Platelet-Derived Microvesicles Promote VSMC Dedifferentiation After Intimal Injury via Src/Lamtor1/mTORC1 Signaling.
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ABSTRACT:
SUBMITTER: Liu JT
PROVIDER: S-EPMC8481604 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rationale: Abnormal migration of vascular smooth muscle cells (VSMCs) from the media to the interior is a critical process during the intimal restenosis caused by vascular injury. Here, we determined the role of platelet-derived microvesicles (PMVs) released by activated platelets in VSMC migration. Methods: A percutaneous transluminal angioplasty balloon dilatation catheter was used to establish vascular intimal injury. Collagen I was used to activate PMVs, mimicking collagen exposure during intimal injury. To determine the effects of PMVs on VSMC migration in vitro, scratch wound healing assays were performed. Fluorescence resonance energy transfer was used to detect variations of calcium dynamics in VSMCs. Results: Morphological results showed that neointimal hyperplasia was markedly increased after balloon injury of the carotid artery in rats, and the main component was VSMCs. PMVs significantly promoted single cell migration and wound closure in vitro. Fluorescence resonance energy transfer revealed that PMVs induced temporal and dynamic calcium oscillations in the cytoplasms of VSMCs. The influx of extracellular calcium, but not calcium from intracellular stores, was involved in the process described above. The channel antagonist GSK219 and specific siRNA revealed that a membrane calcium channel, transient receptor potential vanilloid 4 (TRPV4), participated in the calcium oscillations and VSMC migration induced by PMVs. Conclusions: TRPV4 participated in the calcium oscillations and VSMC migration induced by PMVs. PMVs and the related molecules might be novel therapeutic targets for vascular remodeling during vascular injury.
| S-EPMC7797689 | biostudies-literature
Project description:Reoccurrence of symptoms occurs in 30-50% of coronary artery disease patients receiving vein grafts or bare-metal stents due to intimal thickening (restenosis). Restenosis is caused by vascular smooth muscle cell (VSMC) migration and proliferation. New therapeutic approaches that reduce VSMC migration and proliferation while promoting endothelial cell (EC) coverage are required. We assessed the effect of a soluble form of N-cadherin (SNC-Fc, a fusion of the extracellular portion of N-Cadherin to a mutated Fc fragment of IgG), a cell-cell junction molecule, on human saphenous VSMC proliferation and migration in vitro. We also assessed its effect on intimal thickening in a validated human ex vivo organ culture model. We observed that SNC-Fc significantly inhibited VSMC proliferation and to a lesser extent migration. The anti-proliferative effect of SNC-Fc was mediated by the interaction of SNC-Fc with the FGFR, rather than through inhibition of β-catenin signalling. SNC-Fc also significantly reduced intimal thickening by ~85% in the ex vivo organ culture model. SNC-Fc treatment inhibited proliferation of the intimal cells but did not affect migration. SNC-Fc reduced EC apoptosis, without detrimental effects on EC proliferation and migration in vitro. Importantly SNC-Fc increased EC coverage in the ex vivo model of intimal thickening. In conclusion, we suggest that SNC-Fc may have potential as an anti-proliferative therapeutic agent for reducing restenosis which has no detrimental effects on endothelial cells.
| S-EPMC4749540 | biostudies-literature
Project description:The arterial mechanical microenvironment, including stiffness, is a crucial pathophysiological feature of vascular remodeling, such as neointimal hyperplasia after carotid endarterectomy and balloon dilatation surgeries. In this study, we examined changes in neointimal stiffness in a Sprague-Dawley rat carotid artery intimal injury model and revealed that extracellular matrix (ECM) secretion and vascular stiffness were increased. Once the endothelial layer is damaged in vivo, activated platelets adhere to the intima and may secrete platelet-derived extracellular vesicles (pEVs) and communicate with vascular smooth muscle cells (VSMCs). In vitro, pEVs stimulated VSMCs to promote collagen secretion and cell adhesion. MRNA sequencing analysis of a carotid artery intimal injury model showed that ECM factors, including col8a1, col8a2, col12a1, and elastin, were upregulated. Subsequently, ingenuity pathway analysis (IPA) was used to examine the possible signaling pathways involved in the formation of ECM, of which the Akt pathway played a central role. In vitro, pEVs activated Akt signaling through the PIP3 pathway and induced the production of Col8a1. MicroRNA (miR) sequencing of pEVs released from activated platelets revealed that 14 of the top 30 miRs in pEVs targeted PTEN, which could promote the activation of the Akt pathway. Further research showed that the most abundant miR targeting PTEN was miR-92a-3p, which promoted Col8a1 expression. Interestingly, knockdown of Col8a1 expression in vivo abrogated the increase in carotid artery stiffness and simultaneously increased the degree of neointimal hyperplasia. Our results revealed that pEVs may deliver miR-92a-3p to VSMCs to induce the production and secretion of Col8a1 via the PTEN/PIP3/Akt pathway, subsequently increasing vascular stiffness. Therefore, pEVs and key molecules may be potential therapeutic targets for treating neointimal hyperplasia.
