytes, a newly identified cardiac fibroblast differentiation state. Cardiac fibroblasts of different states have distinct gene expression profiles closely related to their functions. However, the mechanism responsible for the gene expression changes during these activation and differentiation events is still not clear. In this study, the gene expression profiling and genome-wide accessible chromatin mapping of mouse cardiac fibroblasts isolated from the uninjured myocardium and the infarct at multiple time points corresponding to different differentiation states were performed by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. ATAC-seq peaks were highly enriched in the promoter area and the distal area where the enhancers are located. A positive correlation was identified between the expression and promoter accessibility for many dynamically expressed genes, even though evidence showed that mechanisms independent of chromatin accessibility may also contribute to the gene expression changes in cardiac fibroblasts after MI. Moreover, motif enrichment analysis and gene regulatory network construction identified transcription factors that possibly contributed to the differential gene expression between cardiac fibroblasts of different states.

Abstract MP258: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

Background: A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results: The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage. Conclusions: Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation.

BackgroundA hallmark of heart failure is cardiac fibrosis, which results from the injury‐induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early proinflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase–like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation.Methods and ResultsThe role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after transverse aortic constriction surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2 knockout mice displayed a proinflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, transverse aortic constriction surgery led to the death of all mice by day 6 that was associated with myocardial hyperinflammation and vascular leakage.ConclusionsTogether, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

Activation of COX‐2/PGE 2 Pathway Is Not Involved in Dedifferentiation of Cardiac Myofibroblasts Induced by Phorbol 12‐myristate 13‐acetate

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGF-β1 is one of the essential molecules that promotes transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. In our previous studies, Phorbol 12-Myristate 13-Acetate (PMA) induces reversal of myofibroblast differentiation via protein kinase C (PKC)-independent mechanism. Prostaglandin E2 (PGE2) has been shown to reserve differentiation of myofibroblasts of fetal and adult lung fibroblasts. The role of PGE2 in cardiac myofibroblast dedifferentiation remains unknown. Human cardiac fibroblasts were cultured in fibroblast medium (FM)-2. TGF-β1 (2ng/mL) was added to FM-2 for 48 hours to convert fibroblasts into myofibroblasts. PMA (50 ng/mL) or PGE2 (500 nM) was added into cultured cells for 48 hours, respectively. Expression of α-smooth muscle action (SMA), a biomarker of myofibroblasts, and fibroblast specific protein 1 (FSP-1), the biomarker of fibroblasts, were detected by using western blotting and immunofluorescence. To explore the involvement of cyclooxygenase 2 (COX-2)/PGE2 pathway in PMA-induced reversal of cardiac myofibroblast differentiation, NS-398, the selective COX-2 inhibitor, and PF-04418948, a selective PGE2 receptor antagonist, were applied. Endogenous levels of PGE2 in cardiac myofibroblasts were detected by using Elisa assay kit. TGF-β1 promoted conversion of cardiac fibroblasts to myofibroblasts as evidenced by increased expression of α-SMA and reduced expression of FSP-1. Treatment with PMA dose-dependently attenuated expression of de novo myofibroblasts. Both NS-398 and PF-04418948 exerted no effects on PMA-induced reversal of cardiac myofibroblasts. Addition of PGE2 into culture medium had no effect on expression of α-SMA from myofibroblasts. PMA dose-dependently enhanced formation of PGE2 levels in cardiac myofibroblasts. In conclusion, PMA-induced reversal of cardiac myofibroblast is independent of activation of COX-2 and PGE2 pathway. The mechanism in PMA-induced reversal of cardiac myofibroblasts remains to be further explored.

Actin-granule formation is an additional step in cardiac myofibroblast differentiation.

BackgroundAtrial fibrillation is the most common and long-lasting cardiac arrhythmia, and profoundly effects the daily lives of patients. The pathogenesis and persistence of atrial fibrillation is closely related to the cardiac fibroblast and its myofibroblast differentiation as increased collagen synthesis and migration capability. Thus better understanding of myofibroblast differentiation is essential for the prevention and treatment of atrial fibrillationMethodsCardiac fibroblasts were isolated from neonatal rats and its actin structure was analyzed by immunofluorescence staining. Myofibroblast differentiation was induced by Angiotensin II (Ang II) and ROCK signaling related proteins were determined by western blot. Fasudil and Ricolinostat were employed to abrogate ROCK signaling and their effects on myofibroblast differentiation were assessed by IF microscopy and Celigo Image Cytometry.ResultsStress actin fibers similar to actin filaments in myofibroblast differentiation are regulated by ROCK signaling, and our results also suggested Guanine nucleotide exchange factor-H1 (GEF-H1) phosphorylation could be induced by Ang II. In addition, Fasudil could down-regulate RhoA, GEF-H1, and phosphorylated GEF-H1 to inhibit ROCK signaling and further reduce Col I expression and the myofibroblast proportion.ConclusionsAn individual phase characterized by actin-granule formation was identified in cardiac myofibroblast differentiation. In the meanwhile, myofibroblast differentiation and its F-actin assembly could be detained in this phase by Fasudil abrogating the ROCK signaling pathway.

