scholarly journals Liquid Platelet-Rich Fibrin and Heat-Coagulated Albumin Gel: Bioassays for TGF-β Activity

Materials ◽  
2020 ◽  
Vol 13 (16) ◽  
pp. 3466
Author(s):  
Zahra Kargarpour ◽  
Jila Nasirzade ◽  
Layla Panahipour ◽  
Richard J. Miron ◽  
Reinhard Gruber
Keyword(s):  
Target Genes ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Plasma Layer ◽  
Buffy Coat ◽  
Type I ◽  
Platelet Rich Fibrin ◽  
Nadph Oxidase 4 ◽  
Coat Layer ◽  
The Impact

Liquid platelet-rich fibrin (PRF) can be prepared by high centrifugation forces separating the blood into a platelet-poor plasma (PPP) layer and a cell-rich buffy coat layer, termed concentrated PRF (C-PRF). Heating the liquid PPP was recently introduced to prepare an albumin gel (Alb-gel) that is later mixed back with the concentrated liquid C-PRF to generate Alb-PRF. PRF is a rich source of TGF-β activity; however, the overall TGF-β activity in the PPP and the impact of heating the upper plasma layer remains unknown. Here, we investigated for the first time the in vitro TGF-β activity of all fractions of Alb-PRF. We report that exposure of oral fibroblasts with lysates of PPP and the buffy coat layer, but not with heated PPP, provoked a robust increase in the TGF-β target genes interleukin 11 and NADPH oxidase 4 by RT-PCR, and for IL11 by immunoassay. Consistent with the activation of TGF-β signaling, expression changes were blocked in the presence of the TGF-β receptor type I kinase inhibitor SB431542. Immunofluorescence and Western blot further confirmed that lysates of PPP and the buffy coat layer, but not heated PPP, induced the nuclear translocation of Smad2/3 and increased phosphorylation of Smad3. The immunoassay further revealed that PPP and particularly BC are rich in active TGF-β compared to heated PPP. These results strengthen the evidence that not only the cell-rich C-PRF but also PPP comprise a TGF-β activity that is, however, heat sensitive. It thus seems relevant to mix the heated PPP with the buffy coat C-PRF layer to regain TGF-β activity, as proposed during the preparation of Alb-PRF.

scholarly journals Relative Centrifugal Force (RCF; G-Force) Affects the Distribution of TGF-β in PRF Membranes Produced Using Horizontal Centrifugation

2020 ◽  
Vol 21 (20) ◽  
pp. 7629
Author(s):  
Zahra Kargarpour ◽  
Jila Nasirzade ◽  
Layla Panahipour ◽  
Richard J. Miron ◽  
Reinhard Gruber
Keyword(s):  
Centrifugal Force ◽  
High Speed ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Buffy Coat ◽  
Platelet Poor Plasma ◽  
Nadph Oxidase 4 ◽  
Relative Centrifugal Force ◽  
Coat Layer

Solid platelet-rich fibrin (PRF) is produced with centrifugation tubes designed to accelerate clotting. Thus, activated platelets may accumulate within the fibrin-rich extracellular matrix even before centrifugation is initiated. It can thus be assumed that platelets and their growth factors such as transforming growth factor-β (TGF-β) are trapped within PRF independent of their relative centrifugal force (RCF), the gravitation or g-force. To test this assumption, we prepared PRF membranes with tubes where clotting is activated by a silicone-coated interior. Tubes underwent 210 g, 650 g and 1500 g for 12 min in a horizontal centrifuge. The respective PRF membranes, either in total or separated into a platelet-poor plasma and buffy coat fraction, were subjected to repeated freeze-thawing to prepare lysates. Gingival fibroblasts were exposed to the PRF lysates to provoke the expression of TGF-β target genes. We show here that the expression of interleukin 11 (IL11) and NADPH oxidase 4 (NOX4), and Smad2/3 signaling were similarly activated by all lysates when normalized to the size of the PRF membranes. Notably, platelet-poor plasma had significantly less TGF-β activity than the buffy coat fraction at both high-speed protocols. In contrast to our original assumption, the TGF-β activity in PRF lysates produced using horizontal centrifugation follows a gradient with increasing concentration from the platelet-poor plasma towards the buffy coat layer.