| S-EPMC7960786 | biostudies-literature
Project description:Schwann cells (SCs) are endowed with a remarkable plasticity. When peripheral nerves are injured, SCs dedifferentiate and acquire new functions to coordinate nerve repair as so-called repair SCs. Subsequently, SCs redifferentiate to remyelinate regenerated axons. Given the similarities between SC dedifferentiation/redifferentiation in injured nerves and in demyelinating neuropathies, elucidating the signals involved in SC plasticity after nerve injury has potentially wider implications. c-Jun has emerged as a key transcription factor regulating SC dedifferentiation and the acquisition of repair SC features. However, the upstream pathways that control c-Jun activity after nerve injury are largely unknown. We report that the mTORC1 pathway is transiently but robustly reactivated in dedifferentiating SCs. By inducible genetic deletion of the functionally crucial mTORC1-subunit Raptor in mouse SCs (including male and female animals), we found that mTORC1 reactivation is necessary for proper myelin clearance, SC dedifferentiation, and consequently remyelination, without major alterations in the inflammatory response. In the absence of mTORC1 signaling, c-Jun failed to be upregulated correctly. Accordingly, a c-Jun binding motif was found to be enriched in promoters of genes with reduced expression in injured mutants. Furthermore, using cultured SCs, we found that mTORC1 is involved in c-Jun regulation by promoting its translation, possibly via the eIF4F-subunit eIF4A. These results provide evidence that proper c-Jun elevation after nerve injury involves also mTORC1-dependent post-transcriptional regulation to ensure timely dedifferentiation of SCs.SIGNIFICANCE STATEMENT A crucial evolutionary acquisition of vertebrates is the envelopment of axons in myelin sheaths produced by oligodendrocytes in the CNS and Schwann cells (SCs) in the PNS. When myelin is damaged, conduction of action potentials along axons slows down or is blocked, leading to debilitating diseases. Unlike oligodendrocytes, SCs have a high regenerative potential, granted by their remarkable plasticity. Thus, understanding the mechanisms underlying SC plasticity may uncover new therapeutic targets in nerve regeneration and demyelinating diseases. Our work reveals that reactivation of the mTORC1 pathway in SCs is essential for efficient SC dedifferentiation after nerve injury. Accordingly, modulating this signaling pathway might be of therapeutic relevance in peripheral nerve injury and other diseases.
| S-EPMC5956991 | biostudies-literature
Project description:BackgroundTraumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that can contribute to ongoing intracranial hemorrhage. Previous studies have shown adenosine diphosphate (ADP)-induced platelet aggregation to be reduced after TBI. In addition, circulating microvesicles (MVs) are increased following TBI and have been shown to play a role in post-TBI coagulopathy and platelet function. We hypothesized that post-TBI MVs would affect platelet aggregation in a murine head injury model.MethodsModerate TBI was performed using a weight-drop method in male C57BL6 mice. Whole blood, plasma, MVs, and MV-poor plasma were isolated from blood collected 10 minutes following TBI and were mixed separately with whole blood from uninjured mice. Platelet aggregation was measured with Multiplate impedance platelet aggregometry in response to ADP. The ADP P2Y12 receptor inhibitor, R-138727, was incubated with plasma and MVs from TBI mice, and platelet inhibition was again measured.ResultsWhole blood taken from 10-minute post-TBI mice demonstrated diminished ADP-induced platelet aggregation compared with sham mice. When mixed with normal donor blood, post-TBI plasma and MVs induced diminished ADP-induced platelet aggregation compared with sham plasma and sham MVs. By contrast, the addition of post-TBI MV-poor plasma to normal blood did not change ADP-induced platelet aggregation. The observed dysfunction in post-TBI ADP platelet aggregation was prevented by the pretreatment of post-TBI plasma with R-138727. Treatment of post-TBI MVs with R-138727 resulted in similar findings of improved ADP-induced platelet aggregation compared with nontreated post-TBI MVs.ConclusionAdenosine diphosphate-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced in normal blood by the introduction of post-TBI plasma and MVs. Furthermore, observed platelet dysfunction is prevented when post-TBI plasma and MVs are treated with an inhibitor of the P2Y12 ADP receptor. Clinically observed post-TBI platelet dysfunction may therefore be partially explained by the presence of the ADP P2Y12 receptor within post-TBI MVs.Level of evidenceLevel III.
