Extracellular Vesicles in the Skin Microenvironment: Emerging Roles as Biomarkers and Therapeutic Tools in Dermatologic Health and Disease.

The recent discovery of extracellular vesicles (EVs) carrying cargo consisting of various bioactive macromolecules that can modulate the phenotype of recipient target cells has revealed an important new mechanism through which cells can signal their neighbors and regulate their microenvironment. Because EV cargo and composition correlate with the physiologic state of their cell of origin, investigations into the role of EVs in disease pathogenesis and progression have become an area of intense study. The physiologic and pathologic effects of EVs on their microenvironment are incredibly diverse and include the modulation of molecular pathways involved in angiogenesis, inflammation, wound healing, epithelial-mesenchymal transition, proliferation, and immune escape. This review examines recent studies on the role of EVs in diseases of the skin and on how differences in EV composition and cargo can alter cell states and the surrounding microenvironment. We also discuss the potential clinical applications of EVs in skin disease diagnosis and management. We examine their value as an easily isolated source of biomarkers to predict disease prognosis or to monitor patient response to treatment. Given the ability of EVs to modulate disease-specific signaling pathways, we also assess their potential to serve as novel personalized precision therapeutic tools for dermatological diseases.

Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis.

Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis.

Keloid disorder: Fibroblast differentiation and gene expression profile in fibrotic skin diseases.

Keloid disorder, a group of fibroproliferative skin diseases, is characterized by unremitting accumulation of the extracellular matrix (ECM) of connective tissue, primarily collagen, to develop cutaneous tumors on the predilection sites of skin. There is a strong genetic predisposition for keloid formation, and individuals of African and Asian ancestry are particularly prone. The principal cell type responsible for ECM accumulation is the myofibroblast derived from quiescent resident skin fibroblasts either through trans-differentiation or from keloid progenitor stem cells with capacity for multi-lineage differentiation and self-renewal. The biosynthetic pathways leading to ECM accumulation are activated by several cytokines, but particularly by TGF-ß signalling. The mechanical properties of the cellular microenvironment also play a critical role in the cell's response to TGF-ß, as demonstrated by culturing of fibroblasts derived from keloids and control skin on substrata with different degrees of stiffness. These studies also demonstrated that culturing of fibroblasts on tissue culture plastic in vitro does not reflect their biosynthetic capacity in vivo. Collectively, our current understanding of the pathogenesis of keloids suggests a complex network of interacting cellular, molecular and mechanical factors, with distinct pathways leading to myofibroblast differentiation and activation. Keloids can serve as a model system of fibrotic diseases, a group of currently intractable disorders, and deciphering of the critical pathogenetic steps leading to ECM accumulation is expected to identify targets for pharmacologic intervention, not only for keloids but also for a number of other, both genetic and acquired, fibrotic diseases.

Chemical exposure-induced systemic fibrosing disorders: Novel insights into systemic sclerosis etiology and pathogenesis.

Numerous drugs and chemical substances are capable of inducing exaggerated tissue fibrotic responses. The vast majority of these agents cause localized fibrotic tissue reactions or fibrosis confined to specific organs. Although much less frequent, chemically-induced systemic fibrotic disorders have been described, sometimes occurring as temporally confined outbreaks. These include the Toxic Oil Syndrome (TOS), the Eosinophilia-Myalgia Syndrome (EMS), and Nephrogenic Systemic Fibrosis (NSF). Although each of these disorders displays some unique characteristics, they all share crucial features with Systemic Sclerosis (SSc), the prototypic idiopathic systemic fibrotic disease, including vasculopathy, chronic inflammatory cell infiltration of affected tissues, and cutaneous and visceral tissue fibrosis. The study of the mechanisms and molecular alterations involved in the development of the chemically-induced systemic fibrotic disorders has provided valuable clues that may allow elucidation of SSc etiology and pathogenesis. Here, we review relevant aspects of the TOS, EMS, and NSF epidemic outbreaks of chemically-induced systemic fibrosing disorders that provide strong support to the hypothesis that SSc is caused by a toxic or biological agent that following its internalization by endothelial cells induces in genetically predisposed individuals a series of molecular alterations that result in the development of SSc clinical and pathological alterations.

miRNA- and cytokine-associated extracellular vesicles mediate squamous cell carcinomas.

