Disease Fibroblasts Research Articles

Transforming Growth Factor-β Signaling in Fibrotic Diseases and Cancer-Associated Fibroblasts.

Transforming growth factor-β (TGF-β) signaling is essential in embryo development and maintaining normal homeostasis. Extensive evidence shows that TGF-β activation acts on several cell types, including epithelial cells, fibroblasts, and immune cells, to form a pro-fibrotic environment, ultimately leading to fibrotic diseases. TGF-β is stored in the matrix in a latent form; once activated, it promotes a fibroblast to myofibroblast transition and regulates extracellular matrix (ECM) formation and remodeling in fibrosis. TGF-β signaling can also promote cancer progression through its effects on the tumor microenvironment. In cancer, TGF-β contributes to the generation of cancer-associated fibroblasts (CAFs) that have different molecular and cellular properties from activated or fibrotic fibroblasts. CAFs promote tumor progression and chronic tumor fibrosis via TGF-β signaling. Fibrosis and CAF-mediated cancer progression share several common traits and are closely related. In this review, we consider how TGF-β promotes fibrosis and CAF-mediated cancer progression. We also discuss recent evidence suggesting TGF-β inhibition as a defense against fibrotic disorders or CAF-mediated cancer progression to highlight the potential implications of TGF-β-targeted therapies for fibrosis and cancer.

CD47 prevents the elimination of diseased fibroblasts in scleroderma

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the “don’t-eat-me-signal” CD47 and whether blocking CD47 enables the body’s immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated transcription factor JUN and increased promoter accessibilities of both JUN and CD47. Next, we established our scleroderma model, demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1– fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL-6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study demonstrates the efficiency of combining different immunotherapies in treating scleroderma and provides a rationale for combining CD47 and IL-6 inhibition in clinical trials.

Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts.Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes.

Characterization of Collagen Metabolism in Normal and Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Reared in Serum‐free Medium

The structural integrity of the human lung is maintained by a healthy extracellular matrix (ECM). One of the main components, collagen type I is produced by fibroblasts and provides strength and structure to the cells and the function of the lung. A careful balance of collagen degradation and production is controlled by matrix metalloproteases (MMPs). In diseased states such as Chronic Obstructive Pulmonary Disease (COPD), two collagenases, MMP‐8 and MMP‐12, are upregulated. In this study we measure collagen metabolism of human lung fibroblasts (HLFs) reared in serum‐free media (subconfluent normal (NHLF) and COPD diseased (DHLF)). Commercially available early passage human lung fibroblasts established from lung tissue, obtained during biopsy, were cultured and passaged in our laboratory through P4 and P5 for both the NHLF and DHLF. Both normal and diseased HLF cells were viable when cultured at subconfluence for 24 hours in serum free medium. They produced detectable quantities of intact collagen type I secreted and incorporated in the ECM. The amount of intact collagen decreased between passage 4 and 5 (p<0.01) in both cell types indicating there is a limit to the passages that can be utilized to measure ECM alterations. Degraded collagen type I was present in the cell layer fractions of both cell types, but was 10 fold higher in the cell layer of diseased HLF cells. This indicates that the expected ECM profile of diseased HLF can be demonstrated in serum‐free culture. Both cell types produced detectable levels of MMP‐8 and MMP‐12 collagenases. In this study we have demonstrated that normal and diseased HLF can be cultured in serum‐free conditions while maintaining their metabolic profile. The production of MMPs and collagens provides an experimental system in which we can test potential therapeutics that may alter the fibrotic response during COPD.Support or Funding InformationThis work is supported by a grant from the PCOM Alumni and the Division of Research at PCOM.

Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-β and p-SMAD Pathway.

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-β plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-β increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 μM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-β inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-β levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-β signaling. TGF-β inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.

P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test. Results No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C). Conclusion Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.

Engineering clinically-relevant human fibroblastic cell-derived extracellular matrices.

Three-dimensional (3D) culturing models, replicating in vivo tissue microenvironments that incorporate native extracellular matrix (ECM), have revolutionized the cell biology field. Fibroblastic cells generate lattices of interstitial ECM proteins. Cell interactions with ECMs and with molecules sequestered/stored within these are crucial for tissue development and homeostasis maintenance. Hence, ECMs provide cells with biochemical and biomechanical cues to support and locally control cell function. Further, dynamic changes in ECMs, and in cell-ECM interactions, partake in growth, development, and temporary occurrences such as acute wound healing. Notably, dysregulation in ECMs and fibroblasts could be important triggers and modulators of pathological events such as developmental defects, and diseases associated with fibrosis and chronic inflammation such as cancer. Studying the type of fibroblastic cells producing these matrices and how alterations to these cells enable changes in ECMs are of paramount importance. This chapter provides a step-by-step method for producing multilayered (e.g., 3D) fibroblastic cell-derived matrices (fCDM). Methods also include means to assess ECM topography and other cellular traits, indicative of fibroblastic functional statuses, like naïve/normal vs. inflammatory and/or myofibroblastic. For these, protocols include indications for isolating normal and diseased fibroblasts (i.e., cancer-associated fibroblasts known as CAFs). Protocols also include means for conducting microscopy assessments, querying whether fibroblasts present with fCDM-dependent normal or CAF phenotypes. These are supported by discrete semi-quantitative digital imaging analyses, providing some imaging processing advice. Additionally, protocols include descriptions for effective fCDM decellularization, which renders cellular debris-free patho/physiological in vivo-like scaffolds, suitable as 3D substrates for subsequent cell culturing.

