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Recombinant human collagens have attracted intensive interest in the past two decades, demonstrating considerable potential in medicine, tissue engineering, and cosmetics. Several humanized recombinant collagens have been produced, exhibiting similar characteristics as the native species. To get insight into the structural and bioactive properties of different parts of collagen, in this study, the segment of Gly300-Asp329 of type III collagen was first adopted and repeated 18 times to prepare a novel recombinant collagen (named rhCLA). RhCLA was successfully expressed in E. coli, and a convenient separation procedure was established through reasonably combining alkaline precipitation and acid precipitation, yielding crude rhCLA with a purity exceeding 90%. Additionally, a polishing purification step utilizing cation exchange chromatography was developed, achieving rhCLA purity surpassing 98% and an overall recovery of approximately 120 mg/L culture. Simultaneously, the contents of endotoxin, nucleic acids, and host proteins were reduced to extremely low levels. This fragmented type III collagen displayed a triple-helical structure and gel-forming capability at low temperatures. Distinct fibrous morphology was also observed through TEM analysis. In cell experiments, rhCLA exhibited excellent biocompatibility and cell adhesion properties. These results provide valuable insights for functional studies of type III collagen and a reference approach for the large-scale production of recombinant collagens., (Copyright © 2024. Published by Elsevier Inc.)

Alpha-1 Type Ⅲ Collagen (COL3A1) encodes the Collagen alpha-1(Ⅲ) chain, which is a fibrillar collagen that exists in extensile connective tissues. Few studies have reported its role in tumorigenicity. In the present study, we identified that COL3A1 protein and mRNA expression levels were considerably up-regulated in esophageal squamous cell carcinoma (ESCC) cells in comparison with normal esophageal squamous epithelial cells (P < 0.05). Immunohistochemical (IHC) analysis of 114 paraffin-embedded archived ESCC tissues demonstrated that COL3A1 expression was positively correlated with the postoperative T stage. Univariate and multivariable analysis demonstrated that COL3A1 expression was an independent poor prognostic factor for overall survival in the whole cohort. Silencing COL3A1 inhibited, while overexpressing COL3A1 promoted, the proliferation, migration, and invasion of ESCC cells. Furthermore, down-regulation of COL3A1 expression also suppressed the growth of ESCC in subcutaneous xenograft mouse models and inhibited ESCC metastasis in lung metastasis mouse models. In addition, we proved that the tumor-promoting effect of COL3A1 on ESCC cells was related to the activation of NF-κB signaling pathway. These findings indicate that COL3A1 confers a poor prognosis and malignant phenotype by activating the NF-κB pathway in ESCC, potentially representing a novel biomarker and/or providing a new curative target for ESCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The antitumor properties of cruciferous vegetables are mainly due to their high content of isothiocyanates, and sulforaphane (SFA) is the most well-known compound. The aim of this study was to determine the mechanism of SFA inhibiting gastric cancer (GC) progression. After verifying SFA suppressing GC growth in vivo, we utilized the GSE79973 and GSE118916 datasets to identify the GC development signatures that overlap with the RNA-seq analysis in SFA-treated AGS cells. GSEA of the RNA-seq data indicated that SFA regulation of GC progression was related to extracellular matrix and collagens; thus, we identified COL3A1 and COL5A1 as the targets of SFA, which functioned as oncogenes. We found positive correlations between COL3A1 and COL5A1 expression in GC cells, and confirmed that miR-29a-3p is the common regulator of their expression. RNA immunoprecipitation assays based on Ago2, Dicer, and exportin-5 showed that SFA could promote mature miR-29a-3p generation. We also proved that SFA inactivated the Wnt/β-catenin pathway in GC cells in a miR-29a-3p-dependent manner. Overall, SFA boosts miR-29a-3p maturation to downregulate COL3A1 and COL5A1 and inactivate the Wnt/ β -catenin pathway to suppress GC progression., (Copyright © 2021. Published by Elsevier Inc.)

Corneal opacities affect vision for millions of individuals worldwide. Fibrotic scar tissues accumulate in reaction to inflammatory responses and remain permanently in corneal stroma, and conventionally correctable only by donor corneal transplantation. Numerous studies have explored innovative approaches to reverse corneal scarring through non-surgical means; however, existing mouse models limit these studies, due to the lack of visibility of scar tissue in mouse corneas with steep curvature. Here, we reported that corneal scarring was modelled using a transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, in which enhanced green fluorescence protein (EGFP) reporter expression was driven by the promoter of collagen 3a1 (COL3a1), a stromal fibrosis gene. Similar to wildtype, Col3a1-EGFP transgenic corneas developed opacities after wounding by alkali burn and mechanical ablation, respectively, as examined under stereomicroscopy and Spectral Domain optical coherent tomography. The time course induction of EGFP was aligned with Col3a1 upregulation and matched with the elevated expression of other fibrosis genes (α-smooth muscle actin, fibronectin and tenascin C). Measured by flow cytometry and enzyme-linked immunosorbent assay, increased number of EGFP expressing cells and fluorescent intensities were correlated to corneal thickening and scar volume. After treatment with human corneal stromal stem cells or their exosomes, EGFP expression was downregulated together with the reduction of scar volume and fibrosis gene expression. These results have demonstrated that the transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, can be a valuable tool for the detection of corneal fibrosis and scarring in vivo, and will be useful in monitoring the changes of corneal fibrosis over time., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

BACKGROUND Glucocorticoid-induced osteoporosis (GIOP) represents the most frequently seen type of secondary osteoporosis, a systemic skeleton disorder. Numerous factors are associated with GIOP occurrence, but there are no specific diagnostic and therapeutic biomarkers for GIOP so far. MATERIAL AND METHODS In this work, gene modules related to GIOP were screened through weighted gene coexpression network analysis. Moreover, protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were carried out for hub genes. In addition, microarray GSE30159 dataset was used as a training set to analyze gene expression within bone biopsy samples from patients with endogenous Cushing's syndrome with GIOP and from normal controls. GSE129228 was used as the test set for investigating the hub gene involvement within GIOP. RESULTS According to our results, the turquoise module showed clinical significance, and 10 genes (COL3A1, POSTN, COL6A3, COL14A1, SERPINH1, ASPN, OGN, THY1, NID2, and TNMD) were discovered to be the "real" hub genes within coexpression as well as PPI networks. GSEA showed that the interaction of extracellular matrix receptors together with the focal adhesion pathway had significant enrichment within samples with high COL3A1 and COL6A3 expression. After the results from both test and training sets were overlapped, SERPINH1 was also significantly altered between GIOP and normal control samples. CONCLUSIONS COL3A1, COL6A3, and SERPINH1 were identified to be the candidate biomarkers for GIOP.

