Identification of Glucocorticoid Receptor Target Genes That Potentially Inhibit Collagen Synthesis in Human Dermal Fibroblasts.

Over several decades, excess glucocorticoids (GCs) of endogenous or exogenous origin have been recognized to significantly inhibit collagen synthesis and accelerate skin aging. However, little is known regarding their molecular mechanisms. We hypothesized that the action of GCs on collagen production is at least partially through the glucocorticoid receptor (GR) and its target genes, and therefore aimed to identify GR target genes that potentially inhibit collagen synthesis in Hs68 human dermal fibroblasts. We first confirmed that dexamethasone, a synthetic GC, induced canonical GR signaling in dermal fibroblasts. We then collected 108 candidates for GR target genes reported in previous studies on GR target genes and verified that 17 genes were transcriptionally upregulated in dexamethasone-treated dermal fibroblasts. Subsequently, by individual knockdown of the 17 genes, we identified that six genes, AT-rich interaction domain 5B, FK506 binding protein 5, lysyl oxidase, methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, zinc finger protein 36, and zinc fingers and homeoboxes 3, are potentially involved in GC-mediated inhibition of collagen synthesis. The present study sheds light on the molecular mechanisms of GC-mediated skin aging and provides a basis for further research on the biological characteristics of individual GR target genes.

[Industrial development and biomedical application prospect of recombinant collagen].

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.

Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPARγ, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPARγ expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX-PPARγ axis can be considered a novel approach for breast cancer treatment.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Deficiency of vitamin D receptor in keratinocytes augments dermal fibrosis and inflammation in a mouse model of HOCl-induced scleroderma.

Scleroderma, characterized by extensive fibrosis and vascular alterations, involves excessive fibroblast activation, uncontrolled inflammation, and abnormal collagen deposition. Previous studies showed that administrations of either 1,25(OH)2D3 or vitamin D analog effectively decreased or reversed skin fibrosis by regulating the extracellular matrix homeostasis. The actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a transcription regulator crucial for skin homeostasis. Although evidence suggests that keratinocyte-fibroblast interaction influences the development of scleroderma, the role of keratinocytes in scleroderma remains unknown. Here, we demonstrated that the ablation of VDR in keratinocytes greatly exacerbated dermal fibrosis in HOCl-induced scleroderma in mice. The deficiency of VDR in the epidermis marked increased dermal thickness, inflammatory cell infiltration, and severe collagen deposition in comparison to the control group in HOCl-treated skin. Moreover, significant elevations in expression levels of mRNA for collagen overproduction (Col1A1, Col1A2, Col3A1, α-SMA, MMP9, TGF-ß1) and proinflammatory cytokines (IL-1ß, IL-6, CXCL1, CXCL2) were observed in VDR conditional KO versus control mice following HOCl treatment. Collectively, these results suggest that VDR in keratinocytes plays a pivotal role in scleroderma progression, and the interplay between keratinocytes and fibroblasts deserves more attention regarding the exploration of the pathogenesis and treatment for scleroderma.

Calpain inhibition decreases myocardial fibrosis in chronically ischemic hypercholesterolemic swine.

OBJECTIVES: Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content. RESULTS: In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters. CONCLUSIONS: Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.

Targeting cannabinoid receptor 2 (CB2) limits collagen production-An in vitro study in a primary culture of human fibroblasts.

Previous studies showed that cannabinoid 2 (CB2) receptor is involved in skin inflammation, fibrogenesis and re-epithelialization in mice, indicating that this receptor may be implicated in wound healing. Thus, topical use of cannabinoids may have a role in local fibrotic and wound healing diseases such as scars or keloids. We investigate the effect of the CB2 selective receptor agonist (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (JWH133) and the CB2 selective receptor antagonist (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)-methanone (AM630), on primary cultures of human fibroblasts. Primary cultures of adult human fibroblasts were obtained from abdominal human skin samples. Fibroblasts pretreated with JWH133 and/or AM630 were stimulated with TGF-ß (10 ng/ml). Fibroblast activation into myofibroblasts was quantified by the expression of alpha-smooth muscle actin (α-SMA) using Immunocytochemistry and Western Blot assays. Collagen content was quantified with the Sirius red staining assay. Upon human fibroblasts stimulation with TGF-ß, a significant increase on α-SMA and CB2 receptor expression was observed. In these cells, JWH133 decreased α-SMA expression and collagen content. However, this effect was not observed in resting human fibroblasts. AM630 decreased α-SMA expression and collagen content in both resting and activated fibroblasts. This effect was time- and concentration-dependent with an IC50 value of 11 µM. The CB2 receptor appears to be involved in fibroblast repair during skin wound healing in humans, as TGF-ß increases CB2 receptor expression and JWH133 produces an anti-fibrotic effect in human fibroblasts. AM630 also showed an anti-fibrotic effect hypothesizing that other cannabinoid receptors, such as TRPV, may be involved in this response.