Exosomes, or small extracellular vesicles (sEVs), serve as intercellular messengers with key roles in normal and pathological processes. Our previous work had demonstrated that Dsg2 expression in squamous cell carcinoma (SCC) cells enhanced both sEV secretion and loading of pro-mitogenic cargo. In this study, using wild-type Dsg2 and a mutant form that is unable to be palmitoylated (Dsg2cacs), we investigated the mechanism by which Dsg2 modulates SCC tumour development and progression through sEVs. We demonstrate that palmitoylation was required for Dsg2 to regulate sub-cellular localisation of lipid raft and endosomal proteins necessary for sEV biogenesis. Pharmacological inhibition of the endosomal pathway abrogated Dsg2-mediated sEV release. In murine xenograft models, Dsg2-expressing cells generated larger xenograft tumours as compared to cells expressing GFP or Dsg2cacs. Co-treatment with sEVs derived from Dsg2-over-expressing cells increased xenograft size. Cytokine profiling revealed, Dsg2 enhanced both soluble and sEV-associated IL-8 and miRNA profiling revealed, Dsg2 down-regulated both cellular and sEV-loaded miR-146a. miR-146a targets IRAK1, a serine-threonine kinase involved in IL-8 signalling. Treatment with a miR-146a inhibitor up-regulated both IRAK1 and IL-8 expression. RNAseq analysis of HNSCC tumours revealed a correlation between Dsg2 and IL-8. Finally, elevated IL-8 plasma levels were detected in a subset of HNSCC patients who did not respond to immune checkpoint therapy, suggesting that these patients may benefit from prior anti-IL-8 treatment. In summary, these results suggest that intercellular communication through cell-cell adhesion, cytokine release and secretion of EVs are coordinated, and critical for tumour growth and development, and may serve as potential prognostic markers to inform treatment options. ABBREVIATIONS: Basal cell carcinomas, BCC; Betacellulin, BTC; 2-bromopalmitate, 2-Bromo; Cluster of differentiation, CD; Cytochrome c oxidase IV, COX IV; Desmoglein 2, Dsg2; Early endosome antigen 1, EEA1; Epidermal growth factor receptor substrate 15, EPS15; Extracellular vesicle, EV; Flotillin 1, Flot1; Glyceraldehyde-3-phosphate dehydrogenase, GAPH; Green fluorescent protein, GFP; Head and neck squamous cell carcinoma, HNSCC; Interleukin-1 receptor-associated kinase 1, IRAK1; Interleukin 8, IL-8; Large EV, lEV; MicroRNA, miR; Palmitoylacyltransferase, PAT; Ras-related protein 7 Rab7; Small EV, sEV; Squamous cell carcinoma, SCC; Tissue inhibitor of metalloproteinases, TIMP; Tumour microenvironment, TME.

Molecular characteristics and functional differences of anti-PM/Scl autoantibodies and two other distinct and unique supramolecular structures known as "EXOSOMES".

