Scleraxis and fibrosis in the pressure-overloaded heart.

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation.

We have previously shown that overexpression of SKI, an endogenous TGF-ß1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-ß signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI's effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI's capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.

Elimination of endogenous high molecular weight FGF2 prevents pressure-overload-induced systolic dysfunction, linked to increased FGFR1 activity and NR1D1 expression.

Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4-8 weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

Novel factors that activate and deactivate cardiac fibroblasts: A new perspective for treatment of cardiac fibrosis.

Heart disease with attendant cardiac fibrosis kills more patients in developed countries than any other disease, including cancer. We highlight the recent literature on factors that activate and also deactivate cardiac fibroblasts. Activation of cardiac fibroblasts results in myofibroblasts phenotype which incorporates aSMA to stress fibres, express ED-A fibronectin, elevated PDGFRα and are hypersecretory ECM components. These cells facilitate both acute wound healing (infarct site) and chronic cardiac fibrosis. Quiescent fibroblasts are associated with normal myocardial tissue and provide relatively slow turnover of the ECM. Deactivation of activated myofibroblasts is a much less studied phenomenon. In this context, SKI is a known negative regulator of TGFb1 /Smad signalling, and thus may share functional similarity to PPARγ activation. The discovery of SKI's potent anti-fibrotic role, and its ability to deactivate and/or myofibroblasts is featured and contrasted with PPARγ. While myofibroblasts are typically recruited from pools of potential precursor cells in a variety of organs, the importance of activation of resident cardiac fibroblasts has been recently emphasised. Myofibroblasts deposit ECM components at an elevated rate and contribute to both systolic and diastolic dysfunction with attendant cardiac fibrosis. A major knowledge gap exists as to specific proteins that may signal for fibroblast deactivation. As SKI may be a functionally pluripotent protein, we suggest that it serves as a scaffold to proteins other than R-Smads and associated Smad signal proteins, and thus its anti-fibrotic effects may extend beyond binding R-Smads. While cardiac fibrosis is causal to heart failure, the treatment of cardiac fibrosis is hampered by the lack of availability of effective pharmacological anti-fibrotic agents. The current review will provide an overview of work highlighting novel factors which cause fibroblast activation and deactivation to underscore putative therapeutic avenues for improving disease outcomes in cardiac patients with fibrosed hearts.

A small molecule inhibitor of PCSK9 that antagonizes LDL receptor binding via interaction with a cryptic PCSK9 binding groove.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Periostin Reexpression in Heart Disease Contributes to Cardiac Interstitial Remodeling by Supporting the Cardiac Myofibroblast Phenotype.

Cardiac muscle (the myocardium) is a unique arrangement of atria and ventricles that are spatially and electrically separated by a fibrous border. The spirally-arranged myocytes in both left and right ventricles are tethered by the component molecules of the cardiac extracellular matrix (ECM), including fibrillar collagen types I and III. Loss of normal arrangement of the ECM with either too little (as is observed in acute myocardial infarction) or too much (cardiac fibrosis in chronic post-myocardial infarction) is the primary contributor to cardiac dysfunction and heart failure. Matricellular proteins exist as non-structural signaling moieties in the ECM, and in the context of cardiac hypertrophy and heart failure, secreted 90 kDa periostin protein has attracted intense scrutiny during the past decade. Secreted periostin is now recognized for its important role in ECM development and maturation, as well as cellular adhesion. The novel mechanisms of periostin function include its role as a mediator of cell-to-matrix signaling, cell survival, and epithelial-mesenchymal transition (EMT). A number of recent studies have examined the hypothesis that periostin is a major contributor to ECM remodeling in the heart, and a number of very recent studies underscore its important role. This review examines recent developments in the mechanisms of periostin function in the normal heart and vasculature, and discusses recent advances which underpin its putative role in the development of cardiovascular disease. Periostin expression is very low at baseline in healthy tissues, but is re-expressed in damaged heart and in vessel walls after injury, in activated cardiac myofibroblasts and vascular smooth muscle cells, respectively. For this reason, periostin may be exploited for investigation of mechanisms of cardiac fibrosis , and we speculate that data generated from studies utilizing this approach may shed light on the timing for application of periostin-specific therapies to quell cardiac fibrosis and associated cardiac dysfunction.

