_________________________________________________________
Supervisors: Magnus Grenegård, ProfessorDepartment of Clinical Medicine School of Medical Health and Sciences Örebro Univesity, Sweden
Master Thesis in Medicine
(Second Level, 45 Credits)
The role of the acute phase protein α1-acid
glycoprotein on human umbilical vein endothelial
cells and human platelets
2
Abstract
The protein α1-acid glycoprotein (AGP) is an acute phase protein which means that the concentration is elevated during inflammation. The most established physiological function of AGP is its role as a carrier protein in the circulation. AGP is the most glycosylated plasma protein and this glycosylation changes in structure and composition in disease. The chemical structures of AGP are well studied but if it has more physiological effects remain more unclear. In this study the effect of physiological concentrations of AGP on endothelial cells and platelets was investigated. The results show that AGP did not produce significant effects on viable cell number, migration or tube formation of human umbilical vein endothelial cells (HUVEC). However a non-significant trend towards an enhanced migration and tube formation in HUVEC in response to AGP-treatment was indicated as well as a non-significant trend towards enhanced activation of the rho/rho-kinase signaling pathway in platelets. In conclusion there are
indications of a role for AGP in regulating angiogenesis.
Keywords: α1-acid glycoprotein (AGP), endothelial cells, proliferation, migration, tube formation, platelets, rho-kinase signaling.
3
Introduction
The acute phase protein α1-acid glycoprotein (AGP), also called orosomucoid, is present in healthy human plasma with a concentration of approximately 0.5-1 mg/ml (1) and increases several fold during inflammatory conditions. The physiological concentration of AGP is relatively high in comparison to other acute phase proteins. Like other common acute phase proteins such as C-reactive protein (CRP) AGP is primary produced by the hepatocytes in the liver in response to interleukin-1 (IL-1) and IL-6, but discrete amounts of AGP can also be synthesized by other cells like white blood cells and endothelial cells (2,3). Elevated levels of both CRP and AGP are associated to diseases with inflammatory properties such as
atherosclerosis, cancer and rheumatoid arthritis (4). However the exact physiological as well as pathophysiological roles of AGP and other acute phase proteins remain to be determined. Since most studies regarding AGP have focused on chemical structure and changes in glycosylation pattern there are still several possible physiological functions of AGP to investigate. AGP has a molecular weight of 41-43 kDa depending on the constitution and structure of the five N-linked oligosaccharide side chains, the glycans of AGP. Thus is AGP a very highly glycosylated protein, this glycosylation correspond to approximately 45 % of the molecular weight. These glycans can be bi-, tri- or tetra-antennary and have terminal sialic acids which play an important role in the effects of AGP on neutrophil granulocytes. This effect is induced by a cytosolic increase in Ca2+ via sialic acids binding immunoglobulin-like lectins (Siglecs) present on the surface of the neutrophil granulocytes (5,6). Several studies have revealed immunomodulating properties of AGP like generation of reactive oxygen species (ROS) in activated neutrophil granulocytes (6,7), inhibition of neutrophil migration (8) and reduced lymphocyte
responsiveness (9). AGP also affects platelets and induces a shape change via the Rho/Rho-kinase signalling pathway, but this effect of AGP on platelets is not dependent on the
glycosylation pattern of the molecule or the presence of sialic acids as in neutrophils (7). The activation of the Rho/Rho-kinase signaling pathway in platelets initiate several steps of
phosphorylation of different molecules like Myosin phosphatase target subunit 1 (MYPT1) and Myosin Light Chain 2 (MLC2), both included in the contractile mechanism of the platelet (10). Besides the function as an acute phase protein AGP also serves as a transport protein in the circulation (11) and have been characterized as a more important carrier of endogenous
