Do SRC family kinases (SFKs) regulate platelet mediated leukocyte recruitment in atherosclerosis?
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Monocytes contribute to the cycle of inflammation which occurs during the initiation of atherosclerosis. We have previously shown in a monocyte- endothelial cell (EC) coculture, that a mixed monocyte population stimu- late EC so they recruit flowing neutrophils. Here we investigated the contributions of CD14 + CD16 - (CD16 - ) and CD14 + CD16 + (CD16 + ) monocyte subsets to this process. Freshly isolated CD16- and CD16+ monocytes, were cocultured with EC on transwell filters for 24 hours, after which the ability of EC to recruit flowing neutrophils was tested. Expression of EC E-selectin was measured using flow cytometry and qPCR. Cytokine secretion in cocultures was measured by luminex (Millipore). Both CD16 - and CD16 + monocytes stimulated EC to support adhesion of flowing neutrophils, with a marked increase in the presence of CD16 + monocytes. High levels of TNF-a were quantified in supernatants from cocultures of EC with CD16 + , and blockade of TNF-a reduced neutrophil recruitment. Supernatants from CD16 - cocultures contained high concentrations of IL-6. Interestingly, addition of an IL-6 rich CD16 - coculture supernatant to CD16 + co-cultures drastically reduced neutrophil recruitment, and blockade of IL-6 in CD16 - cocultures increased recruit- ment of neutrophils. The adhesion molecule E-selectin was up-regulated in CD16 + cocultures, contributing to the observed increase in neutrophil adhesion. Differences in the ability to modulate the inflammatory responses of EC exist between monocyte subsets. IL-6 plays a central role in this process and the data indicates that IL-6 mediates crosstalk between CD16 - and CD16 + monocytes. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.011 DO SRC FAMILY KINASES (SFKS) REGULATE PLATELET MEDIATED LEUKOCYTE RECRUITMENT IN ATHEROSCLEROSIS? M.J. Harrison * , G.B. Nash, S.P. Watson, G.Ed. Rainger. Cardiovascular Sciences, University of Birmingham, Birmingham, UK * Presenting author The role of platelets in the formation of arterial thrombus on atheroscle- rotic plaque are well established. In early disease, platelets also appear to play a role in enabling the recruitment of inflammatory leukocytes. We have previously described a paradigm where the recruitment of mono- cytes to TGF-b1 stimulated endothelial cells (EC) is mediated by platelet bridges. TGF-b1 promotes the expression of a matrix of Von Willebrand factor (VWF) on the EC surface which recruits platelets from flowing blood. Upon platelet activation at the EC surface by ADP, monocytes are in turn recruited by platelet P-selectin. The SFKs play an important role in platelet signalling during haemostasis and we propose that these molecules could represent therapeutic targets for intervention in platelet mediated monocyte recruitment during atherogenesis. The broad spectrum SFK in- hibitor Dasatinib ablated ADP dependent aggregation of both human and murine platelets, inhibited the activation of platelets and the secondary recruitment of monocytes on substrates of immobilised VWF or on TGF-b1 stimulated EC. In addition, platelets from the Lyn -/- and Fgr -/- mice, showed deficiencies in aggregation in response to ADP, reduced adhesion to VWF and inhibited monocyte recruitment under flow conditions. These data strongly imply that targeting specific SFK members could inhibit platelet dependent recruitment of monocytes, and may represent drugable targets for intervening in the inflammatory processes that support atheroma formation. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.012 LPA NULL MUTATION GENOTYPING AND QPCR ANALYSIS REFINE KRINGLE ISOFORM ANALYSIS OF LP(A) LEVELS Theodosios Kyriakou * , Udo Seedorf, Anuj Goel, Hugh Watkins, Martin Farrall. Department of Cardiovascular Medicine, University of Oxford, UK * Presenting author Introduction: Increased levels of Lp(a) lipoprotein are a highly heritable risk factor for coronary artery disease. The genetic determinants of Lp(a) levels are mainly due to a copy number variation in apolipoprotein(a) (LPA). Copy number has been traditionally assessed by immunoblotting to measure apolipoprotein (a) isoform size, A complementary quantitative polymerase-chain-reaction (qPCR) assay has been developed to assess the relative number of repeats in high-throughput. We have applied these complementary protein and genomic techniques to study 1,865 samples to 1) evaluate their respective merits for large-scale genetic epidemiological studies 2) study the