COL1A1 mRNA Levels Research Articles

Probiotics counteract the expression of hepatic profibrotic genes via the attenuation of TGF-β/SMAD signaling and autophagy in hepatic stellate cells.

Hepatic fibrosis is caused by the increased accumulation and improper degradation of extracellular matrix (ECM) proteins in the liver. Hepatic stellate cells (HSCs) activation is a key process in initiating hepatic fibrosis and can be ameliorated by the administration of probiotic strains. This study hypothesized that LAB strains (Lactiplantibacillus plantarum, Lactobacillus brevis, and Weissella cibaria) might attenuate pro-fibrogenic cytokine TGF-β mediated HSCs activation and induce collagen deposition, expression of other fibrogenic/inflammatory markers, autophagy, and apoptotic processes in vitro. Few studies have evaluated the probiotic effects against fibrogenesis in vitro. In this study, TGF-β exposure increased collagen deposition in LX-2 cells, but this increase was diminished when the cells were pretreated with LAB strains before TGF-β stimulation. TGF-β not only increased collagen deposition, but it also significantly upregulated the mRNA levels of Col1A1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinases-2 (MMP-2), IL-6, CXCL-8, CCL2, and IL-1β in LX-2 cells. Pretreatment of the cells with LAB strains counteracted the TGF-β-induced pro-fibrogenic and inflammatory markers by modulating SMAD-dependent and SMAD-independent TGF-β signaling. In addition, LX-2 cells exposed to TGF-β induced the autophagic and apoptotic associated proteins that were also positively regulated by the LAB strains. These findings suggest that LAB can attenuate TGF-β signaling that is associated with liver fibrogenesis.

NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH.

PTH induces phosphorylation of the transcriptional coregulator NACA on serine 99 through Gαs and PKA. This leads to nuclear translocation of NACA and expression of the target gene Lrp6, encoding a coreceptor of the PTH receptor (PTH1R) necessary for full anabolic response to intermittent PTH (iPTH) treatment. We hypothesized that maintaining enough functional PTH1R/LRP6 coreceptor complexes at the plasma membrane through NACA-dependent Lrp6 transcription is important to ensure maximal response to iPTH. To test this model, we generated compound heterozygous mice in which one allele each of Naca and Lrp6 is inactivated in osteoblasts and osteocytes, using a knock-in strain with a Naca99 Ser-to-Ala mutation and an Lrp6 floxed strain (test genotype: Naca99S/A; Lrp6+/fl;OCN-Cre). Four-month-old females were injected with vehicle or 100 μg/kg PTH(1-34) once daily, 5 days a week for 4 weeks. Control mice showed significant increases in vertebral trabecular bone mass and biomechanical properties that were abolished in compound heterozygotes. Lrp6 expression was reduced in compound heterozygotes vs. controls. The iPTH treatment increased Alpl and Col1a1 mRNA levels in the control but not in the test group. These results confirm that NACA and LRP6 form part of a common genetic pathway that is necessary for the full anabolic effect of iPTH.

Regulatory role of melatonin on epidermal growth factor receptor, Type I collagen α1 chain, and caveolin 1 in granulosa cells of sheep antral follicles.

We investigated the expression of epidermal growth factor receptor (EGFR), Type I collagen α1 chain (COL1A1), and caveolin 1 (CAV1) during follicular development and examined the regulatory role of melatonin (MLT) on EGFR, COL1A1, and CAV1 in sheep antral ovaries. The expression was detected in granulosa and theca cells by immunohistochemistry. Quantitative real-time polymerase chain reaction and Western blotting were used to examine the expression levels of EGFR, COL1A1, and CAV1 in small (≤2mm), medium (2-5mm), and large (≥5mm) follicles. The mRNA and protein levels of EGFR, COL1A1, and CAV1 were found to be the highest in large follicles. Furthermore, cultured granulosa cells were treated with MLT (10-7 -10-11 M), luzindole (nonselective MT1 and MT2 receptor antagonist, 10-7 M), and 4-phenyl-2-propanamide tetraldehyde (4P-PDOT, MT2 selective antagonist, 10-7 M) to detect the regulatory role of MLT on EGFR, COL1A1, and CAV1. Results indicated COL1A1 and CAV1 were at least partially regulated by MLT through MT1 and MT2 pathways, whereas EGFR was not. This study provided a reference for further studies on MLT regulatory role on EGFR, COL1A1, and CAV1 during sheep follicular development and elucidated the physiological mechanism of MLT regulator production.