atherogenic lipids than albumin (12). AGP releases exogenous or endogenous ligands as a result of the conformational change of the protein that occurs in the binding to cell membranes (13,14). This release of ligands might influence the signaling in platelets where the terminal sialic acids did not affect the observed platelet shape change induced by AGP (7). Lysophosphatidic acid (LPA) is one of the potential endogenous ligands transported by AGP. The lysophosphatidic acid
4
receptor 5 (LPA5) is the most abundant of LPA-receptors present on platelets and have
previously been shown to be involved in platelet activation (15). Since AGP can modulate both neutrophil granulocytes and platelets this protein plays a role in the regulation of the innate immune responses and haemostasis that might be associated to events of inflammation and thrombosis in atherosclerosis. Previous studies regarding lipoprotein A, which is considered as risk factor for cardiovascular disease, have also noticed an association between lipoprotein A and circulating levels of AGP (16,17). Another important cell type associated to atherosclerosis is the endothelial cells lining the blood vessels. Endothelial cells have an important role in
angiogenesis, the process of new blood vessel formation, during embryonic development and in response to injury. AGP seems to affect endothelial cells to an increased metabolic rate and this rapid response indicates a receptor signaling pathway (18). One recent study have shown an inhibitory effect by AGP on both injury and tumour necrosis factor-α (TNF-α) induced angiogenesis but also an inducing effect on development angiogenesis (19). The molecular mechanisms responsible for this potentially effect of AGP on endothelial cells remains unclear and these effects have not been further investigated.
Aim
The aim of this present study is to investigate whether physiological concentrations of AGP have any effect on human umbilical vein endothelial cells (HUVEC) and to further investigate the effects of physiological concentrations of AGP on human platelets. Furthermore, the study aims to clarify the underlying molecular mechanisms.
5
Material and methods
Cells
HUVEC were cultured in Vasculife® Basal Medium supplemented with Lifefactors® Vasculife® VEGF including rh VEGF, rh IGF-1, rh FGF-b, Ascorbic acid, rh EGF, Heparin, FBS, L-glutamine and Hydrocort obtained from LifeLine cell technology™ (Stockholm, Sweden) and 0.1 U/ml penicillin and 100 ng/ml streptomycin obtained from Gibco® by Life Technologies™ (Carlsbad, CA, USA). The cells were cultured at a temperature of 37oC in humidified air with 5% CO2. Medium was changed every 48-72 hours and the cells were sub cultured when confluent.
Platelets were isolated from whole blood obtained from 3 healthy volunteers and supplemented with 10 U/ml heparin to prevent coagulation. To prevent pre-activation of the platelets during the preparation 1 part blood was gently mixed with 5 parts acid citrate-dextrose (ACD) solution with pH 4.5. The blood was centrifuged at 220 g for 20 minutes to separate out platelet-rich plasma (PRP). Then the PRP was transferred to conical tubes and 10mM acetylsalicylic acid (ASA) and 1 U/ml apyrase from potato, grade III, both obtained from Sigma Aldrich (ST Louis, MO, USA), were added to further prevent platelet activation. The PRP was incubated in room temperature in a gentle agitation for 30 minutes before next centrifugation at 520 g in 25 minutes to produce a platelet pellet. For Ca2+-measurements the PRP was supplemented with 10mM ASA and 0.5 U/ml apyrase and transferred to conical tubes. This PRP was loaded with 4 µM Fura 2 acetoxymethylester (Fura-2/AM) obtained from Sigma Aldrich (ST Louis, MO, USA) by incubation in room temperature, protected from light during agitation for 60 minutes before it was centrifuged at 480 g for 20 minutes. After the last centrifugation the platelet-pellet was washed 3 times in Krebs-Ringer glucose (KRG) buffer and thereafter resuspended in KRG-buffer supplemented with 1 U/ml apyrase.
