Vascular endothelial injury is a contributing factor to the development of atherosclerosis and the resulting cardiovascular diseases. One particular factor involved in endothelial cell apoptosis and ...atherosclerosis is palmitic acid (PA), which is a long-chain saturated fatty acid. In addition, transient receptor potential melastatin 4 (TRPM4), a non-selective cation channel, plays a significant role in endothelial dysfunction caused by various factors related to cardiovascular diseases. Despite this, the specific role and mechanisms of TRPM4 in atherosclerosis have not been fully understood.
The protein and mRNA expressions of TRPM4, apoptosis - and inflammation-related factors were measured after PA treatment. The effect of TRPM4 knockout on the protein and mRNA expression of apoptosis and inflammation-related factors was detected. The changes of intracellular Ca
, mitochondrial membrane potential, and reactive oxygen species were detected by Fluo-4 AM, JC-1, and DCFH-DA probes, respectively. To confirm the binding of miR-133a-3p to TRPM4, a dual luciferase reporter gene assay was conducted. Finally, the effects of miR-133a-3p and TRPM4 on intracellular Ca
, mitochondrial membrane potential, and reactive oxygen species were examined.
Following PA treatment, the expression of TRPM4 increases, leading to calcium overload in endothelial cells. This calcium influx causes the assemblage of Bcl-2, resulting in the opening of mitochondrial calcium channels and mitochondrial damage, ultimately triggering apoptosis. Throughout this process, the mRNA and protein levels of IL-1β, ICAM-1, and VCAM1 significantly increase. Database screenings and luciferase assays have shown that miR-133a-3p preferentially binds to the 3'UTR region of TRPM4 mRNA, suppressing TRPM4 expression. During PA-induced endothelial injury, miR-133a-3p is significantly decreased, but overexpression of miR-133a-3p can attenuate the progression of endothelial injury. On the other hand, overexpression of TRPM4 counteracts the aforementioned changes.
TRPM4 participates in vascular endothelial injury caused by PA. Therefore, targeting TRPM4 or miR-133a-3p may offer a novel pharmacological approach to preventing endothelial injury.
Abstract Background Vascular smooth muscle cell (VSMC) migration in response to urokinase is dependent on binding of the urokinase molecule to the urokinase plasminogen receptor (uPAR) and cleavage ...of the receptor. The aim of this study was to examine the role of the soluble uPAR (suPAR) in VSMC migration. Methods Human VSMCs were cultured in vitro . Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of suPAR. Inhibitors to G-protein signaling and kinase activation were used to study these pathways. Assays were performed for mitogen-activated protein kinase and epidermal growth factor receptor activation. Results suPAR induced concentration-dependent migration of VSMC, which was G protein-dependent and was blocked by Gαi and Gβγ inhibitors. Removal of the full uPAR molecule by incubation of the cells with a phospholipase did not interfere with this response. suPAR induced ERK1/2, p38MAPK , and c-Jun N-terminal kinase JNK activation in a Gαi/Gβγ-dependent manner, and interruption of these signaling pathways prevented suPAR-mediated migration. suPAR activity was independent of plasmin activity. suPAR did not activate epidermal growth factor receptor. Interruption of the low affinity N-formyl-Met-Leu-Phe receptor ( FPRL1) but not high affinity N-formyl-Met-Leu-Phe receptor ( FPR ) prevented cell migration and activation in response to suPAR. suPAR increased matrix metalloproteinase-2 expression and activity, and this was dependent on the low affinity N-formyl-Met-Leu-Phe receptor ( FPRL1 ) and ERK1/2. Conclusions suPAR induces human smooth muscle cell activation and migration independent of the full uPAR through activation of the G protein-coupled receptor FPRL1 , which is not linked to the plasminogen activation cascade.
Increased Type I IFNs or IFN-I have been associated with human systemic lupus erythematosus. Interestingly augmenting or negating IFN-I activity in murine lupus not only modulates systemic ...autoimmunity, but also impacts lupus nephritis, suggesting that IFN-I may be acting at the level of the end-organ. We find resident renal cells to be a dominant source of IFN-I in an experimental model of autoantibody-induced nephritis. In this model, augmenting IFN-I amplified antibody-triggered nephritis, whereas ablating IFN-I activity ameliorated disease. One mechanism through which increased IFN-I drives immune-mediated nephritis might be operative through increased recruitment of inflammatory monocytes and neutrophils, though this hypothesis needs further validation. Collectively, these studies indicate that an important contribution of IFN-I toward the disease pathology seen in systemic autoimmunity may be exercised at the level of the end-organ.
