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PROVIDER: PRJNA1311473 | ENA |
REPOSITORIES: ENA
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Project description:Proliferative retinopathies are associated with abnormal angiogenesis that can result in visual impairment or vision loss. The tight junction complex regulates blood-retinal barrier integrity; however, its role in proliferative retinopathies is still at an early stage. Here, we employed human retinal endothelial cells (HRMVECs), and a mouse model of oxygen-induced retinopathy (OIR) to investigate the impact of IL-33 signaling on tight junction disintegration and pathological angiogenesis. Our experimental findings demonstrate that IL-33 induces ZO-1 serine/threonine phosphorylation and tight junction disruption in HRMVECs. In addition, mass-spectroscopy (MS) analysis revealed that treating of HRMVECs with IL-33 induces ZO-1 phosphorylation at Thr861 residue. Furthermore, we observed that NOX1-PKC- signaling modulates IL-33-induced ZO-1 phosphorylation and tight junction integrity in HRMVECs. We also observed that IL-33 depletion significantly reduces OIR-induced NOX1-PKC-ZO-1 signaling, vascular leakage, and pathological retinal neovascularization in the ischemic retina. We also observed that the NOX1-specific inhibitor, fluoflavine (ML-090), attenuated OIR-induced NADPH oxidase activity and pathological retinal neovascularization in the ischemic retina. Thus, we infer that IL-33-mediated NOX1-PKC-ZO-1 signaling regulates ischemia-induced retinal endothelial cell tight junction disruption and retinal neovascularization.
2025-10-14 | PXD060282 | Pride
Project description:Rho-kinase signaling and YAP signaling are related to aortic dissection (AD) formation. However, as important biomechanical signaling pathways, their relationships with aortic smooth muscle cell (AoSMC) mechanics have not been investigated in AD formation. This study aims to explore the correlation between RhoA/ROCK1 and YAP, as well as their relationship with intrinsic AoSMC stiffness during AD formation. The expression of RhoA/ROCK1 and YAP as well as F-actin polymerization were analyzed in AoSMCs isolated from normal and AD human aortas. The intrinsic cell stiffness of normal and AD AoSMCs was measured using atomic force microscopy (AFM). The correlations among RhoA/ROCK1, YAP, and intrinsic AoSMC stiffness were explored by manipulating the activity of RhoA and ROCK1, and YAP expression. Finally, the role of RhoA/ROCK1/YAP/F-actin and AoSMC stiffness during AD formation was studied in pharmaceutically induced AD mouse models.Compared to normal human AoSMCs, the expression of RhoA and ROCK1 was downregulated, while the phosphorylation of YAP was increased in AD human AoSMCs, coupled with impaired F-actin polymerization and decreased intrinsic cell stiffness. Pharmaceutical inhibition of RhoA or ROCK1 activities and depletion of YAP all led to impaired F-actin polymerization and decreased intrinsic AoSMC stiffness. Moreover, abnormal collagen deposition and impaired cell-ECM interactions were found when RhoA/ROCK1/YAP/F-actin signaling was inhibited in normal human AoSMCs. We further analyzed the normal human AoSMC treated by Y27632 or not using RNA sequencing to investigate the impact of RhoA/ROCK1/YAP/F-actin on AoSMCs. GO analysis showed that differentially expressed genes (DEGs) related to cAMP-mediated signaling, cytoskeleton organization, cell-matrix adhesion, and extracellular matrix (ECM) organization were affected when ROCK1 activity was inhibited by Y27632 in normal AoSMCs. KEGG analysis showed differences in PI3K-Akt signaling, focal adhesion, MAPK, actin cytoskeleton, and ECM-receptor interaction. Consistently, cAMP, actin cytoskeleton organization, and ECM structural constituents were all downregulated in AoSMCs treated with Y27632 according to GSEA. We also analyzed the downregulated genes in AoSMCs treated with Y27632. Results demonstrated that the downregulated genes were mainly related to the cell-ECM unit, PI3K, MAPK, focal adhesion, and cAMP signaling pathways.