Associations between matrix metalloproteinase, tissue inhibitor of metalloproteinase and collagen expression levels in the adjacent rectal tissue of colorectal carcinoma patients.

As the commonest type of cancer in Europe and the third most common type of cancer worldwide, colorectal carcinoma (CRC) poses a challenge for numerous scientific studies. At present, the cause of this disease is remains to be elucidated, but early diagnosis is only one solution to prevent serious health complications. As a structural scaffold, the extracellular matrix (ECM) is in direct contact with tumour cells and significantly interferes with tumour progression. During the process of tumorigenesis, the ECM undergoes structural changes in which collagens serve an important role. Their life cycle is regulated by proteolytic enzymes called matrix metalloproteinases (MMPs), which are controlled by tissue inhibitors of metalloproteinases (TIMPs). The present study analysed the gene expression of MMPs (MMP1-2-8-10-13), TIMPs (TIMP1-2-4) and collagens (COL1A1 and COL3A1) and the correlation with biochemical parameters in the adjacent rectal tissue (ART) of patients with CRC. The patients who underwent standard neoadjuvant pre-therapy showed increased concentrations of collagen in the normal ART. The mRNA levels of COL3A1, TIMP1 and TIMP2 were significantly higher in the ART of CRC patients (with or without pre-therapy) when compared with the control group. This finding suggested that TIMPs served an important role in the regulation of MMPs and in the modification of collagen content in the ECM. Despite the small data set, the present study provided insights into the transcriptomic relationships between the individual genes that are an integral part of the ECM.

Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality.

Within the Interstitial Lung Diseases (ILD), patients with idiopathic pulmonary fibrosis (IPF) and a subset of those with non-IPF fibrotic ILD have a distinct clinical phenotype of progression despite management. This group of patients has been collectively termed the progressive fibrotic phenotype (PFP). Their early recognition may facilitate access to antifibrotic therapies to prevent or slow progression. Macrophages/monocytes within the lung orchestrate the progression and maintenance of fibrosis. A novel role for monocyte-derived macrophages during tissue damage and wound healing is the expression of collagens. We examined Collagen 1a1 expression in airway macrophages from ILD patients at diagnosis. COL1A1 mRNA levels from BAL cells were elevated in IPF and Non-IPF patients. The presence of a UIP pattern and a subsequent progressive phenotype were significantly associated with the higher BAL COL1A1 levels. In Non-IPF patients, higher COL1A1 levels were associated with a more than twofold increase in mortality. The intracellular localisation of COL1A1 in airway macrophages was demonstrated by confocal microscopy in CD45 and CD163 co-staining assays. Additionally, airway macrophages co-expressed COL1A1 with the profibrotic SPP1 gene product osteopontin. The levels of SPP1 mRNA and OPN in the BAL were significantly higher in IPF and Non-IPF patients relative to healthy. Our results suggest that profibrotic airway macrophages are increased in the BAL of patients with IPF and other ILDs and co-express COL1A1 and OPN. Importantly, COL1A1 expression by pro-fibrotic airway macrophages could be a marker of disease progression and poor survival in ILDs.

Evaluation of the Use of Platelet-Rich Fibrin Xenologous Membranes Derived from Bubaline Blood in Canine Periodontal Defects.

