Pathological Fibroblasts Research Articles

Abstract 108: Exercise Promotes a Cardioprotective Gene Program in Resident Cardiac Fibroblasts

Exercise and disease both induce hypertrophic cardiac growth, but only disease results in fibrosis and compromised heart function. Transcriptional profiling of resident cardiac fibroblasts (CFs), the primary cellular source of fibrosis, was used to define the gene expression programs (GEP) underlying this divergent functional outcome. Bioinformatic analyses revealed distinct transcriptional responses to exercise and disease, including induction of Rho- and SRF-dependent remodeling genes in disease and NRF2-dependent antioxidant genes in exercise. The expression of a number of antioxidant genes, including metallothioneins (Mt1 and Mt2), are specifically maintained in CFs after exercise and lost in disease. Mice lacking Mt1/2 show signs of cardiac dysfunction after exercise, including cardiac fibrosis, vascular rarefaction, and reduced heart function. Importantly, Mt levels are also reduced in human heart failure (HF) patients, suggesting a potentially conserved cardioprotective role in humans. Non-canonical TGF-β1-mediated p38-MAPK signaling has previously been implicated in HF, therefore we tested the role of p38 signaling in Mt regulation. Pharmacological inhibition of p38 in human HF fibroblasts restores Mt1 and Mt2 expression and blunts the pathological fibroblast phenotype. Taken together, our study defines the transcriptional response of CFs to exercise and disease and reveals a cardioprotective mechanism that is lost in disease.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts.

The aim of the present study was to detect the expression of the key molecules, including transforming growth factor-β1 (TGF-β1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) of TGF-β1/mammalian target of rapamycin (mTOR) pathway in pathological scar fibroblasts. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the expression of the key molecules TGF-β1, PI3K, Akt, mTOR in fibroblasts of normal skin tissue and pathological scar tissue. Immunofluorescence showed that the expression of TGF-β1, PI3K and Akt was significantly enhanced (P<0.05) in pathological scar fibroblasts, and mainly expressed in the cell nucleus, but not in normal skin tissue or fibroblasts. RT-PCR and western blot test results revealed that the TGF-β1, PI3K, Akt, and mTOR mRNA and protein expression in pathological scar fibroblasts were significantly higher (P<0.05) than in the normal skin tissue. Expression of the TGF-β1/mTOR signaling pathway in pathological scar fibroblasts was significantly increased. Data suggest that this expression may be an important mechanism for pathological scar formation.

Expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol intervention.

The aim of the study was to examine the expression of mammalian target of rapamycin (mTOR)/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol (Res) intervention. The mTOR and 70S6K in pathological scar and normal skin fibroblasts were detected by immunofluorescence following treatment with different concentrations of Res. RT-PCR and western blot analysis were used to detect the expression of mTOR and 70S6K mRNA and protein, respectively. Immunofluorescence showed that the expression of 70S6K and mTOR was significantly enhanced in pathological scar fibroblasts, and mainly expressed in the nucleus, but not in normal skin fibroblasts. RT-PCR and western blot analysis showed that after different concentrations of Res treatments, the mTOR and 70S6K mRNA and protein expression significantly (P<0.05) decreased in a dose-dependent manner. In conclusion, the expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts was significantly enhanced. Res can downregulate the expression of mTOR and 70S6K to achieve the inhibition of pathological scar fibroblast proliferation.

Effects of resveratrol on the expression of molecules related to the mTOR signaling pathway in pathological scar fibroblasts.

We observed the effects of resveratrol on the expression of molecules involved in the mTOR signaling pathway in pathological scar fibroblasts, including PI3K, Akt and mTOR. We detected the expression of PI3K, Akt and mTOR in pathological scar and normal skin fibroblasts through immunofluorescence. After being treated with different concentrations of resveratrol, the expression of PI3K, Akt and mTOR mRNA and protein were detected by RT-PCR and Western Blot respectively. Results showed that the expression of PI3K, Akt and mTOR were significantly enhanced in pathological scar fibroblasts and mainly expressed in the nucleus, with no expression in normal skin fibroblasts. Results from RT-PCR and Western Blot tests demonstrated that after Res intervention with different concentrations for pathological scar fibroblasts, the relative expression quantity of PI3K mRNA and protein decreased and showed a dose dependent relationship. Compared to the control group, the differences were statistically significant (P<0.05). However, decrease in the expression of PI3K mRNA and protein was not obvious and there were no significant differences in comparison to the control group (P>0.05). The mechanism of resveratrol in the inhibition of the proliferation of pathological scar fibroblasts may be related to its down-regulation in the expression of Akt and mTOR, which are the key molecules of mTOR signaling pathway.