HUVEC viability
The effect of AGP on the number of viable HUVEC after 48 or 72 hours was analysed with the colorimetric WST-8 assay Cell Counting Kit-8 (CCK-8) [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] obtained from Dojindo Laboratories (Kumamoto, Japan) to evaluate any difference in cell redox activity, which is proportional to the number of viable cells. Comparing AGP-treated wells to controls would indicate a final difference in viable cell number in response to AGP treatment. HUVEC in passage 6-10 were seeded with a cell density of 4000 or 8000 cells/cm2 in 96 well TPP®-plates
6
obtained from Sigma Aldrich (ST Louis, MO, USA). The cells were seeded in Vasculife® Basal Medium supplemented with Lifefactors® Vasculife® VEGF plus antibiotics and were incubated over night in 37oC in humidified air with 5% CO2 to adhere. Two hours before treatment with human AGP obtained from Sigma Aldrich (ST Louis, MO, USA) the complete medium was replaced into Vasculife® Basal Medium supplemented with Lifefactors® Vasculife® VEGF but excluding FBS and antibiotics to create a serum-free condition for the experiment. AGP treatment was performed at a dose range between 0.1-500 µg/ml and in combination with10 ng/ml TNF-α. Untreated cells in serum-free medium were used as control and all samples were analyzed in triplicates. After 48 or 72 hours of incubation the substrate in the colorimetric CCK-8 kit was added to each well and the plate was incubated in the dark in 37oC in humidified air with 5% CO2 for 4 hours. The absorbance was measured at 450 nm at a Sunrise Basic
spectrophotometer obtained from Tecan (Austria) with the software Magellan 7.1. To analyze the results of AGP-treatments it was compared to the controls and presented as percent of control.
HUVEC migration assay
To analyse whether AGP affects HUVEC ability to migrate in response to cell injury a scratch was made in the adherent, confluent cell layer in a 6 well TPP®-plate. 300 000 HUVEC in passage 8-10 were seeded in 2 ml Vasculife® Basal Medium supplemented with Lifefactors® Vasculife® VEGF plus antibiotics and incubated overnight in 37oC in humidified air with 5% CO2. When the cells have adhered and become confluent the medium was replaced with
Vasculife® Basal Medium supplemented with Lifefactors® Vasculife® VEGF but excluding FBS to create a serum-free condition. After two hours in serum free medium the scratch was
performed with a pipette tip of the 1000 µl size and the medium was changed into fresh serum free complete medium to reduce the amount of loose cells from the scratch. Then 1 µg/ml or 100 µg/ml AGP was added before pictures of the scratch were taken by the camera Q Imaging Micro publisher 3.3 RTV at 0, 17 and 23 hours. All treatments were done in duplicates. The width of the scratch was measured in µm in the software Q Capture Pro v. 5.1 and the data were presented as mean distance migrated in percent of control.
HUVEC tube formation assay
To investigate if AGP affects the tube formation ability of HUVECs they were treated with AGP in a tube formation assay. 15000 HUVEC in passage 7-10 were seeded/well in OPTI-MEM® (Gibco® by Life Technologies™) on top of a Matrigel obtained from Becton, Dickinson and Company (BD) Biosciences in a µ-slide angiogenesis chamber obtained from Ibidi. Treatment
7
was performed with human AGP at doses of 10 µg/ml or 100 µg/ml. Control was untreated HUVECs in the same tube formation assay. The µ-slide chamber was incubated in 37oC in humidified air with 5% CO2 for 2-6 hours. When the tube formation peaked the cells were stained with Calcein AM Fluorescent Dye obtained from BD and pictures were taken to be able to evaluate changes in tube formation in response to AGP-treatment.Analyses were made by measuring the length and number of tubes in AGP-treated wells and controls in the software Image J. All treatments were performed in duplicates, for number of tubes a mean/treatment was calculated and for length of tubes a mean/well was calculated before a mean/treatment. The data was presented as percent of control.
