Sphingosine-1-phosphate–induced smooth muscle cell migration involves the mammalian target of rapamycin

BackgroundVascular smooth muscle cell (SMC) migration is an important component of the development of intimal hyperplasia. Sphingosine-1-phosphate (S-1-P) is a lipid released from activated platelets with numerous cellular effects including the stimulation of SMC migration in vitro. We examined the role of the mammalian target of rapamycin and ribosomal p70S6 kinase (p70S6K) in S-1-P–induced SMC migration.MethodsRat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P (0.01 to 100 μmol/L) with and without rapamycin (10 nmol/L). Western blotting was performed for phosphorylated and total p70S6K, ERK1/2, and p38MAPK after stimulation with S-1-P (0.1 μmol/L), with and without rapamycin pretreatment. Phosphorylation of p70S6K was also assayed after S-1-P treatment in the presence and absence of inhibitors of PI3 kinase (wortmannin, WN, and LY294002, LY), Akt (AktI), p38MAPK (SB203580), and MEK1 (PD98059).ResultsS-1-P stimulated migration of SMCs in both linear wound and Boyden chamber assays compared to control (P < .05); these responses were inhibited by rapamycin to below the level of control (P < .05 vs S-1-P alone for both assays) in a dose-dependent manner (inhibitory concentration of 50%, 10 nmol/L). S-1-P stimulated phosphorylation of ERK1/2, p38MAPK, and p70S6K, which peaked at 5 minutes for ERK1/2 and p38MAPK and10 minutes for p70S6K (2-fold increase over control for each, P < .05). Rapamycin prevented the phosphorylation of p70S6K at the Thr 389 site (which correlates with enzyme activity), reduced ERK1/2 phosphorylation, but had no effect on the Thr 421/Ser 424 site or on p38MAPK phosphorylation. Wortmannin and LY294002 inhibited phosphorylation of the Thr 389 site of p70S6K. AktI and SB203580 had no effect on p70S6K, whereas PD98059 had a marginal effect.ConclusionsS-1-P–induced SMC migration was completely inhibited by rapamycin, indicating that the p70S6K pathway is involved. This mechanism likely involves modulation of the ERK1/2 pathway. S-1-P stimulates phosphorylation of p70S6K in a MEK1-dependent, PI3 kinase–dependent, but Akt-independent manner.Clinical relevanceS-1-P is released from activated platelets at sites of vessel injury and contributes to the development of intimal hyperplasia after bypass grafting, angioplasty, and stenting. S-1-P is a potent pro-migratory molecule for SMCs. Rapamycin is a commonly used immunosuppressive agent that has most recently been incorporated as the biologic agent in drug eluting stents with good success in the coronary circulation. Rapamycin inhibits the mammalian target of rapamycin, which, in turn, controls the translational mechanisms of the cell. The role of translational control during S-1-P–induced SMC migration is poorly understood. This study identifies a link between the mammalian target of rapamycin translational pathway and S-1-P and demonstrates how rapamycin might interfere with another facet of a vessel's response to injury after a vascular intervention, namely by interfering with the cell signaling of factors released from platelets deposited at the injury site

Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism

BackgroundCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF.ObjectiveTo determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration.MethodsHuman coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, βARKCT were also used.ResultsATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gβγ inhibitor βARKCT inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect.ConclusionATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.Clinical RelevanceCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of ATF of urokinase on a pivotal cross-talk receptor, EGFR. EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target

ESVM Guideline on peripheral arterial disease

International audienc

TNF-α and NF-κB Signaling Play a Critical Role in Cigarette Smoke-induced Epithelial-mesenchymal Transition of Retinal Pigment Epithelial Cells in Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted

Urgent need to clarify the definition of chronic critical limb ischemia - a position paper from the European Society for Vascular Medicine

