Christopher Munsch

Upregulated TRPC1 Channel in Vascular Injury In Vivo and Its Role in Human Neointimal Hyperplasia

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.

Mitral repair best practice: proposed standards

Objectives: To define best practice standards for mitral valve repair surgery. Design: Development of standards for process and outcome by consensus. Setting: Multidisciplinary panel of surgeons, anaesthetists, and cardiologists with interests and expertise in caring for patients with severe mitral regurgitation. Main outcome measures: Standards for best practice were defined including the full spectrum of multidisciplinary aspects of care. Results: 19 criteria for best practice were defined including recommendations on surgical training, intraoperative transoesophageal echocardiography, surgery for atrial fibrillation, audit, and cardiology and imaging issues. Conclusions: 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.

A Sphingosine-1–Phosphate-Activated Calcium Channel Controlling Vascular Smooth Muscle Cell Motility

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.

Interactions, Functions, and Independence of Plasma Membrane STIM1 and TRPC1 in Vascular Smooth Muscle Cells

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.

Rho-kinase inhibitors prevent agonist-induced vasospasm in human internal mammary artery

Vasospasm of arterial conduits used for coronary artery surgery is an important cause of graft failure and is likely to result partly from raised levels of vasoconstrictor substances such as thromboxane A2 and endothelin‐1. Our aim was to find pharmacological agents that could prevent agonist‐induced vasospasm. Isometric tension was recorded from discarded segments of human left internal mammary artery (LIMA). Submaximal contraction evoked by the thromboxane A2 mimetic U46619 (10 nM) was not inhibited by a blocker of store‐ and receptor‐operated Ca2+ channels (30 μM SKF96365) in the presence of diltiazem. Furthermore, contractions to 1 nM U46619 were preserved when extracellular Ca2+ was reduced from 2.5 mM to 60 nM. Thus, sustained U46619‐evoked contraction occurred without Ca2+ influx. We hypothesized that contraction might occur via Rho‐kinase‐mediated Ca2+‐sensitization of myofilaments. Inhibitors of Rho‐kinase (Y27632 and HA1077) were profound relaxants. If contraction was pre‐evoked by 10 nM U46619, Y27632 and HA1077 caused full relaxation with EC50s of 1.67±0.22 μM and 3.58±0.35 μM respectively. Y27632 was also effective if applied before U46619, but was less potent. Y27632 abolished contraction evoked by endothelin‐1 and significantly reduced resting tone in the absence of a vasoconstrictor. Rho‐kinase‐mediated Ca2+‐sensitization appears to be a major mechanism of vasoconstriction in human LIMA. Rho‐kinase inhibitors may have an important role in preventing vasospasm in arterial grafts used for coronary artery surgery.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers.

Aim The aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. Methods and results Blood 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. Conclusion KV1.3 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.

Effect of radial artery harvesting on tissue perfusion and function of the hand.

This study was designed to measure the degree to which hand and forearm blood flow is reduced following harvesting of the radial artery in myocardial revascularization surgery and determine whether there is an effect on hand function. Twenty patients who had the radial artery used for myocardial revascularization underwent bilateral blood flow assessment of hands and forearms using Technetium-99m human serum albumin and clinical evaluation of hand function. Mean tissue perfusion in ml/100 ml/min +/- SD was as follows: donor hand 21.9 +/- 5.6, non-donor hand 25.5 +/- 6.1 (P = 0.00043), donor arm (hand and forearm) 17.5 +/- 3.7 and non-donor arm (hand and forearm) 21 +/- 5.1 (P = 0.000681). No clinical evidence of hand claudication was detected. This study suggests that removal of the radial artery reduces the tissue perfusion of the hand and forearm but does not affect hand function in the short term. The use of radial artery grafts in patients at risk of developing peripheral vascular disease should be carefully considered