Periodontal disease is the most common oral disease in dogs. Platelet-rich fibrin (PRF) is widely utilized to facilitate soft and hard tissue healing and has been proposed in periodontal healing in small animal treatment. However, the quality and amount of autologous PRF is compromised in animals with systemic diseases. The present study aimed to evaluate the efficacy of xenologous bubaline blood-derived PRF (bPRF) on periodontal tissue healing in canine periodontal defects. Split-mouth design was employed in twenty dogs diagnosed with periodontal disease. The defects were divided randomly into two groups: the open-flap debridement (OFD)-treated group and the OFD with bPRF (OFD+bPRF) application group. Results demonstrated that gingival index and periodontal probing depth decreased significantly in the OFD+bPRF group compared with those treated with OFD alone. Application of bPRF in periodontal defects also promoted fibrous tissue formation, as confirmed by the marked increase in fibrosis score. bPRF application significantly increased COL1A1 and PDGFB mRNA levels at day 14 compared with the baseline. Taking this evidence together, bPRF provided a favorable therapeutic modality in canine periodontal defects. bPRF could be an alternative biomaterial for the treatment of periodontal defects in dogs.

The CellBox-2 Mission to the International Space Station: Thyroid Cancer Cells in Space

A spaceflight to the International Space Station (ISS) is a dream of many researchers. We had the chance to investigate the effect of real microgravity (CellBox-2 Space mission) on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells (TCC). The cells had been sent to the ISS by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (United States) and cultured in six automated hardware units on the ISS before they were fixed and returned to Earth. Multicellular spheroids (MCS) were detectable in all spaceflight hardware units. The VCL, PXN, ITGB1, RELA, ERK1 and ERK2 mRNA levels were significantly downregulated after 5 days in space in adherently growing cells (AD) and MCS compared with ground controls (1g), whereas the MIK67 and SRC mRNA levels were both suppressed in MCS. By contrast, the ICAM1, COL1A1 and IL6 mRNA levels were significantly upregulated in AD cells compared with 1g and MCS. The protein secretion measured by multianalyte profiling technology and enzyme-linked immunosorbent assay (AngiogenesisMAP®, extracellular matrix proteins) was not significantly altered, with the exception of elevated angiopoietin 2. TCC in space formed MCS, and the response to microgravity was mainly anti-proliferative. We identified ERK/RELA as a major microgravity regulatory pathway.

Differential behaviour and gene expression in 3D cultures of femoral- and calvarial-derived human osteoblasts under a cyclic compressive mechanical load.

The aim of the study was to compare the response of calvarial and femoral osteoblasts cultured in a 3D hydrogel environment to cyclic compressive mechanical loading. Human foetal femoral and calvarial osteoblasts were encapsulated in a semi-synthetic thiol-modified hyaluronan gelatin polyethylene glycol diacrylate (PEGDA) cross-linked HyStemC hydrogel. Constructs were subjected to a cyclic compressive strain of 33.4kPa force every second for 5 s every hour for 6 h per day using FlexCell BioPress culture plates and compared to non-compressed constructs. Cell viability, mineralisation, and morphological changes were observed over 21 days. BMP2, ALP, COL1A1, COL2A1, and OCN gene expression levels were quantified. Encapsulated osteoblast numbers increased and formed hydroxyapatite over a 21-day period. Cell viability decreased under a cyclical strain when compared to cells under no strain. Femoral osteoblasts under strain expressed increased levels of BMP2 (53.9-fold) and COL1A1 (5.1-fold) mRNA compared to no strain constructs. Surprisingly, no BMP2 mRNA was detected in calvarial osteoblasts. Osteoblasts derived from endochondral (femoral) and intra-membranous (calvarial) processes behaved differently in 3D-constructs. We therefore recommend that site-specific osteoblasts be used for future bone engineering and bone replacement materials and further research undertaken to elucidate how site-specific osteoblasts respond to cyclic compressive loads.

Analysis of Colorectal Carcinogenesis Paradigm between Cold Constitution and Heat Constitution: Earlier ECM Collagen Deposition.