Objective: This study aimed to clarify the underlying mechanism of pathognomonic angiogenesis between the temporal muscle and neocortex after indirect bypass for moyamoya disease by shedding light on the role of platelet-derived growth factor receptor-α (PDGFRα) in angiogenesis., Methods: The gene for PDGFRα was systemically inactivated in adult mice (α-KO mice). The Pdgfra-preserving mice (Flox mice) and α-KO mice were exposed to bilateral common carotid artery stenosis (BCAS) by using microcoils. One week later the animals underwent encephalomyosynangiosis (EMS) on the right side. Cerebral blood flow (CBF) was serially measured using a laser Doppler flowmeter. Histological analysis was performed on the distribution of CD31-positive vessels and collagen deposit at 28 days after BCAS. Reverse transcription polymerase chain reaction (RT-PCR) was performed to assess the expression of collagen mRNA in the skin fibroblasts derived from Flox and α-KO mice., Results: BCAS significantly reduced CBF up to approximately 70% of the control level at 28 days after the onset. There was no significant difference in CBF between Flox and α-KO mice. EMS significantly enhanced the improvement of CBF on the ipsilateral side of Flox mice, but not α-KO mice. EMS significantly induced the development of CD31-positive vessels in both the neocortex and temporal muscle on the ipsilateral side of Flox mice, but not α-KO mice. Deposition of collagen was distinctly observed between them in Flox mice, but not α-KO mice. Expression of mRNA of collagen type 1 alpha 1 (Col1a1) and collagen type 3 alpha 1 (Col3a1) was significantly downregulated in the skin fibroblasts from α-KO mice., Conclusions: This is the first study that denotes the role of a specific growth factor in angiogenesis after EMS for moyamoya disease by inactivating its gene in mice. The findings strongly suggest that PDGFRα signal may play an important role in developing spontaneous angiogenesis between the temporal muscle and neocortex after EMS in moyamoya disease.

Skin fibrosis occurs in a variety of human diseases but the current anti-fibrosis treatments are not sufficient. One major cause of fibrotic diseases shared across diverse organ fibrosis is uncontrolled overexpression of the connective tissue growth factor (CTGF, also known as CCN2). Here, we examine the anti-fibrotic activity of RNAi therapy utilizing siRNA against CTGF with a new drug delivery system (DDS), 'DegradaBALL', which is based on porous nanoparticles, for durable CTGF gene silencing. DegradaBALL is a modular DDS having many favorable properties for RNA delivery such as effective intracellular uptake, convenient drug loading, biocompatibility, sustained release profile and biodegradability. DegradaBALL loaded with siCTGF, named 'LEM-S401', showed highly durable and effective CTGF gene-silencing in TGF-β induced lung fibrosis and skin fibrosis model cells, A549 and HaCaT, respectively. In addition, LEM-S401 induced knockdown of collagen types I and III, which are excess extracellular matrix components in fibrotic skin in addition to CTGF in the mouse wound healing model. Most importantly, we showed that LEM-S401 effectively inhibited the formation of hypertrophic scars in wound-associated dermal fibrosis mouse models, during both the epidermis recovery and tissue remodeling process. Our findings suggest that LEM-S401 could be a highly potent therapeutic option for skin fibrotic diseases.

Introduction: Although it is beneficial to protect the skin from natural aging, especially in an aging society, the approach by which this can be achieved is still not well known. Hochu-ekki-to, a Chinese natural medicine, has various advantageous effects; however, there is no report about its influence on skin aging., Objective: Therefore, we examined the effect of hochu-ekki-to against natural aging., Methods: Hairless mice, bred without ultraviolet ray irradiation and physical stress, were orally administered huchu-ekki-to 3 times per week for 2 years. After that period, degree of skin hydration and permeability were measured. Furthermore, hematoxylin and eosin histochemistry was performed to determine the morphology and condition of the tissues. Lastly, levels of vitamin A, vitamin C, and reactive oxygen species (ROS) in plasma and skin, as well as concentration of hyaluronic acid in the skin, were measured., Results: Signs of skin aging were ameliorated by administration of hochu-ekki-to, such as moisture retention, skin hydration, and the generation of wrinkles. Furthermore, vitamin A, vitamin C, collagen type I, collagen type III, fibroblasts, and hyaluronic acid levels in the skin increased, while levels of ROS decreased after hochu-ekki-to treatment., Conclusion: These results indicated that natural skin aging was ameliorated by treatment with hochu-ekki-to, specifically moisture retention, and skin hydration, and thickening, via the regulation of the vitamin C/fibroblast, collagen type III/collagen type I, and vitamin A/hyaluronic acid signaling pathways., (© 2019 S. Karger AG, Basel.)

Background: Little literature exits on the mechanism of action of implanted polymethylmethacrylate (PMMA) filler., Objective: To characterize PMMA-induced dermal extracellular matrix production in the skin., Materials and Methods: Single-center, open-label prospective study in healthy volunteers undergoing removal of redundant skin was injected intradermally and subdermally with PMMA dermal filler (Bellafill). Punch biopsies were harvested over a time course and evaluated for the deposition of collagen-3 and procollagen-1, proteoglycans and elastin using immunohistochemistry. Blinded histopathologic readings were performed by a dermatopathologist to characterize the nature of the dermal extracellular matrix findings., Results: Normal inflammatory infiltrate was exhibited at all timepoints after PMMA injection with an influx of fibroblasts and new vasculature. Tissue proteoglycans were noted within the injectate beginning at Week 1 and persisted through the study end point. Increased collagen Type 3 was evident following the first week after injection, peaked at Month 2 and diminished through Months 3 through 6. Procollagen-1 was noted at Month 1 and continued to increase in intensity and organization through the study end point (6 months). Elastin staining was inconclusive. Polymethylmethacrylate microspheres remained within the initial injection area and became encapsulated within new collagen fibers. The growth and pattern of new connective tissue mimicked a normal wound healing response., Conclusion: Polymethylmethacrylate-collagen gel filler stimulates collagen-3 and procollagen-1 when injected into human skin. This combination of neocollagenesis followed by microencapsulation of PMMA microspheres in the new tissue provides for long-lasting results.

Renal fibrosis is a key pathological phenomenon of chronic kidney disease (CKD) contributing to the progressive loss of renal function. UK383,367 is a procollagen C proteinase inhibitor that has been selected as a candidate for dermal antiscarring agents, whereas its role in renal fibrosis is unclear. In the present study, UK383,367 was applied to a CKD mouse model of unilateral ureteral obstruction (UUO) and cell lines of renal tubular epithelial cells (mouse proximal tubular cells) and renal fibroblast cells (NRK-49F cells) challenged by transforming growth factor-β 1 . In vivo, bone morphogenetic protein 1, the target of UK383,367, was significantly enhanced in UUO mouse kidneys and renal biopsies from patients with CKD. Strikingly, UK383,367 administration ameliorated tubulointerstitial fibrosis as shown by Masson's trichrome staining in line with the blocked expression of collagen type I/III, fibronectin, and α-smooth muscle actin in the kidneys from UUO mice. Similarly, the enhanced inflammatory factors in obstructed kidneys were also blunted. In vitro, UK383,367 pretreatment inhibited the induction of collagen type I/III, fibronectin, and α-smooth muscle actin in both mouse proximal tubular cells and NRK-49F cells treated with transforming growth factor-β 1 . Taken together, these findings indicate that the bone morphogenetic protein 1 inhibitor UK383,367 could serve as a potential drug in antagonizing CKD renal fibrosis by acting on the maturation and deposition of collagen and the subsequent profibrotic response and inflammation.

The aim of this study was to explore the role of NRON in the atrial fibrosis. The expression of NRON in atrial tissue was detected using qRT-PCR. The protein levels of collagen I, collagen III, NFATc3 and p-NFATc3 were determined by western blot. Immunohistochemistry assay were performed to observe expression and distribution of collagen I in atrial tissues. The atrial fibroblasts were authenticated by vimentin/troponin immunofluorescence staining. Fibroblast proliferation was detected by CCK-8 assay. The morphological changes of cardiac tissue were observed by HE staining. Myocardial fibrosis was detected by masson staining. NRON expression was significantly downregulated in atrial tissues of AF. NRON suppressed fibroblast proliferation; expression of collagen I and collagen III; activation of NFATc3 and nucleu import. NRON promoted p-NFATc3 expression and alleviated atrial fibrosis in vivo. Our data indicated that NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation.

