Abstract 5543: The P2Y 2 Nucleotide Receptor Contributes To Intimal Lesion Growth And Regulates Lymphotoxin-α Secretion

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gary Weisman ◽  
Ningpu Yu ◽  
Cansu Agca ◽  
Rikka Shivaji ◽  
Ying Wan ◽  
...  
Keyword(s):  
Atherosclerotic Lesion ◽  
Actin Binding ◽  
Dependent Manner ◽  
Filamin A ◽  
Nucleotide Receptor ◽  
Transgenic Rats ◽  
Arterial Lumen ◽  
Lesion Growth ◽  
Lymphotoxin Α ◽  
Intimal Lesion

Inflammatory processes play a crucial role in atherosclerotic lesion growth. A number of pro-inflammatory molecules have been implicated in the pathogenesis of atherosclerosis, including lymphotoxin-α (LTA). Our research has shown that activation of a G protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R) expressed in vascular cells mediates inflammatory responses. To examine the role of the P2Y 2 R in lesion growth, we developed P2Y 2 R over-expressing transgenic rats. Collar-induced injury to the carotid artery of P2Y 2 R transgenic rats caused a dramatic increase in intimal lesion growth and significant macrophage accumulation, which nearly blocked the arterial lumen. Immunohistochemical staining showed that LTA and galectin-2 (gal-2) were abundantly expressed in smooth muscle cells (SMC) and macrophages in carotid lesions of P2Y 2 R transgenic rats. We also identified galectin-2 as a P2Y 2 R binding partner using the yeast two-hybrid system and a co-immunoprecipitation assay. P2Y 2 R agonist, UTP, stimulated gal-2 mRNA expression in rat carotid SMC. Transient transfection of SMC with a genomic fragment including the rat galectin (LGALS2) promoter incorporated into a luciferase (pGL-3) reporter vector showed that UTP markedly increased LGALS2 promoter activity in a dose-dependent manner. Moreover, UTP induced LTA secretion in cultured aortic SMC from wild type, but not P2Y 2 R −/− mice. Adenoviral expression of the full length P2Y 2 R in SMC from P2Y 2 R −/− mice fully restored UTP-induced LTA secretion. However, expression of a mutant P2Y 2 R that does not bind filamin A, an actin-binding protein that interacts with the P2Y 2 R, only partially restored UTP-induced LTA secretion in P2Y 2 R −/− SMC. Gal-2 siRNA partially inhibited LTA release into medium of cultured SMC expressing the P2Y 2 R. In contrast, gal-2 siRNA abolished UTP-induced LTA secretion in SMC isolated from P2Y 2 R −/− mice expressing the filamin A binding mutant P2Y 2 R. These results indicate that P2Y 2 Rs regulate gal-2-dependent LTA secretion in SMC via a filamin A-dependent mechanism that likely contributes to vascular inflammation.

Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Cheikh Seye ◽  
Wilbert Derbigny ◽  
Shaomin Qian
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Protein Transport ◽  
Brefeldin A ◽  
Muscle Cells ◽  
Secretory Vesicle ◽  
Actin Binding ◽  
Filamin A ◽  
Artery Disease ◽  
Lymphotoxin Α

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease. Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC). Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y 2 nucleotide receptor (P2Y 2 R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y 2 R -/- SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y 2 R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y 2 R deficient in LTA secretion. Conclusion: These data suggest that P2Y 2 R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission–associated myocardial senescence

2018 ◽  
Vol 11 (556) ◽  
pp. eaat5185 ◽  
Author(s):  
Akiyuki Nishimura ◽  
Tsukasa Shimauchi ◽  
Tomohiro Tanaka ◽  
Kakeru Shimoda ◽  
Takashi Toyama ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Actin Binding ◽  
Dependent Manner ◽  
Filamin A ◽  
Nucleotide Exchange ◽  
Rat Cardiomyocytes ◽  
Nucleotide Exchange Factor ◽  
Starting Point

Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding–dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

scholarly journals Filamin A Regulates Cardiovascular Remodeling

2021 ◽  
Vol 22 (12) ◽  
pp. 6555
Author(s):  
Sashidar Bandaru ◽  
Chandu Ala ◽  
Alex-Xianghua Zhou ◽  
Levent M. Akyürek
Keyword(s):  
Transcription Factors ◽  
Respiratory Diseases ◽  
Cell Function ◽  
Actin Binding ◽  
Cardiovascular Development ◽  
Filamin A ◽  
Cardiovascular Malformations ◽  
Experimental Animal Models ◽  
Actin Networks ◽  
Cardiovascular Remodeling

Filamin A (FLNA) is a large actin-binding cytoskeletal protein that is important for cell motility by stabilizing actin networks and integrating them with cell membranes. Interestingly, a C-terminal fragment of FLNA can be cleaved off by calpain to stimulate adaptive angiogenesis by transporting multiple transcription factors into the nucleus. Recently, increasing evidence suggests that FLNA participates in the pathogenesis of cardiovascular and respiratory diseases, in which the interaction of FLNA with transcription factors and/or cell signaling molecules dictate the function of vascular cells. Localized FLNA mutations associate with cardiovascular malformations in humans. A lack of FLNA in experimental animal models disrupts cell migration during embryogenesis and causes anomalies, including heart and vessels, similar to human malformations. More recently, it was shown that FLNA mediates the progression of myocardial infarction and atherosclerosis. Thus, these latest findings identify FLNA as an important novel mediator of cardiovascular development and remodeling, and thus a potential target for therapy. In this update, we summarized the literature on filamin biology with regard to cardiovascular cell function.

scholarly journals Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

2017 ◽  
Vol 217 (2) ◽  
pp. 779-793 ◽  
Author(s):  
Rebecca C. Adikes ◽  
Ryan A. Hallett ◽  
Brian F. Saway ◽  
Brian Kuhlman ◽  
Kevin C. Slep
Keyword(s):  
Blue Light ◽  
Actin Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Exclusion Zone ◽  
Actin Networks ◽  
Drosophila Melanogaster S2 Cells ◽  
Actin Binding Domain ◽  
Specific Factors ◽  
Insight Into

We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

scholarly journals Oestrogen-Dependent Oxytocin Dynamics in the Hypothalamus of Female Rats

2021 ◽  
Author(s):  
Kazuaki Nishimura ◽  
Kiyoshi Yoshino ◽  
Naofumi Ikeda ◽  
Kazuhiko Baba ◽  
Kenya Sanada ◽  
...  
Keyword(s):  
Systemic Circulation ◽  
Female Rats ◽  
Dependent Manner ◽  
Transgenic Rats ◽  
Dynamic Changes ◽  
Hypothalamic Nuclei ◽  
The Central Nervous System ◽  
Posterior Pituitary Gland ◽  
Dose Dependent ◽  
Ovariectomised Rats

Abstract Oxytocin (OXT) is produced in the hypothalamic nuclei and is secreted into systemic circulation from the posterior pituitary gland (PP). In the central nervous system, OXT regulates behaviours including maternal and feeding behaviours. Our aim was to evaluate whether oestrogen regulates hypothalamic OXT dynamics. Herein, we provide the first evidence that OXT dynamics in the hypothalamus vary with sex and that oestrogen may modulate dynamic changes in OXT levels, using OXT-mRFP1 transgenic rats. The fluorescence intensity of OXT-mRFP1 in the hypothalamic nuclei and PP was most strongly expressed during the oestrus stage in female rats and decreased significantly in ovariectomised rats. Oestrogen replacement caused significant increases in the fluorescent intensities in the hypothalamic nuclei and PP in a dose-dependent manner. This was also demonstrated in feeding behaviour and hypothalamic Fos neurons using immunohistochemistry. Hypothalamic OXT expression was oestrogen dependent and could be enhanced centrally by the administration of oestrogen.