Background Cell migration is an integral part of the development of intimal hyperplasia, and proteases are pivotal components in the process. Cell migration in response to urokinase is mediated ...through the aminoterminal fragment (ATF) of the protein. This study examines the role of NAD(P)H oxidase during epidermal growth factor receptor (EGFR) transactivation by ATF in human vascular smooth muscle cells (VSMC). Methods Human VSMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration in response to ATF were performed in the presence and absence of NAD(P)H oxidase inhibitors (diphenyleneiodonium DPI and apocynin) and small interfering RNA (siRNA) to Nox1. Additional assays were performed to examine the upstream pathways that lead to NAD(P)H oxidase activity. Assays were also performed for EGFR activation. Results ATF produced concentration-dependent VSMC migration, which was inhibited by increasing concentrations of DPI and apocynin. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at 4-fold greater than control. This response was inhibited by DPI and apocynin in a concentration-dependent manner. ATF induced a concentration-dependent increase in intracellular oxygen free radical species, which was mitigated by the presence of DPI and apocynin. Inhibition of Gβγ by βARKCT reduced both NAD(P)H oxidase activity and EGFR activation. Inhibition of rac, which allows the NAD(P)H complex to assemble on the membrane, and inhibition of src, which induces assembly of the complex, both reduced ATF-dependent NAD(P)H oxidase activity and EGFR phosphorylation. siRNA to Nox1 prevented ATF-mediated EGFR activation and cell migration. Conclusion ATF requires NAD(P)H oxidase activity through a Gβγ-, rac-, and src-mediated pathway to facilitate transactivation of EGFR and VSMC migration.
Abstract Lupus nephritis is an immune-mediated disease, where antibodies and T cells both play pathogenic roles. Since spontaneous lupus nephritis in mouse models takes 6–12 months to manifest, there ...is an urgent need for a mouse model that can be used to delineate the pathogenic processes that lead to immune nephritis, over a quicker time frame. We propose that the experimental anti-glomerular basement membrane (GBM) disease model might be a suitable tool for uncovering some of the molecular steps underlying lupus nephritis. This article reviews the current evidence that supports the use of the experimental anti-GBM nephritis model for studying spontaneous lupus nephritis. Importantly, out of about 25 different molecules that have been specifically examined in the experimental anti-GBM model and also spontaneous lupus nephritis, all influence both diseases concordantly, suggesting that the experimental model might be a useful tool for unraveling the molecular basis of spontaneous lupus nephritis. This has important clinical implications, both from the perspective of genetic susceptibility as well as clinical therapeutics.
Abstract Background Metabolic syndrome is now an epidemic in the United States population. Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. ...The aim of this study is to characterize the changes induced in wall morphology in the developing intimal hyperplasia within a murine model in the presence of diabetes (type 1) and metabolic syndrome. Methods Control (wild type B6), Non Obese Diabetic, and metabolic syndrome (RCS-10) mice were used. The murine femoral wire injury model was used in which a micro wire is passed through a branch of the femoral and used to denude the common femoral and iliac arteries. Specimens were perfusion fixed and sections were stained with hematoxylin and eosin and Movat stains such that dimensional and compositional morphometry could be performed using an ImagePro system. Additional stains for proliferation and apoptosis were used. Results In control mice, the injured femoral arteries develop intimal hyperplasia, which is maximal at 28 d and remains stable to day 56. Sham-operated vessels do not produce such a response. In diabetic mice, the intimal response increased 5-fold with a 2-fold increase in proteoglycan deposition, whereas in the metabolic syndrome mice there was a 6-fold increase in the intimal response and a similar increase in proteoglycan deposition. Collagen deposition was different with a 22-fold increase over control in collagen deposition in diabetes and a 100-fold increase over control in collagen deposition in metabolic syndrome as compared with the control injury mice. Maximal vascular smooth muscle cell (VSMC) proliferation was decreased in both diabetes and metabolic syndrome compared with controls, whereas early cell apoptosis in both diabetes and metabolic syndrome was sustained over a longer period of time compared with wild-type mice. Conclusions These data demonstrate that development of intimal hyperplasia is markedly different in diabetes and metabolic syndrome compared with controls, with an increase in collagen deposition, a reduction in VSMC proliferation, and an increase in early VSMC apoptosis. These findings suggest that preventative strategies against restenosis must be tailored for the diabetic and metabolic syndrome patients.