Background: Because of long-term exposure of skin, skin aging is an unavoidable natural law with age. Traditional Vitamin A and novel ablative fractional laser technique both have the effects of skin rejuvenation, and studies have demonstrated both of them have apparent clinical efficacy and histology-improving effects on photo-aging skin., Materials and Methods: 45 female healthy Wistar rats were selected and the depilation areas of every rat were divided into four regions: control region(Region A), Vitamin A acid region(Region B), combination treatment region(Region C), and fractional laser region(Region D). 0.025% Vitamin A acid cream was applied to Region B and C every day for 3 weeks; Region C and D were irradiated once with 10600nm CO 2 fractional laser on the first day of the trail. The skin tissue was dissected and placed into liquid nitrogen according to the design. The real-time quantitative PCR and western blotting methods were taken to detect the expression changes of miR-29a, Akt, TGF-β, and mRNA of type III pre-collagen., Results: It can be seen from the results of the real-time quantitative PCR that the mRNA expression levels of type III pre-collagen, Akt, and TGF-β in the treatment regions are up-regulated and the expression levels of miR-29a mRNA are down-regulated compared to the Region A. The hybridization tests showed that changes of the expression of type III pre-collagen, Akt gene, miR-29a gene, and TGF-β gene across the experiment regions are all significantly different in the third week, and the expression levels of them all achieve the highest value in the third week, the expression level of miR-29a gene achieves the lowest value in the third week, which are consistent with the results of real-time quantitative PCR., Conclusion: It is indicated that the combination region of Vitamin A acid and fractional laser may lead to low expression of miR-29a, thus the inhibition of downstream Akt activation is loss, Akt activation is enhanced, enhancement of the expression of TGF-β is induced, leading to proliferation of fibroblasts, and promotion of the collagen proteins' synthesis in skin. Therefore miR-29a/Akt/TGF-β signal pathway may participate in the skin rejuvenation mechanism of action Vitamin A acid and fractional laser. This may provide a new treatment approach for skin rejuvenation.

Aims: Collagen-derived peptides such as collagen I C-terminal telopeptide (CITP) and procollagen III N-terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression., Methods and Results: In 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = -0.41, P < 0.001 and r = -0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B-type natriuretic peptide (r = 0.32, P = 0.003) and γ-glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end-diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end-diastolic volume index nor with ejection fraction. Each collagen-derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA., Conclusions: Our study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra-cardiac organ injury in heart failure., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

BACKGROUND This study investigated the expression of Bax/Bcl-2, TGF-β1 and type III collagen fiber in sternocleidomastoid of congenital muscular torticollis (CMT), and explored the possible mechanisms of fibrosis in sternocleidomastoid of CMT. MATERIAL AND METHODS The localization and expression of Bax, Bcl-2, TGF-β1, and type III collagen were detected in the control group and experimental group by using immunohistochemical staining method. The RT-PCR assay was used to measure the expression of TGF-β1 in the control group and experimental group. RESULTS HE staining results showed that the collagen fiber in the experimental group had more abundant hyperplasia compared to the control group (p<0.05). Immunohistochemical staining results showed that the expression of Bax, Bax/Bcl-2, TGF-β1, and type III collagen in the experimental group was significantly increased compared to the control group (p<0.01). There were positive correlations between expression of Bax/Bcl-2 and TGF-b1, and between expression of TGF-β1 and type III collagen fiber (p<0.05, r=0.32 and 0.83, respectively). The RT-PCR results showed that the expression of TGF-β1 mRNA was also significantly elevated in the experimental group compared to the control group (p<0.05). CONCLUSIONS Increased muscular apoptosis may aggravate the formation of muscular fibrosis, which may be involved in the pathogenesis of sternocleidomastoid of CMT.

We investigated the effects of a low protein (LP) maternal diet during lactation on type I and III tropocollagen synthesis in infant mouse skin. The LP diet decreased the levels of type I and III tropocollagen proteins and COL1A1 and COL3A1 mRNA. Thus, the protein composition of the maternal perinatal diet may influence the skin health of offspring.

Red grape (Vitis vinifera L.) flavonoids including flavan-3-ols (eg, catechin and epicatechin), flavonols (eg, quercetin) and anthocyanins (eg, malvidin) exert anti-inflammatory and antioxidant activities. In the skin they also have a photoprotective action, and their effects have been extensively investigated in keratinocytes, melanocytes and fibroblasts. Despite their known effects also on blood vasculature, little is known on their activities on human dermal blood endothelial cells (HDBECs), which are critically involved in skin homeostasis as well as in the pathogenesis of neoplastic and inflammatory skin diseases. We sought to study the biological effects of selected red grape flavonoids in preventing the consequences of ultraviolet (UV)-A irradiation in vitro. Our results show that red grape flavonoids prevent UV-A-induced sICAM-1 release in HDBECs, suggesting that this cell type could represent an additional target of the anti-inflammatory activity of flavonoids. In addition, flavonoids effectively inhibited UV-A-induced synthesis of collagen type III at both RNA and protein level, indicating that dermal blood microvasculature could be actively involved in ECM remodelling as a consequence of skin photo-ageing, and that this can be prevented by red grape flavonoids., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Increased ochratoxin A (OTA) or citrinin (CIT) concentrations in food correlate with increased prevalence of tubule-interstitial nephropathy. We tested the hypothesis that co-exposure of human proximal tubule-derived epithelial cells (HK-2) to OTA and CIT promotes synergistic events indicative for inflammation, epithelial-to-mesenchymal-transition (EMT) or fibrosis. We measured markers of inflammation, EMT and fibrosis and investigated the role of MAP-kinases. Only concurrent but not individual exposure to OTA and CIT at nanomolar concentrations led to (i) an increase of TNF protein and mRNA, (ii) a decrease of COX-2 protein and mRNA, (iii) a decrease of E-cadherin protein and (iv) an increase of vimentin and α-SMA protein. Cell shape shifted from a cobblestone- to a spindle-like phenotype indicating EMT. Extra- and intracellular collagen III protein content was increased. Concomitant mRNA expression changes were observed for TNF, COX-2, E-cadherin and α-SMA indicating transcriptional regulation. This was not the case for vimentin and collagen III mRNA indicating posttranscriptional regulation. Inhibition of ERK 1/2 and JNK 1/2 reduced the effect on TNF but not on α-SMA mRNA indicating an involvement of these kinases. Phosphorylation of ERK1/2 was increased by CIT, OTA alone and the mycotoxin combination. In contrast, the phosphorylation of JNK1/2 was unchanged. In conclusion, nanomolar OTA and CIT act synergistically favouring nephropathic processes., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the pathophysiology of osteoarthritis (OA), articular cartilage degeneration exhibits a significant role. Vascular endothelial growth factor (VEGF) is considered to be an effective angiogenic factor and a crucial regulator of articular cartilage degeneration in the development of OA. Therefore, the present study aimed to investigate the underlying influences of exogenous VEGF on articular cartilage degeneration in OA model rat. A total of 24 male Sprague‑Dawley rats were randomly allocated into 3 groups. In the normal saline (NS) and VEGF groups, animals received bilateral anterior cruciate ligament (ACL) transection to establish the OA model; at 4 weeks post‑surgery, the rats received local intra‑articular injections of 100 µl NS or VEGF solution, respectively, every week for 4 weeks. The Control group received neither surgery nor injections. All animals were sacrificed at 12 weeks following surgery. Prominent cartilage degeneration was observed in rats in the NS‑ and VEGF‑injected groups. The extent and the grade of cartilage damage in the VEGF‑injected group were notably more severe compared with those in the NS‑treated group. Western blotting results demonstrated that the expression levels of aggrecan and type II collagen were significantly reduced in OA model rats that were treated with VEGF. In addition, the expression levels of matrix metalloproteinase (MMP)‑3, MMP‑9, MMP‑13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)‑4, ‑5 and ‑12, type III collagen and transforming growth factor‑β1 were significantly increased following VEGF administration. Results from the present study indicated that VEGF may exhibit a promoting role in the development of OA by destroying articular cartilage matrix.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50 mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I-III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