Platelet P-selectin facilitates atherosclerotic lesion development

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2661-2666 ◽  
Author(s):  
Peter C. Burger ◽  
Denisa D. Wagner
Keyword(s):  
Atherosclerotic Lesion ◽  
Soluble Form ◽  
Wild Type ◽  
Lesion Development ◽  
Atherosclerotic Lesions ◽  
Lesion Growth ◽  
Microparticle Formation ◽  
Apoe Deficient Mouse ◽  
Atherosclerotic Lesion Development

P-selectin is an adhesion molecule expressed on activated platelets and endothelium. It is known to play an important role in atherosclerosis. P-selectin also circulates in plasma in a soluble form (sP-selectin), which induces procoagulant microparticle formation. We investigated the role of platelet versus endothelial P-selectin in generating sP-selectin and in the formation of atherosclerotic lesions in the apolipoprotein E (apoE)–deficient mouse model. For this we transplanted apoE−/−P-selectin−/− and apoE−/−P-selectin+/+ lethally irradiated mice with bone marrow of either genotype. Seven months after transplantation, we determined from the chimeric animals that the majority of circulating sP-selectin was of endothelial origin. Thus, in atherosclerosis, the procoagulant sP-selectin reflects endothelial rather than platelet activation. We found that endothelial P-selectin was crucial for the promotion of atherosclerotic lesion growth because in its absence only relatively small lesions developed. However, platelet P-selectin also contributed to the lesion development because lesions in wild-type recipients receiving transplants with wild-type platelets were 30% larger than those receiving P-selectin-deficient platelets (P < .008) and were more frequently calcified (80% versus 44%). In comparison with P-selectin wild-type animals, absence of either endothelial or platelet P-selectin inhibited migration of smooth muscle cells into the lesion. Thus, in addition to endothelium, platelets and their P-selectin also actively promote advanced atherosclerotic lesion development.

Abstract 3760: Atherogenic Affects Of Arsenic: Role Of Endoplasmic Reticulum Stress And Unfolded Protein Response

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Petra Haberzettl ◽  
Elena Vladykovskaya ◽  
Oleg Barski ◽  
Srinivas Sithu ◽  
Stanley D’Souza ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Unfolded Protein Response ◽  
Atherosclerotic Lesion ◽  
The United States ◽  
Hazardous Substance ◽  
Dependent Manner ◽  
Lesion Formation ◽  
Chemical Chaperone ◽  
Unfolded Protein ◽  
Protein Response

Arsenic is a global water contaminant and EPA has listed arsenic as a high priority hazardous substance in the United States. Epidemiological studies suggest that chronic arsenic ingestion increases cardiovascular disease in humans, particularly, carotid atherosclerosis. However, mechanisms of arsenic-induced atherogenesis are unknown. We examined the effect of arsenic exposure on early lesion formation in apoE-null mice maintained on water supplemented with (0, 1, 5 and 50 ppm; 3–16 weeks of age) sodium arsenite. Arsenic, did not affect plasma cholesterol but decreased the triglycerides by 18±4 % (P<0.05). NMR analysis of the lipoproteins showed a significant decrease in the abundance of large VLDL particle (>60 nm diameter). Despite a significant decrease in plasma triglyceride, atherosclerotic lesion formation was significantly increased (2– 4 fold; P<0.05 for all doses) in the aortic sinus and the aortic arch of the arsenic-fed mice in a dose dependent manner. Immunohistochemical analysis showed significant increase in the accumulation of macrophages, expression of MCP-1 and unfolded protein response (UPR) dependent activating transcription factor (ATF)-4 and ATF3, in the lesions of arsenic (1ppm) exposed mice. In vitro , arsenic (5–25 μM), significantly increased the expression of ICAM-1, transmigration of differentiated monocytes and expression of the pro-inflammatory cytokine IL-8 in vascular endothelial cells (vEC). Arsenic, also increased the expression of ER-chaperones Grp 78, HERP and calnexin (2– 6 fold; P<0.01). Examination of the effect of arsenic on UPR showed that arsenic, induced the splicing of IRE-1 dependent, bZIP transcription factor XBP-1(alarm phase) and increased the phosphorylation of eIF2α (PERK mediated adaptive phase) by 3 fold (P<0.01) in vEC. Arsenic also induced the expression of the downstream effecter proteins of eIF2α-ATF3 (8 fold; P<0.01) and pro-apoptotic protein CHOP (4 fold; P<0.01) in vEC. Chemical chaperone, phenyl butyric acid (PBA), attenuated the arsenic-induced expression of ATF3 (>90%; P<0.001) and CHOP (>90%; P<0.001). These data suggest that ER-stress and UPR could exacerbate arsenic-induced vascular inflammation and promote atherogenesis.