Cytosolic Ca2+ measurement in platelets
In the cell suspension of isolated platelets 1 mM CaCl2 was added to increase the extracellular calcium concentration prior to the experiment. The samples were put in 37oC for 2 minutes before they were placed in the Fluorescence Spectrophotometer F7000 from Hitachi at 37oC with stirring for 3 minutes before the UV-light was turned on. After 1 minute 5 µM of the LPA5 receptor antagonist obtained from Tocris bioscience was added and thereafter was 10 µM epinephrine added to increase the subsequent response of AGP. After 5 minutes of incubation with the receptor antagonist and 3 minutes of incubation with epinephrine, 0.5 mg/ml AGP was added. The fluorescence was also measured without prior receptor antagonist incubation. The fluorescence emission was measured at 510 nm during excitation at 340 and 380 nm.
Rho/rho-kinase signalling in platelets
Phosphorylation of MYPT-1 at threonine 696 and phosphorylation of MLC2 at serine 19 occurs during activation of the rho/rho-kinase signalling pathway. These two targets were used to show whether AGP activates the rho/rho-kinase signalling pathway in platelets. Western blot was performed to quantify the phosphorylation of MYPT-1 at threonine 696 and MLC2 at serine 19 in platelets treated with AGP. The platelet concentration was set to 2.5x108/ml supplemented with 1mM CaCl2 and was incubated for 20 minutes in 37oC. Treatment of the platelets with 0.5 mg/ml AGP or 0.1 U/ml thrombin from Sigma Aldrich (ST Louis, MO, USA) was performed in 37oC during 900 rpm agitation for 5-40 seconds before the reaction was stopped by adding 5X sodium dodecyl sulfate (SDS) buffer. The samples were denatured at 96oC for 5 minutes and then stored in -20oC until western blot was performed. For detection of MYPT-1, which is a 130 kDa protein, a NuPAGE® 3-8 % Tris-acetate gel from novex® by Life Technologies was used in combination with Tris-acetate running buffer from novex® by Life Technologies. The proteins were separated on the gel under influence of constant 140 V for 90 minutes in an Invitrogen
8
Xcell Surelock Minicell. As markers on the gel was both SeeBlue® Plus2 Prestained Standard and MagicMark™ XP Western Protein Standard used, both purchased from novex® by Life Technologies. Blotting from the gel to the PVDF-membrane Immobilion®-FLTransfer Membrane obtained from Millipore was performed at 120 mA/gel for 90 minutes in a cool BioRad Mini Trans-Blot Cell. Then the membrane was washed in Tris Buffered Saline
supplemented with 0.1 % Tween (TBS-T) for 5 minutes before blocking the membrane in 5 % dry milk for 1 hour. When the membrane was washed 3x5 minutes in TBS-T the primary antibody Anti-phospho-MYPT1 (Thr696) obtained from Millipore diluted 1:4000 in 5 % milk, was added and incubated in +4oC over night. After the membrane was washed 2x5, 2x10, 2x5 minutes in TBS-T the secondary antibody Anti-rabbit IgG HRP-linked AB 7074 obtained from Cell Signaling Technology, diluted 1:2000, was added and incubated in room temperature for 1 hour. The membrane was washed as previously and covered in Immobilion™ Western
Chemiluminescent HRP Substrate obtained from Millipore before chemiluminescense was measured in the Odyssey Fc Imaging system from LI-COR® Biosciences UK and analyzed in the software Image Studio Lite. For detection of MLC2 the same protocol was followed but the separation of proteins was made in a 12 % Bis-Tris gel from novex® by Life Technologies, this was used in combination with MES SDS running buffer from novex® by Life Technologies. The primary antibody was Phospho-Myosin Light Chain 2 (Ser19) Mouse mAb 3675 obtained from Cell Signaling Technology, diluted 1:1000 and the secondary antibody used for MLC2 was Anti-mouse IgG HRP-linked Antibody 7076 obtained from Cell Signaling Technology, diluted 1:2000.
