Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells

2003 ◽  
Vol 81 (3) ◽  
pp. 301-310 ◽  
Author(s):  
Bernard Abrenica ◽  
Grant N Pierce ◽  
James S.C Gilchrist
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Signal Intensity ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Ca

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY® FL thapsi gargin and BODIPY® FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 μM) and activatory concentrations of ryanodine (1 μM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.Key words: Ca2+, RyR, SERCA, cell nucleus, FK506, thapsigargin, ryanodine.

Altered Cytoskeleton in Smooth Muscle of Aganglionic Bowel

2002 ◽  
Vol 126 (6) ◽  
pp. 692-696
Author(s):  
Laszlo Nemeth ◽  
Udo Rolle ◽  
Prem Puri
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Hirschsprung Disease ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Normal Bowel ◽  
Pull Through

Abstract Context.—Intestinal motility is under the control of smooth muscle cells, enteric plexus, and hormonal factors. In Hirschsprung disease (HD), the aganglionic colon remains spastic or tonically enhanced and unable to relax. The smooth muscle cell's cytoskeleton consists of proteins or structures whose primary function is to link or connect protein filaments to each other or to the anchoring sites. Dystrophin is a subsarcolemmal protein with a double adhesion property, one between the membrane elements and the contractile filaments of the cytoskeleton and the other between the cytoskeletal proteins and the extracellular matrix. Desmin and vinculin are functionally related proteins that are present in the membrane-associated dense bodies in the sarcolemma of the smooth muscle cells. Objective.—To examine the distribution of the cytoskeletal proteins in the smooth muscle of the aganglionic bowel. Design.—Bowel specimens from ganglionic and aganglionic sections of the colon were collected at the time of pull-through surgery from 8 patients with HD. Colon specimens collected from 4 patients at the time of bladder augmentation acted as controls. Anti-dystrophin, anti-desmin, and anti-vinculin antibodies were used for fluorescein immunostaining using confocal laser scanning microscopy. Results.—Moderate to strong dystrophin immunoreactivity was observed at the periphery of smooth muscle fibers in normal bowel and ganglionic bowel from patients with HD, whereas dystrophin immunoreactivity was either absent or weak in the smooth muscle of aganglionic colon. Moderate to strong cytoplasmic immunostaining for vinculin and desmin was seen in the smooth muscle of normal bowel and ganglionic bowel from patients with HD, whereas vinculin and desmin staining in the aganglionic colon was absent or weak. Conclusion.—This study demonstrates that the cytoskeletal proteins are abundant in the smooth muscle of normal bowel, but are absent or markedly reduced in the aganglionic bowel of HD. As cytoskeletal proteins are required for the coordinated contraction of muscle cells, their absence may be responsible for the motility dysfunction in the aganglionic segment.

Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells

1998 ◽  
Vol 274 (2) ◽  
pp. C472-C480 ◽  
Author(s):  
Shinji Naito ◽  
Shunichi Shimizu ◽  
Shigeto Maeda ◽  
Jianwei Wang ◽  
Richard Paul ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Inositol Phosphate ◽  
Muscle Cells ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Intracellular Ca

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid had no effect. However, [Ca2+]irelease alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.

Preparation of recombinant human-like collagen/fibroin scaffold and its promoting effect on vascular cells biocompatibility

2018 ◽  
Vol 33 (4) ◽  
pp. 416-425 ◽  
Author(s):  
Jia Yan ◽  
Kun Hu ◽  
YongHao Xiao ◽  
Fan Zhang ◽  
Lu Han ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Scanning Electron

A novel recombinant human-like collagen/fibroin scaffold has been prepared previously, which has high porosity, controllable pore size, and much better mechanical properties than the reported fibroin-based scaffold. In this research, the cell responses of vascular smooth muscle cells to this blend scaffold were examined in vitro. Cell morphology, adherence, and growth in scaffolds were observed by scanning electron microscopy, laser scanning confocal microscopy after staining of the cells with propidium iodide at 1, 3, 5, and 7 days, respectively. A wide range of measurements, including 3-[4,5–dimethylthiazol-2-yl]-2, 5-diphenyl tetrasodium bromide assay, and total intracellular protein content at the end of 7 days culture, were conducted. An increase of viability and protein content of vascular smooth muscle cells cultured in recombinant human-like collagen/fibroin scaffold was found. The laser scanning confocal microscopy and scanning electron microscopy results confirm that the cells readily adhered and proliferation in the blend than in fibroin scaffold, and indicate a better adhesion process. The positive effects were especially significant for vascular smooth muscle cells. The recombinant human-like collagen/fibroin scaffold could be a promising biomaterial for vascular tissue engineering.