Vasorelaxant properties of nicorandil on human radial artery

OBJECTIVE The radial artery is becoming popular as a conduit for coronary artery surgery but there is concern about its tendency to vasospasm. Diltiazem is used clinically in an effort to prevent vasospasm but there are suggestions that it is relatively ineffective. The first aim of the study was to test the effectiveness of Ca(2+) antagonists against vasospasm evoked by vasoconstrictor agonists. Because a large component of vasospasm was resistant to Ca(2+) antagonists, the second aim was to test if a different class of vasodilator, nicorandil, might relax the residual tone. METHODS Isometric tension was recorded in human radial artery segments harvested from patients undergoing myocardial revascularization surgery. RESULTS Diltiazem at 10 microM, which strongly inhibits L-type voltage-gated Ca(2+) channels, induced partial relaxation (mean+/-SEM, 44.6+/-3.5%, n=31) of phenylephrine-evoked contraction, but only 14.0+/-4.1% (n=10) and 12. 2+/-4.2% (n=10) relaxation of U46619- (a thromboxane A(2) analogue) or endothelin-1-evoked contraction. Strikingly, nicorandil relaxed agonist-evoked contractions that were resistant to diltiazem or nicardipine. In the absence of a Ca(2+) antagonist, nicorandil (30 microM) evoked 74.1+/-5.6% (n=24), 36.8+/-9.3% (n=10) and 64.5+/-7. 9% (n=14) relaxation of phenylephrine-, U46619- and endothelin-1-evoked contractions. CONCLUSIONS Nicorandil has a marked relaxant effect on contractions evoked by three different vasoconstrictor agonists, and relaxes vasospasm that is resistant to conventional Ca(2+) antagonists. These in vitro data suggest that nicorandil might be a useful drug for the inhibition of radial artery vasospasm in myocardial revascularization surgery.

Surgeon length of service and risk-adjusted outcomes: linked observational analysis of the UK National Adult Cardiac Surgery Audit Registry and General Medical Council Register.

Summary Objectives To explore the relationship between in-hospital mortality following adult cardiac surgery and the time since primary clinical qualification for the responsible consultant cardiac surgeon (a proxy for experience). Design Retrospective analysis of prospectively collected national registry data over a 10-year period using mixed-effects multiple logistic regression modelling. Surgeon experience was defined as the time between the date of surgery and award of primary clinical qualification. Setting UK National Health Service hospitals performing cardiac surgery between January 2003 and December 2012. Participants All patients undergoing coronary artery bypass grafts and/or valve surgery under the care of a consultant cardiac surgeon. Main outcome measures All-cause in-hospital mortality. Results A total of 292,973 operations performed by 273 consultant surgeons (with lengths of service from 11.2 to 42.0 years) were included. Crude mortality increased approximately linearly until 33 years service, before decreasing. After adjusting for case-mix and year of surgery, there remained a statistically significant (p = 0.002) association between length of service and in-hospital mortality (odds ratio 1.013; 95% CI 1.005–1.021 for each year of ‘experience’). Conclusions Consultant cardiac surgeons take on increasingly complex surgery as they gain experience. With this progression, the incidence of adverse outcomes is expected to increase, as is demonstrated in this study. After adjusting for case-mix using the EuroSCORE, we observed an increased risk of mortality in patients operated on by longer serving surgeons. This finding may reflect under-adjustment for risk, unmeasured confounding or a real association. Further research into outcomes over the time course of surgeons careers is required.

TRPC1 transcript variants, inefficient nonsense- mediated decay and low up-frameshift-1 in vascular smooth muscle cells

BackgroundTransient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts.ResultsExtensive TRPC1 transcript alternative splicing was observed, with exons 2, 3 and 5-9 frequently omitted, leading to variants containing premature termination codons. Consistent with the predicted sensitivity of such variants to nonsense-mediated decay (NMD) the variants were increased by cycloheximide. However it was notable that control of the variants by NMD was prominent in human embryonic kidney 293 cells but not human vascular smooth muscle cells. The cellular difference was attributed in part to a critical protein in NMD, up-frameshift-1 (UPF1), which was found to have low abundance in the vascular cells. Rescue of UPF1 by expression of exogenous UPF1 was found to suppress vascular smooth muscle cell proliferation.ConclusionsThe data suggest: (i) extensive NMD-sensitive transcripts of TRPC1; (ii) inefficient clearance of aberrant transcripts and enhanced proliferation of vascular smooth muscle cells in part because of low UPF1 expression.