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2−/− mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

Somatic Platelet Derived Growth Factor Receptor Beta Activating Variants in Fusiform Cerebral Aneurysms

Abstract INTRODUCTION The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-yr-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of unrevealing genetic evaluations for known connective-tissue disorders, and served as a somatic mosaic for discovery of novel mutations involved in aneurysm pathogenesis. METHODS Deoxyribonucleic acid (DNA) was extracted from multiple vascular and perivascular tissue samples obtained during surgery on the index patient. Initial variant discovery was carried out via paired-sample exome sequencing between blood and diseased area fibroblasts. A single novel variant was discovered in the platelet derived growth factor receptor beta (PDGFRB), and mutations in this gene were tested in exome sequencing of aneurysm walls from 6 patients with fusiform intracranial aneurysm and 38 patients with saccular aneurysm. RESULTS Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T &gt; C [GRCh37/hg19]; c.1685A &gt; G) change within PDGFRB, a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting postzygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, P &lt; .001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive autophosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated nonligand-dependent autophosphorylation, responsive to the kinase inhibitor sunitinib. CONCLUSION Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.

DNA damage in human skin fibroblasts from patients with dermatitis herpetiformis.

Dermatitis herpetiformis (DH) and celiac disease (CD) are considered to be autoimmune diseases that share a specific trigger (gluten) and a common genetic background (HLA-DQ2/DQ8). However, the pathogenesis of DH is not yet fully understood and no data are available regarding a possible role of fibroblasts in this disease. The aim of this study was to assess baseline DNA damage in fibroblasts in DH-diagnosed patients vs. fibroblasts of controls without DH or CD. Primary fibroblast cultures were derived from dermal biopsies from DH patients and controls (without DH or CD). In vitro genotoxic damage was investigated using the comet assay and ɣH2AX test after different treatments (with 33mer peptide and digested gliadin [DG]) in order to investigate a correlation between oxidative stress (evaluated by reactive oxygen species formation) and glutathione content. Our results demonstrate a difference in baseline DNA damage between cutaneous fibroblasts of controls and DH patients, moreover, DNA damage significantly increased after exposure to gluten (DG and 33mer peptide) in fibroblasts from DH patients. DNA damage in fibroblasts from patients under dapsone treatment was similar to that of the control group. Our data indicate that oxidative stress and DNA damage may be characteristics of fibroblasts from DH patients who are not treated with dapsone, particularly after exposure to gliadin peptides.

Fibroblast activation and inflammation in frozen shoulder.

IntroductionFrozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement.Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,MethodsShoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.ResultsImmunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).ConclusionsThese results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

Renoprotective phenolic meroterpenoids from the mushroom Ganoderma cochlear

Twelve previously undescribed phenolic meroterpenoids, cochlearols N−Y, along with two known analogs, ganocochlearins B and C, were isolated from the fruiting bodies of Ganoderma cochlear. Most of these substances were isolated as racemic mixtures. The structures of cochlearols N−Y were assigned based upon spectroscopic data and theoretical calculations. The renoprotective activities of these compounds were evaluated using normal and diseased rat renal interstitial fibroblast cells (NRK-49F). The results show that ganocochlearins S, U, X and Y display potent inhibitory activities against fibronectin overproduction in TGF-β1-induced NRK-49F cells.

Reversion of in vivo fibrogenesis by novel chromone scaffolds.

BackgroundMyofibroblasts are known to play a key role in the development of idiopathic pulmonary fibrosis (IPF). Two drugs, pirfenidone and nintedanib, are the only approved therapeutic options for IPF, but their applications are limited due to their side effects. Thus, curative IPF drugs represent a huge unmet medical need.MethodsA mouse hepatic stellate cell (HSC) line was established that could robustly differentiate into myofibroblasts upon treatment with TGF-β. Eupatilin was assessed in diseased human lung fibroblasts from IPF patients (DHLFs) as well as in human lung epithelial cells (HLECs). The drug's performance was extensively tested in a bleomycin-induced lung fibrosis model (BLM). Global gene expression studies and proteome analysis were performed.FindingsEupatilin attenuated disease severity of BLM in both preventative and therapeutic studies. The drug inhibited the in vitro transdifferantiation of DHLFs to myofibroblasts upon stimulation with TGF-β. No such induction of the in vitro transdifferantiation was observed in TGF-β treated HLECs. Specific carbons of eupatilin were essential for its anti-fibrotic activity. Eupatilin was capable of dismantling latent TGF-β complex, specifically by eliminating expression of the latent TGF-β binding protein 1 (LTBP1), in ECM upon actin depolymerization. Unlike eupatilin, pirfenidone was unable to block fibrosis of DHLFs or HSCs stimulated with TGF-β. Eupatilin attenuated phosphorylation of Smad3 by TGF-β. Eupatilin induced myofibroblasts to dedifferentiate into intermediate HCS-like cells.InterpretationEupatilin may act directly on pathogenic myofibroblasts, disarming them, whereas the anti-fibrotic effect of pirfenidone may be indirect. Eupatilin could increase the efficacy of IPF treatment to curative levels.