scholarly journals Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells

2021 ◽  
Vol 22 (21) ◽  
pp. 11333
Author(s):  
Zahra Kargarpour ◽  
Jila Nasirzade ◽  
Layla Panahipour ◽  
Richard J. Miron ◽  
Reinhard Gruber
Keyword(s):  
Inflammatory Response ◽  
Inflammatory Mediators ◽  
Nuclear Translocation ◽  
Mesenchymal Cells ◽  
Pathological Process ◽  
Buffy Coat ◽  
Inflammatory Activity ◽  
Platelet Rich Fibrin ◽  
Enhanced Expression ◽  
Anti Inflammatory

Chronic inflammation is a pathological process where cells of the mesenchymal lineage become a major source of inflammatory mediators. Platelet-rich fibrin (PRF) has been shown to possess potent anti-inflammatory activity in macrophages, but its impact on mesenchymal cells has not been investigated. The aim of this study was, therefore, to expose mesenchymal cells to inflammatory cytokines together with lysates generated from liquid platelet-poor plasma (PPP), the cell-rich buffy coat layer (BC; concentrated-PRF or C-PRF), and the remaining red clot layer (RC), following centrifugation of blood. Heating PPP generates an albumin gel (Alb-gel) that when mixed back with C-PRF produces Alb-PRF. Membranes prepared from solid PRF were also subjected to lysis. We report here that lysates of PPP, BC, and PRF decreased the cytokine-induced expression of interleukin 6 (IL6) and nitric oxide synthase (iNOS) in the bone marrow-derived ST2 cells. Consistently, PPP, BC, and PRF greatly decreased the phosphorylation and nuclear translocation of p65 in ST2 cells. The inflammatory response caused by Pam3CSK4 was reduced accordingly. Moreover, PPP, BC, and PRF reduced the enhanced expression of inflammatory mediators IL6 and iNOS in 3T3-L1 pre-adipocyte mesenchymal cells, and iNOS and CCL5 in murine calvarial cells. Surprisingly, PRF lysates were not effective in reducing the inflammatory response of human gingival fibroblasts and HSC2 epithelial cells. The data from the present study suggest that both liquid PRF and solid PRF exert potent anti-inflammatory activity in murine mesenchymal cells.

NP603, a novel and potent inhibitor of FGFR1 tyrosine kinase, inhibits hepatic stellate cell proliferation and ameliorates hepatic fibrosis in rats

2011 ◽  
Vol 301 (2) ◽  
pp. C469-C477 ◽  
Author(s):  
Nan Lin ◽  
Si Chen ◽  
Weidong Pan ◽  
Linan Xu ◽  
Kunpeng Hu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Hepatic Fibrosis ◽  
Hepatic Stellate Cell ◽  
Type I Collagen ◽  
Kinase Inhibitor ◽  
Stellate Cell ◽  
Smooth Muscle Actin ◽  
Type I ◽  
Promising Therapeutic Approach ◽  
The Impact

Fibroblast growth factor 2 (FGF-2) and its main receptor FGFR1 have been shown to promote hepatic stellate cell (HSC) activation and proliferation. However, scant information is available on the anti-fibrogenic activity of FGFR1 inhibitors. The aim of this study was to assess the impact of a selective FGFR1 tyrosine kinase inhibitor NP603 on HSC proliferation and hepatic fibrosis. We demonstrated that rat primary HSCs secreted significant amounts of FGF-2, and its tyrosine phosphorylation of FGFR1 was attenuated by NP603. NP603 inhibited HSC activaton by measuring the expression of α-smooth muscle actin (α-SMA) and the production of type I collagen using ELISA. Furthermore, NP603 (25 μM) in vitro strongly suppressed HSC growth induced by FGF-2 (10 ng/ml) and FCS. This effect correlated with the suppression of extracellular-regulated kinase (ERK) activity and its downstream targets cyclin D1 and p21. In addition, PO NP603 (20 mg·kg−1·day−1) administration significantly decreased hepatic collagen deposition and α-SMA expression in CCl4-treated rats. Collectively, these studies suggest that selective blocking of the FGFR1-mediated pathway could be a promising therapeutic approach for the treatment of hepatic fibrosis.