2024-03-01 | GSE247088 | GEO
Project description:Rho-kinase signaling and YAP signaling are related to aortic dissection (AD) formation. However, as important biomechanical signaling pathways, their relationships with aortic smooth muscle cell (AoSMC) mechanics have not been investigated in AD formation. This study aims to explore the correlation between RhoA/ROCK1 and YAP, as well as their relationship with intrinsic AoSMC stiffness during AD formation. The expression of RhoA/ROCK1 and YAP as well as F-actin polymerization were analyzed in AoSMCs isolated from normal and AD human aortas. The intrinsic cell stiffness of normal and AD AoSMCs was measured using atomic force microscopy (AFM). The correlations among RhoA/ROCK1, YAP, and intrinsic AoSMC stiffness were explored by manipulating the activity of RhoA and ROCK1, and YAP expression. Finally, the role of RhoA/ROCK1/YAP/F-actin and AoSMC stiffness during AD formation was studied in pharmaceutically induced AD mouse models.Compared to normal human AoSMCs, the expression of RhoA and ROCK1 was downregulated, while the phosphorylation of YAP was increased in AD human AoSMCs, coupled with impaired F-actin polymerization and decreased intrinsic cell stiffness. Pharmaceutical inhibition of RhoA or ROCK1 activities and depletion of YAP all led to impaired F-actin polymerization and decreased intrinsic AoSMC stiffness. Moreover, abnormal collagen deposition and impaired cell-ECM interactions were found when RhoA/ROCK1/YAP/F-actin signaling was inhibited in normal human AoSMCs. We further analyzed the normal human AoSMC treated by Y27632 or not using RNA sequencing to investigate the impact of RhoA/ROCK1/YAP/F-actin on AoSMCs. GO analysis showed that differentially expressed genes (DEGs) related to cAMP-mediated signaling, cytoskeleton organization, cell-matrix adhesion, and extracellular matrix (ECM) organization were affected when ROCK1 activity was inhibited by Y27632 in normal AoSMCs. KEGG analysis showed differences in PI3K-Akt signaling, focal adhesion, MAPK, actin cytoskeleton, and ECM-receptor interaction. Consistently, cAMP, actin cytoskeleton organization, and ECM structural constituents were all downregulated in AoSMCs treated with Y27632 according to GSEA. We also analyzed the downregulated genes in AoSMCs treated with Y27632. Results demonstrated that the downregulated genes were mainly related to the cell-ECM unit, PI3K, MAPK, focal adhesion, and cAMP signaling pathways.
2025-03-01 | GSE246021 | GEO
Project description:Dysregulation of vascular stiffness and cellular metabolism occur early in pulmonary hypertension (PH). Yet, the mechanisms by which biophysical properties of extracellular matrix relate to metabolic processes and downstream PH phenotypes remain undefined. In cultured endothelial and smooth muscle cells and confirmed in PH-diseased human samples, we found that ECM stiffening activates the mechanosensitive factors YAP/TAZ to increase glycolysis and induce glutaminase (GLS) expression and glutaminolysis. Glutaminolysis replenishes aspartate for anabolic biosynthesis, thus sustaining proliferation and migration within stiff ECM. In vitro GLS inhibition blocks aspartate production, consequently reprogramming entire cellular proliferative pathways, while aspartate restores proliferation. In a rat model in vivo, GLS inhibition prevents hemodynamic and histologic manifestations of PH. Thus, mechanical ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis â a paradigm that advances our understanding of the connections of mechanical stimuli with dysregulated vascular metabolism and identifies new metabolic drug targets in PH. We used microarrays to decipher the global program of gene expression involved in response to matrix stiffening and determined the implication of glutaminolysis (GLS) in these process PAECs were transfected with an siRNA control (siNC) or a siRNA against GLS (siGLS) and cultivated on soft hydrogel (1kPa) or stiff hydrogel (50kPa). After 48h of transfection cells were lysate and RNA extract for hybridization on Affymetrix microarrays.
2016-09-01 | E-GEOD-75793 | biostudies-arrayexpress
Project description:Mammalian cells are surrounded by the extracellular matrix (ECM), which regulates intercellular signal transduction and provides structural support to cells. Mechanical forces generated by ECM play a key role in regulating cell behaviors including survival, growth, mobility, and differentiation. However, it is unclear how mechanical forces are sensed by cells and transmit biochemical signals to cell fate-determining transcription factors such as YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif). Here we show the identification of Ras-associated protein 2 (RAP2) as an intracellular signal transducer that senses ECM rigidity and modulates cell survival and growth by negatively regulating YAP/TAZ. Activation of RAP2 by low ECM stiffness or contact inhibition triggers YAP/TAZ phosphorylation. Deletion of RAP2 leads to sustained YAP/TAZ activation in cells that grow on soft matrix or undergo contact inhibition, alters transcription programs initiated by low matrix stiffness, and results in cell transformation and malignant growth. Mechanistically, RAP2 can directly bind to Mitogen-activated protein kinase kinase kinase kinase 4/6/7 (MAP4K4/6/7) or Rho GTPase activating protein 29 (ARHGAP29), both of which lead to LATS1/2 activation and YAP/TAZ inhibition. These findings define a new molecular pathway that transmits mechanical cues to their effectors in the nucleus.
