scholarly journals Tissue-Specific Influence of Lamin A Mutations on Notch Signaling and Osteogenic Phenotype of Primary Human Mesenchymal Cells

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 266 ◽  
Author(s):  
Kseniya Perepelina ◽  
Polina Klauzen ◽  
Anna Kostareva ◽  
Anna Malashicheva
Keyword(s):  
Notch Signaling ◽  
Osteogenic Differentiation ◽  
Bone Development ◽  
Umbilical Vein ◽  
Notch Pathway ◽  
Cell Lineage ◽  
Mesenchymal Cells ◽  
Human Cells ◽  
Lamin A ◽  
Umbilical Vein Endothelial Cells

Lamin A is involved in many cellular functions due to its ability to bind chromatin and transcription factors and affect their properties. Mutations of LMNA gene encoding lamin A affect the differentiation capacity of stem cells, but the mechanisms of this influence remain largely unclear. We and others have reported recently an interaction of lamin A with Notch pathway, which is among the main developmental regulators of cellular identity. The aim of this study was to explore the influence of LMNA mutations on the proosteogenic response of human cells of mesenchymal origin and to further explore the interaction of LMNA with Notch pathway. Mutations R527C and R471C in LMNA are associated with mandibuloacral dysplasia type A, a highly penetrant disease with a variety of abnormalities involving bone development. We used lentiviral constructs bearing mutations R527C and R471C and explored its influence on proosteogenic phenotype expression and Notch pathway activity in four types of human cells: umbilical vein endothelial cells (HUVEC), cardiac mesenchymal cells (HCMC), aortic smooth muscle cells (HASMC), and aortic valve interstitial cells (HAVIC). The proosteogenic response of the cells was induced by the addition of either LPS or specific effectors of osteogenic differentiation to the culture medium; phenotype was estimated by the expression of osteogenic markers by qPCR; activation of Notch was assessed by expression of Notch-related and Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. Overall, we observed different reactivity of all four cell lineages to the stimulation with either LPS or osteogenic factors. R527C had a stronger influence on the proosteogenic phenotype. We observed the inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling. Our results suggest that the effect of a LMNA mutation is strongly dependent not only on a specific mutation itself, but also might be influenced by the intrinsic molecular context of a cell lineage.

scholarly journals Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules

2021 ◽  
Vol 8 ◽  
Author(s):  
Victoria L. Messerschmidt ◽  
Uday Chintapula ◽  
Aneetta E. Kuriakose ◽  
Samantha Laboy ◽  
Thuy Thi Dang Truong ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Glycolic Acid ◽  
Umbilical Vein ◽  
Notch Pathway ◽  
Cellular Functions ◽  
Intracellular Domain ◽  
Downstream Signaling ◽  
Notch Intracellular Domain ◽  
Downstream Effects ◽  
Umbilical Vein Endothelial Cells

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. In this study, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles are non-toxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. Lastly, we elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

miR-100 alleviates the inflammatory damage and apoptosis of H2O2-induced human umbilical vein endothelial cells via inactivation of Notch signaling by targeting MMP9

Vascular ◽  
2021 ◽  
pp. 170853812198985
Author(s):  
Chen Wang ◽  
Yanqin Zhang ◽  
Zhenxing Jiang ◽  
Huiling Bai ◽  
Zizhong Du
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Matrix Metalloproteinase ◽  
Notch Signaling ◽  
Umbilical Vein ◽  
Thromboangiitis Obliterans ◽  
Human Umbilical Vein ◽  
Inflammatory Damage ◽  
Umbilical Vein Endothelial Cells ◽  
Metalloproteinase 9