Statistics
Data were statistically tested for significance by using GraphPad Prism (San Diegeo, CA, USA) and performing a one-way ANOVA followed by the post hoc test Dunnett’s multiple comparison test. In experiments with samples in duplicates or triplicates was the mean calculated. Statistical significance was set to p < 0.05. The data was presented as percent of control ± standard error of mean (SEM).
Ethical considerations
No ethical approval is needed in this project since the material consists of a cell-line and commercially purchased AGP. The use of a cell-line is appropriate in this phase of research when the physiological effects by a substance are not well known. There are still a lot of experiments to be done to have enough in vitro results to justify research in vivo in this area.
9
Results
HUVEC viability
In this study, AGP-treatment did not affect HUVEC viability significantly in the colorimetric CCK-8 assay performed in serum free condition. Doses of 0.1 µg/ml – 500 µg/ml of AGP did not significantly affect the number of viable HUVEC during 48 hours, see Figure 1A. Neither after 72 hours was the number of viable cells altered significantly by AGP treatment and showed even less variances between AGP-treated cells in most doses than in the assay for 48 hours, see Figure 1B.
Figure 1. Number of viable HUVEC in response to AGP. Number of viable cells measured by a
colorimetric CCK-8 assay 48 hours (A) or 72 hours (B) after AGP-treatment, data shown as percent of control ± SEM. Each experiment was performed in triplicates, ns = non-significant, i.e. p ≥ 0.05 compared to control (n=4).
The number of viable HUVEC was not significantly altered after 48 hours by 10 ng/ml of TNF-α alone or in combination with 1 µg/ml or 100 µg/ml of AGP as shown in Figure 2A. Neither the number of viable cells after 72 hours was significantly altered by 10 ng/ml of TNF-α alone or in combination with 1 µg/ml or 100 µg/ml of AGP, see Figure 2B.
10
Figure 2. Number of viable HUVEC in response to TNF-α in combination with AGP. Number of
viable cells measured by a colorimetric CCK-8 assay 48 hours (A) or 72 hours (B) after treatment by TNF-α and AGP. Each experiment was performed in triplicates and data is shown as percent of control ± SEM, ns = non-significant, i.e. p ≥ 0.05 compared to control (n=4).
HUVEC migration
The ability of HUVEC to migrate in response to an induced cell injury was not significantly altered 17 or 23 hours after treatment with 1 µg/ml – 10 µg/ml of AGP, see Figure 3. However a non-significant trend of slightly enhanced migration was indicated among AGP-treated cells compared to controls.
Figure 3. HUVEC migration in response to AGP. Distance migrated during 17 hours (A) or 23 hours
(B) after AGP-treatment shown as percent of control ± SEM. Each experiment was performed in duplicates, ns = non-significant, i.e. p ≥ 0.05 compared to control (n=5).
11
HUVEC tube formation
The ability of HUVEC to form tubes at a Matrigel in a µ-slide angiogenesis chamber in response to AGP was measured both in number of tubes formed and the mean length of the tubes. Both 10 µg/ml AGP and 100 µg/ml AGP induced a small but non-significant increase of the number of tubes compared to control, see figure 4A. The mean length of the tubes formed under influence of AGP was slightly but not significantly longer than the tubes formed in the controls, see figure 4B.
Figure 4. HUVEC tube formation in response to AGP. The number of tubes (A) and the mean length
of the tubes (B) formed by HUVEC seeded on a Matrigel in a µ-slide angiogenesis chamber in response to AGP-treatment. Each experiment was performed in duplicates and data is shown as percent of control ± SEM, ns = non-significant, i.e. p ≥ 0.05 compared to control (n=3).
Intracellular Ca2+ increase in platelets
Concentration of intracellular Ca2+ in isolated human platelets pretreated with epinephrine increased in response to 0.5 mg/ml AGP with 10.7 nM. When the platelets were also pre-treated with 5 µM of LPA5 receptor antagonist the Ca2+ increase in response to 0.5 mg/ml AGP was 7.6 nM. This indicate a partial inhibition elicited by the LPA5 receptor antagonist as shown in figure 5.
