From connective tissue builders to cellular architects: the journey of the skin fibroblast.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

The anabolic steroid, stanozolol, is used therapeutically to treat a number of pathological conditions and its clinical effects suggest that it can modulate connective tissue breakdown. The ability of this compound to stimulate prostaglandin E2 (PGE2), collagenase, gelatinase and stromelysin production by human synovial and skin fibroblasts in vitro was examined. The results showed that stanozolol significantly stimulated, in a dose dependent manner, PGE2, collagenase and stromelysin production by skin fibroblasts. However, no stimulation was seen in the synovial cell lines. In contrast, no effect on gelatinase production was seen in either cell type, following exposure to stanozolol. The synovial and skin lines both exhibited a significant stimulation of PGE2 and all three metalloproteinases in response to interleukin-1 beta (IL-1 beta). The anabolic steroids nortestosterone and oxymetholone demonstrated no ability to stimulate PGE2 or collagenase production in either skin or synovial fibroblasts. These results suggest that stanozolol exerts differential effects on skin and synovial fibroblasts in vitro which may enable the elucidation of the mechanism of action of the compound in vivo.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

Background: Autosomal dominant Hyper-IgE syndrome (AD-HIES; Job’s syndrome) is caused by dominant negative mutations in signal transducer and activator of transcription 3 (STAT3). Patients present with immunodeficiency accompanied by severe non-immunological features including poor post infection lung healing, subsequent pulmonary failure, and skeletal, connective tissue, and vascular abnormalities including arterial tortuosity and aneurisms which can lead to myocardial infarction and subarachnoid hemorrhage. HIF1a-dependent deficient angiogenesis has been implicated in the abnormal wound healing response of AD-HIES. Both STAT3 and HIF1a are major regulators of cellular metabolism. In this study we are testing the hypothesis that dysregulation of cellular metabolic pathways may contribute to AD-HIES pathophysiology. Methods: We used skin fibroblasts (SF) from 4 AD-HIES patients and 4 normal volunteers. To investigate ability of the cells to adapt and function in nutrient-deficient conditions (to model tissue damages disrupting normal tissue perfusion) we exposed them to a culture media lacking glucose, pyruvate and glutamine (deficient media). We used a live imaging approach (IncucyteS3 System, Sartorius) to analyze cell proliferation, reactive oxygen species (ROS), cell death, and wound closing ability (scratch-wound assay). Results: In deficient media, both AD-HIES and control SF decreased their proliferation rate. While control SF adapted, AD-HIES SF showed decreased survival after prolong nutrient deprivation. Level of ROS, measured at 12, 24, 48h in deficient media was 25% higher in AD-HIES SF (n=8, P=0.003). We used a scratch-wound assay to assess the ability of fibroblasts to migrate and close the wound after 48h of adaptation to deficient media. In full media, both control and AD-HIES fibroblast completely closed the wound within 36h. In deficient media, wound closure was delayed to much higher degree by AD-HIES fibroblasts (60±14% closure by control SF vs 18±9% by AD-HIES SF, n=4, P=0.016). Conclusions: Adaptive remodeling of cellular metabolism is impaired in AD-HIES SF that may contribute to deficient angiogenesis and poor wound healing, suggesting possible therapeutic targets within cellular metabolic pathways.

