Nanojunctions: Specificity of Ca signaling requires nano-scale architecture of intracellular membrane contact sites.

Spatio-temporal definition of Ca signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na/Ca Exchanger-1 / SR/ER Ca ATPase-2-mediated ER Ca refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture.

Two-Dimensional Interfacial Exchange Diffusion Has the Potential to Augment Spatiotemporal Precision of Ca Signaling.

Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca signals. Herein, we propose that 2D Ca exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca trajectories within nano-junctions are discussed in order to substantiate its possible requirements.

Aortic Dimensions, Biophysical Properties, and Plasma Biomarkers in Children and Adults with Marfan or Loeys-Dietz Syndrome.

Background: Aortic dilation, stiffening, and dissection are common and potentially lethal complications of Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which involve abnormal transforming growth factor beta (TGF-β) signalling. The relation of aortic dimensions, stiffness, and biomarker levels is unknown. The objective of this study was to measure aortic dimensions, stiffness, TGF-β and matrix metalloproteinase (MMP) levels, and endothelial function in patients with MFS, and to compare TGF-β levels in patients with MFS receiving different therapeutic regimens.

Characterization of doxycycline-mediated inhibition of Marfan syndrome-associated aortic dilation by multiphoton microscopy.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root widening and aneurysm if unmanaged. We have previously reported doxycycline, a nonselective matrix metalloproteinases (MMPs) inhibitor, to attenuate aortic root widening and improve aortic contractility and elasticity in MFS mice. We were also first to use multiphoton microscopy, a non-invasive and label-free imaging technique, to quantify and link the aortic ultrastructure to possible changes in the skin dermis.

In vivo characterization of doxycycline-mediated protection of aortic function and structure in a mouse model of Marfan syndrome-associated aortic aneurysm.

Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS.

Neurofibromin haploinsufficiency results in altered spermatogenesis in a mouse model of neurofibromatosis type 1.

The fertility of men with neurofibromatosis 1 (NF1) is reduced. Despite this observation, gonadal function has not been examined in patients with NF1. In order to assess the role of reduced neurofibromin in the testes, we examined testicular morphology and function in an Nf1+/- mouse model.

Tissue Specificity: The Role of Organellar Membrane Nanojunctions in Smooth Muscle Ca Signaling.

In this chapter we examine the importance of cytoplasmic nanojunctions-nanometer scale appositions between organellar membranes including the molecular transporters therein-to the cell signaling machinery, with specific reference to Ca transport and signaling in vascular smooth muscle and endothelial cells. More specifically, we will consider the extent to which quantitative modeling may aid in the development of our understanding of these processes. Testament to the requirement for such approaches lies in the fact that recent studies have provided evermore convincing evidence in support of the view that cytoplasmic nanospaces may be as significant to the process of Ca signaling as the Ca transporters, release channels, and Ca-storing organelles themselves.

Na /Ca exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca via ER nanojunctions in vascular endothelial cells.

We investigated the role of Na/ Ca exchange (NCX) in the refilling of endoplasmic reticulum (ER) Ca in vascular endothelial cells under various conditions of cell stimulation and plasma membrane (PM) polarization. Better understanding of the mechanisms behind basic ER Ca content regulation is important, since current hypotheses on the possible ultimate causes of ER stress point to deterioration of the Ca transport mechanism to/from ER itself. We measured [Ca] temporal changes by Fura-2 fluorescence under experimental protocols that inhibit a host of transporters (NCX, Orai, non-selective transient receptor potential canonical (TRPC) channels, sarco/endoplasmic reticulum Ca ATPase (SERCA), Na/ K ATPase (NKA)) involved in the Ca communication between the extracellular space and the ER.

Mild aerobic exercise blocks elastin fiber fragmentation and aortic dilatation in a mouse model of Marfan syndrome associated aortic aneurysm.

Regular low-impact physical activity is generally allowed in patients with Marfan syndrome, a connective tissue disorder caused by heterozygous mutations in the fibrillin-1 gene. However, being above average in height encourages young adults with this syndrome to engage in high-intensity contact sports, which unfortunately increases the risk for aortic aneurysm and rupture, the leading cause of death in Marfan syndrome. In this study, we investigated the effects of voluntary (cage-wheel) or forced (treadmill) aerobic exercise at different intensities on aortic function and structure in a mouse model of Marfan syndrome.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging.

Maintenance of steady-state calcium (Ca(2+)) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca(2+) content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca(2+) transients, or stress experienced by the cell.

Urinary Retention, Incontinence, and Dysregulation of Muscarinic Receptors in Male Mice Lacking Mras.

Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras-/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly 'layered' with age in Mras-/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras-/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras-/- females were normal.

Drop in endo/sarcoplasmic calcium precedes the unfolded protein response in Brefeldin A-treated vascular smooth muscle cells.

The present study addresses the causal relationship between induction of endo/sarcoplasmic reticulum stress and dysregulation of calcium transport, while examining whether the most widely-used experimental endo/sarcoplasmic reticulum stressors can be considered appropriate for elucidating underlying cellular mechanisms involved during the progression of the unfolded protein response in vascular smooth muscle cells. Brefeldin A is most commonly cited as inducing the stress response through an accumulation of unfolded proteins in the lumen as a result of a blockage of protein transport from the endo/sarcoplasmic reticulum to the Golgi apparatus. We investigated the effects of Brefeldin A on cellular calcium regulation during the the unfolded protein response in cultured rat vascular smooth muscle cells.

Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca (2+) waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca (2+) via L-SR junctions, in a manner that requires initial Ca (2+) release from lysosomes and subsequent Ca (2+)-induced Ca (2+) release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process.

Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored.

Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport.

Calcium oscillations in human mesenteric vascular smooth muscle.

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca(2+) concentration ([Ca(2+)]i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca(2+)]i oscillations in human mesenteric artery smooth muscle.

Waves of calcium depletion in the sarcoplasmic reticulum of vascular smooth muscle cells: an inside view of spatiotemporal Ca2+ regulation.

Agonist-stimulated smooth muscle Ca2+ waves regulate blood vessel tone and vasomotion. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were stimulated by a combination of inositol 1,4,5-trisphosphate- and Ca2+ -induced Ca2+ release from the endo/sarcoplasmic reticulum. Herein, we present the first report of endothelin-1 stimulated waves of Ca2+ depletion from the sarcoplasmic reticulum of vascular smooth muscle cells using a calsequestrin-targeted Ca2+ indicator.

Pan-junctional sarcoplasmic reticulum in vascular smooth muscle: nanospace Ca2+ transport for site- and function-specific Ca2+ signalling.

This review focuses on how smooth muscle sarcoplasmic reticulum (SR), the major releasable Ca(2+) store in these cells, performs its many functions by communicating with the plasma membrane (PM) and other organelles across cytoplasmic nanospaces, defined by membrane-membrane junctions less than 50 nm across. In spite of accumulating evidence in favour of the view that cytoplasmic nanospaces are a prerequisite for effective control of diverse cellular functions, our current understanding of how smooth muscle cells accomplish site- and function-specific Ca(2+) signalling remains in its infancy. We first present evidence in support of the view that effective Ca(2+) signalling depends on the restricted diffusion of Ca(2+) within cytoplasmic nanospaces.

The role of cytoplasmic nanospaces in smooth muscle cell Ca2+ signalling.

We address the importance of cytoplasmic nanospaces in Ca(2+) transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces--nanometre-scale spaces between organellar membranes, hosting cell signalling machinery--are key to Ca(2+) signalling as much as Ca(2+) transporters and Ca(2+) storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces.

The structure and functioning of the couplon in the mammalian cardiomyocyte.

The couplons of the cardiomyocyte form nanospaces within the cell that place the L-type calcium channel (Ca(v)1.2), situated on the plasmalemma, in opposition to the type 2 ryanodine receptor (RyR2), situated on the sarcoplasmic reticulum. These two molecules, which form the basis of excitation-contraction coupling, are separated by a very limited space, which allows a few Ca(2+) ions passing through Ca(v)1.

Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice.

Unlabelled: Traffic related particulate matter air pollution is a risk factor for cardiovascular events; however, the biological mechanisms are unclear. We hypothesize that diesel exhaust (DE) inhalation induces up-regulation of inducible nitric oxide synthase (iNOS), which is known to contribute to vascular dysfunction, progression of atherosclerosis and ultimately cardiovascular morbidity and mortality.

Methods: ApoE knockout mice (30-week) were exposed to DE (at 200 μg/m³ of particulate matter) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week).

Changes in atherosclerotic plaques induced by inhalation of diesel exhaust.

Objective: Exposure to particulate matter air pollution may be an independent risk factor for cardiovascular morbidity and mortality; however, the biological mechanisms are unclear. We hypothesize that exposure to diesel exhaust (DE), an important source of traffic-related particulate air pollution, promotes changes of atherosclerotic plaque component that may lead to plaque vulnerability.

Methods And Results: 30-week old ApoE knockout mice fed with regular chow inhaled DE (at 200 μg/m(3) of particulate) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week) (12 mice/group).

Diosgenin modulates vascular smooth muscle cell function by regulating cell viability, migration, and calcium homeostasis.

In this study, we compared the potencies of diosgenin, a plant-derived sapogenin structurally similar to estrogen and progesterone, on vascular smooth muscle functions ranging from contraction and migration to apoptosis. The effects of diosgenin on vascular smooth muscle cell viability and migration were measured using a primary mouse aortic smooth muscle cell culture. The effects of diosgenin on smooth muscle cell contraction and calcium signaling were investigated in the isolated mouse aorta using wire myography and confocal microscopy, respectively.

Effectiveness of combination of losartan potassium and doxycycline versus single-drug treatments in the secondary prevention of thoracic aortic aneurysm in Marfan syndrome.

Objective: Losartan potassium (INN losartan), an antihypertensive drug, has been shown to prevent thoracic aortic aneurysm in Marfan syndrome through the inhibition of transforming growth factor beta. Recently we reported that doxycycline, a nonspecific inhibitor of matrix metalloproteinases 2 and 9, normalized aortic vasomotor function and suppressed aneurysm growth. We hypothesized that a combination of losartan potassium and doxycycline would offer better secondary prevention treatment than would single-drug therapy to manage thoracic aortic aneurysm.

Integrin-linked kinase functions as a downstream signal of platelet-derived growth factor to regulate actin polymerization and vascular smooth muscle cell migration.

Background: Vascular smooth muscle cell migration and accumulation in response to growth factors extensively contribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown that actin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration. Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 and beta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrix modification, and cell spreading and migration in response to various stimuli.