12
Figure 5. Partial inhibition of intracellular Ca2+ increase in response to AGP in platelets treated
with a LPA5 receptor antagonist. Fluorescence emission in isolated human platelets pre-treated with
epinephrine and treated with AGP, in presence or absence of the LPA5 receptor antagonist, was measured at 510 nm when exitated at 340 and 380 nm in a Fluorescence Spectrophotometer at 37oC. The figure show original traces of the 340/380 nm ratio in one experiment.
Rho/rho-kinase signaling in platelets
Two different targets in the rho/rho-kinase signaling pathway were analyzed to confirm that AGP does induce a rho/rho-kinase signaling in platelets. The phosphorylation of MYPT-1 (Thr696) in response to 0.5 mg/ml AGP was slightly, but not significantly, increased at all time points measured compared to control without treatment but in 900 rpm stirring for 5 seconds, see figure 6. Phosphorylation of MLC-1 (Ser19) was also slightly, but not significantly increased in response to 0.5 mg/ml AGP, see figure 7.
13
Figure 6. Time-course of MYPT-1 Thr696 phosphorylation induced by 0.1 U/ml thrombin and 0.5 mg/ml AGP in platelets. MYPT-1 Thr696 phosphorylation measured by western blot and quantified in
Odyssey Fc Imaging system from LI-COR® Biosciences UK and analyzed in the software Image Studio Lite. Shown as mean ± SEM, ns = non-significant, i.e. p ≥ 0.05 compared to control, a.u. = arbitrary units (n=3).
Figure 7. Time-course of MLC Ser19 phosphorylation induced by 0.1 U/ml thrombin and 0.5 mg/ml AGP in platelets. MLC Ser19 phosphorylation measured by western blot and quantified in Odyssey Fc
Imaging system from LI-COR® Biosciences UK and analyzed in the software Image Studio Lite. Shown as mean ± SEM, ns = non-significant, i.e. p ≥ 0.05 compared to control, a.u. = arbitrary units (n=3).
14
Discussion
Besides its role as a carrier protein the physiological effects of AGP are today poorly
investigated in comparison with the massive extent of studies concerning the chemical structure of this protein and how it is modified during inflammatory disease. Since AGP is present in human serum obtained from healthy individuals at a higher concentration than most plasma proteins and still rises several fold in inflammatory conditions (1) it is necessary to investigate the effects this protein may elicit on different cell types. Endothelial cells, as well as different blood cells, will come in contact with this plasma protein and may be affected of it. From a pathophysiological perspective are both endothelial cells and platelets important in the development of atherosclerosis. Furthermore, atherosclerosis is also characterized by chronic inflammation and elevated levels of acute-phase proteins like AGP (4). Since AGP also have immunomodulating abilities and carries endogenous atherogenic lipids in the circulation the effects of this protein on endothelial cells and platelets are of significance to elucidate (6-9,12). The effect of the acute phase protein AGP on endothelial cells as well as on platelets are poorly investigated but could be of importance in several aspects of atherosclerosis, cancer and/or maybe in maintaining homeostasis in health.
There are quite few previous studies that have investigated how AGP may affect endothelial cells and their functions. What has been shown is that AGP exerts cAMP-dependent effects on endothelial cells (18), supports pro-angiogenic effect elicited by VEGF (19) and inhibits TNF-α induced angiogenesis on endothelial cells (19). These cell responses might be evolved by the glycosylation of AGP as in neutrophil granulocytes, or by lipids carried by AGP as indicated in platelets, or by the protein itself. The action of AGP is induced by different receptors and signaling pathways in platelets and neutrophil granulocytes (5-7) but in endothelial cells there is still no known receptor that mediates these effects by AGP.
