COL1A1 mRNA Research Articles

Treadmill Running Combined With Caloric Restriction Induces Greater Benefits Against Non-alcoholic Fatty Liver Disease

Current guidelines for non-alcoholic fatty liver disease (NAFLD) treatment recommend a restricted diet plus physical activity. However, the underlying mechanisms are not clear. PURPOSE: To investigate the effect of exercise training (EX) plus hypocaloric diet on NAFLD in a high fat diet (HFD)-induced obese mice. METHOD: Fifty mice (C57BL/6) were assigned to standard chow (SC, n=10) or HFD (n=40) for 23 weeks. During the last 8 weeks of the 23-week dietary course, the HFD mice were further assigned to HFD (n=10) or HFD plus exercise training (HFD+EX) (n=10) or HFD+SC (n=10) or HFD+EX+SC (n=10). The HFD+EX and HFD+SC+EX mice were subjected to treadmill running at a moderate intensity for 50 minutes per session with a frequency of 5 days per week. RESULT: Compared to the HFD mice, the HFD+EX, HFD+SC, and HFD+EX+SC mice had significantly lower values in areas under the curves of glucose (HFD: 49230±11353; HFD+EX: 45401±3357; HFD+SC: 25320±4321; HFD+EX+SC: 24466±2335) and insulin (HFD: 6225±1629; HFD+EX: 5871±1070; HFD+SC: 4466±570; HFD+EX+SC: 3784±254) tolerance tests and histological progression of hepatic steatosis (HFD: 3.8±0.4; HFD+EX: 2.5±0.5; HFD+SC: 2.3±0.5; HFD+EX+SC: 0.8±0.4) in conjunction with significantly higher levels of serum adiponectin (HFD: 23.0±1.8; HFD+EX: 29.5±4.4; HFD+SC: 31.6±3.4; HFD+EX+SC: 36.8±7.0) and hepatic adiponectin receptor 1 and 2 mRNAs. Attenuation of whole body insulin resistance (P=0.001 and P=0.001, respectively) hepatic steatosis (P=0.001, respectively), and hypoadiponectinemia (P=0.001, respectively) was significantly higher in the HFD+SC+EX mice than in the HFD+EX or HFD+SC mice. Compared to the HFD mice, the HFD+SC and HFD+EX+SC mice had significantly higher levels of hepatic genes (i.e., Tfam, NRF, COX4i1, SIRT1, CPT-1a, and CYP4a10 mRNAs) involved in mitochondrial function. Compared to the HFD mice, the HFD+EX, HFD+SC, and HFD+EX+SC mice had significantly lower levels of hepatic genes involved in inflammation (i.e., Ly6d and Lgals3 mRNAs) and fibrosis (i.e., Timp1 and Col1a1 mRNAs). CONCLUSION: The current findings suggest that CR+EX has greater effects against NAFLD and its metabolic complications in HFD-induced obese mice. Supported by the National Research Foundation Grant funded by the Korean Government (NRF-2012R1A1A2006180).

Investigating mechanisms of sex bias in lung fibrosis using mice with sex chromosome complement

Abstract Men with systemic sclerosis (SSc) have more end organ damage, an accelerated course, and lower survival than women. Mechanism underlying such sexual dimorphism is unclear. Here, we investigated the role of sex chromosomes in the development of SSc using the four-core genotype (FCG) mouse model that was created to identify effects of the sex chromosome complement without the confounding effects of differences in sex hormones. In this model, findings in the XX females (XXF) can be compared with XY females [XYF = XY−(Sry)], and in XX males [XXM = XX.SryTg] with XY males [XYM = XY−Sry.SryTg]. The FCG C57Bl/6 mice were administered with bleomycin to induce SSc and euthanized 2 weeks later. We found that regardless of the gender, mice with XY sex chromosome complement had more severe disease than mice with XX complement (p = 0.04, n= 37 and 48, respectively). When we compared the groups based on gender, XYF mice had higher fibrosis scores than XXF (p = 0.01). The differences between XY and XX were even more significant in males: XYM animals had much more severe fibrosis than XXM (p < 0.0001). Furthermore, the levels of soluble collagen in bronchoalveolar lavage fluid and of Col1a1 mRNA in lung tissue correlated with the extent of lung fibrosis. Animals with more fibrosis (i.e., XYM) had higher proportions and numbers of myeloid and plasmacytoid dendritic cells in the lungs. XYM mice also had more natural killer T cells, B cells, and T cells in the lungs than XXM mice at 2 weeks post-instillation. Thus, the presence of a Y chromosome, and not the male sex itself, predisposes to lung fibrosis; androgen may play an additive role in causing increased lung fibrosis, whereas the presence of the second X chromosome and/or estrogen might be protective.