Renoprotective meroterpenoids from the fungus Ganoderma cochlear

Nine multifarious new meroterpenoids, cochlearols E-M (1–9), along with seven known meroterpenoids 10–16, were isolated from the fruiting bodies of Ganoderma cochlear. Racemic 1, 6–8, 10 and 13 were separated by chiral HPLC. The structures were elucidated based on detailed spectroscopic analyses (HRESIMS, 1D/2D-NMR) and calculated electronic circular dichroism (ECD). Their biological activities against renal fibrosis were evaluated by using rat normal and diseased renal interstitial fibroblast cells (NRK49F). The results show that compounds 7a, 7b, and 10a exhibit potent proliferation inhibition in TGF-β1-induced NRK-49F cells.

Systems Analysis of Transcriptomic and Proteomic Profiles Identifies Novel Regulation of Fibrotic Programs by miRNAs in Pulmonary Fibrosis Fibroblasts.

Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-β, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct “fibrotic” matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression.

Next generation sequencing of RNA reveals novel targets of resveratrol with possible implications for Canavan disease

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid β-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase.We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial β-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

Pathologic Fibroblasts in Idiopathic Subglottic Stenosis Amplify Local Inflammatory Signals

To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). Basic science. Laboratory. Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. Mechanistically, IL-17A drives iSGS scar fibroblast proliferation ( P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells ( P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor ( P = .02). IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.

Improving knowledge on the activation of bone marrow fibroblasts in MGUS and MM disease through the automatic extraction of genes via a nonnegative matrix factorization approach on gene expression profiles

BackgroundMultiple myeloma (MM) is a cancer of terminally differentiated plasma that is part of a spectrum of blood diseases. The role of the micro-environment is crucial for MM clonal evolution.MethodsThis paper describes the analysis carried out on a limited number of genes automatically extracted by a nonnegative matrix factorization (NMF) based approach from gene expression profiles of bone marrow fibroblasts of patients with monoclonal gammopathy of undetermined significance (MGUS) and MM.ResultsAutomatic exploration through NMF, combined with a motivated post-processing procedure and a pathways analysis of extracted genes, allowed to infer that a functional switch is required to lead fibroblasts to acquire pro-tumorigenic activity in the progression of the disease from MGUS to MM.ConclusionThe extracted biologically relevant genes may be representative of the considered clinical conditions and may contribute to a deeper understanding of tumor behavior.

Regenerative and Protective Actions of the GHK-Cu Peptide in the Light of the New Gene Data.

The human peptide GHK (glycyl-l-histidyl-l-lysine) has multiple biological actions, all of which, according to our current knowledge, appear to be health positive. It stimulates blood vessel and nerve outgrowth, increases collagen, elastin, and glycosaminoglycan synthesis, as well as supports the function of dermal fibroblasts. GHK’s ability to improve tissue repair has been demonstrated for skin, lung connective tissue, boney tissue, liver, and stomach lining. GHK has also been found to possess powerful cell protective actions, such as multiple anti-cancer activities and anti-inflammatory actions, lung protection and restoration of chronic obstructive pulmonary disease (COPD) fibroblasts, suppression of molecules thought to accelerate the diseases of aging such as NFκB, anti-anxiety, anti-pain and anti-aggression activities, DNA repair, and activation of cell cleansing via the proteasome system. Recent genetic data may explain such diverse protective and healing actions of one molecule, revealing multiple biochemical pathways regulated by GHK.

Heparin Functionalized Injectable Cryogel with Rapid Shape-Recovery Property for Neovascularization.

Cryogel based scaffolds have high porosity with interconnected macropores that may provide cell compatible microenvironment. In addition, cryogel based scaffolds can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a carrier for vascular endothelial growth factor (VEGF) and fibroblasts in hindlimb ischemic disease. Our gelatin/heparin cryogel showed gelatin concentration-dependent mechanical properties, swelling ratios, interconnected porosities, and elasticities. In addition, controlled release of VEGF led to effective angiogenic responses both in vitro and in vivo. Furthermore, its sponge-like properties enabled cryogels to be applied as an injectable carrier system for in vivo cells and growth factor delivery. Our heparin functionalized injectable cryogel facilitated the angiogenic potential by facilitating neovascularization in a hindlimb ischemia model.