Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

Caveolae position Ca_V3.2 (T‐type Ca²⁺ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca²⁺ influx to trigger Ca²⁺ sparks and large‐conductance Ca²⁺‐activated K⁺ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca²⁺ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca_v3.2 channel inhibition by Ni²⁺ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca²⁺ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca_v3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca_v1.2^?/? mice, caffeine (RyR activator) and thapsigargin (Ca²⁺ transport ATPase inhibitor), we found that sufficient SR Ca²⁺ load is a prerequisite for the Ca_V3.2‐RyR axis to generate Ca²⁺ sparks. We identified a fraction of Ca²⁺ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd³⁺ (100 µM), but insensitive to Ca_V1.2 and Ca_V3.2 channel blockade. Our data demonstrate that the VSMC Ca_V3.2‐RyR axis is down‐regulated by aging. This defective Ca_V3.2‐RyR coupling is counterbalanced by a Gd³⁺ sensitive Ca²⁺ pathway providing compensatory Ca²⁺ influx for triggering Ca²⁺ sparks in aged VSMCs.     

Role of phosphoinositol 4,5-bisphosphate and diacylglycerol in regulating native TRPC channel proteins in vascular smooth muscle

Stimulation of receptor-operated (ROCs) and store-operated (SOCs) Ca²⁺-permeable cation channels by vasoconstrictors has many important physiological functions in vascular smooth muscle. The present review indicates that ROCs and SOCs with diverse properties in different blood vessels are likely to be explained by composition of different subunits from the canonical transient receptor potential (TRPC) family of cation channel proteins. In addition we illustrate that activation of native TRPC ROCs and SOCs involves different phospholipase-mediated transduction pathways linked to generation of diacylglycerol (DAG). Moreover we describe recent novel data showing that the endogenous phospholipid phosphoinositol 4,5-bisphosphate (PIP₂) has profound and contrasting actions on TRPC ROCs and SOCs. Optimal activation of a native TRPC6 ROC by angiotensin II (Ang II) requires both depletion of PIP₂ and generation of DAG which leads to stimulation of TRPC6 via a PKC-independent mechanism. The data also indicate that PIP₂ has a marked constitutive inhibitory action of TRPC6 and DAG and PIP₂ are physiological antagonists on TRPC6 ROCs. In contrast PIP₂ stimulates TRPC1 SOCs and has an obligatory role in activation of these channels by store-depletion which requires PKC-dependent phosphorylation of TRPC1 proteins. Finally, we conclude that interactions between PIP₂ bound to TRPC proteins at rest, generation of DAG and PKC-dependent phosphorylation of TRPC proteins have a fundamental role in activation mechanisms of ROCs and SOCs in vascular smooth muscle.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

Heparin treatment of vascular smooth muscle cells results in the synthesis of the dual‐specificity phosphatase MKP‐1

The ability of heparin to block proliferation of vascular smooth muscle cells has been well documented. It is clear that heparin treatment can decrease the level of ERK activity in vascular smooth muscle cells that are sensitive to heparin. In this study, the mechanism by which heparin induces decreases in ERK activity was investigated by evaluating the dual specificity phosphatase, MKP‐1, in heparin treated cells. Heparin induced MKP‐1 synthesis in a time and concentration dependent manner. The time‐course of MKP‐1 expression correlated with the decrease in ERK activity. Over the same time frame, heparin treatment did not result in decreases in MEK‐1 activity which could have, along with constitutive phosphatase activity, accounted for the decrease in ERK activity. Antibodies against a heparin receptor also induced the synthesis of MKP‐1 along with decreasing ERK activity. Blocking either phosphatase activity or synthesis also blocked heparin‐induced decreases in ERK activity. Consistent with a role for MKP‐1, a nuclear phosphatase, heparin treated cells exhibited decreases in nuclear ERK activity more rapidly than cells not treated with heparin. The data support MKP‐1 as a heparin‐induced phosphatase that dephosphorylates ERK, decreasing ERK activity, in vascular smooth muscle cells. J. Cell. Biochem. 110: 382–391, 2010. © 2010 Wiley‐Liss, Inc.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The effects of peptides with estrogen‐like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells

Hormone replacement therapy (HRT) for post‐menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post‐menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen‐mimetic peptide 1 (EMP‐1) (VSWFFE) in comparison to estrogen (E₂) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP‐1‐like E₂ inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP‐1 did not affect E₂ stimulation of DNA, but inhibited E₂ inhibition of cell proliferation at high concentration. All effects by the combination of EMP‐1 and E₂ were abolished at hg. A44‐stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co‐incubated with E₂ at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E₂ stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45‐stimulated CK‐specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP‐1. EMP‐1 and A44 similar to E₂‐stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP‐1 provided it acts similarly in vivo can replace E₂ for treatment of post‐menopausal women in hyperglycemia due to diabetes. J. Cell. Biochem. 110: 1142–1146, 2010. Published 2010 Wiley‐Liss, Inc.     