The term "exosome" has been applied to three distinct supramolecular entities, namely the PM/Scl autoantibodies or "RNA exosomes", transforming DNA fragments termed "DNA exosomes", and small size extracellular vesicles knows as "exosomes". Some of the molecular components of the "PM/Scl exosome complex" or "RNA exosome" are recognized by specific autoantibodies present in the serum from some Systemic Sclerosis (SSc), polymyositis (PM) and polymyositis SSc (PM/Scl) overlap syndrome patients. On the other hand, one of the most active focuses of laboratory investigation in the last decade has been the biogenesis and role of extracellular vesicles known as "exosomes". The remarkable ability of these "exosome" vesicles to alter the cellular phenotype following fusion with target cells and the release of their macromolecular cargo has revealed a possible role in the pathogenesis of numerous diseases, including malignant, inflammatory, and autoimmune disorders and may allow them to serve as theranostic agents for personalized and precision medicine. The indiscriminate use of the term "exosome" to refer to these three distinct molecular entities has engendered great confusion in the scientific literature. Here, we review the molecular characteristics and functional differences between the three molecular structures identified as "exosomes". Given the rapidly growing scientific interest in extravesicular exosomes, unless a solution is found the confusion in the literature resulting from the use of the term "exosomes" will markedly increase.

Abrogation of transforming growth factor-ß-induced tissue fibrosis in mice with a global genetic deletion of Nox4.

Excessive connective tissue deposition in skin and various internal organs is characteristic of systemic sclerosis (SSc). The profibrotic growth factor TGF-ß plays a crucial role in SSc pathogenesis. The expression of NADPH oxidase 4 (NOX4), a critical mediator of oxidative stress, is potently stimulated by TGF-ß. Here, we evaluated the effect of NOX4 on the development of TGF-ß-induced tissue fibrosis. C57BL6/J control mice and Nox4 knockout mice were implanted subcutaneously with osmotic pumps containing either saline or 2.5 µg TGF-ß1. After 28 days, skin and lung samples were isolated for histopathologic analysis, measurement of hydroxyproline content and gene expression analysis. Histopathology of skin and lungs from normal C57BL6/J mice treated with TGF-ß1 showed profound dermal fibrosis and peribronchial and diffuse interstitial lung fibrosis. In contrast, TGF-ß-treated Nox4 knockout mice showed normal skin and lung histology. Hydroxyproline levels in TGF-ß-treated C57BL6/J mice skin and lungs demonstrated significant increases, however, hydroxyproline content of TGF-ß-treated Nox4 knockout mice tissues was not changed. Expression of various profibrotic and fibrosis-associated genes was upregulated in skin and lungs of TGF-ß1-treated C57BL6/J mice but was not significantly changed in TGF-ß1-treated Nox4 knockout mice. The induction of skin and lung tissue fibrosis by TGF-ß1 parenteral administration in mice was abrogated by the genetic deletion of Nox4 confirming that NOX4 is an essential mediator of the profibrotic effects of TGF-ß. These results suggest Nox4 inhibition as a potential therapeutic target for SSc and other fibroproliferative disorders.

Existing and novel biomarkers for precision medicine in systemic sclerosis.

The discovery and validation of biomarkers resulting from technological advances in the analysis of genomic, transcriptomic, lipidomic and metabolomic pathways involved in the pathogenesis of complex human diseases have led to the development of personalized and rationally designed approaches for the clinical management of such disorders. Although some of these approaches have been applied to systemic sclerosis (SSc), an unmet need remains for validated, non-invasive biomarkers to aid in the diagnosis of SSc, as well as in the assessment of disease progression and response to therapeutic interventions. Advances in global transcriptomic technology over the past 15 years have enabled the assessment of microRNAs that circulate in the blood of patients and the analysis of the macromolecular content of a diverse group of lipid bilayer membrane-enclosed extracellular vesicles, such as exosomes and other microvesicles, which are released by all cells into the extracellular space and circulation. Such advances have provided new opportunities for the discovery of biomarkers in SSc that could potentially be used to improve the design and evaluation of clinical trials and that will undoubtedly enable the development of personalized and individualized medicine for patients with SSc.

Trametinib prevents mesothelial-mesenchymal transition and ameliorates abdominal adhesion formation.