2018-07-09 | GSE98547 | GEO
Project description:Purpose: Endothelial cells respond to changes in subendothelial stiffness altering their proliferation, migration and barrier integrity but whether that is due to transcriptional reprogramming was largely unknown. Using RNA-Sequencing, we performed gene expression profiling for two endothelial cell types grown on soft or stiff matrices: primary human umbilical vein endothelial cells (HUVEC) and immortalized human microvascular endothelial cells (HMEC-1), to understand whether subendothelial stiffness-dependent changes in endothelial cell mechanics are due to transcriptional regulation. Methods and Results: By analyzing the differentially expressed genes between all samples we found that endothelial cell type rather that subendothelial stiffness is the primary determinant of the endothelial cell transcriptome. Both cell types respond to changes in their subendothelial stiffness by increasing the traction stresses they exert on stiffer as opposed to softer matrices, however it is apparently not the endothelial cell transcriptome that regulates this universal biomechanical response to subendothelial stiffness. Only a handful of genes were differentially expressed in each cell type in a stiffness-dependent manner, and none were shared between the two cell types examined. In contrast, thousands of genes were differentially regulated in HUVEC as compared to HMEC-1. HUVEC (but not HMEC-1) upregulate expression of TGF-2 on stiffer matrices, and also enhance their endogenous TGF-2 expression and their cell-matrix traction stresses in response to application of exogenous TGF-2. Conclusions: Altogether, these findings provide insights into the relationship between subendothelial stiffness, endothelial mechanics and variation of the transcriptome between distinct endothelial cell types, and reveal that subendothelial stiffness while critically impacting endothelial cell mechanics is minimally altering their transcriptome.
2019-07-31 | GSE135123 | GEO
Project description:Type 2 diabetes mellitus (T2DM) is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development, and increased stiffness is known to promote HCC progression in cirrhotic conditions. T2DM is characterized by an accumulation of advanced glycation end products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here, we find that in patients and animal models AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic b-catenin signaling promote HCC induction, while inhibiting AGEs production, reconstituting the clearance receptor AGER1, or breaking AGE-mediated collagen crosslinks reduce viscoelasticity and HCC growth. Matrix analysis and computational modeling demonstrate that lower interconnectivity of AGEs-bundled collagen matrix, marked by shorter fiber length and greater heterogeneity, enhance viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin β1–Tensin 1–YAP mechanotransduction pathway. These results reveal for the first time that AGEs-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can drive cancer progression in vivo, independent of stiffness.
2023-10-14 | GSE245016 | GEO
Project description:Dysregulation of vascular stiffness and cellular metabolism occur early in pulmonary hypertension (PH). Yet, the mechanisms by which biophysical properties of extracellular matrix relate to metabolic processes and downstream PH phenotypes remain undefined. In cultured endothelial and smooth muscle cells and confirmed in PH-diseased human samples, we found that ECM stiffening activates the mechanosensitive factors YAP/TAZ to increase glycolysis and induce glutaminase (GLS) expression and glutaminolysis. Glutaminolysis replenishes aspartate for anabolic biosynthesis, thus sustaining proliferation and migration within stiff ECM. In vitro GLS inhibition blocks aspartate production, consequently reprogramming entire cellular proliferative pathways, while aspartate restores proliferation. In a rat model in vivo, GLS inhibition prevents hemodynamic and histologic manifestations of PH. Thus, mechanical ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis – a paradigm that advances our understanding of the connections of mechanical stimuli with dysregulated vascular metabolism and identifies new metabolic drug targets in PH. We used microarrays to decipher the global program of gene expression involved in response to matrix stiffening and determined the implication of glutaminolysis (GLS) in these process
2016-09-01 | GSE75793 | GEO
Project description:The Rho family GTPases, Rac and Rho, play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac is dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified Activating Transcription Factor 3 (ATF3) as a major target of stiffness/Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.
2023-10-04 | GSE236266 | GEO
Project description:The transcriptional regulator YAP orchestrates important cell functions, determining tissue homeostasis, organ growth control, and tumorigenesis. Mechanical stimuli are a key input to YAP activity, but the mechanisms controlling this regulation remain largely uncharacterized. We show that CAV1 positively modulates the YAP mechanoresponse to substrate stiffness through actin cytoskeleton-dependent and Hippo kinase-independent mechanisms. RHO activity is necessary but not sufficient for CAV1-dependent mechanoregulation of YAP activity. Systematic quantitative interactomic studies and image-based siRNA screenings provide evidence that this actin-dependent regulation is determined by YAP interaction with the 14-3-3 protein YWHAH. Constitutive YAP activation rescued phenotypes associated with CAV1 loss, including defective ECM remodeling. CAV1-mediated control of YAP activity was validated in vivo in a model of pancreatitis-driven acinar-to-ductal metaplasia. We propose that this CAV1-YAP mechanotransduction system controls a significant share of cell programs linked to these two pivotal regulators, with potentially broad physiological and pathological implications.
2018-10-16 | GSE120514 | GEO