scholarly journals Albumin inhibits the activation of hepatic stellate cells by suppressing TGF-β/Smad3 signaling via IL-1β

2019 ◽  
Author(s):  
Ji Hoon Park ◽  
Janghyun Kim ◽  
So-Young Choi ◽  
Kiweon Cha ◽  
Heekyung Park ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Collagen Type I ◽  
Retinol Binding Protein ◽  
Type I ◽  
Dependent Manner ◽  
Promising Therapeutic Approach

AbstractActivated hepatic stellate cells (HSCs) play a key role in liver fibrosis and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative, retinol binding protein (RBP)-albumin domain III fusion protein (named R-III), inhibit HSC activation. Here, we investigate the mode of action of albumin and R-III. NF-κB in activated HSCs was evenly distributed in the cytoplasm, but albumin expression and R-III treatment (albumin/R-III) induced NF-κB nuclear translocation via retinoic acid (RA) sequestration, resulting in increased expression of interleukin-1β (IL-1β). In an IL-1β dependent manner, albumin/R-III inhibited Smad3 nuclear translocation via TAK1-, JNK-mediated Smad3 linker phosphorylation and decreased expression of Smad3 target genes, such as α-smooth muscle actin and collagen type I. Mutation of the Smad3 linker phosphorylation sites abolished R-III effects on Smad3. In conclusion, our data suggest that the anti-fibrotic effects of albumin/R-III are due to RA sequestration which downregulates RAR-mediated signaling and also TGF-β/Smad3 signaling. This mechanistic elucidation of albumin function in HSCs provides clues to understanding the frequent albumin mutations found in hepatocellular carcinoma.

Applied electric fields suppress osimertinib-induced cytotoxicity via inhibiting FOXO3a nuclear translocation through AKT activation

2019 ◽  
Vol 41 (5) ◽  
pp. 600-610
Author(s):  
Li Li ◽  
Chen Hu ◽  
Conghua Lu ◽  
Kejun Zhang ◽  
Rui Han ◽  
...  
Keyword(s):  
Electric Fields ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Transcriptional Analysis ◽  
Akt Inhibitor ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
The Impact

Abstract Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor against T790M-mutant non-small cell lung cancer (NSCLC). Acquired resistance to osimertinib is a growing clinical challenge that is not fully understood. Endogenous electric fields (EFs), components of the tumor microenvironment, are associated with cancer cell migration and proliferation. However, the impact of EFs on drug efficiency has not been studied. In this study, we observed that EFs counteracted the effects of osimertinib. EFs of 100 mV/mm suppressed osimertinib-induced cell death and promoted cell proliferation. Transcriptional analysis revealed that the expression pattern induced by osimertinib was altered by EFs stimulation. KEGG analysis showed that differential expression genes were mostly enriched in PI3K-AKT pathway. Then, we found that osimertinib inhibited AKT phosphorylation, while EFs stimulation resulted in significant activation of AKT, which could override the effects generated by osimertinib. Importantly, pharmacological inhibition of PI3K/AKT by LY294002 diminished EF-induced activation of AKT and restored the cytotoxicity of osimertinib suppressed by EFs, which proved that AKT activation was essential for EFs to attenuate the efficacy of osimertinib. Furthermore, activation of AKT by EFs led to phosphorylation of forkhead box O3a (FOXO3a), and reduction in nuclear translocation of FOXO3a induced by osimertinib, resulting in decreased expression of Bim and attenuated cytotoxicity of osimertinib. Taken together, we demonstrated that EFs suppressed the antitumor activity of osimertinib through AKT/FOXO3a/Bim pathway, and combination of PI3K/AKT inhibitor with osimertinib counteracted the effects of EFs. Our findings provided preliminary data for therapeutic strategies to enhance osimertinib efficacy in NSCLC patients.