Attenuation of the serotonin-induced increase in intracellular calcium in rat aortic smooth muscle cells by sarpogrelate

2003 ◽  
Vol 81 (11) ◽  
pp. 1056-1063 ◽  
Author(s):  
Harjot K Saini ◽  
Sushil K Sharma ◽  
Peter Zahradka ◽  
Hideo Kumamoto ◽  
Nobuakira Takeda ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Receptor Sites ◽  
Intracellular Ca

Although serotonin (5-HT) induced proliferation of vascular smooth muscle cells is considered to involve changes in intracellular Ca2+ ([Ca2+]i), the mechanism of Ca2+ mobilization by 5-HT is not well defined. In this study, we examined the effect of 5-HT on rat aortic smooth muscle cells (RASMCs) by Fura-2 microfluorometry for [Ca2+]i measurements. 5-HT was observed to increase the [Ca2+]i in a concentration- and time-dependent manner. This action of 5-HT was dependent upon the extracellular concentration of Ca2+ ([Ca2+]e) and was inhibited by both Ca2+ channel antagonists (verapamil and diltiazem) and inhibitors of sarcoplasmic reticular Ca2+ pumps (thapsigargin and cyclopia zonic acid). The 5-HT-induced increase in [Ca2+]i was blocked by sarpogrelate, a 5-HT2A-receptor antagonist, but not by different agents known to block other receptor sites. 5-HT-receptor antagonists such as ketanserin, cinanserin, and mianserin, unlike methysergide, were also found to inhibit the 5-HT-induced Ca2+ mobilization, but these agents were less effective in comparison to sarpogrelate. On the other hand, the increase in [Ca2+]i in RASMCs by ATP, angiotensin II, endothelin-1, or phorbol ester was not affected by sarpogrelate. These results indicate that Ca2+ mobilization in RASMCs by 5-HT is mediated through the activation of 5-HT2A receptors and support the view that the 5-HT-induced increase in [Ca2+]i involves both the extracellular and intracellular sources of Ca2+.Key words: sarpogrelate, serotonin, vascular smooth muscle cells, intracellular Ca2+.

Integrin mobilizes intracellular Ca2+ in renal vascular smooth muscle cells

2001 ◽  
Vol 280 (3) ◽  
pp. C593-C603 ◽  
Author(s):  
Wah-Lun Chan ◽  
N.-H. Holstein-Rathlou ◽  
Kay-Pong Yip
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Initial Response ◽  
Rgd Peptides ◽  
Intracellular Ca ◽  
Functional Blocking ◽  
Renal Vascular

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca2+ signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca2+]i rapidly increased from 91 ± 4 to 287 ± 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca2+mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca2+]i, which propagated as Ca2+ waves traveling across the cell and induced a rapid elevation of nuclear [Ca2+]i. Spontaneous recurrence of smaller-amplitude Ca2+ waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca2+ channels with nifedipine or removal of extracellular Ca2+ did not inhibit the RGD-induced Ca2+mobilization. However, pretreatment of 20 μM ryanodine completely eliminated the RGD-induced Ca2+ mobilization. Anti-β1 and anti-β3-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca2+]i. Incubation with anti-β1- or β3-integrin antibodies inhibited the increase in [Ca2+]i induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca2+ from ryanodine-sensitive Ca2+stores in renal VSMC via integrins, which can trigger cytoplasmic Ca2+ waves propagating throughout the cell.

scholarly journals OP0141 HIGH DIMENSIONAL ANALYSIS REVEAL A NETWORK OF CERTAIN TRANSCRIPTION FACTORS THAT LINK VASCULOPATHY AND ORGAN FIBROSIS IN SYSTEMIC SCLEROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 90.2-91
Author(s):  
C. G. Anchang ◽  
B. Matalobos Lawaree ◽  
S. Weber ◽  
S. Rauber ◽  
T. Wohlfahrt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Tube Length ◽  
Confocal Laser ◽  
Research Support ◽  
Organ Fibrosis