Understanding the Role of Stiffness in Pathological Cardiac Fibroblast Signaling

Cardiac fibrosis is characterized by extracellular matrix buildup and tissue stiffening. Fibroblasts in the heart respond to pathological cues by activating to proliferate, upregulate expression of alpha smooth muscle actin, synthesize extracellular matrix protein, and secrete biochemical cues to surrounding cells. The cues that trigger this transformation remain incompletely resolved; however, stiffness appears to play a critical role in this process. In vitro experiments using hydrogels with different stiffnesses show that fibroblasts activate when cultured on stiff hydrogels while they remain unactivated on soft hydrogels as they do in the diseased and healthy heart, respectively. Understanding fibroblast activation through mechanosensitive signaling will provide new therapeutic targets for fibrosis treatment.

Normal versus Pathological Cardiac Fibroblast-Derived Extracellular Matrix Differentially Modulates Cardiosphere-Derived Cell Paracrine Properties and Commitment

Human resident cardiac progenitor cells (CPCs) isolated as cardiosphere-derived cells (CDCs) are under clinical evaluation as a therapeutic product for cardiac regenerative medicine. Unfortunately, limited engraftment and differentiation potential of transplanted cells significantly hamper therapeutic success. Moreover, maladaptive remodelling of the extracellular matrix (ECM) during heart failure progression provides impaired biological and mechanical signals to cardiac cells, including CPCs. In this study, we aimed at investigating the differential effect on the phenotype of human CDCs of cardiac fibroblast-derived ECM substrates from healthy or diseased hearts, named, respectively, normal or pathological cardiogel (CG-N/P). After 7 days of culture, results show increased levels of cardiogenic gene expression (NKX2.5, CX43) on both decellularized cardiogels compared to control, while the proportion and staining patterns of GATA4, OCT4, NKX2.5, ACTA1, VIM, and CD90-positive CPCs were not affected, as assessed by immunofluorescence microscopy and flow cytometry analyses. Nonetheless, CDCs cultured on CG-N secreted significantly higher levels of osteopontin, FGF6, FGF7, NT-3, IGFBP4, and TIMP-2 compared to those cultured on CG-P, suggesting overall a reduced trophic and antiremodelling paracrine profile of CDCs when in contact with ECM from pathological cardiac fibroblasts. These results provide novel insights into the bidirectional interplay between cardiac ECM and CPCs, potentially affecting CPC biology and regenerative potential.

The Exaggerated Collagen Expression in Gvhd-Fibroblasts Is Effectively Inhibited By Therapeutic Concentration of Nilotinib

INTRODUCTIONDermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chronic graft-versus-host disease (Scl-cGVHD) and Systemic Scleroderma (SSc). Moreover, in both diseases stimulating anti-PDGF-R antibodies were found, leading to abnormal collagen production by fibroblasts, eventually contributing to organ damage. Targeted therapy with tyrosine kinase inhibitors (TKI) like Imatinib and Nilotinib demonstrated clinical efficacy in Scl-cGVHD; however, the molecular basis underpinning the clinical effects are not fully elucidated. We investigated here a potential terapeutical target of the dermal cGVHD pathophysiology: the cellular and molecular features of pathological skin fibroblasts (GVHD-Fbs) and the efficacy of Nilotinib on fibrosis modulation.MATERIALS AND METHODSFibroblast cultures (GVHD-Fbs) were obtained from skin biopsies of affected skin from 6 patients with active cGVHD, control fibroblasts are Human Dermal Fibroblasts adult (n-FBS).Fibroblasts were characterized by flow cytometry (FACS CANTO II) for the detection of molecules: CD10, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146.In order to evaluate the adipogenic, osteogenic or chondrogenic differentiation cGVHD-Fbs and n-Fbs (n = 3) were cultured in differentiation medium (respectively NH AdipoDiff, NH OsteoDiff, NH ChondroDiff) after four passages. Intracellular lipid droplets indicated adipogenic lineage differentiation. The differentiation potential in the osteogenic lineage was evaluated by calcium accumulation, as assessed by Alizarin Red. The pellet obtained from chondrogenic lineage differentiation was embedded in paraffin, cut in the microtome and the sections placed on a glass slide were stained with Alcian Blue [Junker JP, Cells Tissues Organs, 2010].For incubation with Nilotinib (Santa Cruz Biotechnology) the 10 mM stock solution was diluted to the final concentration in DMEM supplemented with 0,2% FBS (starvation), added to cell cultures at a concentration of 1 μM or 2 μM for 48h, which covered the mean plasma levels in cGVHD patients after standard doses.In subsets of experiments, after starvation, fibroblasts were stimulated with recombinant TGFβ at 10 ng/ml (GIBCO, Invitrogen).After incubation, total RNA was isolated and reverse transcribed. Gene expression was quantified by real-time PCR using the Sybr Green Mix for qPCR. Specific primer pairs for COL1α1 and COL1α2 were designed with the Primer 3 software. The transcript levels were normalized for the expression of GAPDH constitutive gene. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification.RESULTSGVHD-Fbs are morphologically and phenotypically similar to normal fibroblasts (n-FBS).GVHD-FBS did not show a different immunophenotype from n-Fbs, both in early and late culture passages. Also, no differences were noted between GVHD-Fbs and n-FBS in terms of multilineage differentiation capacity towards the adipogenic, osteogenic and chondrogenic lineage.Gene expression of COL1α1 and COL1α2 in GVHD-Fbs was respectively 4 and 1,6 times higher compared to n-FBS (p = 0.02). However, the increased collagen expression was exclusive of early-passage GVHD-Fbs; in late-passage (>4) GVHD-Fbs, collagen mRNA levels were similar to n-FBS (p=0.6 for COL1α1; p=0.4 for COL1α2). As expected, TGFβ boosted collagen expression in n-FBS, but it did not increase COL1α1 and COL1α2 mRNA levels in GVHD-Fbs.Therapeutic doses of Nilotinib (1μM) were able to reduce expression of COL1α1 and COL1α2 mRNA by 86,5% and 49%, respectively (p <0.01).CONCLUSIONSEarly-passage GVHD-Fbs are a valuable cellular model to study the molecular mechanisms of cGVHD fibrosis in vitro, as they show increased collagen production, which is a strong hallmark of fibrosis. The failure to increase collagen expression in GVHD-Fbs upon TGFβ stimulation indirectly supports a TGFβ-dependent mechanism underpinning the fibrogenesis. Finally Nilotinib inhibits in vitro collagen expression in GVHD-Fbs confirming that the activity of TKI in Scl-cGVHD is mediated, at least in part, by direct antifibrotic effects on the fibroblasts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Desensitization of idiopathic pulmonary fibrosis fibroblasts to Alternaria alternata extract-mediated necrotic cell death.

Alternaria alternata is an allergenic fungus and known to cause an upper respiratory tract infection and asthma in humans with compromised immunity. Although A. alternata's effect on airway epithelial cells has previously been examined, the potential role of A. alternata on lung fibroblast viability is not understood. Since lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) display a distinct phenotype that is resistant to stress and cell death inducing conditions, the investigation of the role of Alternaria on pathological IPF fibroblasts provides a better understanding of the fibrotic process induced by an allergenic fungus. Therefore, we examined cell viability of control and IPF fibroblasts (n = 8 each) in response to A. alternata extract. Control fibroblast cell death was increased while IPF fibroblasts were resistant when exposed to 50–100 μg/mL of A. alternata extract. However, there was no significant difference in kinetics or magnitude of Ca2+ responses from control lung and IPF fibroblasts. In contrast, unlike control fibroblasts, intracellular reactive oxygen species (ROS) levels remained low when IPF cells were treated with A. alternata extracts as a function of time. Caspase 3/7 and TUNEL assay revealed that enhanced cell death caused by A. alternata extract was likely due to necrosis, and 7‐AAD assay and the use of sodium pyruvate for ATP generation further supported our findings that IPF fibroblasts become resistant to A. alternata extract‐induced necrotic cell death. Our results suggest that exposure to A. alternata potentially worsens the fibrotic process by promoting normal lung fibroblast cell death in patients with IPF.

G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.

α-Smooth muscle actin is an inconsistent marker of fibroblasts responsible for force-dependent TGFβ activation or collagen production across multiple models of organ fibrosis.

