Myofibroblast Phenoconversion Research Articles

Alpinetin Suppresses Effects of TGF-β1 on Stimulating the Production and Organization of Fibrotic Markers in Human Primary Dermal Fibroblasts.

Overgrowths of dermal fibroblasts and myofibroblast phenoconversion in response to TGF-β stimulation are the hallmarks of skin fibrosis. Constitutive activation of dermal fibroblasts by TGF-β induces the excessive production of extracellular matrix as well as certain key intracellular proteins which form a complex interaction network. Current therapies include monoclonal anti-bodies against TGF-β and surgery, but these treatments generally elicit a limited effect on certain kinds of skin fibrosis. In the current study, we investigated the effects of alpinetin (AP) on human primary dermal fibroblasts (HPDFs) stimulated with TGF-β1. Results demonstrated that AP exhibited strong inhibitory effects on TGF-β1-induced proliferation and migration of HPDFs. AP also inhibited TGF-β1-induced morphological changes of fibroblasts to myofibroblasts, and these were found to be from its effects on blocking actin stress fiber formation and organization. The expression of major fibrotic molecules including α-SMA and type I collagen upon TGF-β1 stimulation was also inhibited by AP. In addition, AP attenuated TGF-β1-induced production and organization of vimentin, β-catenin, and N-cadherin, important for the pathophysiology of skin fibrosis. In conclusion, we revealed that AP has an ability to reverse the fibrotic effects of TGF-β1 at the cellular level, and this discovery suggests the therapeutic potential of AP for skin fibrosis.

FoxO1 is required for high glucose-dependent cardiac fibroblasts into myofibroblast phenoconversion

In the normal heart, cardiac fibroblasts (CFs) maintain extracellular matrix (ECM) homeostasis, whereas in pathological conditions, such as diabetes mellitus (DM), CFs converse into cardiac myofibroblasts (CMFs) and this CFs phenoconversion increase the synthesis and secretion of ECM proteins, promoting cardiac fibrosis and heart dysfunction. High glucose (HG) conditions increase TGF-β1 expression and FoxO1 activity, whereas FoxO1 is crucial to CFs phenoconversion induced by TGF-β1. In addition, FoxO1 increases CTGF expression, whereas CTGF plays an active role in the fibrotic process induced by hyperglycemia. However, the role of FoxO1 and CTGF in CFs phenoconversion induced by HG is not clear. In this study, we investigated the effects of FoxO1 pharmacological inhibition on CFs phenoconversion in both in vitro and ex vivo models of DM. Our results demonstrate that HG induces CFs phenoconversion and FoxO1 activation. Moreover, AS1842856, a pharmacological inhibitor of FoxO1 activity, prevents CFs phenoconversion and CTGF expression increase induced by HG, whereas these results were corroborated by FoxO1 silencing. Additionally, K252a, a pharmacological blocker of CTGF receptor, prevents HG-induced CFs phenoconversion, which was corroborated with CTGF expression knockdown. Furthermore, through CFs isolation from heart of diabetic rats, we showed that hyperglycemia induces FoxO1 activation, the increase of CTGF expression and CFs phenoconversion, whereas the FoxO1 activity inhibition reverses the effects induced by hyperglycemia on CFs. Altogether, our results demonstrate that FoxO1 and CTGF are necessary for CFs phenoconversion induced by HG and suggest that both proteins are likely to become a potential targeted drug for fibrotic response induced by hyperglycemic conditions.

Mitochondrial Dysfunction and Cellular Senescence Due to Aging Contributes to Prostatic Fibrosis

Aging is the single largest risk factor for many common diseases that burden public health. This is especially true in the prostate; as men age, the prostate undergoes a prototypical aging change, fibrosis. The aged fibrotic prostate causes urinary symptoms which will afflict nearly every man if they live long enough. However, the molecular mechanisms responsible for the aging-dependent promotion of fibrosis are largely unknown and understudied. In this study, we sought to reveal the contribution of mitochondrial dysfunction to fibrosis in the aging prostate, ultimately leading to prostate dysfunction, urinary symptoms, and overall poor health. The demise of mitochondrial function is well established in other aging-associated diseases but has not been investigated in the prostate. Our analysis revealed an increase in cellular senescence and mitochondrial dysfunction in BPH patient tissue compared to normal prostates. Furthermore, selective inhibition of mitochondrial complex I by rotenone in cultured human prostate fibroblasts led to myofibroblast phenoconversion as characterized by increased expression of Col1a1, Col3a1, and ACTA2. To determine the contribution of mitochondrial dysfunction on fibrosis and lower urinary tract dysfunction (LUTD), we examined the prostate lobes in both aging and steroid-induced LUTD in mice. These models have been extensively characterized and exhibit an age-mediated increase in LUTD and fibrosis. We observed a decrease in mitochondrial function and an increase in cellular senescence corresponding to an increase in fibrosis and LUTD. This suggests that normal aging-dependent reductions in mitochondrial complex I function in the prostate may promote fibrosis and contribute to urinary dysfunction.