Exogenous growth factors could accelerate the process of wound healing. However, the underlying mechanisms have not been clearly clarified. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the expression of type I collagen (cola1), type III collagen (cola3), and Elastin in fibroblasts, which are widely expressed in fibroblasts and promote the function of fibroblasts. We measured the levels of cola1 and cola3 in human fibroblast cells cultured in medium containing EGF or bFGF at concentrations ranging from 0.1 to 1000 μg/L by Western blotting and RT-PCR assays, and found that EGF or bFGF enhanced the expression of cola1 and cola3 in a concentration-dependent manner. We further discovered that after stimulation with EGF or bFGF in human fibroblast cells, mitogen-activated protein kinases (MAPK) family members were generally activated, whose expression trend was consistent with that of cola1, cola3, and Elastin. In summary, in this study, we uncovered that exogenous growth factors enhance the expression of cola1, cola3, and Elastin, which is probably regulated via activating MAPK signaling pathway.

Branched-chain amino acids (BCAAs) exhibit many physiological functions. However, the potential link and mechanism between BCAA and skin function are unknown. We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in BCAA catabolism, on type I and III tropocollagen syntheses in mice. Leucine and isoleucine levels were significantly lower in the skin of BDK-KO mice compared with wild-type mice. No changes in valine concentrations were observed. The levels of type I and III tropocollagen proteins and mRNAs (COL1A1 and COL3A1) were significantly lower in the skin of BDK-KO mice compared with wild-type mice. The phosphorylation of p70 S6 kinase, which indicates mammalian target of rapamycin (mTOR) activation, was reduced in the skin of BDK-KO mice compared with wild-type mice. These findings suggest that deficiencies of leucine and isoleucine reduce type I and III tropocollagen syntheses in skin by suppressing the action of mTOR.

Fibrosis of the subsynovial connective tissue (SSCT) in carpal tunnel syndrome (CTS) patients is increasingly recognized as an important aspect of CTS pathophysiology. In this study, we evaluated the effect of blocking profibrotic pathways in fibroblasts from the SSCT in CTS patients. Fibroblasts were stimulated with transforming growth factor β1 (TGF-β1), and then treated either with a specific fibrosis pathway inhibitor targeting TGF-β receptor type 1 (TβRI), platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor (VEGFR). Fibrosis array and quantitative real-time polymerase chain reaction of fibrotic genes were evaluated. Array gene expression analysis revealed significant down-regulation of multiple fibrotic genes after treatment with TβRI, PDGFR, and VEGFR inhibitors. No array fibrotic genes were significantly down-regulated with EGFR inhibition. Further gene expression analysis of known CTS fibrosis markers collagen type I A2 (Col1), collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 showed significantly down-regulation after TβRI inhibition. In contrast, VEGFR inhibition significantly down-regulated CTGF and SERPINE1, whereas, PDGFR and EGFR inhibition significantly down-regulated Col3. Taken together the inhibition of TβRI appears to be the primary mediator of fibrotic gene expression in fibroblasts from CTS patients. TGF-β/Smad activity was further evaluated, and as expected inhibition of Smad activity was significantly down-regulated after inhibition of TβRI, but not with PDGFR, VEGFR, or EGFR inhibition. These results indicate that local therapies specifically targeting TGF-β signaling alone or in combination offer the potential of a novel local antifibrosis therapy for patients with CTS., (© 2017 Wiley Periodicals, Inc.)

Introduction: MiR-195 has been implicated in the development of abdominal aortic aneurysms (AAA). However, the underlying mechanisms have not been fully addressed., Aim: To explore the roles of miR-195 in regulating the proliferation and apoptosis of vascular smooth muscle cells (VSMCs), as well as its molecular basis in vitro., Methods: qRT-PCR was used to determine the expression levels of miR-195 and Smad3 in aortic media specimens or VSMCs. Western blot was performed to detect the protein levels of Smad3, osteopontin (OPN), and collagen III in aortic media specimens and VSMCs. Luciferase reporter assay was applied to confirm the target of miR-195 in VSMCs. Proliferation and apoptosis of VSMCs were measured by MTT and flow cytometry, respectively., Results: In comparison with the normal controls, the levels of miR-195, OPN, and collagen III were significantly increased in AAA tissue. Smad3 was validated to be a direct target of miR-195. miR-195 inhibited proliferation and induced apoptosis of VSMCs, which was abated by Smad3 overexpression. Expression of OPN and collagen III was improved in VSMCs after transfection with miR-195 mimics, while up-regulation of Smad3 reversed this effect., Conclusion: MiR-195 promotes media remodeling by targeting Smad3 in AAA progression. This study suggests that miR-195 contributes to the pathogenesis of AAA and reveals a new targeted therapy strategy for AAA patients., (© 2017 John Wiley & Sons Ltd.)

Importance: Current models of Goltz syndrome cannot estimate the overall neocollagenesis and marked shift in collagen types after ablative fractional laser resurfacing (AFR) within treated areas of focal dermal hypoplasia (FDH)., Objectives: To clinically improve FDH by using AFR to characterize the specific ratio of collagen types associated with observed clinical changes., Design, Setting, and Participants: This case report of a girl with Goltz syndrome used extensive laboratory evaluation and multiple observers blinded to the patient's clinical status. Serial samples of clinically unaffected skin constituted internal control specimens, with clinical and histologic evaluations performed as part of a multicenter investigation. The analysis tested the hypothesis that thermal microtrauma caused by AFR created a unique environment that activated latent genes, inducing neocollagenesis and allowing the patient to adaptively produce the collagen subtype that was specifically deficient at baseline., Interventions: Two AFR treatments were administered within an area of FDH. Histologic comparison of the pretreatment and posttreatment skin was performed using serial internal controls., Main Outcomes and Measures: Histologic changes, including Herovici collagen staining to differentiate between types I and III collagen, within a treated area of mosaically affected FDH compared with clinically unaffected skin., Results: This female patient presented in the second decade of life with self-described red, itchy skin within a large plaque of FDH on her left posterior thigh and calf. After AFR, skin tightening and symptomatic relief were reported. Histologic findings demonstrated objective thickening of the dermal collagen. A marked shift in collagen predominance from type III (fetal/early wound) to type I (adult/mature) was observed., Conclusions and Relevance: Although further study is needed, this report shows promising results and raises important questions about gene expression and the epigenetics of Goltz syndrome-associated mutations and the local effects of AFR. Coupled with more rigorous investigation, this novel technique may help reveal molecular workarounds permitting innovative therapies that take advantage of the subtly different collagens that exist within the skin.