scholarly journals Ubiquitin specific peptidase Usp53 regulates osteoblast versus adipocyte lineage commitment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hadla Hariri ◽  
William N. Addison ◽  
René St-Arnaud
Keyword(s):  
Stromal Cells ◽  
Target Gene ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Maturation Stage ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Immunocompromised Mice ◽  
The Impact

AbstractWe have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (− 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

scholarly journals PYK2 controls intestinal inflammation via activation of IRF5 in macrophages

2020 ◽  
Author(s):  
Grigory Ryzhakov ◽  
Hannah Almuttaqi ◽  
Alastair L. Corbin ◽  
Tariq Khoyratty ◽  
Dorothee Berthold ◽  
...  
Keyword(s):  
Risk Factor ◽  
Genetic Risk ◽  
Protein Tyrosine Kinase ◽  
Target Genes ◽  
Intestinal Inflammation ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Genetic Risk Factor ◽  
Tyrosine Kinase 2

AbstractInflammatory bowel disease (IBD) is a group of inflammatory disorders of the gastro-intestinal tract caused by a complex combination of genetic and environmental factors. Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, which plays a key pathogenic role in mouse colitis models and is a genetic risk factor for IBD. A screen of a protein kinase inhibitor library in macrophages revealed a list of putative IRF5 kinases. Among the top hits validated in multiple in vitro assays, protein-tyrosine kinase 2-beta (PTK2B or PYK2) was identified as the only IBD genetic risk factor, known to impact gene expression in myeloid cells1,2. Phospho-proteomics and mutagenesis analyses established that PYK2 directly phosphorylates and activates IRF5 at tyrosine (Y) 171. IRF5 nuclear translocation and recruitment to target genes was impaired in PYK2-deficient cells or in cells treated with PYK2 inhibitors. Importantly, macrophage transcriptomic signature under PYK2 inhibition phenocopied IRF5 deficiency. Treatment with a PYK2 inhibitor reduced pathology and inflammatory cytokine production in Helicobacter hepaticus + anti-IL-10R antibody induced colitis model. It also decreased levels of pro-inflammatory cytokines in human colon biopsies taken from patients with ulcerative colitis. Thus, we have identified a major role for PYK2 in regulating the inflammatory response and mapped its activity to the IRF5 innate sensing pathway, opening opportunities for therapeutic interference with it in IBD and other inflammatory conditions.

scholarly journals Fas-Associated Factor 1 Negatively Regulates the Antiviral Immune Response by Inhibiting Translocation of Interferon Regulatory Factor 3 to the Nucleus

2016 ◽  
Vol 36 (7) ◽  
pp. 1136-1151 ◽  
Author(s):  
Soonhwa Song ◽  
Jae-Jin Lee ◽  
Hee-Jung Kim ◽  
Jeong Yoon Lee ◽  
Jun Chang ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Antiviral Response ◽  
Innate Immune ◽  
Type I Interferons ◽  
Type I ◽  
Antiviral Immune Response ◽  
Associated Factor

This study is designed to examine the cellular functions of human Fas-associated factor 1 (FAF1) containing multiple ubiquitin-related domains. Microarray analyses revealed that interferon-stimulated genes related to the antiviral response are significantly increased in FAF1-knockdown HeLa cells. Silencing FAF1 enhanced the poly(I·C)- and respiratory syncytial virus (RSV)-induced production of type I interferons (IFNs), the target genes of interferon regulator factor 3 (IRF3). IRF3 is a key transcription factor in IFN-β signaling responsible for the host innate immune response. This study also found that FAF1 and IRF3 physically associate with IPO5/importin-β3 and that overexpression of FAF1 reduces the interaction between IRF3 and IPO5/importin-β3. These findings suggest that FAF1 negatively regulates IRF3-mediated IFN-β production and the antiviral innate immune response by regulating nuclear translocation of IRF3. We conclude that FAF1 plays a novel role in negatively regulating virus-induced IFN-β production and the antiviral response by inhibiting the translocation of active, phosphorylated IRF3 from the cytosol to the nucleus.