Cardiosomal microRNAs Are Essential in Post-Infarction Myofibroblast Phenoconversion.

The inclusion of microRNAs (miRNAs) in extracellular microvesicles/exosomes (named cardiosomes when deriving from cardiomyocytes) allows their active transportation and ensures cell-cell communication. We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Using a murine model of myocardial infarction (MI), we tested our hypothesis by measuring in isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which are upregulated in cardiomyocytes post-MI and involved in myofibroblast phenoconversion. We found that miR-195 was significantly upregulated in cardiosomes and in fibroblasts isolated after MI compared with SHAM conditions. Moreover, primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effect was observed following incubation with cardiosomes or medium from sham cardiomyocytes. Taken together, our findings indicate for the first time that a cardiomyocyte-specific miRNA, transferred to fibroblasts in form of exosomal cargo, is crucial in the activation of myofibroblasts.

P2587Cardiosomal microRNAs are essential in post-infarction myofibroblast phenoconversion

Abstract Background Circulating microRNAs (miRNAs) are actively transported, via inclusion in extracellular microvesicles/exosomes (herein named cardiosomes since they are deriving from cardiomyocytes), allowing protection from degradation and underlying cell-cell communications. After myocardial infarction (MI), quiescent cardiac fibroblasts can acquire an activated phenotype, and a recognized marker of such activation is the expression of α-smooth muscle actin (α-SMA); such cells are commonly known as myofibroblasts. Hypothesis We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Methods Using an established murine model of MI, obtained via permanent ligation of the left anterior descending coronary artery, we tested our hypothesis by measuring in primary isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which we found to be upregulated in cardiomyocytes post-MI and to be involved in myofibroblast phenoconversion. Results Bioinformatic studies validated by biological assays identified three cardiomyocyte-specific miRNAs (namely miR-92a, miR-195, and miR-494) that are upregulated following cardiac ischemia and are also known to regulate α-SMA expression. We demonstrated that these miRNAs are upregulated in fibroblasts post-ischemia and are also contained in cardiosomes; to ensure that these miRNAs were confined inside cardiosomes, the samples were treated with RNase, showing that the level of miRNAs was not affected by RNase treatment, unless in presence of the detergent Triton X-100. The activation of myofibroblasts was confirmed by the increased expression of α-SMA. Using a luciferase assay, we also validated that miR-92a, miR-195, and mir-494 target the 3'UTR of α-SMA and regulate its transcription. Moreover, we observed that primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effects were detected following incubation with cardiosomes or medium from sham cardiomyocytes. Strikingly, myofibroblast phenoconversion was markedly attenuated when GW4869, an inhibitor of neutral sphingomyelinase 2 responsible for cardiosomes secretion, was added to cultured post-MI cardiomyocytes. Conclusion Taken together, our findings indicate that three cardiomyocyte-specific miRNAs (miR-92a, miR-195, and miR-494) are crucial in the activation of myofibroblasts.

Abstract 802: Cardiomyocyte-derived Exosomal MicroRNAs Regulates Post-Infarction Inflammation and Myofibroblast Phenoconversion