Aim: The aim of the present study was to evaluate, compare, and correlate the types of collagen fibers seen in different grades of oral cancer., Methods: Thirty cases of histologically-diagnosed, well-, moderately-, and poorly-differentiated oral squamous cell carcinoma (OSCC) were retrieved from the archives of the Institute. Collagen was evaluated using picrosirius red stain and immunohistochemical analysis of the antibody to type III collagen. A correlation between these findings and the grade of OSCC was evaluated., Results: Collagen fibers showed a change in birefringence ranging from reddish-orange to greenish-yellow in well- to poorly-differentiated oral squamous cell carcinoma. The findings were statistically significant for polarizing colors observed in grades of OSCC (P<.001). Immunohistochemical staining intensity of type III collagen changed from weak to strong as grade increased for OSCC, and was also statistically significant (P<.001)., Conclusion: In the present study, tumor progression reflected a change in collagen present, from type I to type III. Determination of the type of collagen in different grades of OSCC can facilitate therapeutic targeting of molecules responsible for invasion and progression of oral cancer., (© 2016 John Wiley & Sons Australia, Ltd.)

High-mobility group box protein-1 (HMGB-1) plays a central role in the inflammatory network, and uncontrolled chronic inflammation can lead to excessive scarring. The aim of this study was to evaluate the anti-inflammatory effects of polydeoxyribonucleotide (PDRN) on scar formation. Sprague-Dawley rats (n = 30) underwent dorsal excision of the skin, followed by skin repair. PDRN (8 mg/kg) was administered via intraperitoneal injection for three (PDRN-3 group, n = 8) or seven (PDRN-7 group, n = 8) days, and HMGB-1 was administered via intradermal injection in addition to PDRN treatment for three days (PDRN-3+HMGB-1 group; n = 6). The scar-reducing effects of PDRN were evaluated in the internal scar area and by inflammatory cell counts using histology and immunohistochemistry. Western blot, immunohistochemistry and immunofluorescence assays were performed to observe changes in type I and type III collagen and the expression of HMGB-1 and CD45. Treatment with PDRN significantly reduced the scar area, inflammatory cell infiltration and the number of CD45-positive cells. In addition, the increased expression of HMGB-1 observed in the sham group was significantly reduced after treatment with PDRN. Rats administered HMGB-1 in addition to PDRN exhibited scar areas with inflammatory cell infiltration similar to the sham group, and the collagen synthesis effects of PDRN were reversed. In summary, PDRN exerts anti-inflammatory and collagen synthesis effects via HMGB-1 suppression, preventing scar formation. Thus, we believe that the anti-inflammatory and collagen synthesis effects of PDRN resulted in faster wound healing and decreased scar formation., Competing Interests: None of the authors have any financial arrangements or potential conflicts of interest related to this article.

Background: The dynamics of the extracellular matrix (ECM) fibrosis process in dilated cardiomyopathy (DCM) may be assessed non-invasively by means of serum markers of fibrosis., Aim: To explore the kinetics of serum markers of fibrosis during a 12-month follow-up in DCM., Methods: We included 70 consecutive DCM patients (pts) (48±12.1years, EF 24.4±7.4%) with new-onset (n=35, duration <6months) and chronic DCM (n=35, >6months). Markers of collagen type I and III synthesis - procollagens type I and III carboxy- and amino-terminal peptides (PICP, PINP, PIIICP, PIIINP), and ECM metabolism controlling factors - tumor growth factor beta-1 (TGF1-β), and connective tissue growth factor (CTGF) - were measured in serum at baseline, and at 3- and 12-month follow-up. All pts underwent endomyocardial biopsy to determine the presence and extent of ECM fibrosis., Results: Markers of collagen type I synthesis (PICP and PINP) were almost homogenously increased over the 3- and 12-month period, whereas PIIINP values decreased and PIIICP levels were unchanged in new-onset and chronic DCM, and in pts with and without ECM fibrosis. Both TGF-β and CTGF levels decreased over the observation period. Kinetics of serum markers of collagen synthesis and fibrosis controlling factors did not differ between DCM pts categorized according to disease duration and fibrosis status., Conclusions: The kinetics of collagen type I and III synthesis in DCM move in opposite directions, with production of collagen type I consistently increasing, and the synthesis of collagen type III decreasing. Levels of TGF and CTGF, which are proven fibrosis-stimulating factors, had a tendency to decrease. Regardless of disease duration or fibrosis status, the kinetics of serum markers of collagen synthesis, TGF and CTGF were similar in DCM. A better understanding of the kinetics of serum markers of fibrosis in DCM may help to develop more tailored therapeutic approaches to fibrosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (∼3000pgml -1 ) was injected (3dwk -1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Surface modification of materials designed for regenerative medicine may improve biocompatibility and functionality. The application of glycosaminoglycans (GAGs) and chemically sulphated GAG derivatives is a promising approach for designing functional biomaterials, since GAGs interact with cell-derived growth factors and have been shown to support fibroblast growth in two-dimensional (2D) cultures. Here, coatings with artificial extracellular matrix (aECM), consisting of the structural protein collagen I and the GAG hyaluronan (HA) or sulphated HA derivatives, were investigated for their applicability in a three-dimensional (3D) system. As a model, macroporous poly(lactic-co-glycolic acid) (PLGA) scaffolds were homogeneously coated with aECM. The resulting scaffolds were characterized by compressive moduli of 0.9-1.2 MPa and pore sizes of 40-420 µm. Human dermal fibroblasts (dFbs) colonized these aECM-coated PLGA scaffolds to a depth of 400 µm within 14 days. In aECM-coated scaffolds, collagen I(α1) and collagen III(α1) mRNA expression was reduced, while matrix metalloproteinase-1 (MMP-1) mRNA expression was increased within 7 days, suggesting matrix-degradation processes. Stimulation with TGFβ1 generally increased cell density and collagen synthesis, demonstrating the efficiency of bioactive molecules in this 3D model. Thus, aECM with sulphated HA may modulate the effectivity of TGFβ1-induced collagen I(α1) expression, as demonstrated previously in 2D systems. Overall, the tested aECM with modified HA is also a suitable material for fibroblast growth under 3D conditions. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

To improve collagen production by recombinant Pichia pastoris, we applied Placket-Burman and Box-Behnken design to optimize the fermentation medium. Through Placket-Burman design, three variables in the medium (concentration of yeast extract, peptone and glycerol) were selected for having significant effect on cell dry weight. Through Box-Behnken design regression coefficients analysis, a secondary degree polynomial equation was established, and the optimum levels of the three variables were yeast extract 1.13%, peptone 1.61% and glycerol 0.86%. During the growth period, an average cell dry weight of 4.41 g/L was obtained after 12 h fermentation, increased by 26%. Through high density fermentation, the production of recombinant human collagen (Ⅲ) was up to 4.71 g/L in 22 L fermentor. The recombinant human collagen (Ⅲ) exhibited good results to repair acetic acid induced gastric ulcer in rats.

Vacuum sealing drainage (VSD) is an effective technique used to promote wound healing. However, recent studies have shown that it exerts positive pressure (PP) rather than negative pressure (NP) on skin. In this study, we created a homemade device that could maintain NP on the wound, and compared the therapeutic effects of VSD-induced PP to those of our homemade device which induced NP on wound healing. The NP induced by our device required less time for wound healing and decreased the wound area more efficiently than the PP induced by VSD. NP and PP both promoted the inflammatory response by upregulating neutrophil infiltration and interleukin (IL)‑1β expression, and downregulating IL‑10 expression. Higher levels of epidermal growth factor (EGF), transforming growth factor (TGF)‑β and platelet-derived growth factor (PDGF), and lower levels of basic fibroblast growth factor (bFGF) were observed in the wound tissue treated with NP compared to the wound tissue exposed to PP. Proliferation in the wound tissue exposed to NP on day 10 was significantly higher than that in wound tissue exposed to PP. NP generated more fibroblasts, keratinized stratified epithelium, and less epithelia with stemness than PP. The levels of ccollagen Ⅰ and Ⅲ were both decreased in both the NP and PP groups. NP induced a statistically significant increase in the expression of fibronectin (FN) on days 3 and 10 compared to PP. Furthermore, the level of matrix metalloproteinase (MMP)‑13 increased in the NP group, but decreased in the PP group on day 3. NP also induced a decrease in the levels of tissue inhibitor of metalloproteinase (TIMP)‑1 and TIMP‑2 during the early stages of wound healing, which was significantly different from the increasing effect of PP on TIMP‑1 and TIMP‑2 levels at the corresponding time points. On the whole, our data indicate that our homemade device which induced NP, was more efficient than VSD‑induced PP on wound healing by regulating inflammation, secretion, proliferation and the distribution of different cells in wound tissue.

Background: Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1., Methods: The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively., Results: After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole., Conclusion: The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The extracellular matrix (ECM) remodeling represents the pathological substrate of dilated cardiomyopathy (DCM). In this study, we statistically analyzed the immunoexpression of collagen I and III, matrix metalloproteinase-1 (MMP-1) and its tissue inhibitor-1 (TIMP-1) in the myocardial tissue in 18 cases of DCM compared to a control group. We observed a significant increase in the immunoexpression of collagen I and III in patients with DCM and a significant reduction in the immunoexpression of MMP-1 compared with the control group. Also, the collagen I and TIMP-1 expression indicated a positive linear correlation and respectively a negative linear relationship with collagen III and MMP-1. The analyzed markers in this study can be used to quantify the degree of collagen sclerosis from the ECM of DCM.

A known consequence of the large weight loss after bariatric surgery is the appearance of large skinfolds, particularly in the abdomen region of the patients. The balance between the synthesis of extracellular matrix (ECM) components and their proteolysis, mainly by fibrinolytic systems and matrix metalloproteases (MMPs), may be disturbed in these patients. The causes underlying the deregulation of ECM remodeling that occurs in these patients are not, however, clear. We investigated molecular mechanisms responsible for this dysfunction of ECM remodeling process, comparing it to normal skin. Collagen types, MMP2 and MMP9 expression and activity, interleukins 1β (IL1β) and 6 (IL6), and transcription coactivator PGC-1β expression were analyzed in 16 patients. Ex-obese patients presented increased expression of collagen types III and IV mRNA, increased expression of MMP2, decreased expression and activity of MMP9, and increased expression of PGC-1β in the skin. Inflammation markers IL1β and IL6 mRNA were not different. We have demonstrated that obese patients with extensive weight loss after bariatric surgery have increased expression of PGC-1β in the skin, which can result in a decreased expression and activity of MMP9 and increased collagen types III and IV deposition. These molecular changes may contribute for the formation of saggy skinfolds observed in these patients and impair wound healing.

Background and Purpose: Adenosine A 2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A 2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A 2A receptor is Gs-linked and increases cAMP, we determined whether A 2A receptors and Wnt signalling interact., Experimental Approach: Total β-catenin, de-phosphorylated β-catenin (canonical activation, de-phospho β-catenin) and phosphorylated β-catenin at Ser 552 (non-canonical activation, p-Ser 552 β-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A 2A receptor-selective agonist CGS21680, with/without A 2A receptor-selective antagonist (SCH56261) pretreatment. β-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots., Key Results: CGS21680 stimulation rapidly (15 min) increased cellular β-catenin levels. Both de-phospho β-catenin and p-Ser 552 β-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser 552 β-catenin to the nucleus. A 2A receptor-stimulation increased Col1 synthesis similarly in β-catenin knockeddown and scrambled cells. However, β-catenin knockdown abolished the increase in Col3 synthesis induced in A 2A receptor-stimulated fibroblasts., Conclusions and Implications: A 2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical β-catenin, consistent with a role for A 2A receptors in dermal fibrosis and scarring., (© 2016 The British Pharmacological Society.)

Myocardial fibrosis (MF), which typically occurs after a myocardial infarction (MI), is a major factor involved in the process of ventricular remodeling and subsequent progression to heart failure. Current studies have found that various microRNAs (miRNAs), such as miR-125b, play an important role in this process. However, few studies have investigated the specific mechanism of miR-125b. Transfection of miR-125b mimics into cardiac fibroblasts (CFs) resulted in significantly increased expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) and vinculin by Western blot analysis, while transfection of miR-125b inhibitors resulted in the opposite effect. Analysis of putative CF target genes for miR-125b revealed that miR-125b specifically inhibits expression of secreted frizzled-related protein 5 (SFRP5). SFRP5 inhibited expression of α-SMA and collagen I and III in CFs, while miR-125b promoted the expression of these proteins. Cotransfection of the SFRP5 overexpression vector and miR-125b mimics did not result in significant upregulation of SFRP5 expression or downregulation of α-SMA and collagen I and III. Further analysis revealed that miR-125b promotes the proliferation and migration of CFs and inhibits their apoptosis, while SFRP5 exhibits the opposite effects. These results indicate that miR-125b can regulate SFRP5 expression and thus influence the growth and activation of CFs. Hence, this study provides important insight into possible approaches for the prevention and treatment of MF after an MI., (© 2016 International Federation for Cell Biology.)