N-Acetylcysteine Slows Down Cardiac Pathological Remodeling by Inhibiting Cardiac Fibroblast Proliferation and Collagen Synthesis.

OBJECTIVE: By observing the effect of N-acetylcysteine (NAC) on the proliferation and collagen synthesis of rat cardiac fibroblasts (CFs) to explore the effect of NAC on cardiac remodeling (CR). METHODS: In vivo, first, the Sprague Dawley (SD) rat myocardial hypertrophy model was constructed, and the effect of NAC on cardiac structure and function was detected by echocardiography, serological testing, and Masson staining. Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression level of antioxidant enzymes, and flow cytometry was used to detect the intracellular reactive oxygen species (ROS) content. In vitro, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining were used to detect cell proliferation, and the expression level of the NF-κB signaling pathway was detected. RESULTS: Compared with the control group, the model group had disordered cardiac structure, reduced cardiac function, and obvious oxidative stress (OS) response. However, after NAC treatment, it could obviously improve the rat cardiac structure and cardiac function and alleviate redox imbalance and cardiology remodeling. At the same time, NAC can inhibit the activation of the NF-κB signaling pathway and reduce the proliferation level of CFs and the amount of 3H proline incorporated. CONCLUSIONS: NAC can inhibit AngII-induced CF proliferation and collagen synthesis through the NF-κB signaling pathway, alleviate the OS response of myocardial tissue, inhibit the fibrosis of myocardial tissue, and thus slow down the pathological remodeling of the heart.

Dityrosine Crosslinking of Collagen and Amyloid-ß Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans.

(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid ß, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid ß but increased the formation of dityrosine crosslinking in the amyloid ß protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid ß formation, which might promote its stabilization and toxic oligomerization.

Enhanced wound healing properties of guar gum/curcumin-stabilized silver nanoparticle hydrogels.

Biocompatible materials that act as scaffolds for regenerative medicine are of enormous interest. Hydrogel-nanoparticle composites have great potential in this regard, however evaluations of their wound healing and safety in vivo in animal studies are scarce. Here we demonstrate that a guar gum/curcumin-stabilized silver nanoparticle hydrogel composite is an injectable material with exceptional wound healing and antibacterial properties. We show that the curcumin-bound silver nanoparticles themselves exhibit low cytotoxicity and enhance proliferation, migration, and collagen production in in vitro studies of human dermal fibroblasts. We then show that the hydrogel-nanoparticle composite promotes wound healing in in vivo studies on rats, accelerating wound closure by > 40% and reducing bacterial counts by 60% compared to commercial antibacterial gels. Histopathology indicates that the hydrogel composite enhances transition from the inflammation to proliferation stage of healing, promoting the formation of fibroblasts and new blood vessels, while target gene expression studies confirm that the accelerated tissue remodeling occurs along the normal pathways. As such these hydrogel composites show great promise as wound dressing materials with high antibacterial capacity.

Enhancing osteoblast survival through pulsed electrical stimulation and implications for osseointegration.

Electrical stimulation has been suggested as a means for promoting the direct structural and functional bonding of bone tissue to an artificial implant, known as osseointegration. Previous work has investigated the impact of electrical stimulation in different models, both in vitro and in vivo, using various electrode configurations for inducing an electric field with a wide range of stimulation parameters. However, there is no consensus on optimal electrode configuration nor stimulation parameters. Here, we investigated a novel approach of delivering electrical stimulation to a titanium implant using parameters clinically tested in a different application, namely peripheral nerve stimulation. We propose an in vitro model comprising of Ti6Al4V implants precultured with MC3T3-E1 preosteoblasts, stimulated for 72 h at two different pulse amplitudes (10 µA and 20 µA) and at two different frequencies (50 Hz and 100 Hz). We found that asymmetric charge-balanced pulsed electrical stimulation improved cell survival and collagen production in a dose-dependent manner. Our findings suggest that pulsed electrical stimulation with characteristics similar to peripheral nerve stimulation has the potential to improve cell survival and may provide a promising approach to improve peri-implant bone healing, particularly to neuromusculoskeletal interfaces in which implanted electrodes are readily available.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

IL-15 Prevents Renal Fibrosis by Inhibiting Collagen Synthesis: A New Pathway in Chronic Kidney Disease?

