Serum markers of pulmonary epithelial damage in systemic sclerosis-associated interstitial lung disease and disease progression.

BACKGROUND AND OBJECTIVE: The course of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is highly variable, and accurate prognostic markers are needed. KL-6 is a mucin-like glycoprotein (MUC1) expressed by type II pneumocytes, while CYFRA 21-1 is expressed by alveolar and bronchiolar epithelial cells. Both are released into the blood from cell injury. METHODS: Serum KL-6 and CYFRA 21-1 levels were measured in a retrospective (n = 189) and a prospective (n = 118) cohort of SSc patients. Genotyping of MUC1 rs4072037 was performed. Linear mixed-effect models were used to evaluate the relationship with change in lung function parameters over time, while association with survival was evaluated with Cox proportional hazard analysis. RESULTS: In both cohorts, KL-6 and CYFRA 21-1 were highest in patients with lung involvement, and in patients with extensive rather than limited ILD. KL-6 was higher in patients carrying the MUC1 rs4072037 G allele in both cohorts. In patients with SSc-ILD, serum KL-6, but not CYFRA 21-1, was significantly associated with DLCO decline in both cohorts (P = 0.001 and P = 0.004, respectively), and with FVC decline in the retrospective cohort (P = 0.005), but not the prospective cohort. When combining the cohorts and subgrouping by severity (median CPI = 45.97), KL-6 remained predictive of decline in DLCO in both milder (P = 0.007) and more severe disease (P = 0.02) on multivariable analysis correcting for age, gender, ethnicity, smoking history and MUC1 allele carriage. CONCLUSION: Our results suggest serum KL-6 predicts decline in lung function in SSc, suggesting its clinical utility in risk stratification for progressive SSc-ILD.

Endothelial C-Type Natriuretic Peptide Is a Critical Regulator of Angiogenesis and Vascular Remodeling.

BACKGROUND: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia. METHODS: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP-/-, global natriuretic peptide receptor (NPR)-B-/- and NPR-C-/- animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease. RESULTS: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP-/- and NPR-C-/-, but not NPR-B-/-, mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of Gi, ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level. CONCLUSIONS: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.

Functional and phenotypic heterogeneity of Th17 cells in health and disease.

BACKGROUND: Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. DESIGN: In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. RESULTS: The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. CONCLUSION: This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases.

Insights into myofibroblasts and their activation in scleroderma: opportunities for therapy?

PURPOSE OF REVIEW: The persistence of myofibroblasts is a key feature of fibrosis and in fibrotic diseases including scleroderma. This review evaluates the emerging concepts of the origins and cell populations that contribute to myofibroblasts and the molecular mechanisms that govern phenotypic conversion and that highlight opportunities for new interventional treatments in scleroderma. RECENT FINDINGS: Studies have defined heterogeneity in fibroblast-like cells that can develop into myofibroblast in normal wound healing, scarring and fibrosis. Characterizing these distinct cell populations and their behaviour has been a key focus. In addition, the overarching impact of epigenetic regulation of genes associated with inflammatory responses, cell signalling and cell communication and the extracellular matrix (ECM) has provided important insights into the formation of myofibroblast and their function. Important new studies include investigations into the relationship between inflammation and myofibroblast production and further evidence has been gathered that reveal the importance of ECM microenvironment, biomechanical sensing and mechanotransduction. SUMMARY: This review highlights our current understanding and outlines the increasing complexity of the biological processes that leads to the appearance of the myofibroblast in normal functions and in diseased tissues. We also focus on areas of special interest in particular, studies that have therapeutic potential in fibrosis and scleroderma.

Changes in macrophage transcriptome associate with systemic sclerosis and mediate GSDMA contribution to disease risk.