The results of the cell function analyses made in this present study indicate that AGP might affect migration ability and tube formation ability of HUVEC towards an enhanced migration and tube formation in a non-significant trend. However the number of viable cells did not change in response to AGP, indicating that AGP does not affect HUVEC proliferation. This is in line with the data of one previous study which show increased migration and tube formation but no alteration of proliferation in response to AGP (20). The trend of slightly enhanced tube formation in this study does also correlate to some of the results in another study that have investigated the regulatory abilities of AGP in both developmental and injury induced angiogenesis. In that previous study it was shown that AGP blocks the early pro-angiogenic effects elicited by TNF-α,
15
but also enhance the later pro-angiogenic effects mediated by VEGF (17). However in the present study there were no detectable differences when AGP was combined with TNF-α. The diverse results in this study between the slightly enhanced migration and tube formation in comparison with the unaltered proliferation may indicate different molecular mechanisms by which AGP affects these different cell functions. In the models used in this study to investigate angiogenesis in vitro is the ability to migrate much more important to be able to form tubes than the proliferative response.
In platelets, it has previously been shown that AGP induces a shape change and that this effect involves the rho/rho-kinase signalling pathway (7). In the present study there is a non-significant trend towards activated rho/rho-kinase signalling in response to AGP. In neutrophil granulocytes it has previously been shown that AGP results in an increase of intracellular Ca2+ concentration via binding of the sialic acids present on AGP to the Siglecs. In platelets AGP induce a minor intracellular Ca2+ response but the mechanisms which mediate this effect is still unclear (5). In the present study AGP-treatment also resulted in a small intracellular Ca2+ increase in platelets and this was partially inhibited by the LPA5 receptor antagonist which indicate that lipids like LPA, carried by (and released from) AGP in the circulation, might be involved in the interactions between AGP and platelets.
Since the results in this present study only indicate a non-significant trend of enhanced migration and tube formation in HUVEC and a non-significant trend of activated rho/rho-kinase signaling in platelets, more research needs to be done in order to fully evaluate the effect of AGP on endothelial cells and platelets. In this study only physiological or sub-physiological doses of AGP was used so the effects might be different in pathophysiological doses. Furthermore the effects of AGP may be altered in pathophysiological conditions due to changes in glycosylation pattern.
In conclusion this study did not show any significant differences after AGP-treatment in physiological doses on endothelial cells or platelets, but there is still a trend of enhanced migration and tube formation in HUVEC and a trend of enhanced rho/rho-kinase signalling in platelets. In platelets there are also indications of involvement of LPA5-receptor in the raise of cytosolic Ca2+ in response to AGP. Taken together this indicates that AGP might be involved in parts of regulating angiogenesis and haemostasis.
16
Acknowledgements
I would like to thank my supervisor professor Magnus Grenegård for giving me the opportunity to work with this project and for his contribution of wise comments along the road. I would like to give a special thank to Liza Ljungberg who have taught me a lot in the lab, and thank you Knut Fälker for introducing me to your platelet research. I would also like to thank professor Allan Sirsjö and all members of his group that I have shared lab and office space with.
17
References
(1) Blain PG, Mucklow JC, Rawlins MD, Roberts DF, Routledge PA, Shand DG. Determinants of plasma alpha 1-acid glycoprotein (AAG) concentrations in health. Br J Clin Pharmacol 1985 Nov;20(5):500-502.
(2) Fournier T, Medjoubi-N N, Porquet D. Alpha-1-acid glycoprotein. Biochim Biophys Acta 2000 Oct 18;1482(1-2):157-171.
(3) Sorensson J, Matejka GL, Ohlson M, Haraldsson B. Human endothelial cells produce orosomucoid, an important component of the capillary barrier. Am J Physiol 1999 Feb;276(2 Pt 2):H530-4.
(4) Jovicic S, Ignjatovic S, Dajak M, Kangrga R, Majkic-Singh N. Association of lipid and inflammatory markers with C-reactive protein in cardiovascular risk assessment for primary prevention. Clin Lab 2009;55(11-12):411-419.