BACKGROUND: Intra-abdominal adhesions are a major cause of morbidity after abdominal or gynecologic surgery. However, knowledge about the pathogenic mechanism(s) is limited, and there are no effective treatments. Here, we investigated a mouse model of bowel adhesion formation and the effect(s) of an Federal Drug Administration-approved drug (trametinib) in preventing adhesion formation. MATERIALS AND METHODS: C57BL/6 mice were used to develop a consistent model of intra-abdominal adhesion formation by gentle cecal abrasion with mortality rates of <10%. Adhesion formation was analyzed histologically and immunochemically to characterize the expression of pro-fibrotic marker proteins seen in pathologic scaring and included alpha smooth muscle actin (αSMA) and fibronectin EDA (FNEDA) which arises from alternative splicing of the fibronectin messenger RNA resulting in different protein isoforms. Trichrome staining assessed collagen deposition. Quantitative polymerase chain reaction analysis of RNA isolated from adhesions by laser capture microscopy was carried out to assess pro-fibrotic gene expression. To block adhesion formation, trametinib was administered via a subcutaneous osmotic pump. RESULTS: Adhesions were seen as early as post-operative day 1 with extensive adhesions being formed and vascularized by day 5. The expression of the FNEDA isoform occurred first with subsequent expression of αSMA and collagen. The drug trametinib was chosen for in vivo studies because it effectively blocked the mesothelial to mesenchymal transition of rat mesothelium. Trametinib, at the highest dose used (3 mg/kg/d), prevented adhesion formation while at lower doses, adhesions were usually limited, as evidenced by the presence of FNEDA isoform but not αSMA. CONCLUSIONS: Cecal abrasion in mice is a reliable model to study abdominal adhesions, which can be ameliorated using the MEK1/2 inhibitor trametinib. While blocking adhesion formation at the cell and molecular levels, trametinib, at the therapeutic doses utilized, did not impair the wound healing at the laparotomy site.

Abrogation of transforming growth factor-ß-induced tissue fibrosis in TBRIcaCol1a2Cre transgenic mice by the second generation tyrosine kinase inhibitor SKI-606 (Bosutinib).

Transforming growth factor-ß (TGF-ß) plays a crucial role in the pathogenesis of Systemic Sclerosis (SSc) and other fibrotic disorders. TGF-ß-mediated c-Abl and Src kinase activation induces strong profibrotic cascade signaling. The purpose of this study was to test in vivo the antifibrotic activity of Bosutinib (SKI-606), a second generation c-Abl and Src kinase inhibitor, on TGF-ß induced cutaneous and pulmonary fibrosis. For this purpose, we employed the TBRIcaCol1a2Cre transgenic mice expressing an inducible constitutively active TGF-ß receptor 1 constitutively activated by Col1a promoter-mediated Cre recombinase. The mice were treated parenterally with 2.5, 5.0 or 10.0 mg/kg/day of Bosutinib for 42 days. Skin and lungs from control and Bosutinib-treated mice (n = 6 per group) were assessed by histopathology, measurement of tissue hydroxyproline content, PCR analysis of tissue fibrosis associated gene expression, and evidence of myofibroblast activation. Mice with constitutive TGF-ß-1 signaling displayed severe cutaneous and pulmonary fibrosis. Bosutinib administration decreased collagen deposition and hydroxyproline content in the dermis and lungs in a dose-dependent manner. Bosutinib also reversed the marked increase in profibrotic and myofibroblast activation-associated gene expression. These results demonstrate that constitutive TGF-ß-1-signaling-induced cutaneous and pulmonary fibrosis were abrogated in a dose-related manner following parenteral administration of the c-Abl and Src tyrosine kinase inhibitor, Bosutinib. These results indicate that Bosutinib may be a potential therapeutic agent for tissue fibrosis in SSc and other fibroproliferative disorders.

Identification of novel systemic sclerosis biomarkers employing aptamer proteomic analysis.