Colorectal cancer (CRC) is a common malignant tumor around the world. Studying the unique constitution of CRC patients is conducive to the application of personalized medical treatment for CRC. The most common types of constitution in CRC are cold and heat constitution. A previous study has suggested that the malignant progression in cold and heat constitution CRC are different; however, the mechanism remains unclear. The tumor microenvironment (TME) is likely to vary with each individual constitution, which may affect the tumor growth in different constitutions. The extracellular matrix (ECM), the most important component of TME, plays a critical role in disease progression and outcome in patients with CRC. Moreover, collagen, the major component of the ECM, determines the main functional characteristics of ECM and tissue fibrosis caused by collagen deposition, which is one of the signs of CRC malignant progression. This study aimed to explore the mechanisms leading to different colorectal carcinogenesis paradigms between the cold constitution and heat constitution within the context of ECM collagen deposition. We established the CRC rat models and enrolled 30 CRC patients with cold and heat constitution. The collagen-related parameters were detected by using Sirius red staining combined with polarized light microscope, and expressions of collagen (COL I and COL III) and lysyl oxidase (LOX and LOXL2) were determined using immunohistochemistry, while the mRNA levels of COL1A1, COL3A1, LOX, and LOXL2 were measured by qRT-PCR. We found that a higher degree of collagen deposition in the cold-constitution group. The results suggest cold and heat constitution may affect the colorectal carcinogenesis paradigm by influencing the early collagen deposition in colon tissue. The study may provide an effective idea for clinicians to improve the prognosis of CRC patients with different constitutions.

Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis.

BACKGROUND: Hypertension-induced renal damage is a serious and complex condition that has not been effectively treated by conventional blood pressure-lowering drugs. Tengdan capsule (TDC) is a China FDA-approved compound herbal medicine for treating hypertension; however, its chemical basis and pharmacological efficacy have not been fully investigated in a preclinical setting. METHODS: High-performance liquid chromatography (HPLC) was used to identify and quantify the major chemical components of TDC extracted from ultrapure water. Adult spontaneously hypertensive rats (SHR) and age/sex-matched Wistar Kyoto normotensive rats (WKY) were both treated with TDC, losartan, or saline for one month, and their blood pressure (BP) was monitored at the same time by tail-cuff BP system. Biochemical indexes such as urine creatinine (CRE) and blood urea nitrogen (BUN) were determined. Kidney tissue sections were examined with (H&E), and Masson staining to evaluate the pathological effect of TDC on SHR’s kidneys. After TDC treatment, the differentially expressed proteins in the kidneys of SHR were identified by the TMT-based quantitative proteomics analysis, which may provide the targets and possible mechanisms of TDC action. In addition, Western blot analysis, RT-qPCR, and ELISA assays were carried out to further verify the proteomics findings. Finally, two different models involving in vitro renal injuries were established using human kidney HEK293 cells; and the molecular mechanism of TDC kidney protection was demonstrated. RESULTS: Seven chemical compounds, namely Notoginsenoside R1, Ginsenoside RG1, Ginsenoside Re, Ginsenoside Rb1, Sodium Danshensu, Protocatechualdehyde, and Salvianolic acid B, were identified and quantified from the water-soluble extracts of TDC by HPLC. In vivo study using rats showed that TDC effectively reduced BP, BUN, and CRE levels and attenuated renal fibrosis in SHR, and ameliorated damage to the kidneys. Proteomics and subsequent bioinformatics analyses indicated that periostin-mediated inflammatory response and TGFβ/Smad signaling pathway proteins were closely related to the therapeutic effect of TDC in rat kidneys. Western blot analysis and RT-qPCR showed that TDC markedly downregulated the mRNA and protein expression of periostin in renal tissues compared to the untreated SHR. In addition, TGF-β and COL1A1 mRNA levels also decreased in SHR renal tissues following TDC treatment. In vitro studies showed that low to medium doses of TDC down-regulated the expression of periostin in the injury model of HEK293 cell. In addition, medium to high doses of TDC significantly inhibited collagen deposition in TGFβ1-induced HEK293 cell fibrosis. CONCLUSIONS: Major components from the compound herbal medicine Tengdan Capsule are identified and quantified. TDC effectively lowers blood pressure and protects against renal damage caused by hypertension in SHR. Mechanistically, TDC blocks periostin by regulating the TGF-β/Smad signaling pathway in the kidney, both in vivo and in vitro. Preventing periostin-mediated renal fibrosis and inflammation might be a promising strategy for treating a hypertensive renal injury.

Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial–mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients’ lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial–mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-β1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-β1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-β1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

In the quest of the optimal chondrichthyan for the development of collagen sponges for articular cartilage

The limited capability of articular cartilage to self-repair renders its regeneration a formidable challenge. In the quest of the optimal material for cartilage scaffold fabrication, the use of collagen type II has been advocated, but its optimal source still remains elusive. Herein, the potential of four [lesser spotted dogfish ( Scyliorhinus canicula ), thorn back ray ( Raja clavata ), cuckoo ray ( Leucoraja naevus ) and blonde ray ( Raja brachyuran )] cartilaginous fish (chondrichthyes) derived collagen type II preparation in cartilage engineering was assessed. Collagen type II was extracted using the acid-pepsin method, crosslinked with 4-arm poly (ethylene glycol) succinimidyl glutarate sponges were fabricated and their biophysical, biochemical and biological properties were assessed. Pure collagen type II preparations were obtained from all four chondrichthyes, as revealed by gel electrophoresis. Pore size, porosity, mechanical properties, denaturation temperature, free amine content and resistance to enzymatic degradation of the produced sponges were not significantly ( p > 0.05) affected as a function of species. Biological analysis using human adipose derived stem cells revealed no significant ( p > 0.05) differences between the groups in cell viability, DNA concentration, metabolic activity, Alcian blue staining and sulphated glycosaminoglycans synthesis. Human adipose derived stem cells seeded on fish-derived scaffolds expressed lower mRNA levels of COL1A1 (fold change ≥ 2.0) and COL3A1 (apart from lesser spotted dogfish; fold change < 2.0) and higher mRNA levels of COL10A1 (fold change ≥ 2.0), COMP (fold change ≥ 2.0), SOX9 (fold change ≥ 2.0), and ACAN (apart from cuckoo ray; fold change < 2.0) than cells grown on tissue culture plastic. Our data suggest that chondrichthyes derived collagen type II has the potential to be used in cartilage engineering. • High purity collagen type II can be extracted from chondrichthyes. • Chondrichthyan extracted collagen type II scaffolds support cell growth. • Chondrichthyan extracted collagen type II scaffolds can be used in cartilage engineering.

Collagen Type I α-1 Promotes Malignant Glioma Cell Proliferation and Is Associated with Glioma Prognosis

Collagen type I α-1 chain (COL1A1) is closely involved in the advancement of various tumors, yet the role of COL1A1 in the progression of glioma is not clear. Herein, we evaluated the effect of COL1A1 on glioma cell proliferation. The effect of COL1A1 on glioma cell proliferation was assessed through overexpression or knockdown of COL1A1. The CCK-8 and colony formation assays, as well as immunohistochemistry (IHC) were used to detect COL1A1 expression in different glioma grades. U-87MG as well as U-251MG cells were stably-inserted with lentivirus containing COL1A1 through transfection, we additionally used qRT-PCR as well as Western blot assay to validate their overexpression efficiencies. COL1A1 mRNA and protein levels were upregulated in the high-grade glioma (HGG) compared to the low-grade glioma (LGG). COL1A1 IHC score was remarkably higher in HGG than LGG. The staining index (SI) further showed that COL1A1 protein levels were higher in HGG than LGG. The Kaplan–Meier analysis showed that elevated COL1A1 mRNA levels were obviously correlated with lower overall survival (OS) and disease-free survival (DFS) for brain glioma patients. COL1A1 mRNA and protein levels were markedly upregulated in human glioma cell lines when compared with brain astrocyte cell lines. The high expression level of COL1A1 facilitated glioma cell proliferation. COL1A1 knockdown remarkably inhibited glioma cell proliferation. Thus, this research shows that COL1A1 promotes glioma cell proliferation and is closely related to glioma prognosis.