Bone metabolic microarray analysis of ligature-induced periodontitis in streptozotocin-induced diabetic mice.

Periodontal disease is a chronic infectious disease that results in bone loss. Many epidemiological studies have reported the progression of periodontal tissue destruction in patients with diabetes; however, the associated mechanism remains unclear. In this study, we comprehensively investigated how diabetes affects the periodontal tissue and alveolar bone loss using a ligature-induced periodontitis model in streptozotocin-induced diabetic (STZ) mice. Diabetes was induced by intraperitoneal injection with streptozotocin in 6-wk-old C57/BL6J male mice. A silk ligature was tied around the maxillary left second molar in 9-wk-old wild-type (WT) and STZ mice. Bone loss was evaluated at 3 and 7 d after ligation. mRNA expression levels in the gingiva between the two groups were examined by DNA microarray and quantitative polymerase chain reaction at 1, 3 and 7 d post-ligation. Tartrate-resistant acid phosphatase and alkaline phosphatase staining of the periodontal tissue was performed for evaluation of osteoclasts and osteoblasts in histological analysis. In the gingiva, hyperglycemia upregulated the osteoprotegerin (Opg) mRNA expression and downregulated Osteocalcin mRNA expression. In the ligated gingiva, tumor necrosis factor-α (Tnf-α) mRNA expression was upregulated at 1 d post-ligation in STZ mice but not in WT mice. At 3 d post-ligation, alveolar bone loss was observed in STZ mice, but not in WT mice. Significantly severe alveolar bone loss was observed in STZ mice compared to WT mice at 7 d post-ligation. Bone metabolic analysis using DNA microarray showed significant downregulation in the mRNA expression of glioma-associated oncogene homologue 1 (Gli1) and collagen type VI alpha 1 (Col6a1) at the gingiva of the ligated site in STZ mice compared to that in WT mice. Quantitative polymerase chain reaction showed that Gli1 and Col6a1 mRNA expression levels were significantly downregulated in the gingiva of the ligated site in STZ mice compared to WT mice. Histological analysis showed lower alkaline phosphatase activity in STZ mice. In addition, an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the ligated sites in STZ mice. These results suggest that an imbalance of bone metabolism causes osteoclastosis in insulin-deficient diabetes, and that alveolar bone loss could occur at an early phase under this condition.

Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds.

The aim of the present investigation was to compare the effects of cyclic compression, perfusion, dexamethasone (DEX) and bone morphogenetic protein-7 (BMP-7) on the proliferation and differentiation of human bone marrow stromal cells (hBMSCs) in polyurethane scaffolds in a perfusion bioreactor. Polyurethane scaffolds seeded with hBMSCs were cultured under six different conditions, as follows: 10% Cyclic compression at 0.5 and 5 Hz; 10 ml/min perfusion; 100 nM DEX; 100 ng/ml BMP-7; and 1 ml/min perfusion without mechanical and biochemical stimulation (control). On days 7 and 14, samples were tested for the following data: Cell proliferation; mRNA expression of Runx2, COL1A1 and osteocalcin; osteocalcin content; calcium deposition; and the equilibrium modulus of the tissue specimen. The results indicated that BMP-7 and 10 ml/min perfusion promoted cell proliferation, which was inhibited by 5 Hz cyclic compression and DEX. On day 7, the 5 Hz cyclic compression inhibited Runx2 expression, whereas the 0.5 Hz cyclic compression and BMP-7 upregulated the COL1A1 mRNA levels on day 7 and enhanced the osteocalcin expression on day 14. The DEX-treated hBMSCs exhibited downregulated osteocalcin expression. After 14 days, the BMP-7 group exhibited the highest calcium deposition, followed by the 0.5 Hz cyclic compression and the DEX groups. The equilibrium modulus of the engineered constructs significantly increased in the BMP-7, 0.5 Hz cyclic compression and DEX groups. In conclusion, the present results suggest that BMP-7 and perfusion enhance cell proliferation, whereas high frequency cyclic compression inhibits the proliferation and osteogenic differentiation of hBMSCs. Low frequency cyclic compression is more effective than DEX, but less effective compared with BMP-7 on the osteogenic differentiation of hBMSCs seeded on polyurethane scaffolds.