Abstract 254: Kinase-dead Pi3kγ Expression in the Cardiac Myocytes Regulates Fibroblast Signaling and Myofibroblast Differentiation

Phosphoinositide 3 Kinase γ (PI3Kγ) is an anti-apoptotic molecule acting through Akt pathway. Even though, role of PI3Kγ in cardiac fibrosis has been established, the mechanistic details by which PI3Kγ regulates cardiac myofibroblast differentiation are not clear. Myofibroblasts are hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) over-expression. We have previously shown that αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ -/- ) showed significant baseline and pressure overload [Transverse Aortic Constriction (TAC)] induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ -/- exhibited a myofibroblast phenotype with αSMA in stress fibers. Moreover, cardiomyocyte-specific over-expression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- (PI3Kγ inact /PI3Kγ -/- ) reduced αSMA abundance and myofibroblast differentiation suggesting unique kinase-independent function of PI3Kγ in myocyte-initiated pathway that drives CF to become myofibroblasts. Conditioned media experiments showed that PI3Kγ -/- myocytes release pro-fibrotic factors and PI3Kγ inact /PI3Kγ -/- myocytes release fibrosis protective factors. We have previously observed that PI3Kγ regulated MAPK signaling in fibroblasts in a kinase-independent manner by sequestering PP2A association and activity. Previous studies have shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). Consistent with these previous observations, PI3Kγ possibly mediates αSMA and myofibroblast differentiation through regulation of ERK signaling in the fibroblasts. Intriguingly, we observed presence of PI3Kγ when lysates of isolated CF from PI3Kγ inact /PI3Kγ -/- were immunoblotted for PI3Kγ. These data indicate that a unique communication between myocytes and fibroblasts regulated by PI3Kγ, leads to a compensatory mechanism that results in expression of PI3Kγ in the fibroblasts, thereby regulating fibroblast signaling in myofibroblast differentiation.

HIMF (Hypoxia-Induced Mitogenic Factor)-IL (Interleukin)-6 Signaling Mediates Cardiomyocyte-Fibroblast Crosstalk to Promote Cardiac Hypertrophy and Fibrosis.

HIMF (hypoxia-induced mitogenic factor) is a secreted proinflammatory cytokine with a critical role in cardiac hypertrophy development. Loss of HIMF attenuates transverse aortic constriction-induced cardiac hypertrophy and fibrosis, but the underlying mechanisms are unknown. We show that IL (interleukin)-6 production increases following transverse aortic constriction in wild-type mice; this effect is inhibited in HIMF gene knockout ( Himf-/-) mice. IL-6 production also increases in cultured cardiac myocytes overexpressing HIMF and neutralizing IL-6 with an anti-IL-6 antibody prohibits HIMF-induced cardiomyocyte hypertrophy. HIMF expression in cardiac fibroblasts cannot be stimulated by transverse aortic constriction or exposure to prohypertrophic factors, including phenylephrine, Ang II (angiotensin II), TGF (transform growth factor)-β, and hypoxia. However, conditioned medium from cardiomyocytes overexpressing HIMF can increase IL-6 production, and cardiac fibroblast proliferation, migration, and myofibroblast differentiation to a similar level as exposure to exogenous rHIMF (recombinant HIMF). Again, neutralizing IL-6 prevented cardiac fibroblasts activation. Finally, the MAPK (mitogen-activated protein kinase) and CaMKII (Ca2+/calmodulin-dependent protein kinase II)-STAT3 (signal transducers and activators of transcription 3) pathways are activated in HIMF-overexpressing cardiomyocytes and rHIMF-stimulated cardiac fibroblasts; this effect can be inhibited on neutralizing IL-6. These data support that HIMF induces cardiac fibrosis via a cardiomyocyte-to-fibroblast paracrine effect. IL-6 is a downstream signal of HIMF and has a central role in cardiomyocyte hypertrophy and myocardial fibrosis that is mediated by activating the MAPK and CaMKII-STAT3 pathways.

YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.

The Inflammatory Transcription Factor C/EBPβ Plays a Critical Role in Cardiac Fibroblast Differentiation and a Rat Model of Cardiac Fibrosis Induced by Autoimmune Myocarditis

The aim of the present study was to investigate the mechanisms of CCAAT/enhancer-binding protein β (C/EBPβ) in cardiac myofibroblast (CMF) differentiation and in a rat model of cardiac fibrosis induced by experimental autoimmune myocarditis (EAM).In vitro studies performed in primary neonatal rat CMF revealed that silencing of C/EBPβ expression (via lentiviral mediated shRNA strategies) was sufficient to reduce C/EBPβ mRNA and protein levels as well as to decrease the expressions of actin cytoskeletal proteins, cofilin, and filamin A (FLNA). TGFβ increased IL-1β, IL-6 and TNF-a production in cardiac fibroblasts (CF), while C/EBPβ knockdown reduced the secretion of these inflammatory mediators. In vivo studies performed in rats exhibiting EAM revealed that lentiviral-mediated silencing of C/EBPβ was sufficient to reduce the expression of C/EBPβ as well as inflammation and fibrosis in the hearts of EAM rats, when compared to controls. Echocardiography further revealed that C/EBPβ knockdown was sufficient to significantly improve cardiac dimensions and function in EAM rats. Immunohistochemical results showed that C/EBPβ knockdown attenuated the expression of C/EBPβ protein as well as the expressions of collagen I, collagen III, MMP-2, MMP-9, and α-SMA in heart tissue sections from rats in the EAM + Lenti-shC/EBPβ group.Strategies targeted at inhibiting C/EBPβ expression can be potentially exploited to regulate cofilin and FLNA expression, thereby regulating actin polymerization/depolymerization, cytoskeleton rearrangement, and CF differentiation into CMF and the production of inflammatory cytokines. C/EBPβ knock down reduces the degree of inflammation-mediated myocardial fibrosis in a rat model of EAM.

Abstract 528: Cell Specific Signaling Regulated by PI3Kγ Modulates Myofibroblast Differentiation

Phosphoinositide 3 Kinase γ (PI3Kγ) regulates anti-apoptotic Akt signaling. Previous studies have established a role for PI3Kγ in cardiac fibrosis, a key underlying cause of heart failure. However, less is known about the mechanism by which PI3Kγ regulates cardiac myofibroblast differentiation, hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) overexpression. Measurement of αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ -/- ) showed significant baseline and pressure overload induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ -/- exhibited a myofibroblast phenotype with αSMA in stress fibers. Correspondingly, immunoblotting showed significantly higher αSMA in PI3Kγ -/- CF than WT. It has been previously shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). To understand whether PI3Kγ regulates ERK signaling in a cell specific manner and thereby possibly αSMA, the phosphorylation of ERK by insulin stimulation were compared in CF isolated from WT and PI3Kγ -/- . Intriguingly, there was significant loss of ERK phosphorylation in CF from PI3Kγ -/- when compared to CF from WT. However, ERK phosphorylation was not altered in cardiomyocytes (CM) due to the absence of PI3Kγ. Confirming this differential regulation of ERK in CM and CF, there was no change in the association of ERK and protein phosphatase 2A (PP2A) in CM of WT and PI3Kγ -/- . However, there was increased association of PP2A with ERK in CF of PI3Kγ -/- when compared to CF from WT. This is because in the CM, ERK is regulated by Dual Specificity Phosphatase (DUSP8). However, in the CF, PP2A is the major regulator of ERK and thus PI3Kγ plays a major role in ERK signaling in CF. Consistent with previous observations, we found that association of DUSP8 with ERK was not observed in CF. Moreover, ERK-DUSP8 interaction was not dependent on the presence or absence of PI3Kγ. These data indicate that PI3Kγ regulates signaling pathways in a cell specific manner with respect to cardiac remodeling.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Abstract 109: PI3Kγ Regulates Release of Myocyte-derived Factors Responsible for Myofibroblast Differentiation and Cardiac Fibrosis

