scholarly journals MicroRNA-96 inhibits FoxO3a function in IPF fibroblasts on type I collagen matrix

2014 ◽  
Vol 307 (8) ◽  
pp. L632-L642 ◽  
Author(s):  
Richard Seonghun Nho ◽  
Jintaek Im ◽  
Yen-Yi Ho ◽  
Polla Hergert
Keyword(s):  
Type I Collagen ◽  
Collagen Matrix ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Dose Dependent Fashion ◽  
Mrna And Protein Expression

Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by persistent (myo)fibroblasts and the relentless accumulation of collagen matrix. Unlike normal lung fibroblasts, IPF lung fibroblasts have suppressed forkhead box O3a (FoxO3a) activity, which allows them to expand in this diseased environment. microRNA-96 (miR-96) has recently been found to directly bind to the 3′-untranslated region of FoxO3a mRNA, which subsequently inhibits its function. We examined whether aberrantly low FoxO3a expression is in part due to increased miR-96 levels in IPF fibroblasts on polymerized collagen, thereby causing IPF fibroblasts to maintain their pathological properties. miR-96 expression was upregulated in IPF fibroblasts compared with control fibroblasts when cultured on collagen. In contrast, FoxO3a mRNA levels were reduced in most IPF fibroblasts. However, when miR-96 function was inhibited, FoxO3a mRNA and protein expression were increased, suppressing IPF fibroblast proliferation and promoting their cell death in a dose-dependent fashion. Likewise, FoxO3a and its target proteins p21, p27, and Bim expression was also increased in the presence of a miR-96 inhibitor in IPF fibroblasts. However, when control fibroblasts were treated with miR-96 mimic, FoxO3a, p27, p21, and Bim mRNA and protein levels were decreased. In situ hybridization analysis further revealed the presence of enhanced miR-96 expression in cells within the fibroblastic foci of IPF lung tissue. Our results suggest that when IPF fibroblasts interact with collagen-rich matrix, pathologically altered miR-96 expression inhibits FoxO3a function, causing IPF fibroblasts to maintain their pathological phenotype, which may contribute to the progression of IPF.

Abstract 14664: Reduced Activin Like Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Kevin Morine ◽  
Vikram Paruchuri ◽  
Xiaoying Qiao ◽  
Emily Mackey ◽  
Mark Aronovitz ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Remodeling ◽  
Cardiac Fibrosis ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Left Ventricular ◽  
Lv Function ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels

Introduction: Activin receptor like kinase 1 (ALK1) mediates signaling via transforming growth factor beta-1 (TGFb1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. Methods and Results: ALK1 mRNA expression was quantified by RT-PCR in left ventricular (LV) tissue from patients with end-stage heart failure and compared to control LV tissue obtained from the National Disease Research Interchange (n=8/group). Compared to controls, LV ALK1 mRNA levels were reduced by 85% in patients with heart failure. Next, using an siRNA approach, we tested whether reduced ALK1 levels promote TGFb1-mediated collagen production in human cardiac fibroblasts. Treatment with an ALK1 siRNA reduced ALK1 mRNA levels by 75%. Compared to control, TGFb1-mediated Type I collagen and pSmad-3 protein levels were 2.5-fold and 1.7-fold higher, respectively, after ALK1 depletion. To explore a role for ALK1 in heart failure, ALK1 haploinsufficient (ALK1) and wild-type mice (WT; n=8/group) were studied 2 weeks after thoracic aortic constriction (TAC). Compared to WT, baseline LV ALK1 mRNA levels were 50% lower in ALK1 mice. Both LV and lung weights were higher in ALK1 mice after TAC. Cardiomyocyte area and LV mRNA levels of BNP, RCAN, and b-MHC were increased similarly, while SERCa levels were reduced in both ALK1 and WT mice after TAC. Compared to WT, LV fibrosis (Figure) and Type 1 Collagen mRNA and protein levels were higher among ALK1 mice. Compared to WT, LV fractional shortening (48±12 vs 26±10%, p=0.01) and survival (Figure) were lower in ALK1 mice after TAC. Conclusions: Reduced LV expression of ALK1 is associated with advanced heart failure in humans and promotes early mortality, impaired LV function, and cardiac fibrosis in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

scholarly journals Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

1998 ◽  
Vol 331 (2) ◽  
pp. 417-422 ◽  
Author(s):  
David C. RISHIKOF ◽  
Ping-Ping KUANG ◽  
Christine POLIKS ◽  
Ronald H. GOLDSTEIN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Type I Collagen ◽  
State Level ◽  
Lung Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis

2008 ◽  
Vol 294 (1) ◽  
pp. G226-G235 ◽  
Author(s):  
Anne Rullier ◽  
Jennifer Gillibert-Duplantier ◽  
Pierre Costet ◽  
Gaëlle Cubel ◽  
Valérie Haurie ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Blood Coagulation ◽  
Type I Collagen ◽  
Collagen Matrix ◽  
Matrix Metalloproteinase 2 ◽  
Type I ◽  
Mrna Levels ◽  
Protease Activated Receptors ◽  
Thrombin Inhibition ◽  
Β Receptor

Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl4) toxicity was similar in wild-type (WT), PAR-1−/−, and PAR-1+/−mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl4or its solvent for 6 wk (300 μl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl4treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1+/−and PAR-1−/−mice, respectively ( P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1+/−and PAR-1−/−mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-β receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.

scholarly journals Uremic Toxins and Ciprofloxacin Affect Human Tenocytes In Vitro

2020 ◽  
Vol 21 (12) ◽  
pp. 4241
Author(s):  
Erman Popowski ◽  
Benjamin Kohl ◽  
Tobias Schneider ◽  
Joachim Jankowski ◽  
Gundula Schulze-Tanzil
Keyword(s):  
Type I Collagen ◽  
Uremic Toxins ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Cell Matrix ◽  
High Concentrations ◽  
High Doses ◽  
Very High

Tendinopathy is a rare but serious complication of quinolone therapy. Risk factors associated with quinolone-induced tendon disorders include chronic kidney disease accompanied by the accumulation of uremic toxins. Hence, the present study explored the effects of the representative uremic toxins phenylacetic acid (PAA) and quinolinic acid (QA), both alone and in combination with ciprofloxacin (CPX), on human tenocytes in vitro. Tenocytes incubated with uremic toxins +/- CPX were investigated for metabolic activity, vitality, expression of the dominant extracellular tendon matrix (ECM) protein type I collagen, cell-matrix receptor β1-integrin, proinflammatory interleukin (IL)-1β, and the ECM-degrading enzyme matrix metalloproteinase (MMP)-1. CPX, when administered at high concentrations (100 mM), suppressed tenocyte metabolism after 8 h exposure and at therapeutic concentrations after 72 h exposure. PAA reduced tenocyte metabolism only after 72 h exposure to very high doses and when combined with CPX. QA, when administered alone, led to scarcely any cytotoxic effect. Combinations of CPX with PAA or QA did not cause greater cytotoxicity than incubation with CPX alone. Gene expression of the pro-inflammatory cytokine IL-1β was reduced by CPX but up-regulated by PAA and QA. Protein levels of type I collagen decreased in response to high CPX doses, whereas PAA and QA did not affect its synthesis significantly. MMP-1 mRNA levels were increased by CPX. This effect became more pronounced in the form of a synergism following exposure to a combination of CPX and PAA. CPX was more tenotoxic than the uremic toxins PAA and QA, which showed only distinct suppressive effects.

scholarly journals Deletion of Alox15 improves kidney dysfunction and inhibits fibrosis by increased PGD2 in the kidney

2021 ◽  
Vol 25 (5) ◽  
pp. 445-455
Author(s):  
Naohiro Takahashi ◽  
Hiroaki Kikuchi ◽  
Ayaka Usui ◽  
Taisuke Furusho ◽  
Takuya Fujimaru ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Interstitial Fibrosis ◽  
Mrna Level ◽  
Type I ◽  
Renal Tubules ◽  
Mrna Levels ◽  
Metabolizing Enzymes ◽  
Protein Levels ◽  
Lipid Metabolizing Enzymes