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Project description:Accumulating evidence indicates that the lysosomal Ragulator complex is essential for full activation of the mechanistic target of rapamycin complex 1 (mTORC1). Abnormal mTORC1 activation has been implicated in several developmental neurological disorders, including Angelman syndrome (AS), which is caused by maternal deficiency of the ubiquitin E3 ligase UBE3A. Here we report that Ube3a regulates mTORC1 signaling by targeting p18, a subunit of the Ragulator. Ube3a ubiquinates p18, resulting in its proteasomal degradation, and Ube3a deficiency in the hippocampus of AS mice induces increased lysosomal localization of p18 and other members of the Ragulator-Rag complex, and increased mTORC1 activity. p18 knockdown in hippocampal CA1 neurons of AS mice reduces elevated mTORC1 activity and improves dendritic spine maturation, long-term potentiation (LTP), as well as learning performance. Our results indicate that Ube3a-mediated regulation of p18 and subsequent mTORC1 signaling is critical for typical synaptic plasticity, dendritic spine development, and learning and memory.
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Project description:Phenotypic switch of vascular smooth muscle cells (VSMCs) is characterized by increased expressions of VSMC synthetic markers and decreased levels of VSMC contractile markers, which is an important step for VSMC proliferation and migration during the development and progression of cardiovascular diseases including atherosclerosis. Chicoric acid (CA) is identified to exert powerful cardiovascular protective effects. However, little is known about the effects of CA on VSMC biology. Herein, in cultured VSMCs, we showed that pretreatment with CA dose-dependently suppressed platelet-derived growth factor type BB (PDGF-BB)-induced VSMC phenotypic alteration, proliferation and migration. Mechanistically, PDGF-BB-treated VSMCs exhibited higher mammalian target of rapamycin (mTOR) and P70S6K phosphorylation, which was attenuated by CA pretreatment, diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) and nuclear factor-κB (NFκB) inhibitor Bay117082. PDGF-BB-triggered ROS production and p65-NFκB activation were inhibited by CA. In addition, both NAC and DPI abolished PDGF-BB-evoked p65-NFκB nuclear translocation, phosphorylation and degradation of Inhibitor κBα (IκBα). Of note, blockade of ROS/NFκB/mTOR/P70S6K signaling cascade prevented PDGF-BB-evoked VSMC phenotypic transformation, proliferation and migration. CA treatment prevented intimal hyperplasia and vascular remodeling in rat models of carotid artery ligation in vivo. These results suggest that CA impedes PDGF-BB-induced VSMC phenotypic switching, proliferation, migration and neointima formation via inhibition of ROS/NFκB/mTOR/P70S6K signaling cascade.
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Project description:The mechanistic target of rapamycin (mTOR) Complex 1 (mTORC1) controls growth and proliferation of non-neuronal cells, while during neuronal development mTORC1 responds to glutamate and neurotrophins to promote neuronal migration and dendritic arborization. Recent studies reveal that mTORC1 signaling complexes are assembled on lysosomal membranes, but how mTORC1 membrane targeting is regulated is not fully clear. Our examination of palmitoyl-proteomic databases and additional bioinformatic analyses revealed that several mTORC1 proteins are predicted to undergo covalent modification with the lipid palmitate. This process, palmitoylation, can dynamically target proteins to specific membranes but its roles in mTORC1 signaling are not well described. Strikingly, we found that acute pharmacological inhibition of palmitoylation prevents amino acid-dependent mTORC1 activation in HEK293T cells and brain-derived neurotrophic factor (BDNF)-dependent mTORC1 activation in hippocampal neurons. We sought to define the molecular basis for this finding and found that the mTORC1 proteins LAMTOR1 and mTOR itself are directly palmitoylated, while several other mTORC1 proteins are not palmitoylated, despite strong bioinformatic prediction. Interestingly, palmitoylation of LAMTOR1, whose anchoring on lysosomal membranes is important for mTORC1 signaling, was rapidly increased prior to mTORC1 activation. In contrast, mTOR palmitoylation was decreased by stimuli that activate mTORC1. These findings reveal that specific key components of the mTOR pathway are dynamically palmitoylated, suggesting that palmitoylation is not merely permissive for mTOR activation but is instead actively involved in mTORC1-dependent signaling.
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