Caveolin-1 plays a critical role in the differentiation of monocytes into macrophages
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increasing TF expression and activity. Our results suggest a novel role for purinergic P2Y receptors in vascular thrombosis. We propose that P2 receptors are promising therapeutic targets for the regulation of thrombosis associated with inflammation. doi:10.1016/j.vph.2011.08.181 P.13.7 Effects of oxidized and chlorinated phospholipids on signaling pathways in endothelial cells Norsyahida Mohd Fauzi a , Emma Torrance a , Thikryat Neamatallah a , Ho Ka Ho a , Corinne M. Spickett b , Robin Plevin a a Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK b School of Life and Health Sciences, Aston University, Birmingham, UK E-mail address: [email protected] (N. Mohd Fauzi) More than 25 years ago, it was proposed that the inflammatory disease atherosclerosis arises as a response of vascular wall to endothelial injury and many studies showed that this injury could be due to endothelial apoptosis. As a result of vascular inflammation, oxidized and chlorinated lipids are generated and are thought to be a primary risk factor in the progression of atherosclerosis. There are extensive studies on the biological effects of oxidized phospholipids (oxPAPC) in vascular cells including smooth muscle cells and endothelial cells and more recently, analysis of individual oxidized species, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC). For instance, oxPAPC has been shown to induce production of IL-8, MCP-1 /MIP-2 and expression of E-selectin in myeloid and endothelial cells (Erridge et al., 2007), whilst POVPC increases monocyte binding to endothelial cells, by inducing the surface expression of connecting segment-1 domain of fibronectin (Leitinger et al., 1999). Oxidized phospholipids can also have anti-inflammatory effects; OxPAPC prevents TLR-2 and 4 activation by lipopolysacchar- ide and Pam 3 CSK 4 (Erridge et al., 2008). In comparison however, little is known about effects of chlorinated lipids on vascular function. To investigate this, the effect of chlorinated lipids upon NFkB and MAP kinase signaling was studied in HUVECs. Oxidized phospholipids such as oxPAPC, PGPC and POVPC (5–50 μM) and chlorinated lipids including SOPC chlorohydrins and 2-Chlorohexadecanal (5–50 μM) were assessed for IκB-α degradation and phosphorylation of ERK, p38 and JNK, by Western blotting. Results showed that SOPC chlorohydrin and 2-chlorohexadecanal alone did not induce phoshorylation of NFkB and MAPK nor inhibit IκB-α degradation induced by LPS and TNF-α. In contrast, oxidized phospholipids such as oxPAPC inhibited IκB-α degradation induced by LPS but not TNF-α, a result which is in agreement with a previous study (Bochkov et al., 2002). Preliminary studies used microscopy to investigate the effect of oxidized and chlorinated lipids on cellular integrity of endothelial cells. Interest- ingly, treatment of chlorinated lipids (50–100 μM) induced cell death and this was potentiated in the presence of TNF-α. These results demonstrate that chlorinated lipids may produce pro-atherogenic effects but do not appear to regulate endothelial cell function in the same way as oxidized phospholipids. Acknowledgement Malaysian of Higher Education National University of Malaysia References Bochkov et al., 2002. Nature 419(6902): 77–81. Erridge et al., 2007. Atherosclerosis 193(1): 77–85. Erridge et al., 2008. J Biol Chem 283(36): 24748–59. Leitinger et al.,1999. Proc. Natl. Acad. Sci. U. S. A. 96(21):12010–12015. doi:10.1016/j.vph.2011.08.182 P.13.8 Caveolin-1 plays a critical role in the differentiation of monocytes into macrophages Yi Fu, Shirley Moore, Jaye P.F. Chin-Dusting Vascular Pharmacology, Baker IDI Heart and Diabetes Institute, Melbourne, Australia E-mail address: [email protected] (Y. Fu) Monocyte to macrophage differentiation is an essential step in atherogenesis. The structure protein of caveolae, caveolin-1, is involved in protein translocation and integrin signalling. Primary monocytes demonstrate an increase in caveolin-1 expression following adhesion to endothelium. We explore the hypothesis that caveolin-1 plays a role in monocyte differentiation to macrophage. We confirm that PMA treatment of THP-1 cells induces expression of inflammatory genes and macrophage cell surface markers and show that caveolin-1 expression increases in these treated cells. These findings were validated in human and mouse primary monocytes. As well, expres- sion of macrophage markers and inflammatory genes increased following transfection of pEGF-caveolin-1 plasmid, while caveolin-1 knockdown by siRNA reduced the PMA-induced expression of these differentiation markers. Moreover, the nuclear location of the early growth responsor 1 (EGR-1) transcription factor, increased by PMA treatment, was blocked by knocking down caveolin-1. ChIP assay results further showed that caveolin-1 knock-down inhibited the binding of EGR-1 to its target DNA