Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329.

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.

Overexpressed COL3A1 has prognostic value in human esophageal squamous cell carcinoma and promotes the aggressiveness of esophageal squamous cell carcinoma by activating the NF-κB pathway.

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.

miR-29a-3p-dependent COL3A1 and COL5A1 expression reduction assists sulforaphane to inhibit gastric cancer progression.

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/ß-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/ ß -catenin pathway to suppress GC progression.

COL3A1, COL6A3, and SERPINH1 Are Related to Glucocorticoid-Induced Osteoporosis Occurrence According to Integrated Bioinformatics Analysis.

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.

A novel transgenic mouse model for corneal scar visualization.

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.

Critical role of platelet-derived growth factor-α in angiogenesis after indirect bypass in a murine moyamoya disease model.

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.

RNAi nanotherapy for fibrosis: highly durable knockdown of CTGF/CCN-2 using siRNA-DegradaBALL (LEM-S401) to treat skin fibrotic diseases.

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-ß 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.

Ameliorative Effect of Hochu-ekki-to on Natural Skin Aging.

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.

Histologic Characterization of Polymethylmethacrylate Dermal Filler Biostimulatory Properties in Human Skin.

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.

BMP1 inhibitor UK383,367 attenuates renal fibrosis and inflammation in CKD.

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-ß1. 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-ß1. 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.

LncRNA NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation.

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.

The comparison of skin rejuvenation effects of vitamin A, fractional laser, and their combination on rat.

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 CO2 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-ß, 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-ß 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-ß 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-ß is induced, leading to proliferation of fibroblasts, and promotion of the collagen proteins' synthesis in skin. Therefore miR-29a/Akt/TGF-ß 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.

Expression of Bax/Bcl-2, TGF-ß1, and Type III Collagen Fiber in Congenital Muscular Torticollis.

BACKGROUND This study investigated the expression of Bax/Bcl-2, TGF-ß1 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-ß1, 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-ß1 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-ß1, 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-ß1 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-ß1 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.

Circulating markers of collagen types I, III, and IV in patients with dilated cardiomyopathy: relationships with myocardial collagen expression.

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.

Protein-restricted maternal diet during lactation decreases type I and type III tropocollagen synthesis in the skin of mice offspring.

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 down-regulate collagen type III expression after UV-A in primary human dermal blood endothelial cells.

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.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions

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

Synergistic action of the nephrotoxic mycotoxins ochratoxin A and citrinin at nanomolar concentrations in human proximal tubule-derived cells.

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.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions.

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.

Local intra­articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model.

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 Sprague­Dawley 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 post­surgery, the rats received local intra­articular 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 VEGF­injected groups. The extent and the grade of cartilage damage in the VEGF­injected group were notably more severe compared with those in the NS­treated 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, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)­4, ­5 and ­12, type III collagen and transforming growth factor­ß1 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.