Background:Since vascular manifestations such as Raynaud’s phenomenon often precede the onset of other clinical manifestations of systemic sclerosis (SSc), the identification of pathways linking vasculopathy to organ fibrosis might thus provide important insights into early disease mechanisms and allow early targeted intervention for both fibrotic and vascular events.Objectives:In this study we performed high dimensional (HD) analyses to identify mediators that link vasculopathy to organ fibrosis.Methods:HD techniques including RNA-seq, ChIP-seq, ATAC-seq and FISH-seq have been performed to identify mediators in vessels and fibrotic lesions of human skin samples of SSc patients and healthy volunteers. In addition, murine skin and lung tissue samples were analyzed by multi-channel immunofluorescence (IF) and confocal laser scanning microscopy. Microvascular endothelial cells, smooth muscle cells and fibroblasts have been further processed to address their functional attributes with regard to their proliferative, migratory and chemotactic capacity. In vivo models and ex vivomouse fetal metatarsal assays were performed to study fibrotic and angiogenic processes.Results:Bioinformatic HD analyses revealed the ETS transcription factor PU.1 as molecular checkpoint of a network of factors that drive matrix production and fibrotic imprinting in SSc. Within this network ATF3 was significantly upregulated in fibroblasts of skin biopsies of SSc patients and of various organs of fibrosis models. ATF3 deficiency ameliorated fibrosis in various mouse models. Notably, ATF3 was significantly upregulated in vascular cells of fibrotic tissues of SSc patients. Multi-channel IF and confocal laser scanning microscopy of skin and lung biopsies of SSc patients revealed an increased expression of ATF3 especially in microvascular endothelial cells and smooth muscle cells. ATF3 overexpression in smooth muscle cells led to an extensively enhanced proliferation and increased migratory capacity whereas endothelial cells showed a SSc-like phenotype with reduced proliferation and migration. After ATF3 overexpression, tube formation capacity was completely altered as assessed by cumulative tube length, tube numbers and capillary sprouting. To investigate vessel outgrowth from a different perspective, we used theex vivofetal mouse metatarsal assay. ATF3 knockout mice showed a completely altered angiogenic response as assessed by tube length, number of branches and number junctions compared to wildtype controls.Conclusion:We identified PU.1 and ATF3 as key factors in disturbed vasculature and endogenous activated fibroblasts suggesting this axis as a potential therapeutic target intervening both fibrotic and vascular manifestations.Disclosure of Interests:Charles Gwellem Anchang: None declared, Bettina Matalobos Lawaree: None declared, Stefanie Weber: None declared, Simon Rauber: None declared, Thomas Wohlfahrt: None declared, Markus Luber: None declared, Alexander Kreuter: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen

Regulation of calcium-activated chloride channels in smooth muscle cells: a complex picture is emerging

2005 ◽  
Vol 83 (7) ◽  
pp. 541-556 ◽  
Author(s):  
Normand Leblanc ◽  
Jonathan Ledoux ◽  
Sohag Saleh ◽  
Amy Sanguinetti ◽  
Jeff Angermann ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Chloride Channels ◽  
Muscle Cells ◽  
Cell Types ◽  
Dependent Protein Kinase ◽  
Intracellular Ca ◽  
Dependent Protein

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (~100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone. Key words: calcium-activated chloride channels, vascular smooth muscle cells, ion channels, calmodulin-dependent protein kinase II, calcineurin

Ursolic acid suppresses leptin-induced cell proliferation in rat vascular smooth muscle cells

2017 ◽  
Vol 95 (7) ◽  
pp. 811-818 ◽  
Author(s):  
Ya-Mei Yu ◽  
Chiang-Chin Tsai ◽  
Yu-Wen Tzeng ◽  
Weng-Cheng Chang ◽  
Su-Yin Chiang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ursolic Acid ◽  
Vascular Smooth Muscle Cells ◽  
Biological Effects ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Matrix Metalloproteinase 2

Accumulating lines of evidence indicate that high leptin levels are associated with adverse cardiovascular health in obese individuals. Proatherogenic effects of leptin include endothelial cell activation and vascular smooth muscle cell proliferation and migration. Ursolic acid (UA) has been reported to exhibit multiple biological effects including antioxidant and anti-inflammatory properties. In this study, we investigated the effect of UA on leptin-induced biological responses in rat vascular smooth muscle cells (VSMCs). A-10 VSMCs were treated with leptin in the presence or absence of UA. Intracellular reactive oxygen species (ROS) was probed by 2′,7′-dichlorofluorescein diacetate. The expression of extracellular signal-regulated kinase (ERK)1/2, phospho-(ERK)1/2, nuclear factor-kappa B (NF-κB) p65 and p50, and matrix metalloproteinase-2 (MMP2) was determined by Western blotting. Immunocytochemistry and confocal laser scanning microscopy were also used for the detection of NF-κB. The secretion of MMP2 was detected by gelatin zymography. UA exhibited antioxidant activities in vitro. In rat VSMCs, UA effectively inhibited cell growth and the activity of MMP2 induced by leptin. These suppressive effects appeared by decreasing the activation of (ERK)1/2, the nuclear expression and translocation of NF-κB, and the production of ROS. UA appeared to inhibit leptin-induced atherosclerosis, which may prevent the development of obesity-induced cardiovascular diseases.

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