Mechanism of inhibition by alloxan of ATP-driven calcium transport by vascular smooth muscle microsomes

The directin vitro effects of alloxan on the Ca²⁺ handling by microsomal membranes isolated from dog mesenteric arteries were investigated. Preincubation of the vascular muscle microsomal membranes with alloxan showed a suppressive effect on both binding of Ca²⁺ (in the absence of ATP) and ATP-driven Ca²⁺ transport. Such an inhibition was time dependent, dose dependent, and temperature dependent. ATP-driven Ca²⁺ transport was much more susceptible to the inhibitory action of alloxan than Ca²⁺ binding under all experimental conditions examined. Alloxan inhibited ATP-driven Ca²⁺ transport at a comparable level over the entire period of Ca²⁺ uptake, but had no significant effect on the efflux of Ca²⁺ from preloaded microsomal membranes. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca²⁺ transport via its action on the Ca-pump protein rather than the membrane permeability to Ca²⁺. Catalase and mannitol but not superoxide dismutase partially protected against such as inhibition by alloxan. The possible involvement of H₂O₂ mediating the inhibitory action of alloxan was further supported by the finding of a similarin vitro inhibitory effect of H₂O₂ on the ATP-driven Ca²⁺ transport by the vascular smooth muscle microsomes.     

BMP‐9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP‐9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP‐9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre‐dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP‐9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP‐9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP‐9‐induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5‐Dimethoxy‐N‐(quinolin‐3‐yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP‐9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP‐9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4‐siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP‐9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.     

Visualization of stimulus‐specific heterogeneous activation of individual vascular smooth muscle cells in aortic tissues

Intercellular communication among autonomic nerves, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here, we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus‐specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately threefold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO‐induced relaxation is quite high and approximately 10‐fold higher than that of its counterpart, the Wister–Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure.     

Aldosterone up‐regulates 12‐ and 15‐lipoxygenase expression and LDL oxidation in human vascular smooth muscle cells

Several lines of evidence suggest that aldosterone excess may have detrimental effects in the cardiovascular system, independent of its interaction with the renal epithelial cells. Here we examined the possibility that aldosterone modulates 12‐ and/or 15‐lipoxygenase (LO) expression/activity in human vascular smooth muscle cells (VSMC), in vitro, thereby potentially contributing to both vascular reactivity and atherogenesis. Following 24 h treatment of VSMC with aldosterone (1 nmol/L), there was a ～2‐fold increase in the generation rate of 12 hydroxyeicosatetraenoic acid (12‐HETE), 70% increase in platelet type 12‐LO mRNA expression (P < 0.001) along with a ～3‐fold increase in 12‐LO protein expression, which were blocked by the mineralocorticoid receptor (MR) antagonists spironolactone (100 nmol/L) and eplerelone (100 nmol/ml). Additionally, aldosterone (1 nmol/L; 24 h) increased the production of 15‐HETE (50%; P < 0.001) and the expression of 15‐LO type 2 mRNA (50%; P < 0.05) (in VSMC). Aldosterone also increased the 12‐ and 15‐LO type 2 mRNA expression in a line of human aortic smooth muscle cells (T/G HA‐VSMC) (60% and 50%, respectively). Aldosterone‐induced 12‐ and 15‐LO type 2 mRNA expressions were blocked by the EGF‐receptor antagonist AG 1478 and by the MAPK‐kinase inhibitor UO126. Aldosterone‐treated VSMC also showed increased LDL oxidation, (～2‐fold; P < 0.001), which was blocked by spironolactone. In conclusion, aldosterone increased 12‐ and 15‐LO expression in human VSMC, in association with increased 12‐ and 15‐HETE generation and enhanced LDL oxidation and may directly augment VSMC contractility, hypertrophy, and migration through 12‐HETE and promote LDL oxidation via the pro‐oxidative properties of these enzymes. J. Cell. Biochem. 108: 1203–1210, 2009. © 2009 Wiley‐Liss, Inc.     

Muscling in on TRP channels in vascular smooth muscle cells and cardiomyocytes

The human TRP protein family comprises a family of 27 cation channels with diverse permeation and gating properties. The common theme is that they are very important regulators of intracellular Ca²⁺ signaling in diverse cell types, either by providing a Ca²⁺ influx pathway, or by depolarising the membrane potential, which on one hand triggers the activation of voltage-gated Ca²⁺ channels, and on the other limits the driving force for Ca²⁺ entry. Here we focus on the role of these TRP channels in vascular smooth muscle and cardiac striated muscle. We give an overview of highlights from the recent literature, and highlight the important and diverse roles of TRP channels in the pathophysiology of the cardiovascular system.The discovery of the superfamily of Transient Receptor Potential (TRP) channels has significantly enhanced our knowledge of multiple signal transduction mechanisms in cardiac muscle and vascular smooth muscle cells (VSMC). In recent years, multiple studies have provided evidence for the involvement of these channels, not only in the regulation of contraction, but also in cell proliferation and remodeling in pathological conditions.The mammalian family of TRP cation channels is composed by 28 genes which can be divided into 6 subfamilies groups based on sequence similarity: TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipins), TRPV (Vanilloid), TRPP (Policystin) and TRPA (Ankyrin-rich protein). Functional TRP channels are believed to form four-unit complexes in the plasma, each of them expressed with six transmembrane domain and intracellular N and C termini.Here we review the current knowledge on the expression of TRP channels in both muscle types, and discuss their functional properties and role in physiological and pathophysiological processes.     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.