Tumor necrosis factor-α regulates triggering receptor expressed on myeloid cells-1-dependent matrix metalloproteinases in the carotid plaques of symptomatic patients with carotid stenosis

ObjectiveTo determine the relationship between increased triggering receptor expressed on myeloid cells (TREM)-1 and plaque stability in atherosclerotic carotid stenosis. MethodsThe mRNA transcripts and protein for TREM-1, MMP-1, MMP-9, collagen type I (COL1A1) and collagen type III (COL3A1) were analyzed by qPCR and immunofluorescence in both tissues and VSMCs isolated from atherosclerotic carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. ResultsThe TREM-1, MMP-1 and MMP-9 mRNA transcripts were significantly increased (TREM-1, p < 0.01; MMP-1, p < 0.01 and MMP-9, p < 0.001) while COL1A1 and COL3A1 mRNA transcripts were decreased (p < 0.001) in VSMCs isolated from carotid plaques of symptomatic (S) than asymptomatic (AS) patients. Stimulation of cells with TNF-α further increased the mRNA transcripts of TREM-1, MMPs, COL1A1 and COL3A1. Modulation of TREM-1 by treatment with TREM-1 decoy receptor rTREM-1/Fc, and either TREM-1 antibodies or TREM-1 siRNA attenuated the TNF-α-induced expression of MMP-1 and MMP-9 (p < 0.01) and COL1A1 and COL3A1 (p < 0.01) in S compared to AS VSMCs isolated from carotid plaques. Inhibition of NF-kB (BAY 11–7085), JNK (SP600125) and PI3K (LY294002) signaling pathways decreased the expression of TREM-1 (p < 0.01), MMP-1 (p < 0.001) and MMP-9 (p < 0.01) in TNF-α-treated VSMCs isolated from S carotid plaques compared to AS patients. ConclusionIncreased expression of TREM-1 in S compared to AS patients involving MMP-1 and MMP-9 suggest a potential role of TREM-1 in plaque destabilization. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.

Urinary sediment mRNA level of extracellular matrix molecules in adult nephrotic syndrome

BackgroundGlomerular and tubulointerstitial fibrosis play important roles in the progression of chronic kidney disease. We determine whether urinary mRNA levels of extracellular matrix proteins reflect the degree of kidney fibrosis and predict renal function decline in adult nephrotic patients. MethodsWe studied 56 adult nephrotic patients and 20 controls. Urinary mRNA levels of collagen I A1 chain (COL1A1), collagen IV A3 chain (COL4A3), and fibronectin were measured. ResultsUrinary sediment mRNA levels of COL1A1 and fibronectin were significantly higher in nephrotic patients as compared to the control, irrespective to the pathological diagnosis. Urinary COL1A1 mRNA level correlates with proteinuria, glomerular and interstitial fibrosis, and inversely with estimated glomerular filtration rate (GFR); urinary fibronectin mRNA level significantly correlates with glomerular and interstitial fibrosis, and inversely with estimated GFR. After 42.4±12.6months follow-up, the rate of GFR decline inversely correlates with urinary mRNA level of COL1A1 (r=−0.273, p=0.044). Multivariate analysis confirmed that urinary COL1A1 mRNA level is an independent predictor of serum creatinine doubling or progressing to end stage renal disease; for each 10-fold increase in urinary COL1A1 mRNA level, there is 15.1% excess in risk (95% confidence interval, 1.9% to 30.4%, p=0.022). ConclusionsUrinary COL1A1 mRNA level is elevated in nephrotic patients irrespective to the pathological diagnosis, and it correlates with proteinuria, histological scarring, and inversely with renal function. Furthermore, urinary COL1A1 mRNA level predicts renal function loss during follow-up. Our results suggest that urinary COL1A1 mRNA level may be used for risk stratification of adult nephrotic syndrome.