There is an important unmet need for clinically validated non-invasive biomarkers for SSc diagnosis, assessment of disease activity, extent of internal organ involvement, therapeutic response and prognosis. There is also an unmet need for biomarkers to accurately differentiate primary RP from recent onset RP evolving into SSc. The lack of sensitive and specific biomarkers for SSc and SSc-associated RP is a limitation for the optimal clinical management of these patients. The development of highly sensitive and specific proteomic analysis employing aptamers and the expansion in the number of proteins that can be specifically identified by aptamer proteomics have opened new horizons for biomarker discovery. Here, we review the background and rationale for aptamer proteomic analysis for the identification of novel non-invasive biomarkers for SSc and recent onset RP evolving into SSc. Large scale application of aptamer proteomic platforms for this purpose will be of substantial value for the precision and personalized medical care of SSc patients. These studies will be placed in context by comparison with proteomic biomarker studies performed for other rheumatological inflammatory and autoimmune diseases.

Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg2 increased EV release and mitogenic content including epidermal growth factor receptor and c-Src. Inhibiting ectodomain shedding of Dsg2 with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg2 in EVs and reduced EV release. When cocultured with Dsg2/green fluorescence protein-expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/2 and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg2 was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg2 C-terminal fragment and epidermal growth factor receptor. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.-Overmiller, A. M., Pierluissi, J. A., Wermuth, P. J., Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, L. A., Wahl, J. K. III, South, A. P., Mahoney, M. G. Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

Endothelial cell-specific activation of transforming growth factor-ß signaling in mice induces cutaneous, visceral, and microvascular fibrosis.

In this study, we tested the hypothesis that constitutive endothelial cell-specific activation of TGF-ß signaling induces tissue fibrosis and vasculopathy resembling the characteristic fibrotic and vascular alterations of systemic sclerosis. Transgenic mice with inducible expression of a constitutively active TGF-ß receptor I specifically in endothelial cells were generated by intercrossing mice harboring a constitutively active TGF-ß receptor I with a mouse strain containing the endothelial cell-specific Cdh5 gene promoter directing the tamoxifen-inducible expression of the Cre-ERT2 cassette. Administration of tamoxifen to these mice would result in constitutive TGF-ß activation and signaling confined to endothelial lineage cells. The effects of constitutive TGF-ß endothelial cell activation were assessed by histopathological examination of skin and various internal organs, tissue hydroxyproline analysis, and assessment of expression of myofibroblast differentiation and TGF-ß signaling genes employing real-time PCR and immunohistochemical staining of lung vessels for endothelial- and myofibroblast-specific proteins. Constitutive TGFß-1 signaling in endothelial cells resulted in cutaneous and visceral fibrosis with prominent fibrotic involvement of the lungs and severe perivascular and subendothelial fibrosis of small arterioles. A marked increase in the expression of fibrosis-associated genes and of genes indicative of myofibroblast activation was also found. Confocal microscopy of lung vessels showed evidence consistent with the induction of endothelial-to-mesenchymal transition (EndoMT). Taken together, our data indicate that transgenic mice with constitutive endothelial cell-specific activation of TGF-ß signaling display severe cutaneous, pulmonary, and microvascular fibrosis resembling the fibrotic and microvascular alterations characteristic of systemic sclerosis.

Exosomes isolated from serum of systemic sclerosis patients display alterations in their content of profibrotic and antifibrotic microRNA and induce a profibrotic phenotype in cultured normal dermal fibroblasts.