OBJECTIVES: Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc. METHODS: We carried out RNA sequencing and genome-wide genotyping in MDMs from 57 patients with SSc and 15 controls. Our differential expression and expression quantitative trait locus (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes. RESULTS: We identified 602 genes upregulated and downregulated in SSc macrophages that were significantly enriched for genes previously implicated in SSc susceptibility (P=5×10-4), and 270 cis-regulated genes in MDMs. Among these, GSDMA was reported to carry an SSc risk variant (rs3894194) regulating expression of neighbouring genes in blood. We show that GSDMA is upregulated in SSc MDMs (P=8.4×10-4) but not in the skin, and is a significant eQTL in SSc macrophages and lipopolysaccharide/interferon gamma (IFNγ)-stimulated monocytes. Furthermore, we identify an SSc macrophage transcriptome signature characterised by upregulation of glycolysis, hypoxia and mTOR signalling and a downregulation of IFNγ response pathways. CONCLUSIONS: Our data further establish the link between macrophages and SSc, and suggest that the contribution of the rs3894194 risk variant to SSc susceptibility can be mediated by GSDMA expression in macrophages.

Macrophage epoxygenase determines a profibrotic transcriptome signature.

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography-tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.

Endothelial to Mesenchymal Transition Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial cell dysfunction and vascular remodeling. Normally, the endothelium forms an integral cellular barrier to regulate vascular homeostasis. During embryogenesis endothelial cells exhibit substantial plasticity that contribute to cardiac development by undergoing endothelial-to-mesenchymal transition (EndoMT). We determined the presence of EndoMT in the pulmonary vasculature in vivo and the functional effects on pulmonary artery endothelial cells (PAECs) undergoing EndoMT in vitro. Histologic assessment of patients with systemic sclerosis-associated PAH and the hypoxia/SU5416 mouse model identified the presence von Willebrand factor/α-smooth muscle actin-positive endothelial cells in up to 5% of pulmonary vessels. Induced EndoMT in PAECs by inflammatory cytokines IL-1ß, tumor necrosis factor α, and transforming growth factor ß led to actin cytoskeleton reorganization and the development of a mesenchymal morphology. Induced EndoMT cells exhibited up-regulation of mesenchymal markers, including collagen type I and α-smooth muscle actin, and a reduction in endothelial cell and junctional proteins, including von Willebrand factor, CD31, occludin, and vascular endothelial-cadherin. Induced EndoMT monolayers failed to form viable biological barriers and induced enhanced leak in co-culture with PAECs. Induced EndoMT cells secreted significantly elevated proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α, and supported higher immune transendothelial migration compared with PAECs. These findings suggest that EndoMT may contribute to the development of PAH.

Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

Exploring Anti-Fibrotic Effects of Adipose-Derived Stem Cells: Transcriptome Analysis upon Fibrotic, Inflammatory, and Hypoxic Conditioning.

Autologous fat transfers show promise in treating fibrotic skin diseases, reversing scarring and stiffness, and improving quality of life. Adipose-derived stem cells (ADSCs) within these grafts are believed to be crucial for this effect, particularly their secreted factors, though the specific mechanisms remain unclear. This study investigates transcriptomic changes in ADSCs after in vitro fibrotic, inflammatory, and hypoxic conditioning. High-throughput gene expression assays were conducted on ADSCs exposed to IL1-ß, TGF-ß1, and hypoxia and in media with fetal bovine serum (FBS). Flow cytometry characterized the ADSCs. RNA-Seq analysis revealed distinct gene expression patterns between the conditions. FBS upregulated pathways were related to the cell cycle, replication, wound healing, and ossification. IL1-ß induced immunomodulatory pathways, including granulocyte chemotaxis and cytokine production. TGF-ß1 treatment upregulated wound healing and muscle tissue development pathways. Hypoxia led to the downregulation of mitochondria and cellular activity.

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-ß and further enhanced by hypoxia. The effect of TGF-ß was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Mucin 5B promoter polymorphism is associated with idiopathic pulmonary fibrosis but not with development of lung fibrosis in systemic sclerosis or sarcoidosis.