Regulation of cardiac fibroblast MMP2 gene expression by scleraxis.

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFß occurs via a scleraxis-dependent mechanism: TGFß induces recruitment of scleraxis to the MMP2 promoter, and TGFß was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.

Autophagy and the unfolded protein response promote profibrotic effects of TGF-ß1 in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

Periostin in cardiovascular disease and development: a tale of two distinct roles.

Tissue development and homeostasis are dependent upon the concerted synthesis, maintenance, and degradation of extracellular matrix (ECM) molecules. Cardiac fibrosis is now recognized as a primary contributor to incidence of heart failure, particularly heart failure with preserved ejection fraction, wherein cardiac filling in diastole is compromised. Periostin is a cell-associated protein involved in cell fate determination, proliferation, tumorigenesis, and inflammatory responses. As a non-structural component of the ECM, secreted 90 kDa periostin is emerging as an important matricellular factor in cardiac mesenchymal tissue development. In addition, periostin's role as a mediator in cell-matrix crosstalk has also garnered attention for its association with fibroproliferative diseases in the myocardium, and for its association with TGF-ß/BMP signaling. This review summarizes the phylogenetic history of periostin, its role in cardiac development, and the major signaling pathways influencing its expression in cardiovascular pathology. Further, we provide a synthesis of the current literature to distinguish the multiple roles of periostin in cardiac health, development and disease. As periostin may be targeted for therapeutic treatment of cardiac fibrosis, these insights may shed light on the putative timing for application of periostin-specific therapies.

The Functional Role of Zinc Finger E Box-Binding Homeobox 2 (Zeb2) in Promoting Cardiac Fibroblast Activation.

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and α-smooth muscle actin (α-SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.

Chronic expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts.

Inappropriate cardiac interstitial remodeling is mediated by activated phenoconverted myofibroblasts. The synthesis of matrix proteins by these cells is triggered by both chemical and mechanical stimuli. Ski is a repressor of TGFß1/Smad signaling and has been described as possessing anti-fibrotic properties within the myocardium. We hypothesized that overexpression of Ski in myofibroblasts will induce an apoptotic response, which may either be supported or opposed by autophagic flux. We used primary myofibroblasts (activated fibroblasts) which were sourced from whole heart preparations that were only passaged once. We found that overexpression of Ski results in distinct morphological and biochemical changes within primary cardiac myofibroblasts associated with apoptosis. Ski treatment was associated with the expression of pro-apoptotic factors such as Bax, caspase-7, and -9. Our results indicate that Ski triggers a pro-death mechanism in primary rat cardiac myofibroblasts that is mediated through the intrinsic apoptotic pathway. Myofibroblast survival is prolonged by an autophagic response, as the dataset indicate that apoptosis is hastened when autophagy is inhibited. We suggest that the apoptotic death response of myofibroblasts is working in parallel with the previously observed anti-fibrotic properties of Ski within this cell type. As myofibroblasts are the sole mediators of matrix expansion in heart failure, we suggest that Ski, or a putative Ski-mimetic, may induce graded apoptosis in myofibroblasts within the failing heart and may be a novel therapeutic approach towards controlling cardiac fibrosis. Future studies are needed to examine the potential effects of Ski overexpression on other cell types in the heart.

The Ski-Zeb2-Meox2 pathway provides a novel mechanism for regulation of the cardiac myofibroblast phenotype.

Cardiac fibrosis is linked to fibroblast-to-myofibroblast phenoconversion and proliferation but the mechanisms underlying this are poorly understood. Ski is a negative regulator of TGF-ß-Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-ß-mediated cellular processes and is repressed by Zeb2. Here, we investigated whether Ski diminishes the myofibroblast phenotype by de-repressing Meox2 expression and function through repression of Zeb2 expression. We show that expression of Meox1 and Meox2 mRNA and Meox2 protein is reduced during phenoconversion of fibroblasts to myofibroblasts. Overexpression of Meox2 shifts the myofibroblasts into fibroblasts, whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Overexpression of Ski partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. Expression of Zeb2 increased during phenoconversion and Ski overexpression reduces Zeb2 expression in first-passage myofibroblasts. Furthermore, expression of Meox2 is decreased in scar following myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by upregulating the expression of Meox2. This cascade might regulate cardiac myofibroblast phenotype and presents therapeutic options for treatment of cardiac fibrosis.