Epigallocatechin-3-gallate inhibits transforming-growth-factor-β1-induced collagen synthesis by suppressing early growth response-1 in human buccal mucosal fibroblasts

Transforming growth factor (TGF)-β is a key regulator in the pathogenesis of oral submucous fibrosis (OSF). Early growth response (Egr)-1 is essential for fibrotic responses to TGF-β. Because TGF-β signaling is cell-type- and context-dependent, we investigated the signaling involved in TGF-β-induced Egr-1 in primary human buccal mucosal fibroblasts (BMFs). TGF-β-induced Egr-1 and its signaling were assessed by western blotting in BMFs. Egr-1 small interfering RNA was used to define the role of Egr-1 on TGF-β-induced mRNAs of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and acid-soluble collagen production (via Sircol collagen assay). The effects of epigallocatechin-3-gallate (EGCG) on TGF-β-induced Egr-1 protein and acid-soluble collagen were also evaluated. TGF-β1 stimulated Egr-1 production in BMFs. Pretreatment with PD98059, SP600125, SB431542, and SIS3, but not SB203580, significantly reduced TGF-β1-induced Egr-1 protein expression. Genetic targeting of Egr-1 completely inhibited TGF-β1-induced type I collagen mRNAs and collagen protein expression. EGCG fully inhibited TGF-β1-induced Egr-1 and TGF-β1-stimulated production of acid-soluble collagens. We conclude that activin receptor-like kinase (ALK)5, Smad3, extracellular signal-regulated kinase, and c-Jun N-terminal kinase are involved in the TGF-β1-induced Egr-1 protein production in BMFs. Egr-1 mediates TGF-β1-induced COL1A1 and COL1A2 mRNA expression and acid-soluble collagen production in BMFs. EGCG can block TGF-β1-induced collagen production by attenuating Egr-1 expression in BMFs. Egr-1 is a key mediator in TGF-β1-induced pathogenesis of OSF. EGCG may be useful in the prevention or treatment of OSF.

Overexpression of microRNA-210 promotes chondrocyte proliferation and extracellular matrix deposition by targeting HIF-3α in osteoarthritis.

The present study aimed to determine the effect of microRNA (miR)‑210 on osteoarthritis (OA). The expression levels of miR‑210, type I and X collagen (COL1A1 and COL10A1) and matrix metallopeptidase 13 (MMP13) in OA and normal chondrocytes were determined using reverse transcription‑quantitative polymerase chain reaction analysis. The OA chondrocytes were transfected with an miRNA precursor for miR‑210 or a negative control. After 3, 7, 14 and 21 days, the expression levels of miR‑210 were examined, the proliferation of the OA chondrocytes were determined using an XTT assay and the protein levels of Ki67 and HIF‑3α were analyzed by Western blotting. After 21 days, the mRNA and protein levels of COL1A1, COL10A1 and MMP13 were analyzed. Th present study demonstrated that the expression levels of miR‑210 and COL1A1 were lower, and the expression levels of COL10A1 and MMP13 were higher in the OA chondrocytes, compared with the levels of expression in the normal chondrocytes. Overexpression of miR‑210 significantly promoted the proliferation of OA chondrocytes and induced the protein expression of Ki67. In addition, miR‑210 overexpression markedly increased the expression of COL1A1 expression, but decreased the expression levels of COL10A1 and MMP13. A luciferase reporter assay confirmed the direct interaction between miR‑210 and hypoxia‑inducible factor (HIF)‑3α. miR‑210 did not alter the mRNA expression of HIF‑3α, however, it suppressed the protein expression of HIF‑3α. Additionally, HIF‑3α knockdown significantly promoted OA chondrocyte proliferation and increased the mRNA levels of COL1A1, whereas it decreased the mRNA levels of COL10A1 and MMP13. The results of the present study suggested that miR‑210 may be a negative regulator of the progression of OA, which increases chondrocyte proliferation and prompts extracellular matrix deposition by directly targeting HIF‑3α.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

Is Western Diet-Induced Nonalcoholic Steatohepatitis in Ldlr-/- Mice Reversible?