The Prognostic Value of Cytokeratin and Extracellular Collagen Expression in Urinary Bladder Cancer.

Expression levels of collagens have been implicated in the progression of various cancers and interaction with cytokeratins but are not well studied in bladder cancer (BC). Therefore, we analyzed the gene and protein expression levels of collagen 1A1 (Col1a1/COL1A1), collagen 3A1 (col3a1/COL3A1), collagen 5A2 (col5a2/COL5A2), cytokeratin 14 (krt14/CK14), and cytokeratin 17 (krt17/CK17) in urothelial BC samples of different stages. In total, 102 fresh frozen and 190 formalin-fixed and paraffin-embedded (FFPE) samples were tested using immunohistochemistry and RT-qPCR. Expression levels were correlated with clinicopathological and follow-up data. Col1a1, col3a1, col5a2 and krt14 mRNA levels were significantly overexpressed in high-grade and muscle-invasive BC (MIBC) compared to low-grade and non-muscle invasive BC (NMIBC) cases. Disease-specific survival (DSS) was shorter in patients with high expression levels of col1a1 (p = 0.004), col3a1 (p = 0.004), and col5a2 (p = 0.028). CK14 (p = 0.020), COL3A1 (p = 0.006), and Col5A2 (p = 0.006) protein expression levels were significantly higher and protein expression levels of CK17 (p = 0.05) were significantly lower in MIBC compared to NMIBC. Furthermore, CK14 (p = 0.002) and COL5A2 (p = 0.006) protein expression level were significantly higher in high-grade compared to low-grade BC. DSS was shorter in patients with high expression levels of COL5A2 (p = 0.033) and CK14 (p = 0.042). Expression changes of collagens and cytokeratins are univariable prognostic markers in BC.

Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

GDF11 induces mild hepatic fibrosis independent of metabolic health.

Background & aims: Growth Differentiation Factor 11 (GDF11) is an anti-aging factor, yet its role in liver diseases is not established. We evaluated the role of GDF11 in healthy conditions and in the transition from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH).Results: GDF11 mRNA levels positively correlated with NAFLD activity score and with CPT1, SREBP, PPARγ and Col1A1 mRNA levels, and associated to portal fibrosis, in morbidly obese patients with NAFLD/NASH. GDF11-treated mice showed mildly exacerbated hepatic collagen deposition, accompanied by weight loss and without changes in liver steatosis or inflammation. GDF11 triggered ALK5-dependent SMAD2/3 nuclear translocation and the pro-fibrogenic activation of HSC.Conclusions: GDF11 supplementation promotes mild liver fibrosis. Even considering its beneficial metabolic effects, caution should be taken when considering therapeutics that regulate GDF11.Methods: We analyzed liver biopsies from a cohort of 33 morbidly obese adults with NAFLD/NASH. We determined the correlations in mRNA expression levels between GDF11 and genes involved in NAFLD-to-NASH progression and with pathological features. We also exposed wild type or obese mice with NAFLD to recombinant GDF11 by daily intra-peritoneal injection and monitor the hepatic pathological changes. Finally, we analyzed GDF11-activated signaling pathways in hepatic stellate cells (HSC).

OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes.