TGFß1 regulates Scleraxis expression in primary cardiac myofibroblasts by a Smad-independent mechanism.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFß1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFß/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFß1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFß1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFß1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFß1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice.

BACKGROUND AIMS: Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10(6) cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. RESULTS: Blood flow in the MCA-derived MSC-treated animals was higher at each day (P <0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P <0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DISCUSSION: MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

SnoN as a novel negative regulator of TGF-ß/Smad signaling: a target for tailoring organ fibrosis.

Remodeling of the extracellular matrix is beneficial during the acute wound healing stage following tissue injury. In the short term, resident fibroblasts and myofibroblasts regulate the matrix remodeling process through production of matricellular protein components that provide structural support to the damaged tissue. This process is largely governed by the transforming growth factor-ß1 (TGF-ß1) pathway, a critical mediator of the remodeling process. In the long term, chronic activation of the TGF-ß1 pathway promotes excessive synthesis and deposition of matrix proteins, including fibrillar collagens, which ultimately leads to organ failure. SnoN (and its alternatively-spliced isoforms SnoN2, SnoA, and SnoI) is one of four members of a family of negative regulators of TGF-ß1 signaling that includes Ski and functional Smad-suppressing elements on chromosomes 15 and 18. SnoN has been shown to be structurally and functionally similar to Ski and has been demonstrated to directly interact with Ski to abrogate gene expression. Despite this, little progress has been made in delineating a specific role for SnoN in the regulation of myofibroblast phenotype and function. This review outlines the current body of knowledge of what we refer to as the "Ski-Sno superfamily," with a focus on the structural and functional importance of SnoN in mediating the fibrotic response by myofibroblasts following tissue injury.

Assessment of donor heart viability during ex vivo heart perfusion.

Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min(-1)·g(-1)). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R(2) = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R(2) = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R(2) = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R(2) = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R(2) = 0.28; coronary vascular resistance: R(2) = 0.20; lactate concentration: R(2) = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.

Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts.

Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-ß1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.

Increasing bowel cancer screening using SMS in general practice: the SMARTscreen cluster randomised trial.

BACKGROUND: Australia has one of the highest incidences of colorectal cancer (CRC) worldwide. The Australian National Bowel Cancer Screening Program (NBCSP) is a best-practice, organised screening programme, but uptake is low (40.9%) and increasing participation could reduce morbidity and mortality associated with CRC. Endorsement by GPs is strongly associated with increasing screening uptake. AIM: This study (SMARTscreen) aimed to test whether a multi-intervention short message service (SMS) sent by general practices to 50-60-year-old patients who were due to receive the NBCSP kit would increase NBCSP uptake, by comparing it with usual care. DESIGN AND SETTING: A stratified cluster randomised controlled trial was undertaken, involving 21 Australian general practices in Western Victoria, Australia. METHOD: For intervention practices, people due to receive the NBCSP kit within a 6-month study period were sent an SMS just before receiving the kit. The SMS included a personalised message from the person's general practice endorsing the kit, a motivational narrative video, an instructional video, and a link to more information. Control practices continued with usual care, comprising at-home testing with a faecal immunochemical test (FIT) through the NBCSP. The primary outcome was the between-arm percentage difference in uptake of FIT screening within 12 months from randomisation, which was estimated using generalised linear model regression. RESULTS: In total, 39.2% (1143/2914) of people in 11 intervention practices and 23.0% (583/2537) of people in 10 control practices had a FIT result in their electronic health records - a difference of 16.5% (95% confidence interval = 2.02 to 30.9). CONCLUSION: The SMS intervention increased NBCSP kit return in 50-60-year-old patients in general practice. This finding informed a larger trial - SMARTERscreen - to test this intervention in a broader Australian population.

Autophagy regulates trans fatty acid-mediated apoptosis in primary cardiac myofibroblasts.

Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ruminant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200µM, 0-72 h) reduced cell viability in rVF (P<0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-ß lipidation, Atg5-12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 significantly inhibited VA and EA-induced cell death (P<0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.

First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing.

The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing.