This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
The vascular endothelium is an active endocrine organ that controls activities in the vessel wall within the vascular lumen. Hypertension, lipid levels and endothelial lipase (EL) are risk factors for vascular diseases, which can damage the endothelial function this in return can lead to the development of atherosclerotic vessels. EL recently described as a member of triglyceride lipase gene family; is synthesized by endothelial cells (EC). The advent of statins has be proven to have great impact on reducing theses risk factors, which in return cause improvement of endothelial function, thus have beneficial effects in preventing vascular disease. Statins also known as HMG-CoA reductase inhibitors are designed to reduce plasma cholesterol levels. In this study, we investigate wither staitns have an effect on down regulating EL expression in ECs. HUVEC-c cells were cultured in M199 media and treated with TNF-α and/or simvastatin, fluvastatin and pravastatin at different concentrations of 40µM & 20µM. Treatment with TNF-α alone increased EL expression whereas, TNF-α/statins decreased EL expression, this was partially due to the inhibitory effect on NFκB. THP-1 cells were grown in suspension and were treated with PMA, which upregulated EL expression. EL expression was identified by using western blotting analysis which demonstrated EL expression at the molecular weight of 57kDa. From the results obtained, we conclude that statins do have an effect in lowering EL expression by reducing the activation of NFκB.
1.1 Endothelial cells
Endothelial cells (ECs) are specialized squamous ECs that line the interior of the lumen of blood vessels. The EC surface in an adult human is approximately 1 x 1013 forming an almost 1 kg organ and are established as a major organ system (Cines et al., 1998). ECs are heterogeneous population and their properties are dependent on their location (Hogg et al., 2002). They form cobblestone monolayer within large vessels that lines a tube of connective tissues. On the basis of morphological and functional characterization, three types of junctions have been described in EC (Dejana et al., 1995; Schnittler, 1998), tight junctions, adherens junctions and gap junctions.
Endothelium heterogeneity is confirmed in a number of pathological conditions such as, atherosclerotic plaque formation in lager arteries and venule specific leukocyte adhesion (Ribatti et al., 2002). Improvements in understanding the function of ECs have urbanized from the development of technologies that allow the growth of pure cultures of ECs. EC derived from human umbilical veins were first successfully cultured in vitro in 1973 (Nachman and Jaffe, 2004).
1.2 Endothelial cells structure
The vascular endothelium serves as an important autocrine, paracrine and endocrine organ, they are considered to provide a non-thrombogenic lining to blood vessels (He et al., 2003). This is due to the continual production of growth inhibitory substances and anti-coagulants. It has been established that endothelium plays a major role in number of physiological processes for e.g. the regulation of vascular tone, cell growths and haemostasis, also play an important role in pathological process such as immune and inflammatory response (Villar et al., 2006).
1.3 Regulation of vascular tone
EC release vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) which relate to the regulation of blood pressure and blood flow. Vasoconstrictors are also released, including endothelin (ET) and platelet-activating factor (PAF) (Vallance, 1992).
These chemically diverse compounds are regulated by specific receptors on vascular cells, through their rapid metabolism, or at the level of gene transcription. EC constitutively secreted NO, due to the activity of enzyme endothelial nitric oxide synthase (eNOS). But its production is modulated by a number of exogenous chemical and physical stimuli, such as shear stress which has been thought to be responsible for the continual production of NO and the maintenance of vascular tone (Vallance and Chan, 2001). Whereas the other known mediators (PGI2, ET, and PAF) are synthesized mainly in response to changes in the external environment. (Cines et al., 1998) .
1.4 Regulation of haemostasis
The regulation of haemostasis requires coordinated interaction between varied cells types including inflammatory cells, platelets, plasma proteins ect. By release of mediators such as NO, thrombomodulin,PGI2 and heparan sulphate, can maintain endothelium non-thrombogenic integrity (Wu and Thiagarajan, 1996). The endothelium has the ability to convert the pro-thrombin to thrombin, though process of endothelial matrix which contains heparin sulpahte, this helps to promote the activity of plasma anti-thrombin III however, the constitutively expressed thrombomodulin binds to thrombin and limits its action (She et al., 2007).
1.5 Leukocytes recruitment
Up regulation of adhesion molecules on EC surface, is one of the main characteristics of activation of EC. The recruitment of leukocytes to endothelium has been familiarized as the cellular hallmark of the inflammatory response. Inflammatory process is a vital response however, wrong location or timing can lead to serious diseases, such as atherosclerosis. To prevent unnecessary inflammatory responses one mechanism is the regulation of cell adhesion molecules expression. ICAM-1 is an adhesion molecule that can constitutively be expressed, on the other hand molecules such as E-selectin are only expressed to cytokines such as TNF-α (Carlos and Harlan, 1994). EC adhesion molecules can initiate signalling cascade which requires leukocyte transmigration. Selectins however, are weak adhesion molecules and are not sufficient for transmigration as they are responsible for the rolling of leukocytes along endothelium (McEver et al., 1995). It has been proven that expression of EC adhesion molecules has been adjusted by statins, therefore suggesting that it has an inhibitory effect (Greenwood and Mason, 2007). Adhesion molecules have a key role in inflammatory response therefore the ability to modulate they expression by endothelium is a huge clinical interest.
1.6 Endothelial lipase (EL)
Triglyceride lipase gene family have introduced a new member EL, which included lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%) (Choi et al., 2002). EL is synthesized by EC they functions at this site. Like LPL and HL, EL has greater phospholipase activity. Maugeais and group have suggested that EL is an important modulator of HDL concentrations (Maugeais et al., 2003). It is recognized that lipases can form a molecular bridge between lipoproteins and ECs or macrophages through interaction with heparan sulfate proteoglycans. This action of lipases can increase monocyte adhesion (Ishida et al., 2003). It has also been suggested that EL is induced by inflammatory signals raises the possibility that it may play a role in proinflammatory states, such as atherosclerosis (Paradis et al., 2006).
1.7 Endothelial dysfunction & atherosclerosis
Endothelial dysfunction is a physiological damage of endothelium. In 1986, Ludmer first demonstrated that in human there was an imbalance between vasoconstricting and vasodilating substances which was produced by the endothelium. ECs injury is a key of many pathological states including atherosclerosis, thrombosis etc, it releases nitric oxide (NO) however, lack of NO in atherosclerotic vessels contribute to damaged platelet aggregation, increased vascular smooth muscle proliferation, vascular relaxation and enhanced leukocyte adhesion to the endothelium (Endemann and Schiffrin, 2004).
EC has role in maintain vascular homeostasis therefore, any damage of endothelial integrity could be considered a cause of atherosclerosis. Atherosclerosis is known as a chronic inflammatory disease that is initiated by the interaction of monocytes with activated ECs, resulting in monocyte recruitment to the arterial wall. It is a disease of the large arteries and is the primary cause of heart disease and stroke. Epidemiological studies have suggested that atherosclerosis, is the major causes of death in developing countries, and it results in about 50% of all deaths in westernized societies (Strong, 1992; Strong et al., 1995).
Statins are the most powerful and effective drugs available, that safely reduce blood cholesterol levels. Statins exert their action by inhibiting the enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase which is the rate-limiting step in cholesterol synthesis, that catalysis the conversion of HMG-CoA into mevalonate acid. Therapeutic use of staitns have been shown to have reduced the risk of vascular diseases such as, atherosclerosis as they inhibitory effect can decrease circulating LDL-cholesterol by 30-70% (Wierzbicki and Mikhailidis, 2002).
Fluvastatin is the first completely synthetic statin with anti-oxidative properties. Apart from its cholesterol-lowering effect, they can also have other effects such as anti-inflammation and the reduction of myocardial infarct size. It has been suggested that pravastatin may reduce risk of coronary artery disease by reducing inflammation rather than, lowering levels of cholesterol. Simvastatin is one of the most effective drugs for lowering cholesterol levels. It also lowers HDL cholesterol more than the other statins (2009).
In addition to their cholesterol-lowering properties, statins exert a pleiotropic effect (Davignon, 2004) however, these drugs also control other mechanisms, such as glutamate metabolism and NO, angiogenesis, inflammation, platelet aggegration, immune system and apoptosis. In 1997, a study demonstrated that statin treatment can improve endothelial function, by increasing the synthesis of NO from EC.
1.9 AIMS & OBJECTIVES
This study will determine whether statins have an effect in restoring endothelial function by lowering cholesterol levels. THP-1 cell will be used and will be grown in suspension, they will be treated with PMA to differentiate into macrophage. Furthermore, HUVEC-c cells will also be used and grown in a monolayer with stimulation of TNF-α alone and/or with statins. The EL expression in response to TNF-α and/or simvastatin, fluvastatin and pravastatin in HUVEC-c cells, will be studied using western blotting analysis to determine EL expression at a molecular weight of 57kDa. The outcome of this study is to clearly understand, the effects statins have on EL, do they down regulating EL expression?
2.0 MATERIALS AND METHODS
All used tissue culture reagents were of tissue culture grade. Endothelial cell (HUVEC-c) medium M199 (cat no-039k2357) and Phorbol, 2-myristate, 13-acetate (PMA) both purchased from Sigma- Aldrich Company (Ayshire, UK). THP-1 cell medium (RPMI, medium 1640) obtained from Gibco.
The two tissue culture plastics used were 75cm2 flasks and 6- well culture plates was purchased from Corning Costar (High Wycombe, Bucks, UK). Disposable sterile pipettes of volume 5, 10, and 25ml were obtained from Sarstedt (Leicester, UK).
Cell culture was carried out in a Walker class II Laminar flow cabinet. Cultures were maintained in a Heraeus 6000 incubator at 37oC in an atmosphere of 5% C02, 95% air. Inverted microscope (Olympus CK2, Optical CO. LTD, Japan).
Fluvastatin (cat no-344095), pravastatin (cat no-524403) and simvastatin (cat no-567020) were purchased from Calbiochem, UK. Tumour necrosis factor-α (TNF-α) (cat no- T0157) purchased from Sigma Company
All reagents used for SDS-PAGE and western blotting were carried out using a BIORAD mini protean II gel kit and a BioRad mini transblotter. X-ray film was obtained from Amersham (Kodak). Benchmark pre-stained protein ladder (cat no-10748-101) was from Invitrogen. Dye reagent concentrated (cat no-500-00006) was obtained from Heidemannstratebe, Munchen. Nitrocellulose membrane was purchased from Sartorius (Goettinggen, Germany). Rabbit polyclonal anti-human EL antibody (cat no-A1906) was obtained from Santa Cruz, CA, USA. Anti rabbit IgG conjugated to horseradish peroxidise conjugate (NA931V) was obtained from GE healthcare UK Limited, Buckinghamshire, UK.
NaCl, Tris base, glycine, methanol , TEMED, ammonium per sulphate, dH20, hydrogen peroxide, bovine serum albumin (BSA) (cat no-500-0007), N, N'-methylene-bis-acrylamide, Tween 20, trizma hydrochloride, sodium pyrophosphate, EDTA, SDS (sodium lauryl sulphate), bromophenol blue and glycerol.
2.2.1 Cell culture
The human umbilical vein endothelial cell line C (HUVEC-c) was grown in M199 growth medium , with the addition of 10% fetal calf serum (FCS), penicillin (100U/ml), streptomycin (100mg/ml), L- glutamine and fungizone in T75 flasks and was incubated in an atmosphere of 95oC air under 5% CO2 at 37%. After initial plating out the, medium was changed 24h later to remove dead cells, the trypsin traces and was subsequently changed 48h.
The human leukaemic cell line (THP-1) cells grew as a suspension and formed loose clusters. They were maintained at 37oC under 5% CO2 and 95% O2 at concentration of 1.25 x 106 cells/ml in RPMI-1640 containing 10% FCS, penicillin/streptomycin (100U/ml,100mg/ml) , 1mM sodium pyruvate and 15% sodium bicarbonate. Medium was changed twice a week by simply diluting the cells with fresh medium, by adding 10ml of RPMI medium to 20ml of THP-1 cells.
2.2.2 Cell counting
To determine total cell count and viability, 0.2% trypan blue was used. Cells that have taken up the dye were easily identified, when being counted on haemocytomete, due to their intense blue colour. Haemocytometer was loaded (total area 4mm2) and the cells counted immediately. Cell concentration was then determined using the following calculations:
2.2.3 Expansion of cell culture
Cell culture carried out in a Laminar flow cabinet. Upon confluence at a ratio of 1:3 was maintained during the expansion of cultures and each time HUVEC-c were spilt, they were assigned to as passage 1 cells. Medium was removed and the cells were rinsed with 2.5ml of warm (37OC) PBS, then treated with 2-3ml of trpsin/EDTA of 0.05%/0.02% to allow detach of the cells, upon detachment trypsin was removed.12ml of fresh M199 was added, and the cells were equally re-suspended in T-75 flasks and incubated at 37oC. The medium was subsequently changed every 48h.
2.2.4 Effect of cytokines & statins on HUVEC-c
A stock solution of fluvastatin, pravastatin and simvastatin at a concentration of 1mM was prepared in PBS (37oC) and 50µl aliquots were stored at -20oC. Confluent cells (7.2 x 105 cells/ml ) were re-suspended in 2ml of M199, and were transferred to 6-well plate (see appendix 1), and incubator at 37oC for 24h for the cells to form monolayer. The final concentration of the statins was 40µM/well.
After 24h of incubation, 10µl of a stock solution of TNF-α was diluted in 990ml of PBS. 24µl of the mixture was placed into wells 2-5 and were left for 24h in incubator. The final concentration of TNF-α in the wells were 120U/ml.
THP-1 cells grow in suspension in 20ml, 1µl of a stock PMA solution at concentration of 50nM, were mixed with 20ml of THP-1 cell suspension, form this mixture 2ml were pipette out and were plated into 4 wells of a 6-well plate. PMA was left to treat for 48h in incubation. (THP-1-1.25 x 106 cells/ml, 20ml)
2.2.6 Sample preparation for SDS-PAGE
Once the HUVEC-c cells were confluent in the 6-well, the cells were immediately placed on ice to stop the reactions and aspirated. The wells were then rinsed twice with 2.5ml of PBS (-4oC) to detach the cells. 300µl of hot (65oC) Laemmli buffer (see appendix 2) was prepared for western blot analysis and wells 1-5 were used. Clear Laemmli buffer was prepared without bromophenol blue and DTT for protein assay and well 6 (medium alone) was used. The cells were harvested using a cell scraper and collected into Eppendorfs by using a syringe. Samples were boiled (95oC) for 5min and stored at -20oC until analysis.
THP-1 cells were placed on ice and harvested using a cell scraper for 1-2min. They were than centrifuged at 560g for 5min. The supernatant was pipetted out, 1ml of PBS was added to the cell pellet. Cells suspension was centrifuged again. Finally, the cell pellet was re-suspended in Laemmli buffer as described above. Samples were then sonicated (3x) for 5s, then were boiled (95oC) for 5min and stored at -20oC.
2.2.7 Determination of protein concentration
The BioRad dye was diluted at a dilution of 1in 4 in dH20. 2.5ml of dye was prepared by mixing 7.5ml of dH20 and was then filtered. BSA standards were also prepared in dH20 (0-1mg/ml), 2.5ml of diluted dye was added to 50µl of each standard labelled 1-5. 50µl of the samples from each cell lysates were added to 2.5ml of diluted dye. 50µl standard/sample to be determined was assayed in duplicate. They were boiled for 5min then placed back on ice. The absorbances at a wavelength 595nm were determined.
2.2.8 SDS-PAGE & Western blotting
A BioRad mini protean gel electrophoresis kit was used to run all gels.10% resolving gel appendix 3) was poured between two plates and overlaid with 0.1% SDS solution. 10% stacking gel (see appendix 3) was poured on top. Polymerized gels were placed in Perpex tank containing running buffer (see appendix 3). 30µg of protein was loaded per well. 5µl of pre-stained protein ladders were also loaded. Samples were electrophoresed at 100V for 2h.
Gels were blotted onto nitrocellulose membranes for 1 hour at 100 volts. After 1 hour blocking buffer were prepared on membranes and were incubated with anti-human EL polyclonal antibody (1:1000 dilution1 in 0.2% of BSA in NATT) overnight at 4°C and then incubated with anti-rabbit IgG HRP-conjugated antibody (1:1000 dilution in 0.2% of BSA in NATT) for 1 hour (see appendix 3 for more detail).
2.2.9 Detection of protein by enhanced chemiluminescence (ECL)
After 4-5 times of vigorous wash the blot was soaked in equal amounts of ECL 1 and 2 stirred for 90s, then were exposed to X-ray film and incubated in dark for 5-10min. The bands visualized by immersing the x-ray film in developer (see appendix 4), wash with tap water, placed in fixer solution. Finally rinsed with tap water and left for air dry.
Imagej was used to for quantitative measurement of intensity of EL band. Program adapted from: http://rsbweb.nih.gov/ij/download.html
3. 0 RESULTS
3.1 Culture and morphology of THP-1
The human monocytic cell line THP-1 cell was first derived from the peripheral blood of a 1 year old male with acute monocytic leukaemia. THP-1 cells are most widely used cell line with properties similar to the properties of "human monocyte-derived macrophages". In this study THP-1 cells were grown in suspension, and were stimulated with PMA for 48h as shown in 1. The media was changed (section 2.2.1) twice a week. Cell extracts of THP-1 was used as a positive control.
3.2 Culture and morphology of HUVEC-c
HUVEC-c were grown in M199 medium as described in section 2.2.1. The cells were maintained in culture until a confluent monolayer was achieved, this occurred after 48hr. Cell splitting then took place as described in section 2.2.3. The cells displayed a typical confluent homogenous monolayer as shown in 2. Cells appeared polygonal in shape and exhibited cobblestone morphology. Only cells that grew as homogenous monolayers displaying the typical cobblestone morphology were expanded and used for experimental purposes.
2: A confluent monolayer of HUVEC-c after 48h in culture A typical example of confluent homogenous monolayer of HUVEC-c displaying the cobblestone morphology of EC. Cells appear to be densely packed, hyper confluent areas (arrow A) that have failed to exhibit contact inhibition. No EC expressed suggesting dead cell area (arrow B). Magnification x400
3.3 Determination of protein concentration
A protein assay was carried out in order to determine the protein concentration of HUVEC-c and THP-1 cell extracts which were used for western blotting, this was to allow the same amount of each sample to be loaded into each well of the SDS gel. A linear graph was produced as revealed in 3. The absorbance for each concentration of standard was plotted and a line of best fit was drawn. From this the absorbance of HUVEC-c and THP-1 samples were used to determine the protein concentration of each sample.
Result from protein assay using standard concentrations of BSA ranging from 0.05- 1mg/ml. The protein concentration of HUVEC-c obtained from the graph was 0.72mg/ml and for THP-1 the concentration was 0.8mg/ml (not illustrated).
3.4 Effect of TNF-α and statins on HUVEC-c
3.4.1 Analysis of EL expression in HUVEC-c by SDS-PAGE
To assess the expression of EL, HUVEC-c was prepared as described in section 2.2.4. On attaining confluence, the cells were treated to TNF-α (120U/ml) and/or different concentration of statins (40µM & 20µM) for period of 24h. Negative control cells were subjected to the same protocol but were incubated in media alone. In order to study effect of statins on EL in HUVEC-c an appropriate positive control was required to validate the methodology, THP-1 (1.25 X 106 cells/ml, 20ml) was used for this purpose, stimulated with PMA as described in section 2.2-6.
4A shows EL expression was visible in all 5 lanes as demonstrated by the band at 57kDa. Strong EL expression was noticeable in lanes 4 & 5 however, slightly weaker EL bands were shown in lanes 1 & 2. Lane 3 on the other hand, illustrated really weak EL expression when incubated with negative control.
s 4B-5B shows quantitative measurement of intensity of EL band which was performed by using imageJ analysis as described in section 2.2.10. Results are expressed as a percentage and compared to the negative control. The intensity of EL band was measured so that we can compare the intensity of EL expression, when treated with media + TNF-α, media + statin and/or TNF-α + statin.
5A visualizes dominant bands indicating expression of EL at 57kDa. Lanes 4 & 5 showed a strong band which were treated with THP-1 and TNF-α. Lane 3 illustrates a dark band but small amount of the band has not been exposed. A gradual increase in band was observed in lanes 1 & 2.
6A demonstrates a single clear band at 57kDa, with the exception of lanes 3 & 5. Unfortunately, the negative control band did not appear therefore, it was difficult to interpret the data. However, the positive band did come into view in lane 6 which showed a strong band but half of the band was diminished may be due to inappropriate loading. However, lanes 3 and 5 which were HUVEC-c incubated with lower concentration of statins 20µM, both of the bands were very difficult to establish any kind of EL expression as the clarity of the bands were really weak. Whereas, lanes 1, 2 and 4 (40µM) showed a dark band.
It was the aim of this study to investigate the effect of TNF-α and statins (pravastatin , fluvastatin and simvastatin) on EL expression by HUVEC-c. From the result of this study we established that TNF-α alone may be able to up regulate EL expression in HUVEC-c cells. Whereas TNF-α treated with statins, seemed to have down regulated EL expression.
4.1. Culture and characterisation of THP-1 cells
THP-1 cells were maintained in suspension in RPMI-1640 medium. In this study THP-1 cells were induced to differentiate into macrophages by treatment when with PMA for 48h, this has previously been reported by (Qiu and Hill, 2007).
Recent in vitro studies have shown that cell-cell contact between monocytes and EC has been reported to sustain adhesion molecules such as ICAM-1 and VCAM-1 expression for prolonged periods (Alevriadou, 2003). Such findings suggest that monocyte interactions with the endothelium can enhance leucocytes recruitment and activation and hence may have important implications in various in vivo situations which involve inflammatory events, such as aatherosclerosis.
4.2. Culture and characterisation of HUVEC-c cells
HUVEC-c cells were maintained in medium M199 supplemented. This has previously been shown to be essential to maintain the growth of HUVEC-c (Kojima et al., 2010) Kojima reported, an exponential increase in cell number was reported as early as day 1 and the mean doubling time was 48h.
Morphology represents an important criterion for the identification of EC in vitro and has been well documented. Confluent EC monolayers have been shown to display typical “cobblestone” morphology. Typical cytoplasmic architecture has been described for most cultures of EC i.e. round to slight ellipsoid nucleus, dense cytoplasm rich in organelles and a thin sheet of cytoplasm which forms complex interdigitation with neighbouring cells (Baudin et al., 2007).
4.3. EL expression by HUVEC-c
EL has been shown to play a potential role in altering lipoprotein metabolism in pro- inflammatory states, such as atherosclerosis. It was first shown that EL mRNA concentrations in cells were up regulated by TNF-α implicated in vascular diseases (Hirata et al., 2000). A study indicated that the triglyceride lipase and phospholipase activities of EC in response to TNF-α resulted in up regulation of EL expression (Jin et al., 2003).
TNF-α has received the most attention in this study. Results from 4A-6A show that stimulated HUVEC-c illustrated a strong dark band at the molecular weight of 57kDa for EL, compared to non-stimulation cells and this was supported by the bar graphs (5B-6B) that demonstrated that TNF-α increased EL expression up to 200%. Quertermous and colleagues suggested that, NFκB a protein known to play a central role in EC, by activation of TNF-α they can upregulate EL expression (Hirata et al., 2000). Therefore, this might be the suggestion of increased EL expression by TNF-α.
4.4. Effect of statins on HUVEC-C
The non-lipid-lowering effects of statins have been studied in numerous cell models for a wide range of biologic functions. However, very few studies on the effects of statins on lipase expression in EC have been addressed. We demonstrate in the present study by western blot analysis, that EL expression by HUVEC-c was reduced by treatment with different concentrations (40µM & 20µM) of statins.
The primary site of action of all statins is the liver, they block the synthesis of isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) as shown in 7 (Massaro et al., 2009) which have been shown to mediate the prenylation and subsequent activation of Rho proteins, as FPP and GGPP serve as lipid attachments for the posttranslational modification of a variety of proteins, which include small GTP-binding proteins that are part of Ras, Rho, Rap, and Rab GTPases family (Rikitake and Liao, 2005).
Previous reports have indicated that Rho negatively regulates NFκB, which appeared to express EL, which was influenced by NFκB. Statin treatment decreased EL expression by inhibiting NFκB which contributed to the ability of statins to migrate lipid accumulation in macrophage (Qiu and Hill, 2007).
A dominant band was observed at the expected size for EL, approximately 57kDa, in HUVEC-c cells (. 4A-6A). When cell lysates were electrophoresed, transferred to nitrocellulose membrane and probed with primary and secondary antibodies, a band was visualized. These visualized bands confirmed that EL protein was expressed in HUVEC-c and THP-1 cells.
4B suggest that TNF-α /simvastatin and TNF-α/fluvastatin at 40µM for 24h have decreased expression of EL by 40% and 35% compared to the negative control. Similarly 5B demonstrates a comparable effect to that in 4B however; there was a decrease of EL expression with pravastatin by 55%. Overall both suggest that when HUVRC-c were stimulated with TNF-α alone for 24h EL expression had increased but, when the cells were treated with stains at 40µM EL expression reduced, therefore suggesting that statins had an effect on HUVEC-c cells by inhibiting NFκB.
Unfortunately in 6A which represented fluvastatin and pravastatin at different concentrations 40µM & 20µM, the band for the negative control did not seem to have become visible, this may have been due to not accurately loading the sample into the well therefore, the data was difficult to interpret. However, lanes 3 & 5 both treated with 20µM of statins showed really weak expression again suggesting that samples not loaded accurately therefore, firm conclusions cannot be drawn from this results as it is difficult to interpret.
4.6. Limitations & suggestions
During the course of this study there were some limitations which occurred mainly due to western blotting techniques. From observing the bands in 4A, a large number of non- specific bands can be seen. This may have been due to variety of effects such as, non-specific binding of primary or secondary antibodies, inadequate blocking, and insufficient washing or overexposed film. To overcome non-specific binding of the antibodies we could use mono-specific antibodies (such as R&D Systems "AF" designated antibodies). Or we could use blocking in 5% milk powder, instead of 10% BSA, otherwise, we could decrease antibody concentration. To overcome insufficient washing problem we could do the overnight blocking at 4°C which may decrease blocking efficiency since detergents might not be effective at lower temperatures.
Faint bands (weak signal) were observed in 6A (lanes 3 & 5), this may have been due to low protein-antibody binding thus can reduce the number of washes to minimum, insufficient antibody then could overcome by increasing antibody concentration (2-4 fold), inadequate sample loading, so could increase the amount of loading from 30µl-35µl.
Unfortunately there was no band present for negative control in 6A, this may have been due to the fact that poor transfer of nitrocellulose membrane in blocking buffer, to overcome this we must make sure that there is good contact between nitrocellulose membrane and gel. Or it may have been a problem of over transfer therefore can reduce voltage or time of transfer to about 90V for 80min.