refined relationship between apo(a) isoform size and Lp(a) level. Methods: We estimated the size of apolipoprotein(a) isoforms using SDS– agarose gel electrophoresis. A qPCR assay was used to determine the relative number of repeats in the same series of samples. “Null” LPA allele Carrier status (rs41272114) was established using KASP genotyping as- says. Lp(a) lipoprotein levels were measured by a immunoturbidimetric assay. Results: Null allele carriers are common and have significantly lower Lp(a) levels than non-carriers. RQ, a qPCR-derived measure of KIV-II copy number, was strongly correlated with the sum of apo(a) isoform sizes in non-carriers of the null allele. An additive allelic model of KIV-II isoforms, refined by null allele genotype and RQ values, showed a sigmoid rela- tionship with Lp(a) levels with baseline levels for relatively long isoform alleles and high levels of Lp(a) for shorter isoform alleles. Conclusions: Genomic assays complement but cannot replace direct electrophoretic measurement of apo(a) isoform size. A joint genomic and isoform analysis revealed details of the relationship between apo(a) iso- form size and circulating Lp(a) level consistent with a threshold effect on lipoprotein secretion. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.013 LOW DENSITY LIPOPROTEIN AGGREGATED BY SPHINGOMYELINASE IS INTERNALISED BY MACROPHAGES AND OXIDISED IN LYSOSOMES Y. Wen a , L. Satchell a , T.M. Gibson a , P.D. Weinberg b , D.S. Leake a,* . a School of Biological Sciences, University of Reading, Reading, Berkshire, UK; b Department of Bioengineering, Imperial College London, London, UK * Presenting author Background: Large trials of antioxidants to inhibit low density lipoprotein (LDL) oxidation have failed to protect against cardiovascular disease. We have shown that LDL aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes. This raises the novel possibility that antioxi- dants targetting lysosomes might be a therapy for atherosclerosis. Aim: We have now studied sphingomyelinase-aggregated LDL to see if it is oxidised within lysosomes. Methods and results: LDL was incubated for 4 hours with sphingomye- linase, which increased its average particle diameter from 27 to 209 nm. It was then incubated for up to 7 days with human monocyte-derived macrophages. Confocal microscopy revealed the colocalisation of lipids and lysosomes, detected with LipidTOXÔ Red and anti-LAMP2 antibody, respectively. Ceroid was stained by Oil Red O or LipidTOXÔ Red after the soluble lipids had been removed by organic solvents and the cells observed Abstracts / Atherosclerosis 232 (2014) e1–e9 e5
Transcript of Do SRC family kinases (SFKs) regulate platelet mediated leukocyte recruitment in atherosclerosis?
Abstracts / Atherosclerosis 232 (2014) e1–e9 e5
Monocytes contribute to the cycle of inflammation which occurs duringthe initiation of atherosclerosis. We have previously shown in a monocyte-endothelial cell (EC) coculture, that a mixed monocyte population stimu-late EC so they recruit flowing neutrophils. Here we investigated thecontributions of CD14+CD16- (CD16-) and CD14+CD16+ (CD16+) monocytesubsets to this process.
Freshly isolated CD16- and CD16+ monocytes, were cocultured with EC ontranswell filters for 24 hours, after which the ability of EC to recruit flowingneutrophils was tested. Expression of EC E-selectin was measured usingflow cytometry and qPCR. Cytokine secretion in cocultures was measuredby luminex (Millipore).
Both CD16- and CD16+ monocytes stimulated EC to support adhesionof flowing neutrophils, with a marked increase in the presence of CD16+
monocytes. High levels of TNF-a were quantified in supernatantsfrom cocultures of EC with CD16+, and blockade of TNF-a reducedneutrophil recruitment. Supernatants from CD16- cocultures containedhigh concentrations of IL-6. Interestingly, addition of an IL-6 rich CD16-
coculture supernatant to CD16+ co-cultures drastically reduced neutrophilrecruitment, and blockade of IL-6 in CD16- cocultures increased recruit-ment of neutrophils. The adhesion molecule E-selectin was up-regulatedin CD16+ cocultures, contributing to the observed increase in neutrophiladhesion.
Differences in the ability to modulate the inflammatory responses of ECexist between monocyte subsets. IL-6 plays a central role in this processand the data indicates that IL-6 mediates crosstalk between CD16- andCD16+ monocytes.
http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.011
DO SRC FAMILY KINASES (SFKS) REGULATE PLATELET MEDIATEDLEUKOCYTE RECRUITMENT IN ATHEROSCLEROSIS?
M.J. Harrison*, G.B. Nash, S.P. Watson, G.Ed. Rainger.