Classical Ehlers-Danlos syndrome (cEDS) is a dominant inherited connective tissue disorder mainly caused by mutations in the COL5A1 and COL5A2 genes encoding type V collagen (COLLV), which is a fibrillar COLL widely distributed in a variety of connective tissues. cEDS patients suffer from skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. Most of the causative variants result in a non-functional COL5A1 allele and COLLV haploinsufficiency, whilst COL5A2 mutations affect its structural integrity. To shed light into disease mechanisms involved in cEDS, we performed gene expression profiling in skin fibroblasts from four patients harboring haploinsufficient and structural mutations in both disease genes. Transcriptome profiling revealed significant changes in the expression levels of different extracellular matrix (ECM)-related genes, such as SPP1, POSTN, EDIL3, IGFBP2, and C3, which encode both matricellular and soluble proteins that are mainly involved in cell proliferation and migration, and cutaneous wound healing. These gene expression changes are consistent with our previous protein findings on in vitro fibroblasts from other cEDS patients, which exhibited reduced migration and poor wound repair owing to COLLV disorganization, altered deposition of fibronectin into ECM, and an abnormal integrin pattern. Microarray analysis also indicated the decreased expression of DNAJB7, VIPAS39, CCPG1, ATG10, SVIP, which encode molecular chaperones facilitating protein folding, enzymes regulating post-Golgi COLLs processing, and proteins acting as cargo receptors required for endoplasmic reticulum (ER) proteostasis and implicated in the autophagy process. Patients’ cells also showed altered mRNA levels of many cell cycle regulating genes including CCNE2, KIF4A, MKI67, DTL, and DDIAS. Protein studies showed that aberrant COLLV expression causes the disassembly of itself and many structural ECM constituents including COLLI, COLLIII, fibronectin, and fibrillins. Our findings provide the first molecular evidence of significant gene expression changes in cEDS skin fibroblasts highlighting that defective ECM remodeling, ER homeostasis and autophagy might play a role in the pathogenesis of this connective tissue disorder.

Background Systemic sclerosis (SSc) is a connective tissue disorder with progressive fibrosis in multiple organs including skin, lung and the gastrointestinal tract. Fibrosis is thought to be driven by activated fibroblasts. Therefore, inhibition of the profibrotic activity of activated fibroblasts may be a promising therapeutic approach in skin fibrosis in SSc. The autotaxin (ATX)/lysophosphatidic acid (LPA) axis is reportedly involved in fibrotic pathogenesis in SSc1. 2-carba cyclic phosphatidic acid (2ccPA) is a naturally occurring lipid mediator and one of its pleiotropic properties is to inhibit the ATX/LPA axis. Objectives We investigated the anti-fibrotic effect of 2ccPA on human SSc skin fibroblasts and bleomycin-induced skin fibrosis in mice. Furthermore, we searched signaling pathways related to the anti-fibrotic effects of 2ccPA. Methods This study was approved by the ethics committee and the ethical review committee of animal experiments of Tokyo Women’s Medical University. We informed all participants of the content of this study, and written consent was obtained. Skin fibroblasts were taken from SSc patients and adult healthy individuals were purchased. The cells were incubated with 1-10 μM 2ccPA in the presence or absence of 10 ng/ml transforming growth factor-β1 (TGF-β1). Messenger RNA (mRNA) and protein expression for type I collagen, connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), fibronectin (FN) and TGF-β1 were assessed by qRT-PCR or Western blotting. Procollagen type I C-peptide, prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) levels in the supernatant were assessed by ELISA. Intracellular cyclic adenosine monophosphate (cAMP) levels were calculated using a commercially available EIA kit. Forskolin was used to increase intracellular cAMP levels in cultured SSc skin fibroblasts. An inhibitor of adenylate cyclase (AC), 2’-deoxyadenosine, was used to investigate whether the anti-fibrotic effect of 2ccPA was mediated via the AC/cAMP pathway. Furthermore, we used a mouse model of bleomycin-induced skin fibrosis to investigate the safety and anti-fibrotic effects of 2ccPA. Results Ten μM 2ccPA significantly reduced mRNA and protein expression for type I collagen, CTGF and αSMA in SSc skin fibroblasts and adult healthy skin fibroblasts treated with TGF-β1. PGE2 and HGF levels in the supernatant of SSc skin fibroblasts were also reduced by 2ccPA treatment. 2ccPA increased intracellular cAMP levels as well as the AC stimulator, forskolin. In addition, forskolin decreased the mRNA expression of profibrotic markers. Reduction of COL1A1 mRNA expression by 2ccPA was blocked by treatment with 2’-deoxyadenosine in cultured SSc skin fibroblasts, suggesting that the anti-fibrotic activity of 2ccPA was partially mediated via AC stimulation. In mouse experiments, intraperitoneal injection of 10 mg/kg 2ccPA significantly reduced the development of skin thickness, collagen content and αSMA-positive cell counts. Conclusion 2ccPA suppressed the profibrotic activity of SSc skin fibroblasts and the development of bleomycin-induced skin fibrosis. Our experiments suggested that the anti-fibrotic property of 2ccPA was at least in part due to increased intracellular cAMP levels in skin fibroblasts. 2ccPA has been reported to be well tolerated in clinical trials of other diseases and may be expected for the treatment of fibrotic lesions in SSc. Reference [1] Castelino FV et al. Arthritis Rheumatol. 2016;68:2964-2974. Disclosure of Interests Tomoaki Higuchi: None declared, Kae Takagi: None declared, Akiko Tochimoto: None declared, Yuki Ichimura: None declared, Takanari Norose: None declared, Yasuhiro Katsumata: None declared, Ikuko Masuda: None declared, Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Toshihiro Moroboshi: None declared, Yasushi Kawaguchi: None declared

A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.