Phosphoinositide 3 Kinase γ (PI3Kγ) is a lipid kinase that regulates downstream anti-apoptotic Akt signaling. Thus, pressure overload in PI3Kγ null (PI3Kγ -/- ) mice leads to significant cardiac fibrosis, a key underlying cause of fatal heart failure. Classical hallmark of tissue fibrosis is differentiation of fibroblasts to myofibroblasts characterized by smooth muscle α-actin (αSMA) overexpression. However, less is known about the role of PI3Kγ in cardiac myofibroblast differentiation. Assessment of αSMA expression in cardiac lysates from WT and PI3Kγ -/- showed significant baseline upregulation in PI3Kγ -/- showing that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ -/- cardiac fibroblasts (CF) exhibit a myofibroblast phenotype, CF were isolated from hearts of WT and PI3Kγ -/- and assessed by immunostaining for αSMA in stress fibers. Greater number of CF from PI3Kγ -/- exhibited αSMA in stress fibers than CF from WT. Correspondingly, immunoblotting showed significantly higher expression of αSMA in PI3Kγ -/- CF compared to WT showing enhanced myofibroblast differentiation by PI3Kγ -/- fibroblasts. Surprisingly, abundance of αSMA protein is significantly reduced in the hearts of mice with cardiomyocyte-specific expression of kinase-dead PI3Kγ (PI3Kγ inact ) in the PI3Kγ -/- (PI3Kγ inact /PI3Kγ -/- ) suggesting that myocytes derived factors responsible for myofibroblast differentiation are regulated by kinase-independent function of PI3Kγ. To directly evaluate the PI3Kγ-dependent cardiomyocyte derived factors responsible for myofibroblast differentiation; fibroblasts were treated with conditioned media derived from primary adult cardiomyocytes from WT, PI3Kγ -/- and PI3Kγ inact /PI3Kγ -/- mice. Conditioned media derived from PI3Kγ -/- showed pro-fibrotic effects, while that from PI3Kγ inact /PI3Kγ -/- showed fibrosis protective biological activity compared to WT. These findings reveal that kinase-independent function of PI3Kγ is a key regulator of the myocyte-initiated pathway that ultimately drives myofibroblast conversion. Proteomic analysis of conditioned media identified several pro-fibrotic factors that are regulated by PI3Kγ, the results of which will be discussed.

Paracrine Effects of Adipose-Derived Stem Cells on Matrix Stiffness-Induced Cardiac Myofibroblast Differentiation via Angiotensin II Type 1 Receptor and Smad7.

Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.

Oxymatrine inhibits aldosterone-induced rat cardiac fibroblast proliferation and differentiation by attenuating smad-2,-3 and-4 expression: an in vitro study.

BackgroundWe previously demonstrated oxymatrine, an alkaloid from the Chinese medicine radix Sophorae flavescentis, ameliorates hemodynamic disturbances and cardiac fibrosis; however, the underlying mechanisms are unclear. Here, we investigated the effect and mechanism of action of oxymatrine on aldosterone-induced cardiac fibroblast to myofibroblast differentiation in vitro.MethodsCardiac fibroblasts were isolated purified from neonatal Sprague Dawley rats. The optimal concentration of aldosterone to stimulate cardiac fibroblast proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cardiac fibroblasts were pretreated with 7.57 × 10−4 mol/L or 3.78 × 10−4 mol/L oxymatrine or without oxymatrine for 2 h, and then coincubated with 1 × 10−8 mol/L aldosterone for 48 h. The MTT assay and Masson staining were used to detect the cardiac fibroblast proliferation and myofibroblast differentiation. The secretion of type I and III collagen was measured by commercial ELISA kits, and the hydroxyproline content was determined by the colorimetric assay. Western blotting assayed the Smad-2, Smad-3, and Smad-4 protein expression in cardiac fibroblasts.Results The present results confirmed that aldosterone induced cardiac fibroblast to myofibroblast proliferation and differentiation. The MTT assay and Masson staining indicated oxymatrine significantly inhibited aldosterone-induced cardiac fibroblast proliferation and myofibroblast differentiation. Oxymatrine significantly inhibited aldosterone-induced secretion of type I and III collagen, as indicated by commercial ELISA kits, and aldosterone-induced increase in hydroxyproline content, as indicated by a colorimetric assay. Western blotting revealed oxymatrine attenuated aldosterone-induced Smad-2, Smad-3, and Smad-4 expression in cardiac fibroblasts.ConclusionOxymatrine can inhibit cardiac fibroblast proliferation and differentiation into myofibroblasts via a mechanism linked to attenuation of the Smad signaling pathway.

