Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1−/− SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1−/− mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization

Potentiation of large conductance, Ca2+-activated K+ (BK) channels by α5β1 integrin activation in arteriolar smooth muscle

Injury/degradation of the extracellular matrix (ECM) is associated with vascular wall remodelling and impaired reactivity, a process in which altered ECM–integrin interactions play key roles. Previously, we found that peptides containing the RGD integrin-binding sequence produce sustained vasodilatation of rat skeletal muscle arterioles. Here, we tested the hypothesis that RGD ligands work through α5β1 integrin to modulate the activity of large conductance, Ca2+-activated K+ (BK) channels in arteriolar smooth muscle. K+ currents were recorded in single arteriolar myocytes using whole-cell and single-channel patch clamp methods. Activation of α5β1 integrin by an appropriate, insoluble α5β1 antibody resulted in a 30–50% increase in the amplitude of iberiotoxin (IBTX)-sensitive, whole-cell K+ current. Current potentiation occurred 1–8 min after bead–antibody application to the cell surface. Similarly, the endogenous α5β1 integrin ligand fibronectin (FN) potentiated IBTX-sensitive K+ current by 26%. Current potentiation was blocked by the c-Src inhibitor PP2 but not by PP3 (0.1–1 μm). In cell-attached patches, number of open channels × open probability (NPo) of a 230–250 pS K+ channel was significantly increased after FN application locally to the external surface of cell-attached patches through the recording pipette. In excised, inside-out patches, the same method of FN application led to large, significant increases in NPo and caused a leftward shift in the NPo–voltage relationship at constant [Ca2+]. PP2 (but not PP3) nearly abolished the effect of FN on channel activity, suggesting that signalling between the integrin and channel involved an increase in Ca2+sensitivity of the channel via a membrane-delimited pathway. The effects of α5β1 integrin activation on both whole-cell and single-channel BK currents could be reproduced in HEK 293 cells expressing the BK channel α-subunit. This is the first demonstration at the single-channel level that integrin signalling can regulate an ion channel. Our results show that α5β1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca2+- and c-Src-dependent mechanisms. This mechanism is likely to play a role in the arteriolar dilatation and impaired vascular reactivity associated with ECM degradation