OBJECTIVES: Exosomes are lipid bilayer-bound microvesicles containing various macromolecules including numerous microRNA (miRNA). Exosomes mediate intercellular communication by fusing and releasing their macromolecular content into target cells. Here, we analysed the content of profibrotic and antifibrotic miRNAs in exosomes isolated from the serum of systemic sclerosis (SSc) patients and tested their ability to induce a profibrotic phenotype in normal human dermal fibroblasts in vitro. METHODS: Exosomes were isolated from serum from patients with limited cutaneous or diffuse cutaneous SSc and were characterised by Nanosight Particle Tracking Analysis, exosome antibody arrays, and transmission electron microscopy. The content of nine profibrotic and eighteen antifibrotic miRNA was assessed in the isolated exosomes by semiquantitative real time PCR. The effects of the isolated exosomes on cultured normal human dermal fibroblasts were assessed by real time PCR and Western blotting. RESULTS: The isolated serum exosomes displayed the expected exosome size and morphology and contained characteristic exosome proteins. Six profibrotic miRNAs were increased and ten antifibrotic miRNAs were decreased in SSc serum exosomes compared to normal serum exosomes. The levels of eight miRNA were significantly different between exosomes from limited and diffuse SSc. Exosomes isolated from both limited or diffuse SSc patients caused dose-dependent stimulation of profibrotic gene expression and type I collagen and fibronectin production and secretion in normal human dermal fibroblasts in vitro. CONCLUSIONS: Serum exosomes from SSc patients contain miRNA displaying a markedly profibrotic profile and induce a profibrotic phenotype in target normal fibroblasts in vitro suggesting a plausible mechanism for the extension of the fibrotic SSc process to non-affected tissues.

Stimulation of Transforming Growth Factor-ß1-Induced Endothelial-To-Mesenchymal Transition and Tissue Fibrosis by Endothelin-1 (ET-1): A Novel Profibrotic Effect of ET-1.

TGF-ß-induced endothelial-to-mesenchymal transition (EndoMT) is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role in the pathogenesis of various fibrotic processes. Endothelin-1 (ET-1) has been implicated in the development of tissue fibrosis but its participation in TGF-ß-induced EndoMT has not been studied. Here we evaluated the role of ET-1 on TGF-ß1-induced EndoMT in immunopurified CD31+/CD102+ murine lung microvascular endothelial cells. The expression levels of α-smooth muscle actin (α-SMA), of relevant profibrotic genes, and of various transcription factors involved in the EndoMT process were assessed employing quantitative RT-PCR, immunofluorescence histology and Western blot analysis. TGF-ß1 caused potent induction of EndoMT whereas ET-1 alone had a minimal effect. However, ET-1 potentiated TGF-ß1-induced EndoMT and TGF-ß1-stimulated expression of mesenchymal cell specific and profibrotic genes and proteins. ET-1 also induced expression of the TGF-ß receptor 1 and 2 genes, suggesting a plausible autocrine mechanism to potentiate TGF-ß-mediated EndoMT and fibrosis. Stimulation of TGF-ß1-induced skin and lung fibrosis by ET-1 was confirmed in vivo in an animal model of TGF-ß1-induced tissue fibrosis. These results suggest a novel role for ET-1 in the establishment and progression of tissue fibrosis.

The significance of macrophage polarization subtypes for animal models of tissue fibrosis and human fibrotic diseases.

The systemic and organ-specific human fibrotic disorders collectively represent one of the most serious health problems world-wide causing a large proportion of the total world population mortality. The molecular pathways involved in their pathogenesis are complex and despite intensive investigations have not been fully elucidated. Whereas chronic inflammatory cell infiltration is universally present in fibrotic lesions, the central role of monocytes and macrophages as regulators of inflammation and fibrosis has only recently become apparent. However, the precise mechanisms involved in the contribution of monocytes/macrophages to the initiation, establishment, or progression of the fibrotic process remain largely unknown. Several monocyte and macrophage subpopulations have been identified, with certain phenotypes promoting inflammation whereas others display profibrotic effects. Given the unmet need for effective treatments for fibroproliferative diseases and the crucial regulatory role of monocyte/macrophage subpopulations in fibrogenesis, the development of therapeutic strategies that target specific monocyte/macrophage subpopulations has become increasingly attractive. We will provide here an overview of the current understanding of the role of monocyte/macrophage phenotype subpopulations in animal models of tissue fibrosis and in various systemic and organ-specific human fibrotic diseases. Furthermore, we will discuss recent approaches to the design of effective anti-fibrotic therapeutic interventions by targeting the phenotypic differences identified between the various monocyte and macrophage subpopulations.