fragments (CSF1R and TNFα) induced by monocyte differentiation thus decreasing EGR-1 transcrip- tional activity. Finally, caveolin-1 transfection altered the transmigra- tion ability of THP-1 (monocyte) cells to one closer to that observed in PMA treated THP-1 (macrophage) cells. We conclude that caveolin-1 promotes monocyte to macrophage differentiation through regulation of EGR-1 transcriptional activity and suggest that monocytic caveolin- 1 is a potential therapeutic target for atherosclerosis. doi:10.1016/j.vph.2011.08.183 P.13.9 Nod1 ligands induce coronary arteritis Toshiro Hara, Hisanori Nishio, Shunsuke Kanno, Sagano Onoyama, Kazuyuki Ikeda Departments of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan E-mail address: [email protected] (T. Hara) There is a line of evidence that activation of innate immune Toll- like receptors (TLRs) contributes to the development and progression of cardiovascular diseases. With respect to nucleotide-binding domain, leucine-rich repeat-containing (NLRs) protein family, an exact role in the cardiovascular system remains to be clarified. We investigated the effects of stimulants for NLRs on human artery endothelial cells in vitro and murine arteries in vivo. Human coronary artery endothelial cells (HCAEC) were chal- lenged in vitro with microbial components that stimulate NLRs or TLRs. We found the stimulatory effects of NLR and TLR ligands on the adhesion molecule expression and cytokine secretion by HCAEC. Based on these in vitro results, we examined the in vivo effects of these ligands in mice. Among NLR and TLR ligands, FK565, one of the nucleotide-binding oligomerization domain (Nod)1 ligands, induced strong site-specific inflammation in the aortic root. Furthermore, Abstracts 373
Transcript of Caveolin-1 plays a critical role in the differentiation of monocytes into macrophages
increasing TF expression and activity. Our results suggest a novel rolefor purinergic P2Y receptors in vascular thrombosis. We propose thatP2 receptors are promising therapeutic targets for the regulation ofthrombosis associated with inflammation.
doi:10.1016/j.vph.2011.08.181
P.13.7Effects of oxidized and chlorinated phospholipids on signalingpathways in endothelial cellsNorsyahida Mohd Fauzia, Emma Torrancea, Thikryat Neamatallaha,Ho Ka Hoa, Corinne M. Spickettb, Robin Plevina
aStrathclyde Institute of Pharmacy and Biomedical Sciences,University of Strathclyde, UKbSchool of Life and Health Sciences, Aston University, Birmingham, UKE-mail address: [email protected] (N. Mohd Fauzi)
More than 25 years ago, it was proposed that the inflammatorydisease atherosclerosis arises as a response of vascular wall toendothelial injury and many studies showed that this injury could bedue to endothelial apoptosis. As a result of vascular inflammation,oxidized and chlorinated lipids are generated and are thought to be aprimary risk factor in the progression of atherosclerosis. There areextensive studies on the biological effects of oxidized phospholipids(oxPAPC) in vascular cells including smooth muscle cells andendothelial cells and more recently, analysis of individual oxidizedspecies, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine(POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine(PGPC). For instance, oxPAPC has been shown to induce productionof IL-8, MCP-1 /MIP-2 and expression of E-selectin in myeloid andendothelial cells (Erridge et al., 2007), whilst POVPC increasesmonocyte binding to endothelial cells, by inducing the surfaceexpression of connecting segment-1 domain of fibronectin (Leitingeret al., 1999). Oxidized phospholipids can also have anti-inflammatoryeffects; OxPAPC prevents TLR-2 and 4 activation by lipopolysacchar-ide and Pam3CSK4 (Erridge et al., 2008). In comparison however, littleis known about effects of chlorinated lipids on vascular function. Toinvestigate this, the effect of chlorinated lipids upon NFkB and MAPkinase signaling was studied in HUVECs. Oxidized phospholipids suchas oxPAPC, PGPC and POVPC (5–50 μM) and chlorinated lipidsincluding SOPC chlorohydrins and 2-Chlorohexadecanal (5–50 μM)were assessed for IκB-α degradation and phosphorylation of ERK, p38and JNK, by Western blotting. Results showed that SOPC chlorohydrinand 2-chlorohexadecanal alone did not induce phoshorylation ofNFkB and MAPK nor inhibit IκB-α degradation induced by LPS andTNF-α. In contrast, oxidized phospholipids such as oxPAPC inhibitedIκB-α degradation induced by LPS but not TNF-α, a result which is inagreement with a previous study (Bochkov et al., 2002). Preliminarystudies used microscopy to investigate the effect of oxidized andchlorinated lipids on cellular integrity of endothelial cells. Interest-ingly, treatment of chlorinated lipids (50–100 μM) induced cell deathand this was potentiated in the presence of TNF-α. These resultsdemonstrate that chlorinated lipids may produce pro-atherogeniceffects but do not appear to regulate endothelial cell function in thesame way as oxidized phospholipids.