BACKGROUND: A polymorphism (rs35705950) 3 kb upstream of MUC5B, the gene encoding Mucin 5 subtype B, has been shown to be associated with familial and sporadic idiopathic pulmonary fibrosis (IPF). We set out to verify whether this variant is also a risk factor for fibrotic lung disease in other settings and to confirm the published findings in a UK Caucasian IPF population. METHODS: Caucasian UK healthy controls (n=416) and patients with IPF (n=110), sarcoidosis (n=180) and systemic sclerosis (SSc) (n=440) were genotyped to test for association. The SSc and sarcoidosis cohorts were subdivided according to the presence or absence of fibrotic lung disease. To assess correlation with disease progression, time to decline in forced vital capacity and/or lung carbon monoxide transfer factor was used in the IPF and SSc groups, while a persistent decline at 4 years since baseline was evaluated in patients with sarcoidosis. RESULTS: A significant association of the MUC5B promoter single nucleotide polymorphism with IPF (p=2.04 × 10(-17); OR 4.90, 95% CI 3.42 to 7.03) was confirmed in this UK population. The MUC5B variant was not a risk factor for lung fibrosis in patients with SSc or sarcoidosis and did not predict more rapidly progressive lung disease in any of the groups. Rather, a trend for a longer time to decline in forced vital capacity was observed in patients with IPF. CONCLUSIONS: We confirm the MUC5B variant association with IPF. We did not observe an association with lung fibrosis in the context of SSc or sarcoidosis, potentially highlighting fundamental differences in genetic susceptibility, although the limited subgroup numbers do not allow a definitive exclusion of an association.

Microarray profiling reveals suppressed interferon stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease.

BACKGROUND: Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options. Progression of pulmonary fibrosis involves expanding populations of fibroblasts, and the accumulation of extracellular matrix proteins. Characterisation of SSc lung fibroblast gene expression profiles underlying the fibrotic cell phenotype could enable a better understanding of the processes leading to the progressive build-up of scar tissue in the lungs. In this study we evaluate the transcriptomes of fibroblasts isolated from SSc lung biopsies at the time of diagnosis, compared with those from control lungs. METHODS: We used Affymetrix oligonucleotide microarrays to compare the gene expression profile of pulmonary fibroblasts cultured from 8 patients with pulmonary fibrosis associated with SSc (SSc-ILD), with those from control lung tissue peripheral to resected cancer (n=10). Fibroblast cultures from 3 patients with idiopathic pulmonary fibrosis (IPF) were included as a further comparison. Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered. Microarray expression data was verified by qRT-PCR and/or western blot analysis. RESULTS: A total of 843 genes were identified as differentially expressed in pulmonary fibroblasts from SSc-ILD and/or IPF compared to control lung, with a large overlap in the expression profiles of both diseases. We observed increased expression of a TGF-ß response signature including fibrosis associated genes and myofibroblast markers, with marked heterogeneity across samples. Strongly suppressed expression of interferon stimulated genes, including antiviral, chemokine, and MHC class 1 genes, was uniformly observed in fibrotic fibroblasts. This expression profile includes key regulators and mediators of the interferon response, such as STAT1, and CXCL10, and was also independent of disease group. CONCLUSIONS: This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung. The data suggests that the repressed expression of interferon-stimulated genes may underpin critical aspects of the profibrotic fibroblast phenotype, identifying an area in pulmonary fibrosis that requires further investigation.

Unravelling morphoea aetiopathogenesis by next-generation sequencing of paired skin biopsies.

BACKGROUND: Morphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho's lines of epidermal development, providing potential pathogenic clues. OBJECTIVE: The first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk. METHODOLOGY: Skin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results. RESULTS: Sixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFß, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal 'damage' signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt. CONCLUSION: This study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies.

Single-Cell Analysis of ADSC Interactions with Fibroblasts and Endothelial Cells in Scleroderma Skin.

Adipose-derived stem cells (ADSCs) as part of autologous fat grafting have anti-fibrotic and anti-inflammatory effects, but the exact mechanisms of action remain unknown. By simulating the interaction of ADSCs with fibroblasts and endothelial cells (EC) from scleroderma (SSc) skin in silico, we aim to unravel these mechanisms. Publicly available single-cell RNA sequencing data from the stromal vascular fraction of 3 lean patients and biopsies from the skin of 10 control and 12 patients with SSc were obtained from the GEO and analysed using R and Seurat. Differentially expressed genes were used to compare the fibroblast and EC transcriptome between controls and SSc. GO and KEGG functional enrichment was performed. Ligand-receptor interactions of ADSCs with fibroblasts and ECs were explored with LIANA. Pro-inflammatory and extracellular matrix (ECM) interacting fibroblasts were identified in SSc. Arterial, capillary, venous and lymphatic ECs showed a pro-fibrotic and pro-inflammatory transcriptome. Most interactions with both cell types were based on ECM proteins. Differential interactions identified included NTN1, VEGFD, MMP2, FGF2, and FNDC5. The ADSC secretome may disrupt vascular and perivascular inflammation hubs in scleroderma by promoting angiogenesis and especially lymphangiogenesis. Key phenomena observed after fat grafting remain unexplained, including modulation of fibroblast behaviour.

Endothelin-1 stimulates colon cancer adjacent fibroblasts.

Endothelin-1 (ET-1) is produced by and stimulates colorectal cancer cells. Fibroblasts produce tumour stroma required for cancer development. We investigated whether ET-1 stimulated processes involved in tumour stroma production by colonic fibroblasts. Primary human fibroblasts, isolated from normal tissues adjacent to colon cancers, were cultured with or without ET-1 and its antagonists. Cellular proliferation, migration and contraction were measured. Expression of enzymes involved in tumour stroma development and alterations in gene transcription were determined by Western blotting and genome microarrays. ET-1 stimulated proliferation, contraction and migration (p < 0.01 v control) and the expression of matrix degrading enzymes TIMP-1 and MMP-2, but not MMP-3. ET-1 upregulated genes for profibrotic growth factors and receptors, signalling molecules, actin modulators and extracellular matrix components. ET-1 stimulated colonic fibroblast cellular processes in vitro that are involved in developing tumour stroma. Upregulated genes were consistent with these processes. By acting as a strong stimulus for tumour stroma creation, ET-1 is proposed as a target for adjuvant cancer therapy.

Expression of integrin beta1 by fibroblasts is required for tissue repair in vivo.

In tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin ß1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of α-smooth muscle actin (α-SMA). Integrin-ß1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced α-SMA stress fiber formation. Loss of integrin ß1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-ß1-deficient fibroblasts showed reduced activation of latent TGFß. Addition of active TGFß alleviated the phenotype of integrin-ß1-deficient mice. Thus integrin ß1 is essential for normal wound healing, where it acts, at least in part, through a TGFß-dependent mechanism in vivo.

Focal adhesion kinase and reactive oxygen species contribute to the persistent fibrotic phenotype of lesional scleroderma fibroblasts.

OBJECTIVE: Fibrotic diseases such as SSc (systemic sclerosis, scleroderma) are characterized by the abnormal presence of the myofibroblast, a specialized type of fibroblast that overexpresses the highly contractile protein α-smooth muscle actin. Myofibroblasts display excessive adhesive properties and hence exert a potent mechanical force. We aim to identify the precise contribution of adhesive signalling, which requires integrin-mediated activation of focal adhesion kinase (FAK)/src, to fibrogenic gene expression in normal and fibrotic SSc fibroblasts. METHODS: We subject either FAK wild-type and knockout fibroblasts or normal and SSc fibroblasts treated with FAK/src inhibitors to real-time polymerase chain, western blot, cell migration and collagen gel contraction analyses. RESULTS: FAK operates downstream of both integrin ß1 and reactive oxygen species (ROS) to promote the expression of genes involved in matrix production and remodelling, including CCN2, α-smooth muscle actin and type I collagen. Blocking either FAK/src with PP2 or ROS with N-acetyl cysteine alleviates the elevated contractile and migratory capability of lesional SSc dermal fibroblasts. CONCLUSIONS: Excessive adhesive signalling is intimately involved with the fibrotic phenotype of lesional SSc fibroblasts; blocking adhesive signalling or ROS generation may be beneficial in controlling the fibrosis observed in SSc.

Morphometric analysis of intralobular, interlobular and pleural lymphatics in normal human lung.

In spite of their presumed relevance in maintaining interalveolar septal fluid homeostasis, the knowledge of the anatomy of human lung lymphatics is still incomplete. The recent discovery of reliable markers specific for lymphatic endothelium has led to the observation that, contrary to previous assumptions, human lymphatic vessels extend deep inside the pulmonary lobule in association with bronchioles, intralobular arterioles or small pulmonary veins. The aim of this study was to provide a morphometric characterization of lymphatic vessels in the periphery of the human lung. Human lung sections were immunolabelled with the lymphatic marker D2-40, followed by blood vessel staining with von Willebrand Factor. Lymphatic vessels were classified into: intralobular (including those associated with bronchovascular bundles, perivascular, peribronchiolar and interalveolar), pleural (in the connective tissue of the visceral pleura), and interlobular (in interlobular septa). The percentage area occupied by the lymphatic lumen was much greater in the interlobular septa and in the subpleural space than in the lobule. Most of the intralobular lymphatic vessels were in close contact with a blood vessel, either alone or within a bronchovascular bundle, whereas 7% were associated with a bronchiole and < 1% were not connected to blood vessels or bronchioles (interalveolar). Intralobular lymphatic size progressively decreased from bronchovascular through to peribronchiolar, perivascular and interalveolar lymphatics. Lymphatics associated with bronchovascular bundles had similar morphometric characteristics to pleural and interlobular lymphatics. Shape factors were similar across lymphatic populations, except that peribronchiolar lymphatics had a marginally increased roundness and circularity, suggesting a more regular shape due to increased filling, and interlobular lymphatics had greater elongation, due to a greater proportion of conducting lymphatics cut longitudinally. Unsupervised cluster analysis confirmed a marked heterogeneity of lymphatic vessels both within and between groups, with a cluster of smaller vessels specifically represented in perivascular and interalveolar lymphatics within the alveolar interstitium. Our data indicate that intralobular lymphatics are a heterogeneous population, including vessels surrounding the bronchovascular bundle analogous to the conducting vessels present in the pleural and interlobular septa, many small perivascular lymphatics responsible for maintaining fluid balance in the alveolar interstitium, and a minority of intermediate lymphatics draining the peripheral airways. These lymphatic populations could be differentially involved in the pathogenesis of diseases preferentially involving distinct lung compartments.

Cyclic mechanical stress downregulates endothelin-1 and its responsive genes independently of TGFß1 in dermal fibroblasts.

Mechanical forces are highly variable ranging from the ubiquitous gravity force to compression, fluid shear, torsion, tension and other forms. Mechanical forces act on cells and modulate their biological responses by regulating gene transcription, enzyme and growth factor activity. In soft connective tissues, formation of myofibroblasts strictly requires a mechanically loaded environment in addition to local transforming growth factor (TGF)-ß activity, which itself can be modulated by the mechanical status of the environment. The aim of this study was to monitor the adaptive responses of primary dermal fibroblasts towards cyclic mechanical stress under conditions of high force to better understand the regulation of gene expression in normal skin and mechanisms of gene regulation in mechanically altered fibrotic skin. Primary murine dermal fibroblasts were exposed to equi-biaxial tensile strain. Cyclic mechanical tension was applied at a frequency of 0.1 Hz (6× /min) for 24 h with a maximal increase in surface area of 15%. This treatment resulted in downregulation of alpha smooth muscle actin (αSMA) and connective tissue growth factor (CTGF) but not of TGFß1 expression. Cyclic strain also strongly reduced endothelin-1 (ET-1) expression and supplementing strained cultures with exogenous ET-1 rescued αSMA and CTGF levels. Of note, no biologically significant levels of TGFß1 activity were detected in strained cultures. We provide evidence for a novel, TGFß1-independent mechanism regulating ET-1 expression in dermal fibroblasts by biomechanical forces. Modulation of ET-1-dependent activities regulates downstream fibrotic marker genes; this pathway might therefore provide an approach to attenuate myofibroblast differentiation.

CCN2 is required for bleomycin-induced skin fibrosis in mice.

OBJECTIVE: No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast-specific deletion of CCN2 are resistant to bleomycin-induced skin scleroderma. METHODS: Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell-specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α-SMA messenger RNA in response to transforming growth factor ß (TGFß) was impaired. RESULTS: The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant α-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFß, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment. CONCLUSION: These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.