Mechanoregulation of cardiac myofibroblast differentiation: implications for cardiac fibrosis and therapy

Cardiac myofibroblast differentiation, as one of the most important cellular responses to heart injury, plays a critical role in cardiac remodeling and failure. While biochemical cues for this have been extensively investigated, the role of mechanical cues, e.g., extracellular matrix stiffness and mechanical strain, has also been found to mediate cardiac myofibroblast differentiation. Cardiac fibroblasts in vivo are typically subjected to a specific spatiotemporally changed mechanical microenvironment. When exposed to abnormal mechanical conditions (e.g., increased extracellular matrix stiffness or strain), cardiac fibroblasts can undergo myofibroblast differentiation. To date, the impact of mechanical cues on cardiac myofibroblast differentiation has been studied both in vitro and in vivo. Most of the related in vitro research into this has been mainly undertaken in two-dimensional cell culture systems, although a few three-dimensional studies that exist revealed an important role of dimensionality. However, despite remarkable advances, the comprehensive mechanisms for mechanoregulation of cardiac myofibroblast differentiation remain elusive. In this review, we introduce important parameters for evaluating cardiac myofibroblast differentiation and then discuss the development of both in vitro (two and three dimensional) and in vivo studies on mechanoregulation of cardiac myofibroblast differentiation. An understanding of the development of cardiac myofibroblast differentiation in response to changing mechanical microenvironment will underlie potential targets for future therapy of cardiac fibrosis and failure.

Abstract 329: Simvastatin Inhibits TGF-β1-induced Proliferation and Activation of Cardiac Fibroblasts Through Inhibition of SMAD Pathway

Background: HMG-CoA reductase inhibitors (statins) have been shown to reduce the incidence of atrial fibrillation (AF) and its progression but the underlying mechanisms are not fully elucidated. Since atrial fibrosis plays a major role in the development of the substrate for AF progression, we hypothesized that statins antagonize the effect of profibrotic cytokines, reducing their stimulatory effect on fibroblast proliferation, differentiation and activation. Methods: The effect of TGF-β1, a major profibrotic cytokine, on fibroblast proliferation and activation of myofibroblasts was assessed by expression of alpha smooth muscle actin (α-SMA) message (qPCR) and proteins (western and immunofluorocytochemistry) in the presence and absence of simvastatin (1-10μM). The inhibitory effect of simvastatin on SMAD 2/3 phosphorylation (western) and its nuclear translocation by TGF-β1 (5ng) was determined by immunofluorescence antibodies using fluorescent microscopy. Results: TGF-β1 treatment increased fibroblast proliferation (cell count) by 63% compared to control (p&lt;0.001) at 96 hours, which was inhibited by simvastatin by 61% (p&lt;0.001) (Fig a). Simvastatin also reduced TGF-β1-mediated myofibroblast differentiation (α-SMA positive) by 75% (p=0.02); (Fig b and c). TGF-β1 increased SMAD2/3 phosphorylation with increase in nuclear localization was inhibited by simvastatin. Conclusion: Simvastatin inhibits TGFβ-1-mediated cardiac fibroblast proliferation and myofibroblast differentiation by antagonizing SMAD phosphorylation and its translocation into the nucleus.

Roles of transforming growth factor-β1 and OB-cadherin in porcine cardiac valve myofibroblast differentiation.

Calcific aortic stenosis is a common disease, and some of its early causes are the activation and differentiation of resident fibroblasts to myofibroblasts in response to transforming growth factor β1 (TGF-β1). The aim of this study was to understand how TGF-β1 and its downstream effector, OB-cadherin [cadherin 11 (CDH11)], regulate porcine myofibroblast phenotypes. Based on whole-genome microarrays, 95 and 107 genes are up- and down-regulated at both the early (8 h) and the late (24 h) time points of TGF-β1 treatment. Gene functions related to cell adhesion, skeletal system development, and extracellular matrix are up-regulated by TGF-β1, whereas oxidation-reduction and steroid metabolic process are down-regulated. Notably, one of the cell adhesion molecules, CDH11, is up-regulated by ∼2-fold through both the Smad2/3 and the ERK pathways elicited by TGF-β1. CDH11 mediates cell-cell contacts in both valvular fibroblasts and myofibroblasts. Knockdown of CDH11 by small interfering RNA increases the myofibroblast phenotype, including an ∼2-fold increase in α-smooth muscle actin (α-SMA) expression and stress fiber formation. In contrast, increased binding of CDH11 through antibody treatment inhibits α-SMA expression. This study presents gene functional changes in response to TGF-β1 at the systems level and supports an inhibitory role of CDH11 in myofibroblast differentiation.