AcknowledgementMalaysian of Higher EducationNational University of Malaysia
ReferencesBochkov et al., 2002. Nature 419(6902): 77–81.Erridge et al., 2007. Atherosclerosis 193(1): 77–85.Erridge et al., 2008. J Biol Chem 283(36): 24748–59.
Leitingeret al.,1999. Proc.Natl. Acad. Sci. U. S. A. 96(21):12010–12015.
doi:10.1016/j.vph.2011.08.182
P.13.8Caveolin-1 plays a critical role in the differentiation of monocytesinto macrophagesYi Fu, Shirley Moore, Jaye P.F. Chin-DustingVascular Pharmacology, Baker IDI Heart and Diabetes Institute,Melbourne, AustraliaE-mail address: [email protected] (Y. Fu)
Monocyte to macrophage differentiation is an essential step inatherogenesis. The structure protein of caveolae, caveolin-1, is involvedin protein translocation and integrin signalling. Primary monocytesdemonstrate an increase in caveolin-1 expression following adhesionto endothelium.We explore the hypothesis that caveolin-1 plays a rolein monocyte differentiation to macrophage. We confirm that PMAtreatment of THP-1 cells induces expression of inflammatory genesand macrophage cell surface markers and show that caveolin-1expression increases in these treated cells. These findings werevalidated in human and mouse primary monocytes. As well, expres-sion of macrophage markers and inflammatory genes increasedfollowing transfection of pEGF-caveolin-1 plasmid, while caveolin-1knockdown by siRNA reduced the PMA-induced expression of thesedifferentiation markers. Moreover, the nuclear location of the earlygrowth responsor 1 (EGR-1) transcription factor, increased by PMAtreatment, was blocked by knocking down caveolin-1. ChIP assayresults further showed that caveolin-1 knock-down inhibited thebinding of EGR-1 to its target DNA fragments (CSF1R and TNFα)induced bymonocyte differentiation thus decreasing EGR-1 transcrip-tional activity. Finally, caveolin-1 transfection altered the transmigra-tion ability of THP-1 (monocyte) cells to one closer to that observed inPMA treated THP-1 (macrophage) cells. We conclude that caveolin-1promotes monocyte to macrophage differentiation through regulationof EGR-1 transcriptional activity and suggest that monocytic caveolin-1 is a potential therapeutic target for atherosclerosis.
doi:10.1016/j.vph.2011.08.183
P.13.9Nod1 ligands induce coronary arteritisToshiro Hara, Hisanori Nishio, Shunsuke Kanno,Sagano Onoyama, Kazuyuki IkedaDepartments of Pediatrics, Graduate School of Medical Sciences,Kyushu University, Fukuoka, JapanE-mail address: [email protected] (T. Hara)
There is a line of evidence that activation of innate immune Toll-like receptors (TLRs) contributes to the development and progressionof cardiovascular diseases. With respect to nucleotide-bindingdomain, leucine-rich repeat-containing (NLRs) protein family, anexact role in the cardiovascular system remains to be clarified.
We investigated the effects of stimulants for NLRs on humanartery endothelial cells in vitro and murine arteries in vivo.
Human coronary artery endothelial cells (HCAEC) were chal-lenged in vitro with microbial components that stimulate NLRs orTLRs. We found the stimulatory effects of NLR and TLR ligands on theadhesion molecule expression and cytokine secretion by HCAEC.Based on these in vitro results, we examined the in vivo effects ofthese ligands in mice. Among NLR and TLR ligands, FK565, one of thenucleotide-binding oligomerization domain (Nod)1 ligands, inducedstrong site-specific inflammation in the aortic root. Furthermore,
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