Drebrin attenuates atherosclerosis by limiting smooth muscle cell transdifferentiation

2021 ◽  
Author(s):  
Jiao-Hui Wu ◽  
Lisheng Zhang ◽  
Igor Nepliouev ◽  
Leigh Brian ◽  
Taiqin Huang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Foam Cell ◽  
Atherosclerotic Lesion ◽  
Foam Cells ◽  
Actin Binding ◽  
Cross Sectional ◽  
Cell Transdifferentiation

Abstract Aims The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation and pro-inflammatory signaling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis. Methods and results SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a Western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn-/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 wk, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs was reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. Conclusions We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells. Translational perspective Drebrin is abundantly expressed in vascular smooth muscle cells (SMCs) and is up-regulated in human atherosclerosis. A hallmark of atherosclerosis is the accumulation of foam cells that secrete pro-inflammatory cytokines and contribute to plaque instability. A large proportion of these foam cells in humans derive from SMCs. We found that SMC Drebrin limits atherosclerosis by reducing SMC transdifferentiation to macrophage-like foam cells in a manner dependent on Nox1 and KLF4. For this reason, strategies aimed at augmenting SMC Drebrin expression in atherosclerotic plaques may limit atherosclerosis progression and enhance plaque stability by bridling SMC-to-foam-cell transdifferentiation.

Abstract 16440: Cu Transporter ATP7A Protects Against Fibrosis Through Limiting Endothelial to Mesenchymal Transition

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Seock-Won Youn ◽  
Sudhahar Varadarajan ◽  
Archita Das ◽  
Ronald D McKinney ◽  
Tohru Fukai ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Inflammatory Diseases ◽  
Shape Change ◽  
Atherosclerotic Lesion ◽  
Transport Proteins ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition

Background: Endothelial to mesenchymal transition (EndMT) is induced by inflammation and contributes to fibrosis; however, underlying mechanism is poorly understood. Cu plays an important role in physiological processes and pathophysiologies associated with inflammatory diseases. Since excess Cu is toxic, bioavailability of Cu is tightly controlled by Cu exporter ATP7A, which obtains Cu via Cu chaperone, Atox1, and exclude Cu. We reported that Atox1 also functions as a Cu dependent transcription factor. However, role of Cu transport proteins in EndMT is entirely unknown.[[Unable to Display Character: &#8232;]] Results: Here we show that TNFα stimulation for 24hr in HUVEC significantly decreased ATP7A protein (80%) and increased intracellular Cu and Atox1 in nucleus, which was associated with shape change forming EndMT. ATP7A depletion with shRNA in EC significantly reduced EC markers (VE-cadherin and VEGFR2) and increased mesenchymal markers (αSMA, Calponin, SM22α, Collagen I/II). ATP7A siRNA also increased intracellular Cu and nuclear Atox1. These ATP7A knockdown-induced phenotype changes were inhibited by Cu chelators BCS and TTM. Mechanistically, microarray and qPCR based screening revealed that ATP7A knockdown in EC significantly increased miR21 (2.5 fold) and miR125b (1.5 fold) which induce EndMT in a Cu-dependent manner. Of note, promoters of both miR21 and miR125b have Cu dependent transcription factor Atox1 binding sites. Consistent with this, overexpression of Atox1 increased miR21 and miR125b expression as well as promoted EndMT. In vivo, ATP7A mutant (ATP7Amut) mice with reduced Cu export function showed impaired blood flow recovery and reduced arteriogenesis while increased αSMA+ cells and fibrosis in capillary network after ischemic injury. Moreover, ATP7Amut mice crossed with ApoE-/- mice with high fat diet (HFD) induced robust fibrosis and enhanced atherosclerotic lesion vs ApoE-/-/HFD mice.[[Unable to Display Character: &#8232;]] Conclusions: ATP7A protects against fibrosis by preventing EndMT via nuclear Atox1-mediated upregulation of miR21 and miR125b which induce EndMT, in Cu dependent manner. These findings provide the foundation for novel protective role of Cu transport proteins against EndMT- and fibrosis-mediated cardiovascular diseases.

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