Abstract Background Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. Methods To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. Results In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15−/− mice. Alox15−/− CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15−/− CKD mice. Mediator lipidomics revealed that Alox15−/− CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-β1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. Conclusion Increased PGD2 in Alox15−/− CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD.

scholarly journals Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices

2015 ◽  
Vol 309 (6) ◽  
pp. L552-L561 ◽  
Author(s):  
Jintaek Im ◽  
Polla Hergert ◽  
Richard Seonghun Nho
Keyword(s):  
Cell Death ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Type I Collagen ◽  
Collagen Matrix ◽  
Dominant Negative ◽  
Luciferase Assay ◽  
Type I ◽  
Protein Levels ◽  
Autophagic Activity

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirmed that FoxO3a binds to the promoter region of LC3B and transcriptionally activates LC3B. The overexpression of wild-type FoxO3a increased LC3B mRNA and protein expression in IPF fibroblasts, whereas the dominant negative FoxO3a decreased the LC3B level in control fibroblasts. The inhibition of autophagic activity sensitized control fibroblasts to collagen matrix-induced cell death. In contrast, enhanced viability was found when autophagic function was inhibited in IPF fibroblasts. Our study showed that aberrantly low FoxO3a expression participates in reducing autophagic activity via transcriptional suppression of LC3B in IPF fibroblasts on collagen. This suggests that low autophagic activity by the alteration of FoxO3a may contribute to IPF progression.

scholarly journals Phorbol-ester-mediated expression of the collagen type I pro-α2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant

1991 ◽  
Vol 278 (2) ◽  
pp. 369-373 ◽  
Author(s):  
I Stuiver ◽  
Y Shimizu ◽  
N Shimizu
Keyword(s):  
Steady State ◽  
Type I Collagen ◽  
Culture Media ◽  
Sequence Similarity ◽  
Collagen Type I ◽  
Cdna Clones ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mitogenic Signal Transduction

We have previously isolated a phorbol 12-myristate 13-acetate (PMA)-non-responsive variant line (VT-1) from mouse 3T3-L1 cells [Shimizu, Fujiki, Sugimura & Shimizu (1986) Cancer Res. 46, 4027-4031]. Differential hybridization of cDNAs obtained from PMA-treated 3T3-L1 and VT-1 cells resulted in the isolation of a number of unique cDNA clones, including one with a high degree of sequence similarity to the type I pro-alpha 2 collagen gene (COL1A2) [Amagai, Inokuchi, Nishikawa, Shimizu & Shimizu (1989) Somat. Cell Mol. Genet. 15, 153-158]. Here we examined the expression of type I collagen pro-alpha 2 [pro-alpha 2(I)] mRNA and production of type I collagen in these two cell lines. In quiescent cells, the pro-alpha 2(I) steady-state mRNA levels were four times greater in 3T3-L1 cells than in VT-1 cells. PMA addition caused the steady-state levels of pro-alpha 2(I) mRNA to be six times greater in 3T3-L1 cells than in VT-1 cells, with a maximum at 30-60 min. The pro-alpha 2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by PMA treatment, but no significant increase was detected in VT-1 cells. The correlation of collagen expression with a PMA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction.