Duchenne muscular dystrophy (DMD) is a lethal muscle disease involving progressive loss of muscle regenerative capacity and increased fibrosis. We tested whether epigenetic silencing of the klotho gene occurs in the mdx mouse model of DMD and whether klotho silencing is an important feature of the disease. Our findings show that klotho undergoes muscle-specific silencing at the acute onset of mdx pathology. Klotho experiences increased methylation of CpG sites in its promoter region, which is associated with gene silencing, and increases in a repressive histone mark, H3K9me2. Expression of a klotho transgene in mdx mice restored their longevity, reduced muscle wasting, improved function and greatly increased the pool of muscle-resident stem cells required for regeneration. Reductions of fibrosis in late, progressive stages of the mdx pathology achieved by transgene expression were paralleled by reduced expression of Wnt target genes (axin-2), transforming growth factor-beta (TGF-β1) and collagens types 1 and 3, indicating that Klotho inhibition of the profibrotic Wnt/TGFβ axis underlies its anti-fibrotic effect in aging, dystrophic muscle. Thus, epigenetic silencing of klotho during muscular dystrophy contributes substantially to lost regenerative capacity and increased fibrosis of dystrophic muscle during late progressive stages of the disease., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fibrostrictures (FS) are a major complication of Crohn's disease (CD). Pathogenesis of FS is not fully understood, but activation of fibroblasts and excessive collagen deposition are crucial in the development of FS. Here, we investigated the role of aryl hydrocarbon receptor (AhR) in intestinal fibrosis. AhR RNA and protein expression were evaluated in intestinal fibroblasts of CD patients and controls. CD fibroblasts were stimulated with TGF-β1 or TNF-α in the presence or absence of the AhR activator Ficz, an AhR antagonist CH223191, or a specific AhR-silencing RNA. In CD fibroblasts, TGF-β1 and TNF-α increased Col1A1, Col3A1 and α-SMA transcripts and collagen secretion and this effect was reduced by Ficz and upregulated by CH22319. TGF-β1 or TNF-α induced activation of p38 and ERK1/2 MAP kinases was decreased by Ficz and increased by CH223191. The inhibitory effect of Ficz on Map kinase activation and collagen induction was abolished by AhR silencing. To assess the role of AhR in vivo, mice with trinitrobenzene-sulfonic-acid induced colonic fibrosis were given Ficz or CH223191. Mice given either Ficz or CH223191 produced less or more collagen respectively as compared with control mice. Our results indicate that AhR is a negative regulator of profibrotic signals in the gut., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To investigate the role of plasminogen activator inhibitor-1 (PAI-1) in the inhibition of rosiglitazone on the transformation and collagen synthesis of rats' embryo lung fibroblasts and to examine the signal pathways in the process., Methods: Fibroblasts from rats' embryo lung tissues were divided into 3 groups: Control, Rosiglitazone (Rosiglitazone 30 mmol/L) and PAI-1 (Rosiglitazone 30 mmol/L and PAI-1 20 mmol/L) groups. The fibroblasts were collected at 24, 48 and 72 h, and stored at -80 °C. RT-PCR was used to determine the expressions of collagen type-1 and type-3 at 24 h. Western blot analysis was used to determine the expressions of PAI-1, a-smooth muscle actin (α-SMA), p-AKT and p-ERK at 24, 48 and 72 h., Results: There was a significant decrease in the protein expression of PAI-1 in the Rosiglitazone group (0.732±0.015, 0.583±0.005, 0.762±0.032) at 24, 48 and 72 h compared with the Control group (1.116±0.046). There was a significant increase in the protein expression in the PAI-1 group (0.923±0.042, 1.024±0.009, 1.070±0.011) compared with the Rosiglitazone group (F=78.609, P<0.01). The α-SMA protein expressions were significantly reduced in the Rosiglitazone group (0.209±0.012, 0.280±0.140, 0.254±0.025) compared with the Control group (0.340±0.026), while the expressions were significantly increased in the PAI-1 group (0.386±0.042, 0.400±0.037, 0.385±0.026) compared with the Rosiglitazone group (F=35.009, P<0.01). The collagen type-1 (1.065±0.004) and type-3 (1.282±0.001) mRNA expressions were significantly reduced in the Rosiglitazone group compared with the Control group (1.279±0.013, 1.690±0.005), while the expressions were significantly increased in the PAI-1 groups (1.390±0.029, 1.350±0.044) compared with the Rosiglitazone group (type-1: F=12.429, P<0.01; type-3: F=127.456, P<0.01). The expressions of p-AKT showed no differences among the 3 groups, but there were significant differences in the expressions of p-ERK in the Rosiglitazone group (0.288±0.010, 0.311±0.034, 0.336±0.038) compared with the Control group (0.506±0.032), and in PAI-1 groups (0.561±0.101, 0.448±0.022, 0.406±0.003) compared with the Rosiglitazone group (F=153.548, P<0.01)., Conclusions: Rosiglitazone inhibits PAI-1 expression in fibroblasts from rats' embryo lung tissues and activates the fibrinolytic system. The up-regulation of PAI-1 expression alleviates the inhibition effect of rosiglitazone on the transformation and collagen synthesis of fibroblasts. The cross-talk between PAI-1 and ERK signal pathway may play an important role in the regulation of rosiglitazone on fibrosis.

Surgical repairs of rotator cuff tears have high re-tear rates and many scaffolds have been developed to augment the repair. Understanding the interaction between patients' cells and scaffolds is important for improving scaffold performance and tendon healing. In this in vitro study, we investigated the response of patient-derived tenocytes to eight different scaffolds. Tested scaffolds included X-Repair, Poly-Tape, LARS Ligament, BioFiber (synthetic scaffolds), BioFiber-CM (biosynthetic scaffold), GraftJacket, Permacol, and Conexa (biological scaffolds). Cell attachment, proliferation, gene expression, and morphology were assessed. After one day, more cells attached to synthetic scaffolds with dense, fine and aligned fibres (X-Repair and Poly-Tape). Despite low initial cell attachment, the human dermal scaffold (GraftJacket) promoted the greatest proliferation of cells over 13 days. Expression of collagen types I and III were upregulated in cells grown on non-cross-linked porcine dermis (Conexa). Interestingly, the ratio of collagen I to collagen III mRNA was lower on all dermal scaffolds compared to synthetic and biosynthetic scaffolds. These findings demonstrate significant differences in the response of patient-derived tendon cells to scaffolds that are routinely used for rotator cuff surgery. Synthetic scaffolds promoted increased cell adhesion and a tendon-like cellular phenotype, while biological scaffolds promoted cell proliferation and expression of collagen genes. However, no single scaffold was superior. Our results may help understand the way that patients' cells interact with scaffolds and guide the development of new scaffolds in the future.

The aim of the study was to learn the degree of biocompatibility of synthetic herniological prostheses «Progrip», with collagen-stimulating effect of ascorbic acid on the growth of connective tissue in the experiment. We examined characteristics of collagenogenesis in the anterior abdominal wall depending on the effect of vitamin C using the method of polarization microscopy. We suggest a new way to stimulate reparation process by ascorbic acid which would help to optimize forming functionally matured structures of connective tissue of the anterior abdominal wall surrounding endoprosthesis. Due to the results of our investigation it has been clear that modification effect of vitamin C is positive for synthesis of collagen in the periprosthetic capsule for any type of endoprosthesis. It has been proves by statistically reliable increase in ratio of collagen types I and III when vitamin C is used in the diet of experimental animals.

The purpose of this article is to investigate the effect of Opuntia stricta H (Cactaceae) extract on suppression of hypertrophic scar on ventral surface wounds of rabbit ears. Full thickness skin defection was established in a rabbit ear to simulate hypertrophic scar. Opuntia extract was sprayed on the wounds in the experimental group, and normal saline was used in the control group. After the wounds healed with scar formation, the hypertrophic scar tissue was harvested on days 22, 39, and 54 for histological analysis. The expression of type I and type III collagen and matrix metalloproteinase-1 (MMP-1) were evaluated by immunohistochemistry and real-time quantitative polymerase chain reaction. The results indicated that the scar of the control group is more prominent compared with the opuntia extract group. The expression of type I collagen in the opuntia extract group was lower than the control group, while type III collagen in opuntia extract group gradually increased and exceeded control group. The expression of MMP-1 decreased in the opuntia extract group, while the control group increased over time, but the amount of MMP-1 was much higher than that in the control group on day 22. In conclusion, opuntia extract reduces hypertrophic scar formation by means of type I collagen inhibition, and increasing type III collagen and MMP-1.T he novel application of opuntia extract may lead to innovative and effective antiscarring therapies., (© The Author(s) 2015.)

Leukemia inhibitory factory (LIF), as a member of the IL-6 family, has been reported to ameliorate myocardial fibrosis and myocardial cell death. The purpose of the present study was to investigate the effect of LIF on renal fibrosis and its underlying mechanism. Our results showed, first, that LIF inhibited collagen type 1 and collagen type 3 expression induced by ANG II in NRK-49F (rat kidney fibroblast) cells and in mice with unilateral ureteral obstruction. Second, LIF induced Stat3 Tyr(705) phosphorylation and inhibited Stat3 Tyr(705) and Ser(727) phosphorylation induced by ANG II in NRK-49F cells. Third, LIF exerted an antirenal fibrosis effect mainly through activation of Stat3 Tyr(705) phosphorylation in NRK-49F cells. These effects of LIF were not observed in Stat3(-/-) cells. Finally, LIF-Stat3 upregulated microRNA-29c expression, and the latter downregulated collagen type 1 and collagen type 3 expression in NRK-49F cells and in mice with unilateral ureteral obstruction. In conclusion, LIF played a role in antirenal fibrosis by competitively activating Stat3 Tyr(705) phosphorylation, which upregulated microRNA-29c to suppress collagen expression., (Copyright © 2015 the American Physiological Society.)