Objective Thromboangiitis obliterans is a nonatherosclerotic segmental inflammatory disease, and miR-100 plays an anti-inflammatory role in chronic inflammation. Therefore, we hypothesized that miR-100 might alleviate the inflammatory damage and apoptosis of H2O2-induced ECV304 cells and aimed to investigate the relationship between miR-100 and thromboangiitis obliterans and the related molecular mechanism. Methods Cell counting kit-8 was used to detect cell viability, and the expression of inflammatory factors and oxidative stress was measured by ELISA. TUNEL assay was used to detect the apoptosis of human umbilical vein endothelial cells after induction by H2O2. Furthermore, the interaction between miR-100 and matrix metalloproteinase-9 was verified by dual-luciferase assay. Quantitative reverse transcription polymerase chain reaction and western blot were used to detect the expression of the adhesion factors, apoptosis-related proteins and Notch pathway-related protein. Results The results revealed that miR-100 was decreased in H2O2-induced human umbilical vein endothelial cells. Overexpression of miR-100 attenuated inflammatory response and cell apoptosis in H2O2-induced human umbilical vein endothelial cells. The overexpression of miR-100 inhibited matrix metalloproteinase-9 expression in H2O2-induced human umbilical vein endothelial cells. miR-100 inhibited H2O2-induced human umbilical vein endothelial cell inflammation, oxidative stress, and cell apoptosis via inactivation of Notch signaling by targeting matrix metalloproteinase. Conclusions Our study demonstrated that miR-100 reduced the inflammatory damage and apoptosis of H2O2-induced human umbilical vein endothelial cells via inactivation of Notch signaling by targeting matrix metalloproteinase. These findings suggested that miR-100 might be a novel therapeutic target for the prevention of thromboangiitis obliterans.

scholarly journals MicroRNA-10a Influences Osteoblast Differentiation and Angiogenesis by Regulating β-Catenin Expression

2015 ◽  
Vol 37 (6) ◽  
pp. 2194-2208 ◽  
Author(s):  
Jun Li ◽  
Yongqing Zhang ◽  
Qingxia Zhao ◽  
Jianghua Wang ◽  
Xijing He
Keyword(s):  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Molecular Mechanisms ◽  
Bone Cells ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Angiogenic Activity ◽  
Osteoblast Cell Line ◽  
Umbilical Vein Endothelial Cells

Background/Aims: Elucidation of the molecular mechanisms governing osteoblast differentiation and angiogenesis are of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-10a in the differentiation of MC3T3-E1 cells and pro angiogenic activity of mouse umbilical vein endothelial cells (MUVECs). Methods: The murine pre-osteoblast cell line MC3T3-E1 and MUVECs were used in the experiment. After transfected with miR-10a mimics or inhibitors, with or without LiCl pretreatment, the miR-10a, ALP, Runx2, Osx, OC and Dlx5 expression were assessed by RT-PCR. MC3T3-E1 cells were cultured with BMP2 to differentiate into bone cells, osteogenic differentiation of MC3T3-E1 cells were detected by ALP and ARS staining. Cell viability were analyzed by MTT and the protein expression of β-catenin, LEF1, cyclinD1, MMP2, and VEGF were detected by Western blotting; VEGF and VE-cadherin release were assessed by ELISA, and the migration of MUVECs, as well as tube formation were also detected. Results: MiR-10a expression was obviously down-regulated during osteogenic differentiation. Overexpression of miR-10a inhibited osteogenic differentiation of MC3T3-E1 cells, effectively decreasing MUVECs proliferation, migration, VEGF expression, VE-cadherin concentrations, and tube formation in vitro, whereas miR-10a silence enhanced those processes. Further mechanism assays demonstrated that overexpression of miR-10a reduced the β-catenin at both protein and transcription level, while pretreatment with Wnt signaling activator Licl partially attenuated the suppression effects of miR-10a overexpression on osteoblast differentiation and angiogenesis. Conclusion: Our findings imply that miR-10a plays a suppressive role in osteoblast differentiation of MC3T3-E1 cells and pro angiogenic activity of MUVECs by regulating the β-catenin expression, representing a novel and potential therapeutic target for the treatment of bone regeneration-related diseases.

scholarly journals Notch Intracellular Domain Plasmid Delivery via Poly(lactic-co-glycolic acid) Nanoparticles to Upregulate Notch Signaling

2021 ◽  
Author(s):  
Victoria Messerschmidt ◽  
Aneetta Kuriakose ◽  
Uday Chintapula ◽  
Samantha Laboy-Segarra ◽  
Thuy Truong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Notch Signaling ◽  
Glycolic Acid ◽  
Umbilical Vein ◽  
Genetic Material ◽  
Human Umbilical Vein ◽  
Notch Intracellular Domain ◽  
Downstream Effects ◽  
Umbilical Vein Endothelial Cells ◽  
Plasmid Delivery