Objectives A recent work has reported that the elevated osteopontin (OPN) levels in the articular cartilage and synovial fluid are correlated with the progressive osteoarthritis (OA) joint damage, and OPN has a protective effect against OA by suppressing the expressions of OA-associated genes. The present study examined whether the OPN deficiency was susceptible to OA through the regulation of chondrocyte senescence and apoptosis and the expressions of OA-associated genes. Methods The mRNA levels of COL2A1 and OPN were compared between human OA chondrocytes and normal chondrocytes. The effects of OPN siRNA on the SA-β-Gal expressions and the percentage of apoptotic chondrocytes were examined by using SA-β-Gal staining and apoptosis assay, and the effects on the expressions of COL2A1 and OA-associated genes (COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5) were examined by western blot analysis and quantitative real-time RT-PCR. Furthermore, an in vivo OA model was established to examine the effects of OPN siRNA on the senescence and apoptosis of OA chondrocytes and the expressions of OA-associated genes. Results The mRNA levels of COL2A1 and OPN were decreased in knee OA chondrocytes in comparison with those in normal chondrocytes. The OPN deficiency enhanced the senescence and apoptosis of OA chondrocytes and increased the expressions of COL10A1, IL-1β, TNF-ɑ, MMP-13, and ADAMTS5 but decreased the expression of COL2A1. Meanwhile, OPN deficiency could result in severe, accelerated OA in vivo, which was also associated with enhanced senescence and apoptosis of chondrocytes and elevated expressions of OA-associated genes. Conclusions The findings of this study suggest that the OPN deficiency can result in accelerated OA, which is associated with enhanced senescence and apoptosis of OA chondrocytes and the upregulated expressions of OA-associated genes.

Glucagon-Like Peptide-2 Analogue ZP1849 Augments Colonic Anastomotic Wound Healing

Background The enteroendocrine hormone glucagon-like peptide- (GLP-) 2 is a potent trophic factor in the gastrointestinal tract. The GLP-2 receptor (GLP-2R) is expressed in the stroma of the large bowel wall, which is the major therapeutic target area to prevent anastomotic leakage. We investigated the efficacy of the long-acting GLP-2 analogue ZP1849 on colonic anastomotic wound healing. Methods Eighty-seven male Wistar rats were stratified into four groups and received daily treatment with vehicle or ZP1849 starting one day before (day -1) end-to-end anastomosis was constructed in the left colon on day 0, and on days 0 (resected colon segment), 3, and 5, gene expressions of GLP-2R, Ki67, insulin-like growth factor- (IGF-) 1, type I (COL1A1) and type III (COL3A1) procollagens, cyclooxygenase- (COX-) 1, COX-2, and matrix metalloproteinase- (MMP-) 7 were quantified by RT-qPCR. Breaking strength, myeloperoxidase (MPO), transforming growth factor- (TGF-) β1, and soluble collagen proteins were measured on days 3 and 5. Results ZP1849 treatment increased Ki67 (P < 0.0001) and IGF-1 (P < 0.05) mRNA levels in noninjured colon day 0, and postoperatively in the anastomotic wounds compared to vehicle-treated rats. ZP1849-treated rats had increased (P = 0.042) anastomotic breaking strength at day 5 compared with vehicle. COL1A1 and COL3A1 mRNA levels (P < 0.0001) and soluble collagen proteins (P < 0.05) increased from day 3 to day 5 in ZP1849-treated rats, but not in vehicle-treated rats. COX-2 mRNA and MPO protein levels decreased from day 3 to day 5 (P < 0.001) in both groups. ZP1849 treatment reduced TGF-β1 protein levels on day 5 (P < 0.001) but did not impact MMP-7 transcription. Conclusions The GLP-2 analogue ZP1849 increased breaking strength, IGF-1 expression, and cell proliferation, which may be beneficial for colonic anastomotic wound healing.

Suppressing PLCγ1 enhances osteogenic and chondrogenic potential of BMSCs

Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-β3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.

Remodeling process in bone of aged rats in response to resistance training

AimsWe investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. Main methodsMale rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. Key findingsAging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). SignificanceRT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes.