Oscar Busnadiego

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation

OBJECTIVE Enhanced adhesive signaling, including activation of focal adhesion kinase (FAK), is a hallmark of fibroblasts from lung fibrosis patients, and FAK has therefore been hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. METHODS FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor PF-562,271, or with small interfering RNA (siRNA)-mediated silencing of FAK were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and lungs were harvested for histologic and biochemical analysis. Using endothelin 1 (ET-1) as a stimulus, cell adhesion and contraction, as well as profibrotic gene expression, were studied in fibroblasts isolated from wild-type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild-type and β1 integrin-deficient mouse fibroblasts. RESULTS FAK expression and activity were up-regulated in fibroblast foci and remodeled vessels from lung fibrosis patients. Pharmacologic or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis in mice. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by JNK activation through β1 integrin/FAK signaling. CONCLUSION These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

Role of endothelin in the cardiovascular system

The endothelin (ET) system consists of three peptide ligands (ET-1, ET-2 and ET-3) and two G-protein-coupled receptors, ET(A) and ET(B). In the cardiovascular system, ETs, particularly ET-1, are expressed in smooth muscle cells, cardiomyocytes, fibroblasts, and notably in vascular endothelial cells. Intense research over the last 10 years has changed the original view of ET-1 as mainly a vasoconstrictor regulating blood pressure, into a biological factor regulating processes such as vascular remodeling, angiogenesis or extracellular matrix synthesis. The advent of specific (and type-selective) ET receptor antagonists has greatly fostered our knowledge of the biological function of ET-1, and has offered a potential therapeutic approach for numerous diseases including hypertension, atherosclerosis or fibrosis. In this article, we review the regulation of the expression of vascular ET-1, as well as the contribution of ET-1 to endothelial, smooth muscle and fibroblast cell function, with particular interest in the role of ET-1 in the development of cardiovascular diseases.

Endothelin 1 contributes to the effect of transforming growth factor β1 on wound repair and skin fibrosis

OBJECTIVE To characterize the pathways induced by transforming growth factor beta1 (TGFbeta1) that lead to the expression of endothelin 1 (ET-1) in human dermal fibroblasts, and to study the effects of TGFbeta1 and ET-1 on the acquisition of a profibrotic phenotype and assess the contribution of the TGFbeta1/ET-1 axis to skin wound healing and fibrosis in vivo. METHODS The mechanism of induction of ET-1 expression by TGFbeta1 and its effect on the expression of alpha-smooth muscle actin and type I collagen were studied in human dermal fibroblasts, in experiments involving the TGFbeta receptor inhibitor GW788388 and the ET receptor antagonist bosentan, by real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, immunofluorescence, Western blotting, and promoter/reporter transient transfection analyses. Experiments assessing dermal wound healing in mice were performed with adenovirus-driven overexpression of active TGFbeta1 and ET-1, with or without treatment with bosentan. The contributions of TGFbeta1 and ET-1 to the fibrotic response were also assessed in a mouse model of bleomycin-induced skin fibrosis, by histologic, immunohistochemical, RT-PCR, and protein analyses. RESULTS TGFbeta1 induced ET-1 expression in human dermal fibroblasts through Smad- and activator protein 1/JNK-dependent signaling. The ability of TGFbeta1 to induce the expression of profibrotic genes was dependent on ET-1. Adenovirus-mediated overexpression of TGFbeta1 and ET-1 in mouse skin was associated with accelerated wound closure, increased fibrogenesis, and excessive scarring. Treatment with bosentan prevented the effects of TGFbeta1. In the bleomycin-induced fibrosis model, treatment with GW788388 and bosentan prevented the fibrotic response. CONCLUSION Our results strongly support the notion that the TGFbeta1/ET-1 axis has a role in wound repair and skin fibrosis. ET-1 receptor antagonists, such as bosentan, may represent a useful therapeutic tool in the treatment of excessive scarring and fibrosis-related diseases.

Vascular Smooth Muscle Cell Phenotypic Changes in Patients With Marfan Syndrome

Objective— Marfan’s syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-&bgr; signaling. TGF-&bgr; is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-&bgr; signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan’s syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results— Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (&agr;-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-&bgr; pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. Conclusions— In Marfan VSMC, both in tissue and in culture, there are variable TGF-&bgr;-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan’s syndrome aneurysm formation.

LOXL4 Is Induced by Transforming Growth Factor β1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

ABSTRACT Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis.

The profibrotic role of endothelin-1: is the door still open for the treatment of fibrotic diseases?

The endothelin (ET) system consists of two G-protein-coupled receptors (ETA and ETB), three peptide ligands (ET-1, ET-2 and ET-3), and two activating peptidases (endothelin-converting enzyme-, ECE-1 and ECE-2). While initially described as a vasoregulatory factor, shown to influence several cardiovascular diseases, from hypertension to heart failure, ET-1, the predominant form in most cells and tissues, has expanded its pathophysiological relevance by recent evidences implicating this factor in the regulation of fibrosis. In this article, we review the current knowledge of the role of ET-1 in the development of fibrosis, with particular focus on the regulation of its biosynthesis and the molecular mechanisms involved in its profibrotic actions. We summarize also the contribution of ET-1 to fibrotic disorders in several organs and tissues. The development and availability of specific ET receptor antagonists have greatly stimulated a number of clinical trials in these pathologies that unfortunately have so far given negative or inconclusive results. This review finally discusses the circumstances underlying these disappointing results, as well as provides basic and clinical researchers with arguments to keep exploring the complex physiology of ET-1 and its therapeutic potential in the process of fibrosis.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

AIMS After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery.

miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor‐β1 (TGF‐β1) stimulates the production of NADPH oxidase 4 (NOX4)‐dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR‐9‐5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF‐β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR‐9‐5p abrogates TGF‐β1‐dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin‐induced LF, miR‐9‐5p dramatically reduces fibrogenesis and inhibition of miR‐9‐5p and prevents its anti‐fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR‐9‐5p are found. In omentum‐derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR‐9‐5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF‐β1 induces miR‐9‐5p expression as a self‐limiting homeostatic response.

Matrix Stiffening and β1 Integrin Drive Subtype-Specific Fibroblast Accumulation in Lung Cancer

The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non–small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal- or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by β1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in β1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC–TAFs exhibited higher FAK (pY397), β1 expression, and ERK1/2 (pT202/Y204) than ADC–TAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC–TAFs (>50%) compared with ADC–TAFs (10%–20%). In contrast, SCC–TAFs were largely serum desensitized, whereas ADC–TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC–TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or β1 integrin-dependent mechano regulation may be effective against SCC–TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC–TAFs. Implications: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells. Mol Cancer Res; 13(1); 161–73. ©2014 AACR.

A Pathogenetic Role for Endothelin-1 in Peritoneal Dialysis-Associated Fibrosis

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-β1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-β1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-β1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-β1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-β1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.