Cartilage Regeneration with Cell-free Type 1 Collagen Matrix – Past, Present and Future (Part 1 – Clinical Aspects)

2020 ◽  
Author(s):  
Philip Peter Roessler ◽  
Turgay Efe ◽  
Dieter Christian Wirtz ◽  
Frank Alexander Schildberg
Keyword(s):  
Type I Collagen ◽  
Case Series ◽  
Cartilage Regeneration ◽  
Collagen Matrix ◽  
Treatment Method ◽  
Collagen Type I ◽  
Legal Framework ◽  
Clinical Aspects ◽  
Type I ◽  
Collagen Hydrogel

AbstractCartilage regeneration with cell-free matrices has developed from matrix-associated autologous cartilage cell transplantation (MACT) over ten years ago. Adjustments to the legal framework and higher hurdles for cell therapy have led to the procedures being established as an independent alternative to MACT. These procedures, which can be classified as matrix-induced autologous cartilage regeneration (MACR), all rely on the chemotactic stimulus of a cross-linked matrix, which mostly consists of collagens. Given the example of a commercially available type I collagen hydrogel, the state of clinical experience with MACR shall be summarized and an outlook on the development of the method shall be provided. It has been demonstrated in the clinical case series summarized here over the past few years that the use of the matrix is not only safe but also yields good clinical-functional and MR-tomographic results for both small (~ 10 mm) and large (> 10 mm) focal cartilage lesions. Depending on the size of the defect, MACR with a collagen type I matrix plays an important role as an alternative treatment method, in direct competition with both: microfracture and MACT.

scholarly journals Transcriptomics Study to Determine the Molecular Mechanism by which sIL-13Rα2-Fc Inhibits Caudal Intervertebral Disc Degeneration in Rats

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xin Wang ◽  
Jianshi Tan ◽  
Junhao Sun ◽  
Pengzhong Fang ◽  
Jinlei Chen ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Model Group ◽  
Expression Levels ◽  
Protein Levels ◽  
Collagen Protein

Background. Intervertebral disc degeneration is related to tissue fibrosis. ADAMTS can degrade the important components of the ECM during the process of intervertebral disc degeneration, ultimately resulting in the loss of intervertebral disc function. sIL-13Rα2-Fc can inhibit fibrosis and slow down the degeneration process, but the mechanism involved remains unclear. Objective. To determine the mechanism by which sIL-13Rα2-Fc inhibits ECM degradation and reduces intervertebral disc tissue fibrosis using a transcriptomics analysis. Methods. A rat model of caudal intervertebral disc degeneration was established, and Sirius red staining was used to observe the pathological changes in the caudal intervertebral disc. Transcriptome sequencing was employed to assess the gene expression profiles of the intervertebral disc tissues in the model group and the sIL-13Rα2-Fc-treated group. Differentially expressed genes were identified and analyzed using GO annotation and KEGG pathway analyses. Real-time fluorescence quantitative PCR was used to verify the expression levels of candidate genes. The levels of GAG and HA were quantitatively assessed by ELISA, and the levels of collagen I and collagen II were analyzed by western blotting. Results. Sirius red staining showed that in the model group, the annulus fibrosus was disordered, the number of breaks increased, and the type I collagen protein levels increased, whereas in the sIL-13Rα2-Fc group, the annulus fibrosus was ordered, the number of breaks decreased, and the type II collagen protein levels increased. In comparison with the model group, we identified 58 differentially expressed genes in the sIL-13Rα2-Fc group, and these were involved in 35 signaling pathways. Compared with those in the model group, the mRNA expression levels of Rnux1, Sod2, and Tnfaip6 in the IL-13Rα2-Fc group were upregulated, and the mRNA expression levels of Aldh3a1, Galnt3, Fgf1, Celsr1, and Adamts8 were downregulated; these results were verified by real-time fluorescence quantitative PCR. TIMP-1 (an ADAMTS inhibitor) and TIMP-1 combined with the sIL-13Rα2-Fc intervention increased the levels of GAG and HA, inhibited the expression of type I collagen, and promoted the expression of type II collagen. Conclusion. Adamts8 may participate in the degradation of ECM components such as GAG and HA and lead to an imbalance in the ECM of the intervertebral disc, resulting in intervertebral disc degeneration. sIL-13Rα2-Fc promoted anabolism of the ECM and increased the levels of ECM components by inhibiting the expression of Adamts8, thus maintaining the dynamic equilibrium of the ECM and ultimately delaying intervertebral disc degeneration.

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