Caveolin-1 deficiency induces spontaneous endothelial-to-mesenchymal transition in murine pulmonary endothelial cells in vitro.

It was previously demonstrated that transforming growth factor ß (TGF-ß) induces endothelial-to-mesenchymal transition (EndoMT) in murine lung endothelial cells (ECs) in vitro. Owing to the important role of caveolin-1 (CAV1) in TGF-ß receptor internalization and TGF-ß signaling, the participation of CAV1 in the induction of EndoMT in murine lung ECs was investigated. Pulmonary ECs were isolated from wild-type and Cav1 knockout mice using immunomagnetic methods with sequential anti-CD31 and anti-CD102 antibody selection followed by in vitro culture and treatment with TGF-ß1. EndoMT was assessed by semiquantitative RT-PCR for Acta2, Col1a1, Snai1, and Snai2; by immunofluorescence for α-smooth muscle actin; and by Western blot analysis for α-smooth muscle actin, SNAIL1, SNAIL2, and the α2 chain of type I collagen. The same studies were performed in Cav1(-/-) pulmonary ECs after restoration of functional CAV1 domains using a cell-permeable CAV1 scaffolding domain peptide. Pulmonary ECs from Cav1 knockout mice displayed high levels of spontaneous Acta2, Col1A, Snai1, and Snai2 expression, which increased after TGF-ß treatment. Spontaneous and TGF-ß1-stimulated EndoMT were abrogated by the restoration of functional CAV1 domains using a cell-permeable peptide. The findings suggest that CAV1 regulation of EndoMT may play a role in the development of fibroproliferative vasculopathies.

Gadolinium compounds signaling through TLR4 and TLR7 in normal human macrophages: establishment of a proinflammatory phenotype and implications for the pathogenesis of nephrogenic systemic fibrosis.

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB-inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R-associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-ß, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

Effect of protein kinase C delta (PKC-δ) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.

NFκB activation and stimulation of chemokine production in normal human macrophages by the gadolinium-based magnetic resonance contrast agent Omniscan: possible role in the pathogenesis of nephrogenic systemic fibrosis.

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a generalised fibrotic disorder occurring in certain individuals with renal insufficiency exposed to gadolinium-based contrast agents (GdBCA) for MRI. Histopathological examination of affected tissues shows increased numbers of activated macrophages. To elucidate the mechanisms responsible for macrophage activation, the effects of the GdBCA Omniscan on normal human macrophage global gene expression, chemokine production and nuclear factor κB (NFκB) activation was examined. METHODS: Normal human monocyte-derived macrophages were incubated with Omniscan (50 mM) and their gene expression analysed by microarrays and real-time PCR. Macrophage chemokine production was assayed by multiplex ELISA. NFκB activation was assessed by NFκB nuclear localisation and quantitation of intracellular levels of inducible nitric oxide synthase (iNOS) protein. A specific cell-permeable NFκB peptide inhibitor was used to abrogate NFκB stimulation of chemokine and iNOS protein levels. CCL8/MCP-2 in affected skin of patients with NSF was examined by indirect immunofluorescence. RESULTS: Omniscan caused a profound change in the transcriptome of differentiated human normal macrophages in vitro, including a large increase in the expression of genes encoding CC and CXC chemokines. It induced rapid nuclear localisation of NFκB and stimulation of iNOS protein levels and chemokine production which were blocked by an NFκB inhibitory peptide. CCL8/MCP-2, the most upregulated chemokine following in vitro macrophage exposure to Omniscan, was strongly increased in NSF-affected skin. CONCLUSION: The GdBCA Omniscan induces potent stimulation of macrophage gene expression, NFκB activation and increased NFκB-mediated production of CC and CXC chemokines and iNOS. These alterations may play a crucial role in the pathogenesis of NSF.