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Liposomes and retroviruses are most commonly used to deliver genetic material to cells. However, there are many drawbacks to these systems such as increased toxicity, nonspecific delivery, short half-life, and stability after formulation. We utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domaincontaining plasmid in nanoparticles. In this study, we show that primary human umbilical vein endothelial cells readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles also are nontoxic, stable over time, and compatible with blood. We also determined that we can successfully transfect primary human umbilical vein endothelial cells (HUVECs) with our nanoparticles in static and dynamic environments. Lastly, we elucidated that our transfection upregulates the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

A Regulatory Network Between Notch and AKT Signaling Pathways Differentially Controls Megakaryocyte Development From Hematopoietic Stem or Committed Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 384-384
Author(s):  
Melanie G Cornejo ◽  
Stephen M Sykes ◽  
Cristina Lo Celso ◽  
Zuzana Tothova ◽  
Jon Aster ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Notch Pathway ◽  
Cell Lineage ◽  
Notch Signaling Pathway ◽  
Akt Pathway ◽  
Hematopoietic Stem ◽  
Akt Activation ◽  
Pathway Activation

Abstract Abstract 384 The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment to a specific lineage is unclear. During hematopoiesis, the Notch signaling pathway is known to play an important role in T cell lineage development. Recently, we demonstrated that the Notch signaling pathway is also a positive regulator of megakaryocyte lineage specification from hematopoietic stem cells (HSC). The importance of a tight regulation of this latter role is highlighted by the aberrant activation of the canonical Notch pathway transcription factor RBPJ by OTT-MAL, a fusion oncogene specifically associated with infant acute megakaryoblastic leukemia (AMKL). Here, we report a crosstalk between the Notch and PI3K/AKT pathways that provides new insights into the mechanism through which Notch signaling pathway regulates HSC differentiation into the erythro-megakaryocytic lineages. First, we observed that cells expressing a constitutively active Notch mutant had an increased level of phosphorylation of AKT compared to controls, indicating an association between Notch and AKT pathway activation. Using a Notch-GFP reporter mouse line, we confirmed that phosphorylation of AKT was increased in wild-type bone marrow cells upon physiological Notch stimulation (i.e. GFP+ cells) compared to control cells (i.e. GFP- cells) in vivo. Next, we assessed whether PI3K/AKT activation could replace or mimic Notch signaling during megakaryocyte development by transducing Lineage-Sca-1+cKit+ (LSK) cells or committed common myeloid progenitors (CMP) with a constitutively activated myristoylated AKT (myrAKT) mutant, followed by plating with or without Notch pathway stimulation on OP9-DL1 stroma or OP9 control stroma, respectively. MyrAKT-expressing LSK cells did not efficiently give rise to CD41+ megakaryocytic cells in the absence of Notch pathway stimulation, whereas myrAKT-expressing CMP showed partial rescue of development of megakaryocytes. Conversely, expression of a kinase-dead AKT mutant resulted in a pronounced reduction in megakaryocyte development from CMP, but had only a modest effect on LSK differentiation. Similar results were obtained with a chemical inhibitor of the AKT pathway. These results indicate that PI3K/AKT activation acts as an essential effector of the Notch pathway and can mimic Notch stimulation in CMP, whereas Notch-induced megakaryopoiesis from LSK cells is largely independent of the status of the PI3K/AKT pathway. To investigate the role of PI3K-AKT pathway on megakaryocyte development in vivo, we used FoxO1/3/4-deficient and PTEN-deficient mice, and observed that both mouse lines had significantly increased megakaryopoiesis compared to control animals both in vivo and ex vivo after culture on OP9-DL1 stroma. Importantly, FoxO1/3/4-deficient progenitors had upregulation of Nrarp and Hes1, two Notch pathway targets, and chromatin immunoprecipitation assays revealed the presence of FoxO factors at the Hes1 promoter, indicating a feedback control of the PI3K/AKT pathway on Notch pathway activation. Taken together, these data demonstrate a complex regulatory network between the Notch and PI3K/AKT pathways during megakaryopoiesis. In addition, our results annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of two distinct developmental pathways. Disclosures: Gilliland: Merck: Employment.

Sign in / Sign up

Export Citation Format

Share Document