(5) Gunnarsson P, Levander L, Pahlsson P, Grenegard M. The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs). FASEB J 2007 Dec;21(14):4059-4069.
(6) Gunnarsson P, Fornander L, Pahlsson P, Grenegard M. Sialic acid residues play a pivotal role in alpha(1)-acid glycoprotein (AGP)-induced generation of reactive oxygen species in
chemotactic peptide pre-activated neutrophil granulocytes. Inflamm Res 2010 Feb;59(2):89-95. (7) Gunnarsson P, Levander L, Pahlsson P, Grenegard M. alpha(1)-acid glycoprotein (AGP)-induced platelet shape change involves the Rho/Rho kinase signalling pathway. Thromb Haemost 2009 Oct;102(4):694-703.
(8) Mestriner FL, Spiller F, Laure HJ, Souto FO, Tavares-Murta BM, Rosa JC, et al. Acute-phase protein alpha-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism. Proc Natl Acad Sci U S A 2007 Dec 4;104(49):19595-19600. (9) Chiu KM, Mortensen RF, Osmand AP, Gewurz H. Interactions of alpha1-acid glycoprotein with the immune system. I. Purification and effects upon lymphocyte responsiveness.
Immunology 1977 Jun;32(6):997-1005.
(10) Aburima A, Wraith KS, Raslan Z, Law R, Magwenzi S, Naseem KM. cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway. Blood 2013 Nov 14;122(20):3533-3545.
(11) Schonfeld DL, Ravelli RB, Mueller U, Skerra A. The 1.8-A crystal structure of alpha1-acid glycoprotein (Orosomucoid) solved by UV RIP reveals the broad drug-binding activity of this human plasma lipocalin. J Mol Biol 2008 Dec 12;384(2):393-405.
(12) Ojala PJ, Hermansson M, Tolvanen M, Polvinen K, Hirvonen T, Impola U, et al. Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a
complementary role to albumin in the scavenging of lysophosphatidylcholine. Biochemistry 2006 Nov 28;45(47):14021-14031.
18
(13) Nishi K, Maruyama T, Halsall HB, Handa T, Otagiri M. Binding of alpha1-acid
glycoprotein to membrane results in a unique structural change and ligand release. Biochemistry 2004 Aug 17;43(32):10513-10519.
(14) Nishi K, Sakai N, Komine Y, Maruyama T, Halsall HB, Otagiri M. Structural and drug-binding properties of alpha(1)-acid glycoprotein in reverse micelles. Biochim Biophys Acta 2002 Dec 16;1601(2):185-191.
(15) Williams JR, Khandoga AL, Goyal P, Fells JI, Perygin DH, Siess W, et al. Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation. J Biol Chem 2009 Jun 19;284(25):17304-17319.
(16) Rantapaa-Dahlqvist S, Wallberg-Jonsson S, Dahlen G. Lipoprotein (a), lipids, and lipoproteins in patients with rheumatoid arthritis. Ann Rheum Dis 1991 Jun;50(6):366-368. (17) Seman LJ, DeLuca C, Jenner JL, Cupples LA, McNamara JR, Wilson PW, et al. Lipoprotein(a)-cholesterol and coronary heart disease in the Framingham Heart Study. Clin Chem 1999 Jul;45(7):1039-1046.
(18) Sorensson J, Ohlson M, Bjornson A, Haraldsson B. Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine. Am J Physiol Heart Circ Physiol 2000 May;278(5):H1725-31.
(19) Ligresti G, Aplin AC, Dunn BE, Morishita A, Nicosia RF. The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time- and context-dependent inhibitory and stimulatory properties. PLoS One 2012;7(8):e41387.
(20) Irmak S, Oliveira-Ferrer L, Singer BB, Ergun S, Tilki D. Pro-angiogenic properties of orosomucoid (ORM). Exp Cell Res 2009 Nov 1;315(18):3201-3209.