Chronic kidney disease (CKD), secondary to renal fibrogenesis, is a public health burden. The activation of interstitial myofibroblasts and excessive production of extracellular matrix (ECM) proteins are major events leading to end-stage kidney disease. Recently, interleukin-15 (IL-15) has been implicated in fibrosis protection in several organs, with little evidence in the kidney. Since endogenous IL-15 expression decreased in nephrectomized human allografts evolving toward fibrosis and kidneys in the unilateral ureteral obstruction (UUO) model, we explored IL-15's renoprotective role by pharmologically delivering IL-15 coupled or not with its soluble receptor IL-15Rα. Despite the lack of effects on myofibroblast accumulation, both IL-15 treatments prevented tubulointerstitial fibrosis (TIF) in UUO as characterized by reduced collagen and fibronectin deposition. Moreover, IL-15 treatments inhibited collagen and fibronectin secretion by transforming growth factor-ß (TGF-ß)-treated primary myofibroblast cultures, demonstrating that the antifibrotic effect of IL-15 in UUO acts, in part, through a direct inhibition of ECM synthesis by myofibroblasts. In addition, IL-15 treatments resulted in decreased expression of monocyte chemoattractant protein 1 (MCP-1) and subsequent macrophage infiltration in UUO. Taken together, our study highlights a major role of IL-15 on myofibroblasts and macrophages, two main effector cells in renal fibrosis, demonstrating that IL-15 may represent a new therapeutic option for CKD.

Curcumin inhibition of bleomycin-induced changes in lung collagen synthesis, deposition and assembly.

BACKGROUND: Idiopathic pulmonary fibrosis is characterized by progressive lung tissue remodeling and disproportionate deposition of collagenous proteins with limited therapeutic interventions. The purpose of this study was to determine whether curcumin inhibits bleomycin (BLM)-induced increases in synthesis, degradation and cross-linking of lung collagen in rats. METHODS AND RESULTS: Following a single intratracheal instillation of BLM to rats (0.75 U/100 g, sacrificed 3, 5, 7, 14 and 28 days post-BLM), lung collagen synthesis (determined by incorporation of 3H-proline) and deposition (determined by lung hydroxyproline content) progressively increased at days 7, 14 and 28 post-BLM injection. Lung lavage fluid hydroxyproline and collagenase levels (a measure of collagen turnover) were increased in BLM rats compared with control groups. In addition, BLM instillation resulted in increased concentrations of collagenase and collagenolytic cathepsin in the lungs. Furthermore, increased cross-linking (as determined by aldehyde content of acid soluble collagen), and decreased susceptibility of fibrotic lung insoluble collagen to denaturing agents occurred in BLM-injured lungs. Significant increases in alveolar macrophage (AM) release of transforming growth factor-ß1 (TGF-ß1) were noted at various time points (days 3, 5, 7, 14 and 28 post-BLM) during the development and progression of lung fibrosis in rats. Curcumin treatment to BLM rats (300 mg/kg 10 days before and daily thereafter throughout the experimental time period) was associated with marked reductions in lung collagen synthesis and deposition, BALF and lung collagenase activity, BALF hydroxyproline content and lung collagenolytic levels. Additionally, reduced levels of collagen cross-linking and enhanced susceptibility of insoluble lung collagen to denaturing agents were observed in curcumin-treated BLM rats. Finally, curcumin inhibited BLM-induced increases in AM production of TGF-ß1. CONCLUSIONS: Our data demonstrate for the first time that curcumin prevents fibrotic deposits by modulating collagen turnover, assembly and deposition in BLM-instilled rat lungs, and that curcumin treatment protects against BLM activation of macrophages by suppressing the release of TGF-ß1.

The effects of collagen peptide supplementation on body composition, collagen synthesis, and recovery from joint injury and exercise: a systematic review.

Collagen peptide supplementation (COL), in conjunction with exercise, may be beneficial for the management of degenerative bone and joint disorders. This is likely due to stimulatory effects of COL and exercise on the extracellular matrix of connective tissues, improving structure and load-bearing capabilities. This systematic review aims to evaluate the current literature available on the combined impact of COL and exercise. Following Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, a literature search of three electronic databases-PubMed, Web of Science and CINAHL-was conducted in June 2020. Fifteen randomised controlled trials were selected after screening 856 articles. The study populations included 12 studies in recreational athletes, 2 studies in elderly participants and 1 in untrained pre-menopausal women. Study outcomes were categorised into four topics: (i) joint pain and recovery from joint injuries, (ii) body composition, (iii) muscle soreness and recovery from exercise, and (iv) muscle protein synthesis (MPS) and collagen synthesis. The results indicated that COL is most beneficial in improving joint functionality and reducing joint pain. Certain improvements in body composition, strength and muscle recovery were present. Collagen synthesis rates were elevated with 15 g/day COL but did not have a significant impact on MPS when compared to isonitrogenous higher quality protein sources. Exact mechanisms for these adaptations are unclear, with future research using larger sample sizes, elite athletes, female participants and more precise outcome measures such as muscle biopsies and magnetic imagery.

Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells).