Background: The inclusion of microRNAs (miRNAs) in extracellular microvesicles/exosomes (named cardiosomes when deriving from cardiomyocytes) allows their active transportation and ensures cell-cell communication. Aim: We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Methods: Using bioinformatic approaches and an established murine model of myocardial infarction (MI, achieved via ligation of the left anterior descending coronary artery), we tested our hypothesis by measuring in isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, upregulated in cardiomyocytes post-MI and involved in myofibroblast phenoconversion. Results: We found that miR-92a and miR-195 were significantly upregulated (absolute values) in cardiosomes as well as in fibroblasts isolated at different time points after MI compared with SHAM conditions. These miRs were demonstrated to be both predicted (bioinformatics) and biologically validated (luciferase assays) targets of SMAD7. Moreover, primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effects were observed following incubation with cardiosomes or medium from sham cardiomyocytes. Myofibroblast activation was assessed measuring the expression of aSMA, periostin, collagen I/III, FAP, fibronectin ED-A. Both miRs also regulated the inflammatory response (evaluated in terms of IL-6 and CXCL1). The addition of an inhibitor of exosome release (GW4869 10 μM for 12 h) significantly attenuated all these responses. The mechanistic contribution of miR-92a and miR-195 was further confirmed using specific miR mimics and inhibitors. Conclusions: Taken together, our findings indicate for the first time that two cardiomyocyte-specific miRNAs (miR-92a and miR-195), transferred to fibroblasts in form of exosomal cargo, are essential in the activation of myofibroblasts in MI.

Inhibition of the CXCL12/CXCR4 axis prevents periurethral collagen accumulation and lower urinary tract dysfunction in vivo.

Several studies show that prostatic fibrosis is associated with male lower urinary tract dysfunction (LUTD). Development of fibrosis is typically attributed to signaling through the transforming growth factor β (TGF-β) pathway, but our laboratory has demonstrated that in vitro treatment of human prostatic fibroblasts with the C-X-C motif chemokine ligand 12 (CXCL12) chemokine stimulates myofibroblast phenoconversion and that CXCL12 has the capacity to activate profibrotic pathways in these cells in a TGF-β-independent manner. We have previously reported that feeding mice high-fat diet (HFD) results in obesity, type II diabetes, increased prostatic fibrosis, and urinary voiding dysfunction. The purpose of this study was to test the hypothesis that in vivo blockade of the CXCL12/CXCR4 axis would inhibit the development of fibrosis-mediated LUTD in HFD-fed mice. Two-month-old male senescence-accelerated mouse prone-6 mice were fed either a HFD or low-fat diet (LFD) for 8 months. Half of each dietary group were given constant access to normal water or water that contained the C-X-C chemokine receptor type 4 (CXCR4; CXCL12 receptor) antagonist CXCR4AIII. At the conclusion of the study, mice were weighed, subjected to oral glucose tolerance testing and cystometry, and lower urinary tract tissues collected and assessed for collagen content. HFD-fed mice became significantly obese, insulin resistant, and hyperglycemic, consistent with acquisition of metabolic syndrome, compared with LFD-fed mice. Anesthetized cystometry demonstrated that HFD-fed mice experienced significantly longer intercontractile intervals and greater functional bladder capacity than LFD-fed mice. Immunohistochemistry demonstrated high levels of CXCR4 and CXCR7 staining in mouse prostate epithelial and stromal cells. Picrosirius red staining indicated significantly greater periurethral collagen deposition in the prostates of HFD than LFD-fed mice. Treatment with the CXCR4 antagonist CXCR4AIII did not affect acquisition of metabolic syndrome but did reduce both urinary voiding dysfunction and periurethral prostate collagen accumulation. This is the first study to report that obesity-induced lower urinary tract fibrosis and voiding dysfunction can be repressed by antagonizing the activity of the CXCR4 chemokine receptor in vivo. These data suggest that targeting the CXCL12/CXCR4 signaling pathway may be a clinical option for the prevention or treatment of human male LUTD.

Toll-like receptor 4 contributes to a myofibroblast phenotype in cardiac fibroblasts and is associated with autophagy after myocardial infarction in a mouse model

Background and aimsCardiac fibrosis after myocardial infarction (MI) is involved in fibroblast transforming and differentiating into myofibroblast phenoconversion, however, the underlying mechanisms are poorly understood. Toll-like receptor 4 (TLR4)-mediated pathogen-associated molecular patterns are key factors that deteriorate cardiac remodelling after MI. Moreover, autophagy has dual roles in cell survival in myocardial tissues after MI. We evaluated the relationship between TLR4 signalling and cardiac myofibroblast transformation-differentiation after MI in vivo and in vitro and analysed the role of autophagy. MethodsWe reproduced a model of MI by the permanent ligation of the left anterior descending coronary artery of Tlr4-knockout (Tlr4−/−) and wild-type (WT) male mice. We evaluated scar formation, myofibroblast phenoconversion, LC3 dot formation, autophagy related proteins and α-smooth muscle actin (SMA) in cardiac tissues, 7, 14, and 28 days after myocardial infarction. Cardiac fibroblasts were cultured from Tlr4−/− or WT mice. Vimentin, α-SMA, bilayer membrane vesicle structures of autophagosomes, and autophagy related proteins were observed after treatment with lipopolysaccharide (LPS) or 3-methyladenine (3-MA) at 24 h. ResultsAfter MI on 7, 14, and 28 days, Tlr4−/− mice showed that heart tissue fibrosis and expression of α-SMA, a marker of myofibroblasts, were decreased compared to WT mice. Additionally, levels of LC3II, Atg5, Atg7, and Beclin-1, which are involved in autophagy, were lower than those in WT mice. Further, p62 expression, which is negatively correlated with autophagy levels, was higher in Tlr4−/− mice. Moreover, LC3-labelled autophagosomes in cardiac tissues were reduced in these animals. In vitro, LPS, a ligand of TLR4, stimulated α-SMA expression in cardiac fibroblasts, enhanced autophagic flux, and increased autophagosome numbers. In contrast, these effects were not obvious in Tlr4−/− cardiac fibroblasts. LC3II, Atg5, Atg7, and Beclin-1 were upregulated, and p62 was downregulated in cardiac fibroblasts of WT mice stimulated with LPS. However, these effects were blocked by 3-methyladenine, an inhibitor of autophagy. ConclusionsThese results suggest that TLR4 signalling executes the development of a myofibroblast phenotype after MI via autophagy and could be therapeutically exploited to improve outcome after myocardial injury.

CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation

Tissue fibrosis is mediated by the actions of multiple pro-fibrotic proteins that can induce myofibroblast phenoconversion through diverse signaling pathways coupled predominantly to Smads or MEK/Erk proteins. The TGFβ/TGFβR and CXCL12/CXCR4 axes induce myofibroblast phenoconversion independently through Smads and MEK/Erk proteins, respectively. To investigate these mechanisms at the genetic level, we have now elucidated the TGFβ/TGFβR and CXCL12/CXCR4 transcriptomes in human fibroblasts. These transcriptomes are largely convergent, and up-regulate transcripts encoding proteins known to promote myofibroblast phenoconversion. These studies also revealed a molecular signature unique to CXCL12/CXCR4 axis activation for COPII vesicle formation, ubiquitination, and Golgi/ER localization/targeting. In particular, both CUL3 and KLHL12, key members of the Cullin-RING (CRL) ubiquitin ligase family of proteins involved in procollagen transport from the ER to the Golgi, were highly up-regulated in CXCL12-, but repressed in TGFβ-, treated cells. Up-regulation of CUL3 and KLHL12 was correlated with higher procollagen secretion by CXCL12-treated cells, and this affect was ablated upon treatment with inhibitors specific for CXCR4 or CUL3 and repressed by TGFβ/TGFβR axis activation. The results of these studies show that activation of the CXCL12/CXCR4 axis uniquely facilitates procollagen I secretion through a COPII-vesicle mediated mechanism to promote production of the ECM characteristic of fibrosis.

The non-neuronal cyclin-dependent kinase 5 is a fibrotic mediator potentially implicated in systemic sclerosis and a novel therapeutic target.

The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-β fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-β responses in fibroblast monolayers and in ex vivo human skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in two distinct mouse models of SSc. Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.

MP17-09 CXCL12-CXCR4 AXIS ACTIVATION PROMOTES COP II VESICLE-MEDIATED SECRETION OF COLLAGEN BY PROSTATE MYOFIBROBLASTS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2017MP17-09 CXCL12-CXCR4 AXIS ACTIVATION PROMOTES COP II VESICLE-MEDIATED SECRETION OF COLLAGEN BY PROSTATE MYOFIBROBLASTS Susan Patalano-Salsman, Jose A. Rodriguez-Nieves, Diego Almanza, Amy Avery, Andrew Judell-Halfpenney, Todd Riley, and Jill Macoska Susan Patalano-SalsmanSusan Patalano-Salsman More articles by this author , Jose A. Rodriguez-NievesJose A. Rodriguez-Nieves More articles by this author , Diego AlmanzaDiego Almanza More articles by this author , Amy AveryAmy Avery More articles by this author , Andrew Judell-HalfpenneyAndrew Judell-Halfpenney More articles by this author , Todd RileyTodd Riley More articles by this author , and Jill MacoskaJill Macoska More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.598AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Factors that promote lower urinary tract voiding dysfunction (LUTD) in aging men include excessive prostatic proliferation and muscle contraction. Recent studies suggest that tissue fibrosis, e.g., peri-urethral collagen accumulation, may also contribute to LUTD. Our group recently reported that activation of the CXCL12/CXCR4 axis and downstream MEK/ERK activation, as well as canonical TGFβ/TGFβR axis activation and downstream Smad3 activation, promote prostate fibroblast to myofibroblast phenoconversion and collagen production. Based on this finding, we hypothesized that downstream MEK/ERK and Smad signaling would converge at the transcriptional level to promote the activation of genes encoding pro-fibrotic proteins. To test this, RNA sequencing analysis was performed on prostate fibroblasts treated with CXCL12 or TGFβ. METHODS Prostate fibroblasts were treated with 4ng/ml TGFβ or 100pM CXCL12 for 12 hrs (RNA) or 24, 48 and 72 hrs (protein). Total RNA was purified and subjected to qRT-PCR or RNASeq. Sequence data was pipelined through the Bowtie/TopHat/Cufflinks/CummeRbund (Tuxedo) packages. Pathview v1.10.1 and Cytoscape were used to perform Kegg pathway and network analysis, respectively. Immunoblot analysis was performed using whole protein lysates or concentrated conditioned media. RESULTS RNASeq analysis showed that prostate fibroblasts treated with CXCL12 up-regulated the transcript levels of genes that encoded members of the Cullin-RING 3 (CRL3) ubiquitin ligase family of proteins. These proteins form COPII vesicles that transport procollagen from the endoplasmic reticulum to the cell membrane and extracellular space. Western blot analysis showed that CXCL12-treated cells up-regulated CRL3 proteins and secreted higher levels of procollagen compared to vehicle and TGFβ-treated cells. Procollagen secretion was ablated upon treatment with AMD3100, a CXCR4 antagonist, demonstrating that the observed increased collagen secretion was specifically coupled to CXCL12/CXCR4 axis activation. CONCLUSIONS The results of these studies are consistent with our hypothesis and present a major new discovery: Activation of the CXCL12/CXCR4 axis promotes fibrosis by increasing both the expression and secretion of collagen. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e214-e215 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Susan Patalano-Salsman More articles by this author Jose A. Rodriguez-Nieves More articles by this author Diego Almanza More articles by this author Amy Avery More articles by this author Andrew Judell-Halfpenney More articles by this author Todd Riley More articles by this author Jill Macoska More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibition of autophagy inhibits the conversion of cardiac fibroblasts to cardiac myofibroblasts.

The incidence of heart failure with concomitant cardiac fibrosis is very high in developed countries. Fibroblast activation in heart is causal to cardiac fibrosis as they convert to hypersynthetic cardiac myofibroblasts. There is no known treatment for cardiac fibrosis. Myofibroblasts contribute to the inappropriate remodeling of the myocardial interstitium, which leads to reduced cardiac function and ultimately heart failure. Elevated levels of autophagy have been linked to stress-induced ventricular remodeling and other cardiac diseases. Previously, we had shown that TGF-β1 treatment of human atrial fibroblasts both induced autophagy and enhanced the fibrogenic response supporting a linkage between the myofibroblast phenotype and autophagy. We now demonstrate that with in vitro culture of primary rat cardiac fibroblasts, inhibition of autophagy represses fibroblast to myofibroblast phenoconversion. Culturing unpassaged cardiac fibroblasts for 72 hours on plastic tissue culture plates is associated with elevated α-smooth muscle actin (α-SMA) expression. This activation parallels increased microtubule-associated protein 1A/1B-light chain 3 (LC-3β II) protein expression. Inhibition of autophagy with bafilomycin-A1 (Baf-A1) and chloroquine (CQ) in cardiac fibroblasts significantly reduces α-SMA and extracellular domain A fibronectin (ED-A FN) protein vs untreated controls. Myofibroblast cell migration and contractility were significantly reduced following inhibition of autophagy. These data support the possibility of a causal link between cardiac fibroblast-to-myofibroblast phenoconversion and autophagy.

CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling.

Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis.

Resveratrol-Mediated Repression and Reversion of Prostatic Myofibroblast Phenoconversion.

BackgroundResveratrol, a phytoalexin found in berries, peanuts, grapes, and red wine, inhibits oxidation, inflammation, and cell proliferation and collagen synthesis in multiple cell types and or animal models. It represses collagen deposition in the vasculature, heart, lung, kidney, liver, and esophagus in animal models and may have some utility as an anti-fibrotic. Recent studies have shown that increased collagen deposition and tissue stiffness in the peri-urethral area of the prostate are associated with lower urinary tract dysfunction (LUTD) and urinary obstructive symptoms. The aim of this study was to determine whether Resveratrol might be useful to inhibit or revert TGFβ- and/or CXCL12-mediated myofibroblast phenoconversion of prostate fibroblasts in vitro, and therefore whether the use of anti-fibrotic therapeutics might be efficacious for the treatment of LUTD.MethodsPrimary prostate and lung tissues were explanted and fibroblast monolayers expanded in vitro. Primary and N1 immortalized prostate stromal fibroblasts, as well as primary fibroblasts cultured from a normal lung and one affected by idiopathic pulmonary fibrosis (IPF) for comparison, were grown in serum–free defined media supplemented with vehicle, TGFβ or CXCL12, pre- or post-treatment with Resveratrol, and were evaluated using immunofluorescence for alpha smooth muscle actin (αSMA) and collagen I (COL1) protein expression and assessed for cell proliferation, apoptosis, and COL1 and EGR1 transcript expression.ResultsThis study showed that low concentrations of Resveratrol (≤50 μM) had no effect on N1 or primary prostate fibroblast cell proliferation, apoptosis, or COL1 or EGR1 gene transcription but repressed and reversed myofibroblast phenoconversion. As expected, these same effects were observed for IPF lung fibroblasts though higher levels of Resveratrol (≥100uM) were required. Taken together, these data suggest that, like lung fibroblasts, prostate fibroblast to myofibroblast phenoconversion can be both repressed and reversed by Resveratrol treatment. Thus, anti-fibrotic therapeutics might be efficacious for the treatment of LUTD.

Zeb2: A novel regulator of cardiac fibroblast to myofibroblast transition

Cardiac fibroblast to myofibroblast phenoconversion is a critical step in cardiovascular disease progression. Myofibroblasts contribute to cardiac fibrosis by chronic extracellular matrix remodelling and scar formation which subsequently results in myocardial stiffening, impaired contraction, cardiac dysfunction and eventually to heart failure. Previously we showed that Meox2, a homeobox transcription factor, is highly expressed in fibroblasts compared to myofibroblasts. As well we demonstrated that Meox2 can inhibit myofibroblast phenoconversion by down‐regulating a set of myofibroblast markers. Here we report for the first time that Zeb2, a repressor of Meox2 and an epithelial‐mesenchymal transition inducer, may play a crucial role during this phenoconversion process. Using rat cardiac fibroblasts, we have shown that Zeb2 overexpression causes an up‐regulation of a set of myofibroblast markers‐ SMemb, ED‐A fibronection and α‐SMA. We showed that Zeb2 is highly expressed in myofibroblast nuclei whereas it is only minimally expressed in fibroblasts as observed by immunoblotting and confocal microscopy. Moreover, ectopic Zeb2 expression results in a less migratory phenotype‐ characteristic of mature myofibroblasts. We also showed that Zeb2 overexpression represses Meox2 expression in endothelial cells. We will investigate the role of Zeb2 in myofibroblast proliferation, apoptosis and function in vitro. Thus findings from our study will provide insight to understand the mechanism behind myofibroblast phenoconversion and may provide a basis for developing a Zeb2‐based novel anti‐fibrotic drug in the future.

Control of Cardiac Fibroblast Phenotype by the Meox2/Zeb2 Signalling Switch (LB47)

The fibroblast to myofibroblast phenoconversion, which is induced by TGF‐β, is a crucial step during cardiac fibrosis. Activated myofibroblasts increase extracellular matrix (ECM) synthesis, which impairs contraction that results in myocardial stiffening, and eventually heart failure. Meox2, a regulator of cell proliferation and senescence, can block TGF‐β mediated epithelial to mesenchymal transition (EMT). In contrast, Zeb2, a repressor of Meox2, enhances EMT in concert with TGF‐β. However, their roles in the fibroblast to myofibroblast phenoconversion remain elusive. We have shown that Meox2 is more highly expressed in fibroblasts than in myofibroblasts. Conversely, Zeb2 expression is lower in fibroblasts but higher in myofibroblasts. This suggests that Meox2/Zeb2 may regulate the fibroblast phenotype during cardiac fibrosis. We have shown that ectopic expression of Meox2 represses a set of myofibroblast markers. We have successfully knocked down Meox2 protein in primary cells. By confocal microscopy, Meox2 protein was found to be confined to the nuclei of fibroblasts whereas a cytoplasmic distribution was seen in myofibroblasts. By immunoblotting, Zeb2 expression was found to be higher in the nuclei of myofibroblasts. To test if ectopic Zeb2 expression enhances this phenoconversion, we have recently generated an adenoviral Zeb2 expression construct. We anticipate that the Meox2/Zeb2 signalling switch plays a critical role in cardiac fibrosis and findings from this study may provide a basis for developing Meox2 or Zeb2 based novel anti‐fibrotic drugs in the future.Grant Funding Source: Supported by Canadian Institutes of Health Research & Heart and Stroke Foundation of Canada

Autophagy in phenoconversion of differentiated and undifferentiated fibroblasts

Cardiac fibroblasts phenoconvert to hypersecretory myofibroblasts and deposit matrix resulting in cardiac fibrosis and autophagy that is associated to fibroproliferative events. Autophagy inhibition was studied in both primary (P0) rat cardiac fibroblasts (PF) and mouse embryonic fibroblasts (MEF). Autophagy was inhibited in PF using Baf‐A1 or 3‐MA while MEF ATG3 and ATG5 KO were also used. Western blot analysis, Immunofluorescence, and transmission electron microscopy were used to investigate autophagy and fibroblast to myofibroblast phenoconversion. α‐SMA and ED‐A fibronectin were assessed as phenoconversion markers. Inhibition of autophagy was revealed by punctate LC3‐II staining and double membrane autophagosomes in combination with decreased myofibroblast markers in PF vs. controls. Inhibition of autolysosome formation and accumulation of autophagosomes were found in Baf‐A1 treated cells. Conversely, ATG3 and ATG5 KO MEF cells showed increased myofibroblast markers vs. WT controls. Increased α‐SMA was seen in MEF ATG3 and ATG5 KO vs. controls while inhibition of autophagy in PFs showed diminished myofibroblastic phenotype vs. controls. A link between fibroblast phenoconversion and autophagy is established. This effect is dependent on cell type eg, PFs vs. MEFs. This finding illustrates that autophagy functions in a variable manner in differentiated vs. non‐differentiated fibroblasts.

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-β-Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-β-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.

CXC-type chemokines promote myofibroblast phenoconversion and prostatic fibrosis.

Recent studies from our group suggest that extracellular matrix (ECM) deposition and fibrosis characterize the peri-urethral prostate tissues of some men suffering from Lower Urinary Tract Symptoms (LUTS) and that fibrosis may be a contributing factor to the etiology of LUTS. Fibrosis can generally be regarded as an errant wound-healing process in response to chronic inflammation, and several studies have shown that the aging prostate tissue microenvironment is rich with inflammatory cells and proteins. However, it is unclear whether these same inflammatory proteins, particularly CXC-type chemokines, can mediate myofibroblast phenoconversion and the ECM deposition necessary for the development of prostatic tissue fibrosis. To examine this, immortalized and primary prostate stromal fibroblasts treated with TGF-β1, CXCL5, CXCL8, or CXCL12 were evaluated morphologically by microscopy, by immunofluorescence and qRT-PCR for αSMA, collagen 1, vimentin, calponin, and tenascin protein and transcript expression, and by gel contraction assays for functional myofibroblast phenoconversion. The results of these studies showed that that immortalized and primary prostate stromal fibroblasts are induced to express collagen 1 and 3 and αSMA gene transcripts and proteins and to undergo complete and functional myofibroblast phenoconversion in response to CXC-type chemokines, even in the absence of exogenous TGF-β1. Moreover, CXCL12-mediated myofibroblast phenoconversion can be completely abrogated by inhibition of the CXCL12 receptor, CXCR4. These findings suggest that CXC-type chemokines, which comprise inflammatory proteins known to be highly expressed in the aging prostate, can efficiently and completely mediate myofibroblast phenoconversion and may thereby promote fibrotic changes in prostate tissue architecture associated with the development and progression of male lower urinary tract dysfunction.