Abstract-In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation. Key Words: vascular smooth muscle Ⅲ vein Ⅲ sphingosine-1-phosphate Ⅲ transient receptor potential Ⅲ calcium channel S phingosine-1-phosphate (S1P) has emerged as a major endogenous signaling phospholipid with diverse roles in yeast, plants, and mammals. 1 Proposed functions include the regulation of cell proliferation, migration, programmed death, and pathological processes including cancer, asthma, inflammation, and trauma. There has been particular interest in the role of S1P in the cardiovascular system, where it accumulates in atherosclerotic lesions and plays a role in ischemic preconditioning of the heart. 2-5 S1P is derived from the phosphorylation of sphingosine catalyzed by sphingosine kinase, sphingosine being from ceramide and ceramide from sphingomyelin, a constituent lipid of signaling microdomains of plasma membrane lipid rafts and caveolae. 3 S1P is detected in serum at almost 1 mol/L, although protein binding impacts on the available concentration and local concentrations may vary substantially. 6 S1P is quite unusual among signaling molecules in having separate intracellular and extracellular effects. 1,4,7,8 It affects vascular smooth muscle cell migration, 9,10 evokes contraction of rat mesenteric artery, 11 and slows pacemaker activity of the sino-atrial node of the heart. 12 The underlying mechanisms are only partially worked out, but vascular smooth muscle cells respond to S1P with transient followed by sustained elevation of the cytosolic Ca 2ϩ concentration, 10,11,13,14 whereas cardiac myocytes show activation of potassium current and S1P-evoked "Ca 2ϩ deregulation," depending on extracellular Ca 2ϩ . 12,15 Despite positive effects on Ca 2ϩ signaling, the molecular basis of a Ca 2ϩ channel stimulated by S1P is unknown. L-type voltage-gated Ca 2ϩ channels are inhibited by S1P. 12 The Drosophila transient receptor potential ...

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Induction of K(Ca)3.1 (IKCa) potassium channel plays an important role in vascular smooth muscle cell proliferation. Here, we report that the gene encoding K(Ca)3.1 (KCNN4) contains a functional repressor element 1-silencing transcription factor (REST or NRSF) binding site and is repressed by REST. Although not previously associated with vascular smooth muscle cells, REST is present and recruited to the KCNN4 gene in situ. Significantly, expression of REST declines when there is cellular proliferation, showing an inverse relationship with functional K(Ca)3.1. Downregulated REST and upregulated K(Ca)3.1 are also evident in smooth muscle cells of human neointimal hyperplasia grown in organ culture. Furthermore, inhibition of K(Ca)3.1 suppresses neointimal formation, and exogenous REST reduces the functional impact of K(Ca)3.1. Here, we show REST plays a previously unrecognized role as a switch regulating potassium channel expression and consequently the phenotype of vascular smooth muscle cells and human vascular disease.

Abstract-Stromal interaction molecule 1 (STIM1) is a predicted single membrane-spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane-spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation. (Circ Res. 2008;103:e97-e104.)Key Words: vascular smooth muscle Ⅲ calcium channel Ⅲ stromal interaction molecule 1 Ⅲ transient receptor potential canonical 1 V ascular smooth muscle cells (VSMCs) in their contractile phenotype determine the caliber of most blood vessels and thus regulate blood pressure and local tissue perfusion. Throughout life, however, VSMCs also retain capacity for plasticity, which enables switching to a noncontractile modulated phenotype that is important for blood vessel formation, vascular adaptation, and response to injury, as well as contributing substantially to the vascular diseases of atherosclerosis, neointimal hyperplasia and in-stent restenosis. 1,2 Critical events regulating these VSMC properties are plasma membrane ion fluxes, such as Ca 2ϩ entry through Ca 2ϩ -permeable ion channels. [3][4][5][6][7][8] Voltage-gated Ca 2ϩ channels, especially the dihydropyridine-sensitive L-type channel (Ca V 1.2), have established pharmacological importance and roles in contractile VSMCs. There are, however, other types of Ca 2ϩ -permeable channels that are not voltage-gated and have importance in both VSMC phenotypes. 4 -8 One type is coupled to Ca 2ϩ store depletion: the store-operated channels, which are Ca 2ϩ -and ...

Cardiopulmonary bypass (CPB) involves the use of either an occlusive roller pump or centrifugal pump. Damage to blood elements, including haemolysis, may arise from occlusion when using a roller pump; the appropriate degree of occlusion has not yet been determined scientifically. Centrifugal and nonocclusive roller pumps are reputed to reduce haemolysis. The objective of this study was to compare haemolysis caused by a standard roller pump with a dynamically set nonocclusive roller pump and with a centrifugal pump. We prospectively randomized 60 patients undergoing routine coronary artery surgery into three groups: standard roller pump (STD, n = 20), dynamically set roller pump (DYN, n = 20), or centrifugal pump (CEN, n = 20). The level of plasma free haemoglobin (FHb) was measured preoperatively, and the rate of formation of FHb (in mg/dl/min) was determined at the end of the ischaemic phase and at the end of CPB. Cardiotomy suction blood was isolated for the ischaemic phase and returned before the end of CPB. It was found that there were no differences between the groups in demographic or operative variables. The rate of formation of FHb at the end of the ischaemic phase was similar for all groups (STD 0.108 +/- 0.10, DYN 0.117 +/- 0.08, CEN 0.129 +/- 0.07). At the end of CPB, after return of the cardiotomy suction blood, there was a significant (< 0.001) increase in the rate of formation of FHb in all groups. The increase was similar for each of the groups (STD 0.424 +/- 0.17, DYN 0.481 +/- 0.20, CEN 0.471 +/- 0.18). We conclude that the rates of haemolysis are similar for each of the pump types, and no benefit is conferred by the use of either a dynamically set roller pump or a centrifugal pump compared with the standard roller pump. The return of the cardiotomy suction blood to the circulation is the principal source of plasma free haemoglobin.

Standards for best practice in mitral valve repair were defined by multidisciplinary consensus. This study gives centres undertaking mitral valve repair an opportunity to benchmark their care against agreed standards that are challenging but achievable. Working towards these standards should act as a stimulus towards improvements in care.

AimThe aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia.Methods and resultsBlood vessels were obtained from patients or mice and studied in culture. Reverse transcriptase–polymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function. KV1.3 was unique among the KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC50 of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia.ConclusionKV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events.

As a consequence, there has been an increased focus upon faculty development, while some of the assessments have been redesigned in line with the PBA. A global rating scale has been introduced that uses clinical anchors. The rating scales have also been altered with a reduction in the number of ratings while an enhanced description of the complexity of the case has been introduced within the Case-based Discussion and the Clinical Evaluation Exercise. A re-evaluation will take place in the near future.