scholarly journals Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2301 ◽  
Author(s):  
Eun-Jin An ◽  
Yumi Kim ◽  
Seung-Hyeon Lee ◽  
Hyun Min Ko ◽  
Won-Seok Chung ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Plant Medicine ◽  
Anti Cancer ◽  
M Phase

As a plant medicine, Oxalidaceae has been used to treat various diseases in Korea. However, there is little data on the anti-cancer efficacy of Oxalidaceae, particularly O. obtriangulata. This study aimed to investigate the anti-cancer effect of O. obtriangulata methanol extract (OOE) and its regulatory actions on pancreatic carcinoma. OOE showed anti-proliferative effects and induced cell death in the colony formation and cell viability assays, respectively. The Fluorescence-activated cell sorting (FACS) data confirmed that OOE significantly induced cell cycle accumulation at the G2/M phase and apoptotic effects. Additionally, OOE inhibited the activated ERK (extracellular-signal-regulated kinase)/Src (Proto-oncogene tyrosine-protein kinase Src)/STAT3 (signal transducers and activators of transcription 3) pathways including nuclear translocation of STAT3. Furthermore, suppression of Ki67, PARP(Poly ADP-ribose polymerase), caspase-3, P27(Cyclin-dependent kinase inhibitor 1B), and c-Myc as well as the STAT3 target genes CDK(cyclin-dependent kinase)1, CDK2, Cyclin B1, VEGF-1(vascular endothelial growth factor-1), MMP-9(Matrix metallopeptidase 9), and Survivin by OOE was observed in BxPC3. We speculate that these molecular actions might support an anti-cancer effect of OOE. In this study, we demonstrated that OOE may be a promising anti-cancer material and may serve as a natural therapy and alternative remedy for pancreatic cancer treatment.

scholarly journals Nrf2-Keap-1 imbalance under acute shear stress induces inflammatory response in venous endothelial cells

Perfusion ◽  
2021 ◽  
pp. 026765912110125
Author(s):  
Alexander O Ward ◽  
Graciela B Sala-Newby ◽  
Shameem Ladak ◽  
Gianni D Angelini ◽  
Massimo Caputo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Vascular Endothelial Cell ◽  
High Shear ◽  
High Shear Stress ◽  
Protective Environment ◽  
The Impact

Vascular endothelial cell stimulation is associated with the activation of different signalling pathways and transcription factors. Acute shear stress is known to induce different pro-inflammatory mediators such as IL-8. Nrf2 is activated by prolonged high shear stress promoting an antiinflammatory and athero-protective environment. However, little is known about the impact of acute shear stress on Nrf2 and Keap1 function and its role in IL-8 regulation. We aimed to examine Nrf2-Keap1 complex activation in-vitro and its role in regulating IL-8 transcripts under acute arterial shear stress (12 dyn/cm2) in venous endothelial cells (ECs). We note that acute high shear stress caused a significant upregulation of Nrf2 target genes, HO-1 and GCLM and an increased IL-8 upregulation at 90 and 120 minutes. Mechanistically, acute high shear did not affect Nrf2 nuclear translocation but resulted in reduced nuclear Keap1, suggesting that the reduction in nuclear Keap1 may result in increased free nuclear nrf2 to induce transcription. Consistently, the suppression of Keap1 using shRNA (shKeap1) resulted in significant upregulation of IL-8 transcripts in response to acute shear stress. Interestingly; the over expression of Nrf2 using Nrf2-Ad-WT or Sulforaphane was also associated with significant upregulation of IL-8 compared to controls. This study highlights the role of Keap1 in Nrf2 activation under shear stress and indicates that activation of Nrf2 may be deleterious in ECs in the context of acute haemodynamic injury.

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