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

Tetrahexyldecyl Ascorbate (THDC) Degrades Rapidly under Oxidative Stress but Can Be Stabilized by Acetyl Zingerone to Enhance Collagen Production and Antioxidant Effects.

Tetrahexyldecyl Ascorbate (THDC) is an L-ascorbic acid precursor with improved stability and ability to penetrate the epidermis. The stability and transdermal penetration of THDC, however, may be compromised by the oxidant-rich environment of human skin. In this study, we show that THDC is a poor antioxidant that degrades rapidly when exposed to singlet oxygen. This degradation, however, was prevented by combination with acetyl zingerone (AZ) as a stabilizing antioxidant. As a standalone ingredient, THDC led to unexpected activation of type I interferon signaling, but this pro-inflammatory effect was blunted in the presence of AZ. Moreover, the combination of THDC and AZ increased expression of genes associated with phospholipid homeostasis and keratinocyte differentiation, along with repression of MMP1 and MMP7 expression, inhibition of MMP enzyme activity, and increased production of collagen proteins by dermal fibroblasts. Lastly, whereas THDC alone reduced viability of keratinocytes exposed to oxidative stress, this effect was completely abrogated by the addition of AZ to THDC. These results show that AZ is an effective antioxidant stabilizer of THDC and that combination of these products may improve ascorbic acid delivery. This provides a step towards reaching the full potential of ascorbate as an active ingredient in topical preparations.

Iloprost Attenuates Oxidative Stress-Dependent Activation of Collagen Synthesis Induced by Sera from Scleroderma Patients in Human Pulmonary Microvascular Endothelial Cells.

Endothelial cell injury is an early event in systemic sclerosis (SSc) pathogenesis and several studies indicate oxidative stress as the trigger of SSc-associated vasculopathy. Here, we show that circulating factors present in sera of SSc patients increased reactive oxygen species (ROS) production and collagen synthesis in human pulmonary microvascular endothelial cells (HPMECs). In addition, the possibility that iloprost, a drug commonly used in SSc therapy, might modulate the above-mentioned biological phenomena has been also investigated. In this regard, as compared to sera of SSc patients, sera of iloprost-treated SSc patients failed to increased ROS levels and collagen synthesis in HPMEC, suggesting a potential antioxidant mechanism of this drug.

Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

Tissue expansion is widely used to obtain new skin tissue for repairing defects in the clinical practice of plastic surgery. One major complication can be dermal thinning during expansion, which usually leads to skin rupture. Collagen synthesis can determine dermal thickness and can be influenced by macrophage polarization during expansion. The aim of the study was to test whether pigment epithelium-derived factor (PEDF) could be a modulator of collagen synthesis in fibroblasts by regulating macrophage polarization during skin expansion. Our results showed that PEDF mRNA expression was increased in expanded human and mouse epidermis. PEDF protein levels were elevated in the subcutaneous exudates of a rat skin expansion model. Increased PEDF mRNA expression was accompanied by dermal thinning during a three-week expansion protocol. Subcutaneous injection of PEDF in vivo further resulted in dermal thinning and cell number increase of M1 macrophage in the expanded skin. PEDF also promoted macrophage polarization in vitro to the M1 subtype under hypoxic conditions. PEDF did not influence collagen gene expression in fibroblasts directly, but attenuated collagen synthesis in a macrophage-mediated manner. Additionally, blockage of PEDF receptors on macrophages with inhibitors rescued collagen synthesis in fibroblasts. Our research demonstrated PEDF elevation in expanded skin leads to dermal thinning through M1 macrophage-mediated collagen synthesis inhibition in fibroblasts. Our results could form a basis for the development of novel strategies to improve skin integrity in expanded skin by using PEDF.

ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6.

Excessive deposition of extracellular matrix, mainly collagen protein, is the hallmark of organ fibrosis. The molecular mechanisms regulating fibrotic protein biosynthesis are unclear. Here, we find that chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a plasma membrane receptor for prostaglandin D2, is trafficked to the endoplasmic reticulum (ER) membrane in fibroblasts in a caveolin-1-dependent manner. ER-anchored CRTH2 binds the collagen mRNA recognition motif of La ribonucleoprotein domain family member 6 (LARP6) and promotes the degradation of collagen mRNA in these cells. In line, CRTH2 deficiency increases collagen biosynthesis in fibroblasts and exacerbates injury-induced organ fibrosis in mice, which can be rescued by LARP6 depletion. Administration of CRTH2 N-terminal peptide reduces collagen production by binding to LARP6. Similar to CRTH2, bumetanide binds the LARP6 mRNA recognition motif, suppresses collagen biosynthesis, and alleviates bleomycin-triggered pulmonary fibrosis in vivo. These findings reveal a novel anti-fibrotic function of CRTH2 in the ER membrane via the interaction with LARP6, which may represent a therapeutic target for fibrotic diseases.