Abstract Background Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets that stimulates migration of vascular smooth muscle cells (VSMC) in vitro . S-1-P is ...associated with oxidized low-density lipoprotein (oxLDL) and is important in vessel remodeling. S-1-P will activate multiple G protein-coupled receptors (S-1-PR 1 to 5), which can regulate multiple cellular functions, including cell migration. The aim of this study is to examine the role of S-1-PR signaling during smooth muscle cell migration in response to S-1-P. Methods Human VSMCs were cultured in vitro . Expression of S-1-PR 1 to 5 was determined in conditions mirroring diabetes (40 mM glucose) and metabolic syndrome (25 mM glucose with 20 μM linoleic acid and 20 μM oleic acid). Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P with and without siRNA against S-1-PR 1 to 5. Assays were performed for activation of ERK1/2, p38MAPK and JNK. Results Human VSMCs express S-1-PR1, S-1-PR2, and S-1-PR3. There was no significant expression of S-1-PR4 and S-1-PR5. The expression of S-1-PR1 and S-1-PR3 is enhanced under high glucose conditions and metabolic syndrome conditions. Migration of VSMC in response to S-1-P is enhanced 2-fold by diabetes and 4-fold by metabolic syndrome. In diabetes, S-1-PR1 expression is enhanced, while S-1-PR2 and S-1-PR3 expression are both maintained. In metabolic syndrome, S-1-PR1 and 3 expressions are enhanced and that of S-1-PR2 is reduced. siRNA to S-1-PR1 results in a 2-fold reduction in S-1-P-mediated cell migration under all conditions. siRNA to S-1-PR2 enhanced cell migration only under normal conditions, while siRNA S-1-PR3 decreased migration in metabolic syndrome only. Down-regulation of S-1-PR1 reduced ERK1/2 activation in response to S-1-P, while that of S-1-PR2 had no effect under normal conditions. In diabetes, down-regulation of S-1-PR1 reduced activation of all three MAPKs. In metabolic syndrome, down-regulation of S-1-PR1 and S-1-PR3 reduced activation of all three MAPKs. Conclusion S-1-PR 1, 2, and 3 regulate human VSMC migration and their expression level and function are modulated by conditions simulating diabetes and metabolic syndrome.
Abstract Background Urokinase (uPA) modulates cellular and extracellular matrix responses within the microenvironment of the vessel wall and has been shown to activate the epidermal growth factor ...receptor (EGFR). This study examines the role of the protease domain of uPA during EGFR activation in human vascular smooth muscle cells (VSMC). Methods Human coronary VSMC were cultured in vitro . Assays of cell proliferation and EGFR phosphorylation were examined in response to the carboxyterminal fragment of uPA (CTF) in the presence and absence of the plasmin, metalloprotease and a disintegrin and metalloproteinase (ADAM) inhibitors, heparin-bound epidermal growth factor (HB-EGF), and EGFR inhibitors, and small interfering RNA to EGFR and ADAMs. Results CTF produced a dose-dependent increase in DNA synthesis and cell proliferation in human VSMC, which was blocked in a dose-dependent manner by both plasmin inhibitors and the EGFR inhibitor, AG1478. CTF induced time-dependent EGFR phosphorylation, which was blocked by inhibitors of plasmin and metalloproteinases activity. The presence of urokinase plasminogen activator receptor was not required. Inhibition of ADAM-10 and -12, and of HB-EGF blocked EGFR activation in response to CTF. CTF-mediated activation of EGFR was mediated through Gβγ, src, and NAD(P)H oxidase. Conclusions In human coronary VSMC, uPA induces uPAR-independent, domain-dependent smooth muscle cell proliferation through transactivation of EGFR by a plasmin-mediated, ADAM-induced, and HB-EGF–dependent process, which is mediated by the intracellular pathways involving Gαi, Gβγ, src, and NAD(P)H oxidase.
Abstract Background Vessels heal after injury and G protein–coupled receptors are involved in the vascular smooth muscle cell proliferation required to form intimal hyperplasia. We have previously ...identified the role of Gαq in vascular smooth muscle cell proliferation in vitro . This study now examines the role of Gαq in the developing intimal hyperplasia in a murine model and the impact of disruption of Gαq signaling on intimal hyperplasia development. Methods We employed a murine femoral wire injury model in which a micro-wire is passed through a branch of the femoral artery and used to denude the common femoral artery. We perfusion-fixed specimens and stained sections with hematoxylin-eosin and Movat's stains such that morphometric analysis could be performed using an Image-Pro system. We also harvested additional specimens of femoral artery and snap-froze them for Western blotting or zymography, to allow for the study of G protein expression and both protease expression and activity. We used contralateral vessels as controls. We immersed additional vessels in pluronic gel containing the chemical Gαq G protein inhibitors GP-2A, siRNA to Gαq or adenovirus containing mutant inactive Gαq. Results Gαq expression increased in a time-dependent manner after femoral artery injury. Sham-operated vessels did not produce such a response. Inhibition of Gαq reduced cell proliferation without affecting cell migration. Interruption of Gαq signaling also inhibited the development of intimal hyperplasia. Inhibition of Gαq did not alter peak urinary-type plasminogen activator activity and expression, but did increase early plasminogen activator inhibitor-1 activity and expression. Inhibition of Gαq reduced peak metalloproteinase (MMP)-9 activity at Day 3 but did not influence peak MMP-2 activity at Day 7. Protein expression for MMP-9 was also decreased, but that of MMP-2 was not affected. There were no changes in the expression or the activity of the respective inhibitors for MMP-9 and MMP-2, and tissue inhibitor of metalloproteinases-1 and -2. Conclusions These data demonstrate that femoral wire injury in the mouse is associated with a time-dependent increase in Gαq expression. Inhibition of Gαq alters cell proliferation and is associated with decreased MMP-9 expression and activity.
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