The balance between ER-alpha and ER-beta in fibroblasts may be crucial in the physiological response to ligands. Up- or down-regulation of the ERs in response to different compounds could mediate the reversal of certain age-related changes in skin and connective tissue. The time-dependent effects of 17-beta estradiol, raloxifene and tamoxifen on ER-alpha and ER-beta mRNA expression in the skin fibroblast cultures were performed. Experiments were carried out in primary cultures of human skin fibroblasts obtained from postmenopausal women. The cells were cultured in medium containing: 2 micromol/l estradiol (E2), 4 micromol/l tamoxifen (Tx) or 4 micromol/l raloxifene (Rx) for 7, 24 and 32 h. ER-alpha and ER-beta mRNAs were measured by quantitative assays based on reverse transcription (RT) of the mRNA and polymerase chain reaction (PCR) amplification of the cDNA. We suggest that ER-alpha and ER-beta are co-expressed in human postmenopausal skin fibroblast and documented that the level of mRNA expression of ERs in this tissue is estradiol, raloxifene or tamoxifen regulated as a mechanism to control the action of those ligands on the cell. On the basis of ER mRNA expression levels, fibroblast response to estradiol appears to be modulated by up-regulation of ER-beta rather than ER-alpha. Two of the examined SERMs appear to have different response to modulation of ERs: response of raloxifen is modulated by up-regulation of ER-beta, and no changes in expression of ER-alpha and tamoxifen response seem to be modulated by ER down-regulation in short-term or up-regulation during longer treatment.

The ability of the anabolic steroid, stanozolol, to stimulate procollagenase production by human synovial and skin fibroblasts was examined in an in vitro assay system. Stanozolol is used therapeutically to treat a variety of connective tissue and vascular disorders and its clinical effects suggest that it can modulate connective tissue breakdown. The results showed that stanozolol was capable, in a dose dependent manner, of significantly stimulating procollagenase production by skin fibroblasts. However, in three synovial fibroblast lines no evidence was found of increased collagenase production following treatment with stanozolol; although the synovial fibroblasts secreted significantly increased amounts of procollagenase in response to IL-1. These results may shed some light on the mechanism of action in vivo of stanozolol in the treatment of connective tissue disorders.

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures.To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues.

Background: Autosomal dominant Hyper-IgE syndrome (AD-HIES) is a rare primary immunodeficiency caused by dominant negative mutations in signal transducer and activator of transcription 3 (STAT3 ) , a mediator of widespread physiological processes. It is characterized by dermatitis, recurrent infections, elevated IgE, poor post-surgical healing, and connective tissue abnormalities. How STAT3 deficiency leads to this phenotype, however, is not known. Current treatment options are limited to antimicrobials for infection control. The aim of this study was to investigate which of STAT3’s many functions are dis-regulated in AD-HIES, and where potential targets for therapy may lie. Methods: We used skin fibroblasts (SF) from 3 AD-HIES patients and 3 normal volunteers. To evaluate potentially affected pathways, we utilized RNA- Seq and subsequent Gene Set Enrichment (GSEA) and pathway analysis (Pathway Studio, GeneGo Metacore). Endothelial cell tube formation assay was used to assess ability of AD-HIES SFs to support angiogenesis. Results: GSEA and pathway analysis showed deficiencies in signaling pathways linked to wound healing, extracellular matrix remodeling and angiogenesis including targets of Hypoxia Inducible Factor 1a (HIF1a) (P values for enrichments < 0.001) . Therefore, we hypothesized that AD-HIES SFs have impaired ability to support angiogenesis due to deficient Hif1a-dependent secretion of matrix proteases and growth factors. Indeed, AD-HIES SF secreted up to 5 times less matrix metalloprotease 1, 3, and 9, placental growth factor and fibroblast growth factors 1 and 2 (Luminex Multiplex, n=3-9, P<0.05). Culture medium from AD-HIES SFs failed to fully support tube formation by endothelial cells resulting in lower number of junctions, meshes, and total tubule length (n=6, P<0.005). Stabilization of Hif1a in AD-HIES SFs by prolyl hydroxylase inhibitor dimethyl fumarate restored its transcriptional activity leading to increased number of junctions, meshes, and tubule length (n=12, P<0.05) Conclusion: AD-HIES SFs have deficiencies in pro-angiogenic signaling pathways that lead to decreased growth factor secretion and angiogenesis. Stabilization of HIF1a corrects this deficiency and is an enticing target for future therapy.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF(+/-)) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF(+/-), may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.

Extensive research has shown that connective tissues such as tendons and ligaments are able to respond to mechanical forces by changing their structure, composition, and function. This mechanical adaptation is made possible largely by fibroblasts, the major cell types responsible for maintaining, repairing, and remodeling extracellular matrix (ECM) in connective tissues. This review focuses on mechanobiological responses of tendon, ligament, and skin fibroblasts in terms of their ECM gene expression and protein synthesis and secretion. The mechanobiological responses of fibroblasts in tissue engineering constructs and in wound healing are also discussed, followed by a review of the roles of major cellular components including integrins, the cytoskeleton, and stretch-activated ion channels in cellular mechanotransduction mechanisms. Finally, the review concludes with a brief discussion of future research directions in fibroblast mechanobiology.

The extensive proliferation of connective tissue in muscular dystrophy caused Duchenne1 to term it ‘paralysie myosclerosique’. Surprisingly, there has been little interest in the pathogenesis of this marked fibrosis or of the fat replacement in dystrophic muscle. The fibrosis has generally been considered secondary, with various hypotheses2 to explain the fundamental cause of muscular dystrophy. Several authors have commented on the increased endomysial stroma early in the disease before any apparent muscle degeration3,4 and have suggested that there might be an aberration in the production of connective tissue in muscular dystrophy and that the thickening pericellular reticulum would adversely affect muscle nutrition5,6. lonasescu and his collaborators found an over-production of connective tissue with a concomitant decrease in muscle protein synthesis both by polyribosomes from skeletal muscle and by tissue culture of muscle tissue and skin fibroblasts from patients suffering from Duchenne muscular dystrophy7–9. Thomson et al.10 have observed in tissue culture that dissociated muscle from patients with Duchenne and Becker muscular dystrophy will form unusual clusters of ‘sticky’ cells, which they suggested may reflect an abnormal collagen production. These findings have cast doubts on the interpretation that the extensive connective tissue proliferation characteristic of Duchenne muscular dystrophy, and also seen in limb-girdle, Becker and congenital dystrophies, is simply due to a compensatory replacement of the wasting muscle11 but infers a more primary role for the connective tissue collagen. Only recently has the role of connective tissue in developing muscle come to be appreciated12–14. Moreover, immunofluorescent techniques14–16, now allow investigation of the various types of collagen present in skeletal muscle. We report here on the localisation and change in proportion of collagen types I, III, IV and V in muscle from patients with various forms of neuromuscular disease, and propose a more positive role for collagen.

A protein factor in human urine which has the ability to activate connective tissue cells has been identified and partially purified; it appears to be different from epidermal growth factor and IgG. This urinary connective tissue activating factor (CTAP-U) is nondialyzable, labile to protease, stable to thiols, heat, and acid, and has an acidic isoelectric point. Purified preparations of CTAP-U have biologic activities that cause human connective tissue cells to synthesize incremental amounts of 14C-hyaluronic acid, 35S-proteoglycans, and 3H-DNA in vitro. The cell spectrum responsive to this substance includes human synovial cells, human chondrocytes, and skin fibroblasts. CTAP-U does not react with antisera to connective tissue activating peptide-III or to antibodies against IgG or its Fc and Fab fragments. Furthermore, CTAP-U does not cross-react in a radioreceptor assay for insulin, basic somatomedin, or epidermal growth factor-urogastrone. Utilizing standardized isolation conditions, CTAP-U preparations with these properties have been isolated from the urine of 6 normal individuals.