Chronic critical lower limb ischemia (CLI) has been defined as ischemia that endangers the leg. An attempt was made to give a precise definition of CLI, based on clinical and hemodynamic data (Second European Consensus). CLI may be easily defined from a clinical point of view as rest pain of the distal foot or gangrene or ulceration. It is probably useful to add leg ulcers of other origin which do not heal because of severe ischemia, and to consider the impact of frailty on adverse outcome. From a hemodynamic viewpoint there is no consensus and most of the existing classifications are not based upon evidence. We should thus propose a definition and then validate it in a prospective cohort in order to define the patients at major risk of amputation, and also to define the categories of patients whose prognosis is improved by revascularisation. From today\u27s available data, it seems clear that the patients with a systolic toe pressure (STP) below 30 mmHg must be revascularised whenever possible. However other patients with clinically suspected CLI and STP above 30 mmHg must be evaluated and treated in specialised vascular units and revascularisation has to be discussed on a case by case basis, taking into account other data such as the WiFi classification for ulcers.In conclusion, many useful but at times contradictory definitions of CLI have been suggested. Only a few have taken into account evidence, and none have been validated prospectively. This paper aims to address this and to give notice that a CLI registry within Europe will be set up to prospectively validate, or not, the previous and suggested definitions of CLI

UEMS Training Requirements for Angiology and Vascular Medicine: European Standards of Postgraduate Medical Specialist Training (ETR Document)

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Cyclin D1 promotes neurogenesis in the developing spinal cord in a cell cycle-independent manner

Neural stem and progenitor cells undergo an important transition from proliferation to differentiation in the G1 phase of the cell cycle. The mechanisms coordinating this transition are incompletely understood. Cyclin D proteins promote proliferation in G1 and typically are down-regulated before differentiation. Here we show that motoneuron progenitors in the embryonic spinal cord persistently express Cyclin D1 during the initial phase of differentiation, while down-regulating Cyclin D2. Loss-of-function and gain-offunction experiments indicate that Cyclin D1 (but not D2) promotes neurogenesis in vivo, a role that can be dissociated from its cell cycle function. Moreover, reexpression of Cyclin D1 can restore neurogenic capacity to D2-expressing glial-restricted progenitors. The neurogenic function of Cyclin D1 appears to be mediated, directly or indirectly, by Hes6, a proneurogenic basic helic-loop-helix transcription factor. These data identify a cell cycle-independent function for Cyclin D1 in promoting neuronal differentiation, along with a potential genetic pathway through which this function is exerted

Ultrasonography of the reticulum in 30 healthy Saanen goats

Background: The reticulum plays a crucial role in the ruminant digestive tract because the primary cycle of rumen motility always starts with a reticular contraction. In contrast to cattle, there are only few results on the ultrasonographic examination of the reticulum in goats. Therefore, it was the goal of the present study, to describe the results of ultrasonography of the reticulum of 30 healthy Saanen goats. Methods: Ultrasonography was carried out on standing, non-sedated animals using a 5.0 MHz linear transducer. The shape, contour and motility of the reticulum were investigated. A nine-minute video recording of the reticulum was made for each goat and the frequency, duration and amplitude of reticular contractions were calculated as described for cattle. Results: The reticulum appeared as a crescent-shaped structure with a smooth contour located immediately adjacent to the diaphragm. 0.8 to 2.1 (1.41 ± 0.31) reticular contractions were seen per minute. In all goats, biphasic reticular contractions were observed. 90% of the goats also had monophasic reticular contractions, and two had triphasic contractions. During the nine-minute observation periods, there were 0 to 6 monophasic reticular contractions and 6 to 15 biphasic contractions per goat. The duration of the biphasic contractions was 6.56 ± 0.74 s, which was significantly longer than the monophasic contractions at 4.31 ± 0.81 s. The average interval between two reticular contractions was 45.06 ± 12.57 s. Conclusion: Ultrasonography of the reticulum in goats is a valuable tool to characterise the appearance and motility of this organ. In addition to the biphasic motility pattern seen in cattle the reticular motility of goats is characterized by monophasic reticular contractions. The results of the present study are an important contribution for better understanding of the reticular motility in goats

ESVM guidelines:the diagnosis and management of Raynaud's phenomenon

Regarding the clinical diagnosis of Raynaud's phenomenon and its associated conditions, investigations and treatment are substantial, and yet no international consensus has been published regarding the medical management of patients presenting with this condition. Most knowledge on this topic derives from epidemiological surveys and observational studies; few randomized studies are available, almost all relating to drug treatment, and thus these guidelines were developed as an expert consensus document to aid in the diagnosis and management of Raynaud's phenomenon. This consensus document starts with a clarification about the definition and terminology of Raynaud's phenomenon and covers the differential and aetiological diagnoses as well as the symptomatic treatment