Cardiovascular Sciences, University of Birmingham, Birmingham, UK* Presenting author
The role of platelets in the formation of arterial thrombus on atheroscle-rotic plaque are well established. In early disease, platelets also appear toplay a role in enabling the recruitment of inflammatory leukocytes. Wehave previously described a paradigm where the recruitment of mono-cytes to TGF-b1 stimulated endothelial cells (EC) is mediated by plateletbridges. TGF-b1 promotes the expression of a matrix of Von Willebrandfactor (VWF) on the EC surface which recruits platelets from flowing blood.Upon platelet activation at the EC surface by ADP, monocytes are in turnrecruited by platelet P-selectin. The SFKs play an important role in plateletsignalling during haemostasis and we propose that these molecules couldrepresent therapeutic targets for intervention in platelet mediatedmonocyte recruitment during atherogenesis. The broad spectrum SFK in-hibitor Dasatinib ablated ADP dependent aggregation of both human andmurine platelets, inhibited the activation of platelets and the secondaryrecruitment of monocytes on substrates of immobilised VWF or on TGF-b1stimulated EC. In addition, platelets from the Lyn-/- and Fgr-/- mice, showeddeficiencies in aggregation in response to ADP, reduced adhesion to VWFand inhibited monocyte recruitment under flow conditions. These datastrongly imply that targeting specific SFK members could inhibit plateletdependent recruitment of monocytes, and may represent drugable targetsfor intervening in the inflammatory processes that support atheromaformation.
http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.012
LPA NULL MUTATION GENOTYPING AND QPCR ANALYSIS REFINEKRINGLE ISOFORM ANALYSIS OF LP(A) LEVELS
Theodosios Kyriakou*, Udo Seedorf, Anuj Goel, Hugh Watkins, MartinFarrall.
Department of Cardiovascular Medicine, University of Oxford, UK* Presenting author
Introduction: Increased levels of Lp(a) lipoprotein are a highly heritablerisk factor for coronary artery disease. The genetic determinants of Lp(a)levels are mainly due to a copy number variation in apolipoprotein(a)(LPA). Copy number has been traditionally assessed by immunoblotting tomeasure apolipoprotein (a) isoform size, A complementary quantitativepolymerase-chain-reaction (qPCR) assay has been developed to assess therelative number of repeats in high-throughput. We have applied thesecomplementary protein and genomic techniques to study 1,865 samples to1) evaluate their respective merits for large-scale genetic epidemiologicalstudies 2) study the refined relationship between apo(a) isoform size andLp(a) level.
Methods: We estimated the size of apolipoprotein(a) isoforms using SDS–agarose gel electrophoresis. A qPCR assay was used to determine therelative number of repeats in the same series of samples. “Null” LPA alleleCarrier status (rs41272114) was established using KASP genotyping as-says. Lp(a) lipoprotein levels were measured by a immunoturbidimetricassay.Results: Null allele carriers are common and have significantly lower Lp(a)levels than non-carriers. RQ, a qPCR-derived measure of KIV-II copynumber, was strongly correlated with the sum of apo(a) isoform sizes innon-carriers of the null allele. An additive allelic model of KIV-II isoforms,refined by null allele genotype and RQ values, showed a sigmoid rela-tionship with Lp(a) levels with baseline levels for relatively long isoformalleles and high levels of Lp(a) for shorter isoform alleles.Conclusions: Genomic assays complement but cannot replace directelectrophoretic measurement of apo(a) isoform size. A joint genomic andisoform analysis revealed details of the relationship between apo(a) iso-form size and circulating Lp(a) level consistent with a threshold effect onlipoprotein secretion.
http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.013
LOW DENSITY LIPOPROTEIN AGGREGATED BY SPHINGOMYELINASE ISINTERNALISED BY MACROPHAGES AND OXIDISED IN LYSOSOMES
Y. Wen a, L. Satchell a, T.M. Gibson a, P.D. Weinberg b, D.S. Leake a,*.
a School of Biological Sciences, University of Reading, Reading, Berkshire, UK;bDepartment of Bioengineering, Imperial College London, London, UK* Presenting author
Background: Large trials of antioxidants to inhibit low density lipoprotein(LDL) oxidation have failed to protect against cardiovascular disease.We haveshown that LDL aggregated by vortexing is internalised by macrophages andoxidised by iron in lysosomes. This raises the novel possibility that antioxi-dants targetting lysosomes might be a therapy for atherosclerosis.
Aim:We have now studied sphingomyelinase-aggregated LDL to see if it isoxidised within lysosomes.Methods and results: LDL was incubated for 4 hours with sphingomye-linase, which increased its average particle diameter from 27 to 209 nm. Itwas then incubated for up to 7 days with human monocyte-derivedmacrophages. Confocal microscopy revealed the colocalisation of lipidsand lysosomes, detected with LipidTOX� Red and anti-LAMP2 antibody,respectively. Ceroid was stained by Oil Red O or LipidTOX� Red after thesoluble lipids had been removed by organic solvents and the cells observed
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