scholarly journals Platelet-Rich Fibrin Increases BMP2 Expression in Oral Fibroblasts via Activation of TGF-β Signaling

2021 ◽  
Vol 22 (15) ◽  
pp. 7935
Author(s):  
Zahra Kargarpour ◽  
Jila Nasirzade ◽  
Layla Panahipour ◽  
Goran Mitulović ◽  
Richard J. Miron ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Mesenchymal Cells ◽  
Buffy Coat ◽  
Bmp Signaling ◽  
Bone Morphogenetic Protein 2 ◽  
Gingival Fibroblasts ◽  
Platelet Rich Fibrin ◽  
Morphogenetic Protein ◽  
Β Receptor ◽  
Tgf Β1

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

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.

scholarly journals Bone Morphogenetic Protein-2 Antagonizes Renal Interstitial Fibrosis by Promoting Catabolism of Type I Transforming Growth Factor-β Receptors

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 727-740 ◽  
Author(s):  
Yu-Lin Yang ◽  
Yi-Shiuan Liu ◽  
Lea-Yea Chuang ◽  
Jinn-Yuh Guh ◽  
Tao-Chen Lee ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Ureteral Obstruction ◽  
Time Course ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Bone Morphogenetic Protein 2 ◽  
Type I ◽  
Morphogenetic Protein ◽  
Tgf Β1

TGF-β is a therapeutic target for renal fibrosis. Scientists have long sought ways to antagonize TGF-β to ameliorate diabetic nephropathy. Bone morphogenetic protein (BMP-2) is a member of the TGF-β superfamily and is highly regulated in the kidney. Thus, the role of BMP-2 was investigated in NRK-49F cells (rat fibroblasts). We showed that TGF-β1 induces an increase in fibronectin. Treatment with exogenous BMP-2 or pCMV-BMP-2 significantly reversed the TGF-β1-induced increase in fibronectin concomitant with a significant decrease in type I TGF-β receptors (TGF-β RI). Moreover, BMP-2 significantly shortened the half-life of TGF-β RI. These results are related to proteosomal activation because MG132, a proteasome inhibitor, abolished BMP-2-mediated degradation of TGF-β RI. This was confirmed because BMP-2 time course dependently enhanced the ubiquitination level of TGF-β RI. In addition, Smads would seem to be involved in the interaction of BMP-2 and TGF-β. We demonstrated that BMP-2 significantly reversed the TGF-β1-induced increase in pSmad2/3 and reversed the TGF-β1-induced decrease in inhibitory Smad7. Most importantly, Smad7 small interfering RNA abolished the BMP-2-induced decrease in TGF-β RI. We evaluated the clinical efficacy of BMP-2 using unilateral ureteral obstruction rats. BMP-2 was administered ip for 7 d. In the unilateral ureteral obstruction kidneys, interstitial fibrosis was prominent. However, treatment with BMP-2 dramatically reduced Masson’s trichrome staining (collagen) in the interstitial and tubular areas of the kidneys concomitantly with a reduction in TGF-β RI. These results suggest that BMP-2 acts as a novel fibrosis antagonizing cytokine partly by down-regulating TGF-β RI and Smads. Bone morphogenetic protein-2 can antagonize TGF-β-inducing cellular fibrosis by intervening post-receptors signaling, thus disclosing an application of therapeutical potential against fibrosis disorders.

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 Effect of Carbonate Apatite Hydrogel-Advanced Platelet-Rich Fibrin Injection on Osteoblastogenesis during Orthodontic Relapse in Rabbits

2020 ◽  
Author(s):  
Ananto Ali Alhasyimi ◽  
Sri Suparwitri ◽  
Christnawati Christnawati
Keyword(s):  
Transforming Growth Factor ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carbonate Apatite ◽  
Histomorphometric Analysis ◽  
Platelet Rich Fibrin ◽  
Morphogenetic Protein ◽  
Post Hoc ◽  
Autologous Platelet ◽  
Tgf Β1

Abstract Objective The study aimed to determine the effect of carbonate apatite (CHA) hydrogel-aPRF on osteoblastogenesis during relapse in rabbits. Materials and Methods Forty-five rabbits were divided into three groups (n = 15): the control, CHA, and CHA-autologous platelet-rich fibrin (aPRF) groups. An open-coil spring was compressed between brackets to distalize the lower incisors of the rabbits by delivering a force of 50 cN for 1 week. The new position of the teeth was retained for 14 days, and CHA hydrogel-aPRF was injected every 7 days. The appliances were then debonded to allow relapse. On days 0, 3, 7, 14, and 21 after debonding, transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 expression was examined using immunohistochemistry staining and Runx-2 levels were analyzed by enzyme-linked immunosorbent assay. The data collected were analyzed using analysis of variance and a post hoc Tukey’s test (p < 0.05). Results Histomorphometric analysis revealed that TGF-β1 expression in the CHA-aPRF group is statistically higher than that in other groups on days 0, 3, and 7 after debonding (p < 0.05). BMP-2 expression in the CHA-aPRF group was also statistically higher than that in the other groups on days 3, 14, and 21 after debonding (p < 0.05). ELISA showed that Runx-2 levels are slightly higher in the CHA-aPRF group than in the other groups (p > 0.05). Conclusion Although injection of CHA-aPRF aids in osteoblastogenesis associated with enhancing TGF-β1 and BMP-2 expressions, it does not significantly upregulate Runx-2 levels.

scholarly journals Bone morphogenetic protein (BMP) receptor inhibitor JL5 synergizes with Ym155 to induce AIF-caspase independent cell death.

2020 ◽  
Author(s):  
Arindam Mondal ◽  
Rachel NeMoyer ◽  
Elaine Langenfeld ◽  
Danea Glover ◽  
Michael Scott ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Bmp Signaling ◽  
Lung Carcinomas ◽  
Bmp Receptor ◽  
Cell Death Pathway ◽  
Morphogenetic Protein ◽  
Receptor Inhibitor

Abstract Background: BMP is an evolutionary conserved morphogen that is reactivated in lung carcinomas. BMP receptor inhibitors promote cell death of lung carcinomas by mechanisms not fully elucidated. The studies here reveal novel mechanisms by which the “survivin” inhibitor Ym155 in combination with the BMP inhibitor JL5 synergistically induces death of lung cancer cells.Methods: This study examines the mechanism by which Ym155 in combination with JL5 downregulates BMP signaling and induces cell death of non-small cell lung carcinomas (NSCLC) cell lines. Validation experiments were performed on five passage 0 primary NSCLC.Results: We found that Ym155, which is reported to be a survivin inhibitor, potently inhibits BMP signaling by causing BMPR2 mislocalization into the cytoplasm and its decreased expression. Ym155 mediated cell death is not caused by the inhibition of survivin but involves Ym155 binding to mitochondrial DNA leading to depletion of ATP. The combination of Ym155 and the BMP receptor inhibitor JL5 synergistically causes the downregulation of BMP Smad-1/5 dependent and independent signaling and the induction of cell death of lung cancer cell lines and primary lung tumors. Cell death involves the nuclear translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus causing DNA double stranded breaks independent of caspase activation, which occurs only when JL5 and Ym155 are used in combination. Knockdown of BMPR2 together with Ym155 also induced AIF localization to the nucleus.Conclusions: These studies suggest that inhibition of BMPR2 together with Ym155 can induce AIF caspase-independent cell death. AIF caspase-independent cell is an evolutionary conserved cell death pathway that has never been targeted to induce cell death in cancer cells. These studies provide mechanistic insight how to target AIF caspase-independent cell death using BMP inhibitors.

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