BackgroundNonalcoholic fatty liver disease (NAFLD) is a major public health burden in western societies. The progressive form of NAFLD, nonalcoholic steatohepatitis (NASH), is characterized by hepatosteatosis, inflammation, oxidative stress, and hepatic damage that can progress to fibrosis and cirrhosis; risk factors for hepatocellular carcinoma. Given the scope of NASH, validating treatment protocols (i.e., low fat diets and weight loss) is imperative.MethodsWe evaluated the efficacy of two diets, a non-purified chow (NP) and purified (low-fat low-cholesterol, LFLC) diet to reverse western diet (WD)-induced NASH and fibrosis in Ldlr-/- mice.ResultsMice fed WD for 22–24 weeks developed robust hepatosteatosis with mild fibrosis, while mice maintained on the WD an additional 7–8 weeks developed NASH with moderate fibrosis. Returning WD-fed mice to the NP or LFLC diets significantly reduced body weight and plasma markers of metabolic syndrome (dyslipidemia, hyperglycemia) and hepatic gene expression markers of inflammation (Mcp1), oxidative stress (Nox2), fibrosis (Col1A, LoxL2, Timp1) and collagen crosslinking (hydroxyproline). Time course analyses established that plasma triglycerides and hepatic Col1A1 mRNA were rapidly reduced following the switch from the WD to the LFLC diet. However, hepatic triglyceride content and fibrosis did not return to normal levels 8 weeks after the change to the LFLC diet. Time course studies further revealed a strong association (r2 ≥ 0.52) between plasma markers of inflammation (TLR2 activators) and hepatic fibrosis markers (Col1A, Timp1, LoxL2). Inflammation and fibrosis markers were inversely associated (r2 ≥ 0.32) with diet-induced changes in hepatic ω3 and ω6 polyunsaturated fatty acids (PUFA) content.ConclusionThese studies establish a temporal link between plasma markers of inflammation and hepatic PUFA and fibrosis. Low-fat low-cholesterol diets promote reversal of many, but not all, features associated with WD-induced NASH and fibrosis in Ldlr-/- mice.

A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair.

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.

Introduction: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.Methods: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.Results: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.Conclusions: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

Development of novel real-time PCR methodology for quantification of COL11A1 mRNA variants and evaluation in breast cancer tissue specimens.

BackgroundCollagen XI is a key structural component of the extracellular matrix and consists of three alpha chains. One of these chains, the α1 (XI), is encoded by the COL11A1 gene and is transcribed to four different variants at least (A, B, C and E) that differ in the propensity to N-terminal domain proteolysis and potentially in the way the extracellular matrix is arranged. This could affect the ability of tumor cells to invade the remodeled stroma and metastasize. No study in the literature has so far investigated the expression of these four variants in breast cancer nor does a method for their accurate quantitative detection exist.MethodsWe developed a conventional PCR for the general detection of the general COL11A1 transcript and real-time qPCR methodologies with dual hybridization probes in the LightCycler platform for the quantitative determination of the variants. Data from 90 breast cancer tissues with known histopathological features were collected.ResultsThe general COL11A1 transcript was detected in all samples. The developed methodologies for each variant were rapid as well as reproducible, sensitive and specific. Variant A was detected in 30 samples (33 %) and variant E in 62 samples (69 %). Variants B and C were not detected at all. A statistically significant correlation was observed between the presence of variant E and lymph nodes involvement (p = 0.037) and metastasis (p = 0.041).ConclusionsWith the newly developed tools, the possibility of inclusion of COL11A1 variants as prognostic biomarkers in emerging multiparameter technologies examining tissue RNA expression should be further explored.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1725-8) contains supplementary material, which is available to authorized users.

YAP1 Is a Driver of Myofibroblast Differentiation in Normal and Diseased Fibroblasts

Dupuytren disease is a fibrotic disorder characterized by contraction of myofibroblast-rich cords and nodules in the hands. The Hippo member Yes-associated protein 1 (YAP1) is activated by tissue stiffness and the profibrotic transforming growth factor-β1, but its role in cell fibrogenesis is yet unclear. We hypothesized that YAP1 regulates the differentiation of dermal fibroblasts into highly contractile myofibroblasts and that YAP1 governs the maintenance of a myofibroblast phenotype in primary Dupuytren cells. Knockdown of YAP1 in transforming growth factor-β1-stimulated dermal fibroblasts decreased the formation of contractile smooth muscle α-actin stress fibers and the deposition of collagen type I, which are hallmark features of myofibroblasts. Translating our findings to a clinically relevant model, we found that YAP1 deficiency in Dupuytren disease myofibroblasts resulted in decreased expression of ACTA2, COL1A1, and CCN2 mRNA, but this did not result in decreased protein levels. YAP1-deficient Dupuytren myofibroblasts showed decreased contraction of a collagen hydrogel. Finally, we showed that YAP1 levels and nuclear localization were elevated in affected Dupuytren disease tissue compared with matched control tissue and partly co-localized with smooth muscle α-actin-positive cells. In conclusion, our data show that YAP1 is a regulator of myofibroblast differentiation and contributes to the maintenance of a synthetic and contractile phenotype, in both transforming growth factor-β1-induced myofibroblast differentiation and primary Dupuytren myofibroblasts.

TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-β1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-β1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-β1. TGF-β1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-β1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-β1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-β1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-β1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.

Tissue Inhibitor of Metalloproteinase-2 Suppresses Collagen Synthesis in Cultured Keloid Fibroblasts.

Background:Keloids are defined as a kind of dermal fibroproliferative disorder resulting from the accumulation of collagen. In the remodeling of extracellular matrix, the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is as critical as the proper production of extracellular matrix. We investigate the role of TIMPs and MMPs in the pathogenesis of keloids and examine the therapeutic potential of TIMP-2.Methods:The expression of TIMPs and MMPs in most inflamed parts of cultured keloid fibroblasts (KFs) and peripheral normal skin fibroblasts (PNFs) in the same individuals and the reactivity of KFs to cyclic mechanical stretch were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (n = 7). To evaluate the effect of treating KFs with TIMP-2, collagen synthesis was investigated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and microscopic analysis was used to examine the treatment effects of TIMP-2 on ex vivo cultures of keloid tissue (n = 6).Results:TIMP-2 was downregulated in cultured KFs compared with PNFs in the same individuals, and the reduction in TIMP-2 was exacerbated by cyclic mechanical stretch. Administration of TIMP-2 (200 or 300 ng/mL) significantly suppressed expression of Col1A2 and Col3A1 mRNA and collagen type I protein in KFs. TIMP-2 also significantly reduced the skin dermal and collagen bundle thickness in ex vivo cultures of keloid tissue.Conclusion:These results indicated that downregulation of TIMP-2 in KFs is a crucial event in the pathogenesis of keloids, and the TIMP-2 would be a promising candidate for the treatment of keloids.

Abstract 5134: Pharmaceutical effect of vitamin C (ascorbate) on B16 melanoma in vitro and in vivo

Abstract In 2014, 76,100 new cases of melanoma were diagnosed in the United States. Treatment consists of surgery and chemotherapy, but efficacy is low. Indeed, five-year survival rates for patients with metastatic melanoma remain below 25%, indicating the need for new therapeutic strategies. In parallel, some studies indicate that vitamin C (ascorbic acid, ascorbate) only in pharmacologic doses may have a beneficial anti-tumor effect. Although ascorbate is an essential nutrient for humans, who are unable to synthesize it, pharmacologic ascorbate concentrations are achieved by parenteral administration. Although pharmacologic ascorbate in cancer treatment in humans is unproven, many studies report a selective cytotoxic effect in cancer cells in vitro. Cytotoxicity is thought to be mediated by hydrogen peroxide formation. To investigate the effect of high dose ascorbate in melanoma, we first used a murine melanoma cell line B16F10. We confirmed that ascorbate induced cell death in a dose-dependent manner (up to 40mM) in this cell line which is mostly reversed by adding catalase into the media, reinforcing the hydrogen peroxide dependent effect. We next investigated the role of ascorbic acid in vivo using a murine B16 melanoma model. Tumor-bearing C57BL/6 mice were treated with pharmacological ascorbic acid (4g/kg mice/twice a day) or hypertonic NaCl solution. We observed that the tumor size was decreased by 63% in the vitamin C treated group compared to the control group. We also used this model to analyze collagen expression in B16 tumors from ascorbate or saline-treated mice by qPCR and ELISA. Vitamin C treated tumors showed increased expression of various types of collagen. Indeed, we observed a five-fold increase in Col1A1, Col1A2, Col1A3, Col8A1 and Col28A1 mRNA expression and we also observed an increase in the Prolyl 4-hydroxylase, alpha peptide III (P4HA3) expression, an enzyme involved in collagen formation. No side effects were evident from administration of high dose ascorbate, which is fully consistent with previously published clinical data. We are currently investigating the role of hydrogen peroxide in the murine model by generating tumors with B16F10 cells that overexpress catalase. Taken together, these results showed that pharmacologic ascorbate is cytotoxic to B16F10 melanoma cells, with cytotoxicity in vitro mediated by hydrogen peroxide. Ascorbate is effective in reducing melanoma tumor size without apparent host toxicity. These data support the idea that vitamin C should be studied in humans both for combination chemotherapy and also as a stand-alone treatment. Citation Format: Pierre-Christian Violet, Nermi L. Parrow, Serrano L. Oscar, Jacqueline Yang, Mark A. Levine. Pharmaceutical effect of vitamin C (ascorbate) on B16 melanoma in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5134. doi:10.1158/1538-7445.AM2015-5134