Unlabelled: In this study, we co-expressed the human prolyl 4-hydroxylases (P4H) with human collagen α1 (III) (COL3A1) in an inducible system: Pichia pastoris (pPICZB), and one constitutive system: P. pastoris (pGAPZαB). The P4H catalyses the post-translational hydroxylation of proline residues in collagen strands. Conventional protein expression system such as bacteria and yeasts, which lack endogenous P4H, are not efficient for the production of recombinant collagen. In this study, the P4H gene was constructed in pGAPZαB plasmid and pPICZB plasmid respectively. These two plasmids were transformed in P. pastoris #1 that carrying COL3A1. Colony PCR analysis and sequencing after electroporation P. pastoris GS115 showed that the target gene had inserted successfully. The results of reverse transcript-qPCR, SDS-PAGE, Western blotting and LC-MS/MS analysis of the rhCOL3A1 demonstrated that the P4H was expressed successfully. Besides, it is noted that low copy number, constitutive system was suitable for hydroxylated rhCOL3A1., Significance and Impact of the Study: Successful co-expression of recombinant human collagen α1 (III) (rhCOL3A1) and human prolyl 4-hydroxylases (P4H) in Picha pastoris GS115, simultaneously results in the acquisition of rhCOL3A1 with hydroxylation of proline (Hyp). Further, this experiment also discusses that the high or low copy numbers and different promoters affect the Hyp degree of rhCOL3A1. Selecting more appropriate strains can express high degree Hyp of rhCOL3A1. This work will be helpful to the collagen structure study., (© 2015 The Society for Applied Microbiology.)

Objective: to study a change in the content of main components of the internal carotid artery (ICA) wall in pathological tortuosity (PT) resulting from fibromuscular dysplasia, using immunohistochemistry and confocal laser scanning microscopy., Material and Methods: Immunohistochemical (IHC) analysis using antibodies to elastin, collagen types I and III, and smooth muscle actin was made. The levels of elastin and matrix metalloproteinase 9 (MMP-9) were determined by confocal laser scanning microscopy. The relative area of expression and the area of co-location of these markers were measured., Results: IHC examination of the expression of elastin revealed that the patients with PT of ICA had its higher content than the controls, but they were observed to have fiber fragmentation. Comparison of collagen types I and III expressions showed no significant differences between the groups. The found significantly lower smooth muscle actin expression in the patients with PT of ICA than in the controls was suggestive of the decreased levels of smooth muscle cells. Confocal microscopy analysis showed high elastin and low MMP-9 expressions in the control group and, on the contrary, low elastin and high proteinase levels in the PT group (р<0.05)., Conclusion: One of the causes of PT is impairment in vascular elastic properties due to the destruction of elastic fibers and to their fragmentation, as well as to the decreased count of smooth muscle cells, which in turn causes enhanced MMP-9 activity and tissue matrix degradation.

Decorin is a natural transforming growth factor-β1 (TGF-β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid-derived fibroblasts (KFs) were transduced with decorin-expressing adenovirus (dE1-RGD/GFP/DCN), and we examined the therapeutic potential of decorin-expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF-β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) mRNA levels were measured by real-time RT-PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1-RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1-RGD/GFP/DCN, secreted TGF-β1 and EGFR protein expressions were decreased in TGF-β1-treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP-1 and MMP-3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1-RGD/GFP/DCN-transduced keloid spheroids. These results support the utility of decorin-expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recombinant collagen and gelatin have been applied in biomedical materials field because of products from genetically engineered microorganisms with improved safety, traceability, reproducibility, and homogeneous quality. To obtain high-level secretory expression of single-chain full-length human collagen α1(III) chain (COL3A1) without the N and C telopeptides, the cDNA coding for the human COL3A1 gene was cloned into the secretory expression vector pPIC9K and integrated into Pichia pastoris GS115. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis of culture supernatant from the recombinant methylotrophic yeast suggested that the unhydroxylated recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium, and exhibited an apparent molecular mass of approximately 130 kDa, which is 1.4 times higher than the theoretical one. Finally, the unhydroxylated rhCOL3A1 was purified to greater than 90% purity using a four-step approach. In addition, methylthiazolydiphenyl-tetrazolium bromide experiments indicated that low concentration of rhCOL3A1 could promote Baby hamster kidney cell (BHK21) proliferation effectively. The production and purification of rhCOL3A1 described in this study offer a new method for obtaining high level of rhCOL3A1 in relatively pure form, which is suitable for biomedical materials application., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

Objective: To explore the expression of miR-21 in lung tissues of mice with pulmonary fibrosis, and its role in proliferation, trans-differentiation and collagen synthesis of pulmonary fibroblasts., Methods: Thirty C57BL/6 mice were randomly divided into control group (n=5) and model group (n=25). Mice in the latter group were intratracheally administered with bleomycin to establish pulmonary fibrosis model. Five animals in the model group were sacrificed on days 3, 7, 14, 28 and 56, while all ones of the control group were killed on day 56 as total control. The expression of miR-21 in lung tissues was then measured through fluorescence real-time quantitative PCR. Pulmonary fibroblasts were treated with Lipofectamine™2000 (blank control group) as well as miR-21 negative control, mimics and inhibitor, respectively. Cell proliferative ability was analyzed by MTT assay. The mRNA and protein expressions of α-smooth muscle actin (α-SMA), a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1), collagen type I (Col1) and collagen type III (Col3) were detected using fluorescence real-time quantitative PCR and Western blotting, respectively., Results: The expression levels of miR-21 in lung tissues of the model group on days 7-56 were higher than those in the control group, and reached the peak on day 28. After 24, 48, 72 and 96 hours of co-culture, cell viability significantly increased in miR-21 mimics group but decreased in miR-21 inhibitor group as compared with blank control group. In comparison with blank control group, miR-21 mimics group showed up-regulated mRNA and protein expression levels of α-SMA, Col1 and Col3 while down-regulated ADAMTS-1 mRNA and protein expression levels; however, miR-21 inhibitor group presented with reduced mRNA and protein expression levels of α-SMA, Col1 and Col3 but increased ADAMTS-1 mRNA and protein expression levels. There was no significant difference in the above indicators between negative control group and blank control group., Conclusion: miR-21 expression is raised in lung tissues of mice with pulmonary fibrosis. Up-regulation of miR-21 facilitates the proliferation, trans-differentiation and collagen synthesis of pulmonary fibroblasts.

Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

Cardiac fibroblasts (CFs) activation plays a vital role in cardiac fibrosis. There are some studies demonstrate that norepinephrine (NE, an α1-adrenoceptor agonist) induced CFs proliferation. But whether Ca2+-calcineurin, a signaling concerned with growth and differentiation in various cell types, is participated in NE-induced CFs activation is unclear. In present study, we determined NE-induced CFs proliferation and differentiation, synthesis of collagen, and calcineurin (CaN) activity, and the effects of phentolamine (Phen, an α1-adrenoceptor antagonist), verapamil (Ver, a calcium channel blocker) and cyclosporine A (CsA, an inhibitor of CaN) on NE-induced CFs activation. The results showed that NE induced CFs proliferation and differentiation, increased α-SMA protein expression, increased collagen I, collagen III and fibronectin production, promoted ECM expression, activated CaN and increased CaN protein expression, which were inhibited by Phen, Ver and CsA. In vivo, more collagen deposition could be observed and total collagen volume fraction (CVF) was significantly increased in NE group. Phen, Ver and CsA decreased NE-induced collagen deposition, reduced cardiac fibrosis. Thus, our results demonstrate that Ca2+/CaN is involved in NE-induced CFs proliferation and collagen synthesis.