Fibrosis is a common pathological sequela of tissue injury or inflammation, and is a major cause of organ failure. Subsets of fibroblasts contribute to tissue fibrosis in multiple ways, including generating contractile force to activate integrin-bound, latent TGFβ and secreting excess amounts of collagens and other extracellular matrix proteins (ECM) that make up pathologic scar. However, the precise fibroblast subsets that drive fibrosis have been poorly understood. In the absence of well-characterized markers, α-smooth muscle actin (αSMA) is often used to identify pathologic fibroblasts, and some authors have equated αSMA(+) cells with contractile myofibroblasts and proposed that these cells are the major source of ECM. Here, we investigated how well αSMA expression describes fibroblast subsets responsible for TGFβ activation and collagen production in three commonly used models of organ fibrosis that we previously reported could be inhibited by loss of αv integrins on all fibroblasts (using PDGFRβ-Cre). Interestingly, αSMA-directed deletion of αv integrins protected mice from CCl4-induced hepatic fibrosis, but not bleomycin-induced pulmonary or unilateral ureteral obstruction-induced renal fibrosis. Using Col-EGFP/αSMA-RFP dual reporter mice, we found that only a minority of collagen-producing cells coexpress αSMA in the fibrotic lung and kidney. Notably, Col-EGFP(+)αSMA-RFP(-) cells isolated from the fibrotic lung and kidney were equally capable of activating TGFβ as were Col-EGFP(+)αSMA-RFP(+) cells from the same organ, and this TGFβ activation was blocked by a TGFβ-blocking antibody and an inhibitor of nonmuscle myosin, respectively. Taken together, our results suggest that αSMA is an inconsistent marker of contractile and collagen-producing fibroblasts in murine experimental models of organ fibrosis.

Resveratrol Inhibits Proliferation and Induces Apoptosis of Pathological Scar Fibroblasts Through the Mechanism Involving TGF-β1/Smads Signaling Pathway.

The objective of this study was to determine the effect of resveratrol (Res) treatment on pathological scar fibroblasts and the changes in TGF-β1/Smads signaling pathway. For this purpose, cultured pathological scar fibroblasts were treated with various concentrations of Res (10, 50, and, 100 µmol/l), and the morphological changes in target cells were studied using scanning electron microscopy (SEM). The cellular proliferation was assessed by MTT assay; the mRNA and protein expressions of TGF-β1 and Smad-2,3,4,7 were determined by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay, respectively. We found that Res-treated fibroblasts exhibited the typical apoptotic morphological changes. As shown by MTT assay, the OD values of Res-treated fibroblasts, as a measure of cell growth, were significantly lower than those of controls (P < 0.05). In addition, as compared to controls, TGF-β1 and Smad-2,3,4 mRNA/protein expression decreased but those of Smad7 increased in a dose-dependent manner (P < 0.05). It was, therefore, concluded that Res treatment inhibited the pathological scar fibroblast proliferation and induced cell apoptosis through the mechanism involving downregulation of TGF-β1, Smad-2,3,4, and upregulation of Smad7.

IPF fibroblasts are desensitized to type I collagen matrix-induced cell death by suppressing low autophagy via aberrant Akt/mTOR kinases.

Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal interstitial lung disease in which the aberrant PTEN/Akt axis plays a major role in conferring a survival phenotype in response to the cell death inducing properties of type I collagen matrix. The underlying mechanism by which IPF fibroblasts become desensitized to polymerized collagen, thereby eluding collagen matrix-induced cell death has not been fully elucidated. We hypothesized that the pathologically altered PTEN/Akt axis suppresses autophagy via high mTOR kinase activity, which subsequently desensitizes IPF fibroblasts to collagen matrix induced cell death. We found that the autophagosome marker LC3-2 expression is suppressed, while mTOR activity remains high when IPF fibroblasts are cultured on collagen. However, LC3-2 expression increased in response to IPF fibroblast attachment to collagen in the presence of rapamycin. In addition, PTEN over-expression or Akt inhibition suppressed mTOR activity, thereby increasing LC3-2 expression in IPF fibroblasts. Furthermore, the treatment of IPF fibroblasts over-expressing PTEN or dominant negative Akt with autophagy inhibitors increased IPF fibroblast cell death. Enhanced p-mTOR expression along with low LC3-2 expression was also found in myofibroblasts within the fibroblastic foci from IPF patients. Our data show that the aberrant PTEN/Akt/mTOR axis desensitizes IPF fibroblasts from polymerized collagen driven stress by suppressing autophagic activity, which produces a viable IPF fibroblast phenotype on collagen. This suggests that the aberrantly regulated autophagic pathway may play an important role in maintaining a pathological IPF fibroblast phenotype in response to collagen rich environment.

Abstract 301: Targeting Mixed Lineage Kinases in pathological cardiac fibroblast activation and intercellular communication

Cardiovascular disease (CVD) and the final clinical iteration, heart failure (HF), affect more than 82 million Americans yearly, affects &gt;5.7 million). Although the cardiac myocyte has long been the focus of cardiac cellular research, our lab and others have demonstrated that pathological activation of cardiac fibroblasts (CF) after onset of disease or cardiac injury is also a key player in HF pathogenesis. Pathologic activation of CFs causes a release of various paracrine and autocrine factors that target cardiomyocytes (CM), local inflammatory cells and the CFs themselves. Elucidating the mechanisms involved in pathological ‘support cell (CF)’- ‘functional cell (CM)’ communication may hold therapeutic promise. Mixed lineage kinase 3 (MLK3) is a stress activated mitogen-activated protein kinase kinase kinase (MAPKKK) involved in pro-apoptotic pathways. Inhibition of MLK3 in HAND results in the attenuation of microglial cell activation, preservation of neuronal function and synaptic structures. The parity between microglia-neuron communication and CF-CM communication and the effects of pathological activation on the respective support cells for each tissue type suggested a role for MLK3 in aberrant CF-CM cross-talk associated with the development and progression of HF. Preliminary data in mice subjected to a pharmacological model of HF (chronic isoproterenol) that were treated with a small molecule inhibitor (URMC-099) of MLK3 demonstrated a reduction in the development of myocardial fibrosis compared to vehicle treated animals. Additionally, small molecule MLK3 inhibition attenuated the development of myocardial hypertrophy as measured by heart weight: body weight and heart weight: tibia length ratios. Studies in cultured neonatal rat ventricular fibroblasts (NRVF) demonstrated that small molecule MLK3 inhibition attenuated CF activation and transition to a myofibroblast phenotype, including reductions of pathologic CF markers such as α-SMA, IL-6, IL-1β, and others. Our data demonstrate that MLK3 plays an important role in pathologic CF-CM communication and myocardial hypertrophy, and suggest that small molecule inhibition of MLK3 holds therapeutic promise for HF.

Abstract 253: Modeling of Pathologic Hypertophy with Cardiac Microtissues

Introduction: Pathological myocyte hypertrophy and fibroblast activation are key features of myocardial remodeling in hypertension and heart failure. Crosstalk between myocytes and fibroblasts contributes to the structural, mechanical, and electrical remodeling involved in various forms of heart failure. Replicating these responses in traditional 2D cell culture conditions has proven difficult. In this study, we utilized a novel platform to examine the cellular crosstalk and biological processes that underlie functional and structural changes in the 3D engineered tissues when exposed to pathological factors. We hypothesized that engineered cardiac tissues can report structural and functional responses to pathologic biochemical stimuli. Methods: These studies employ microfabricated polydimethylsiloxane (PDMS) templates to engineer arrays of 3D cardiac microtissues (CMT). Cantilevers within templates provide physiologically-relevant biomechanical loading to the CMTs, promote the appropriate organization of neonatal rat cardiac myocytes and fibroblasts, and report active and passive force generation in real time. Data was collected using fluorescent and confocal microscopy. Results: CMTs in a controlled mechanical environment demonstrate mechanical and structural changes within 24 hours of exposure to a cocktail of factors involved in maladaptive remodeling, including Transforming Growth Factor-β, Endothelin-1, and Angiotensin II. Analysis of 40 microtissues revealed that the percent change in passive tension was 45% higher (16.4 ± 10.3% vs. 61.1 ± 11.8%, p&lt;0.01) and in active tension 170% higher (24.9 ± 9.5% vs.196.1 ± 76.0%, p&lt;0.001) in treated CMTs compared to untreated CMTs. The percentage change in cross-sectional area of CMTs treated with pathological factors was 35% higher (-24.5 ± 15.6% vs.11.6 ± 7.9%, p&lt;0.001) than untreated samples. Conclusions: These findings demonstrate that the CMT model can be used to study and dissect the dynamics of pathological hypertrophy. Ongoing experiments are examining the distinct contributions of the pathologic growth factors, the relative contributions of fibroblasts and cardiomyocytes to the pathologic growth response, and the interactions between biomechanical and humoral inputs.

Synthesis and Biological Evaluation of New Creatine Fatty Esters Revealed Dodecyl Creatine Ester as a Promising Drug Candidate for the Treatment of the Creatine Transporter Deficiency

The creatine transporter deficiency is a neurological disease caused by impairment of the creatine transporter SLC6A8, resulting in mental retardation associated with a complete absence of creatine within the brain and cellular energy perturbation of neuronal cells. One of the therapeutic hypotheses was to administer lipophilic creatine derivatives which are (1) thought to have better permeability through the cell membrane and (2) would not rely on the activity of SLC6A8 to penetrate the brain. Here, we synthesized creatine fatty esters through original organic chemistry process. A screening on an in vitro rat primary cell-based blood-brain barrier model and on a rat primary neuronal cells model demonstrated interesting properties of these prodrugs to incorporate into endothelial, astroglial, and neuronal cells according to a structure-activity relationship. Dodecyl creatine ester showed then a 20-fold increase in creatine content in pathological human fibroblasts compared with the endogenous creatine content, stating that it could be a promising drug candidate.

SAT0027 Pharmacological blockade of canonical wnt signaling inhibits experimental dermal fibrosis

BackgroundFibrosis is a major socioeconomic burden, but effective anti-fibrotic therapies are not available in clinical routine. Fibrosis emerges from excessive release of extracellular matrix components by pathologically activated fibroblasts, which...

Tacrolimus fails to regulate collagen expression in dermal fibroblasts

BackgroundThe purpose of this study was to investigate the effects of tacrolimus on human fibroblasts derived from unwounded skin, hypertrophic scars (HTS), and keloids. We hypothesized that tacrolimus, a potent anti-inflammatory and immunosuppressant drug known to attenuate solid organ transplant fibrosis, would block collagen expression in human dermal fibroblasts. MethodsWe performed genomewide microarray analysis on human dermal fibroblasts treated with tacrolimus in vitro. We used principal component analysis and hierarchical clustering to identify targets regulated by tacrolimus. We performed quantitative polymerase chain reaction to validate the effect of tacrolimus on collagen 1 and 3 expression. ResultsWe identified 62, 136, and 185 gene probes on microarray analysis that were significantly regulated (P < 0.05) by tacrolimus in normal, HTS, and keloid fibroblasts, respectively. Collagen pathways were not blocked after tacrolimus exposure in any of the fibroblast groups; we validated these findings using quantitative polymerase chain reaction for collagen 1 and 3. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 were significantly downregulated (P < 0.05) by tacrolimus in both HTS and keloid fibroblast populations but not normal fibroblasts. ConclusionsTacrolimus does not modulate the expression of collagen 1 or 3 in human dermal fibroblasts in vitro. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 are blocked by tacrolimus in pathologic fibroblasts but not normal fibroblasts, and may represent novel genes underlying HTS and keloid pathogenesis. Tacrolimus-based anti-fibrotics might prove more effective if non-fibroblast populations such as inflammatory cells and keratinocytes are targeted.

The Dynamic Role of Cardiac Fibroblasts in Development and Disease

Cardiac fibroblasts are the most abundant cell in the mammalian heart. While they have been historically overlooked in terms of functional contributions to development and physiology, cardiac fibroblasts are now front and center. They are currently recognized as key protagonists during both normal development and cardiomyopathy disease, and work together with cardiomyocytes through paracrine, structural, and potentially electrical interactions. However, the lack of specific biomarkers and fibroblast heterogeneous nature currently convolutes the study of this dynamic cell lineage; though, efforts to advance marker analysis and lineage mapping technologies are ongoing. These tools will help elucidate the functional significance of fibroblast-cardiomyocyte interactions in vivo and delineate the dynamic nature of normal and pathological cardiac fibroblasts. Since therapeutic promise lies in understanding the interface between developmental biology and the postnatal injury response, future studies to understand the divergent roles played by cardiac fibroblasts both in utero and following cardiac insult are essential.

Resveratrol inhibits transforming growth factor-β–induced proliferation and differentiation of ex vivo human lung fibroblasts into myofibroblasts through ERK/Akt inhibition and PTEN restoration

ABSTRACTThe authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-β (TGF-β)–induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-β–induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-β–induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-β–induced fibrosis. The authors show that RV inhibited TGF-β–induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-β–induced decrease in phosphatase and tensin homolog (PTEN) expression levels.