Abstract 4423: Chemoresistance regulation by COL11A1 in ovarian cancer

Abstract Epithelial ovarian carcinoma remains the most lethal gynecologic malignancy. While initial response to first-line therapy (consisting of surgical cytoreduction and combination platinum/taxane therapy) is usually effective, the majority of patients will ultimately recur with chemotherapy-resistant cancer and succumb to disease. Biomarkers that predict patient's responsiveness to chemotherapy might assist the development of better therapeutic strategies for aggressive cancers, such as ovarian cancer. Here, we investigated the molecular mechanisms of collagen type XI alpha 1 (COL11A1) in chemo-resistance of ovarian cancer. We analyzed the expression profiles of 60 epithelial ovarian cancer tissue samples obtained at first cytoreductive surgery to identify genes linked with chemotherapy response. Among the 47 selected genes, collagen type XI alpha 1 (COL11A1) was the most highly elevated in chemo-resistance tumors. Small interfering RNA-mediated specific reduction of COL11A1 protein levels increased the sensitivity of ovarian chemoresistant cells to cisplatin and paclitaxel. A combination of experimental approaches, including real-time RT-PCR, western blotting, luciferase assay and chromatin immunoprecipitation (ChIP) assays, showed that the c/EBPβ binding site on the COL11A1 promoter was identified as the major determinant of cisplatin and paclitaxel-dependent COL11A1 activation. Furthermore, COL11A1 silencing suppressed phosphorylated Akt expression, which was in concert with increased PDK1 degradation in chemoresistant cells. Analysis of 104 EOC patients showed that high COL11A1 mRNA levels are significantly associated with poor chemoresponse and poor clinical outcome. In conclusion, activation of COL11A1 by anticancer drugs confers chemoresistance on ovarian cancer cells through the activation of Akt/c/EBPβ pathway and PDK1 stabilization. Note: This abstract was not presented at the meeting. Citation Format: Yi-Hui Wu. Chemoresistance regulation by COL11A1 in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4423. doi:10.1158/1538-7445.AM2015-4423

Simvastatin prevents cardiac hypertrophy in a rat model via modulation of the JAK/STAT pathway

Simvastatin prevents cardiac hypertrophy in a rat model via modulation of the JAK/STAT pathway

COL11A1 confers chemoresistance on ovarian cancer cells through the activation of Akt/c/EBPβ pathway and PDK1 stabilization.

Chemoresistance to anticancer drugs substantially reduces survival in epithelial ovarian carcinoma (EOC). Here, microarray analysis showed that collagen type XI alpha 1 (COL11A1) is a chemotherapy response-associated gene. Chemoresistant cells expressed higher COL11A1 and c/EBPβ than chemosensitive cells. COL11A1 or c/EBPβ downregulation suppressed chemoresistance, whereas COL11A1 overexpression attenuated sensitivity to cisplatin and paclitaxel.The c/EBPβ binding site in the COL11A1 promoter was identified as the major determinant of cisplatin- and paclitaxel-induced COL11A1 expression. Immunoprecipitation and immunofluorescence showed that in resistant cells, Akt and PDK1 were highly expressed and that anticancer drugs enhanced binding activity between COL11A1 and PDK1 binding and attenuated PDK1 ubiquitination and degradation. Conversely, chemosensitive cells showed decreased activity of COL11A1 binding to PDK1 and increased PDK1 ubiquitination, which were reversed by COL11A1 overexpression. Analysis of 104 EOC patients showed that high COL11A1 mRNA levels are significantly associated with poor chemoresponse and clinical outcome.