4.7. Conclusion & further studies
Results of pervious work will open the way for more experiments to study the possible mechanism behind such effects of statins on EL in HUVEC-c cells. As we know from previous studies, EL is a major determinant of HDL metabolism and is found in human atherosclerotic plaques (Jin et al., 2003). Statins have dramatically improved the treatment of dyslipidaemia and the prevention of atherosclerosis over the past 10 years. This statement has been proven by many studies, which has also been proven in present study, demonstrating the fact that statins can down regulate EL expression in EC.
In addition, other cholesterol lowering drugs, which have lead to further improvements in vascular functions such as fibrate which, are drugs that are used in the treatment of patients with hypertriglyceridaemia. Fibrate therapy results have shown to significantly decrease triglycerides and increase HDL-c levels (Elisaf, 2002). Therefore, indicating that it may have potentials to down regulate EL expression in ECs. However, there has not been a significant amount of evidence provided to support this therefore, more work and research needs to be done in order to prove that expression of EL mRNA in ECs can down regulate after fibrate treatment.
To determine the cellular expression of EL mRNA in EC we can perform RT-PCR, one of its major uses is to quantify gene expression by analysing mRNA levels. EL can be achieved by detection of a fluorescent reporter such as, SYBR® green dye or TaqMan® probes. Northern blotting technique can also be used which similarly recognises mRNA by homologous base-pairing. Finally immunoprecipitation can be taken in to account to study the amount of EL expression. These techniques could help explain how fibrate drug works and may provide ways to improve similar drugs in the future.
Perhaps future clinical trials comparing the effects of statins alone versus statins with fibrate may help address the question regarding the relative contribution of lipid lowering by statins to vascular protection. Overall, it remains to be determined, which of these effects are more major, in terms of clinical outcome, in patients with low cholesterol levels. Therefore more epidemiology work needs to be done in order to demonstrate this point.
Overall, in agreement with other investigators, we proved that it may be possible to use statins to down regulate EL expression in HUVEC-c cells, by reducing the activation of NFκB which in return preventing vascular disease states.
(2009). Statin therapy may do much more than lower cholesterol. Research shows statins can lower the odds of cardiovascular events among low-risk heart patients, but the drugs aren't for everyone. Heart Advis 12, 1, 11.
Alevriadou, B.R. (2003). CAMs and Rho small GTPases: gatekeepers for leukocyte transendothelial migration. Focus on "VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration". Am J Physiol Cell Physiol 285, C250-252.
Baudin, B., Bruneel, A., Bosselut, N., and Vaubourdolle, M. (2007). A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2, 481-485.
Carlos, T.M., and Harlan, J.M. (1994). Leukocyte-endothelial adhesion molecules. Blood 84, 2068-2101.
Choi, S.Y., Hirata, K., Ishida, T., Quertermous, T., and Cooper, A.D. (2002). Endothelial lipase: a new lipase on the block. J Lipid Res 43, 1763-1769.
Cines, D.B., Pollak, E.S., Buck, C.A., Loscalzo, J., Zimmerman, G.A., McEver, R.P., Pober, J.S., Wick, T.M., Konkle, B.A., Schwartz, B.S., et al. (1998). Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91, 3527-3561.
Davignon, J. (2004). Beneficial cardiovascular pleiotropic effects of statins. Circulation 109, III39-43.
Dejana, E., Corada, M., and Lampugnani, M.G. (1995). Endothelial cell-to-cell junctions. FASEB J 9, 910-918.
Elisaf, M. (2002). Effects of fibrates on serum metabolic parameters. Curr Med Res Opin 18, 269-276.
Endemann, D.H., and Schiffrin, E.L. (2004). Endothelial dysfunction. J Am Soc Nephrol 15, 1983-1992.
Greenwood, J., and Mason, J.C. (2007). Statins and the vascular endothelial inflammatory response. Trends Immunol 28, 88-98.
He, H., Shirota, T., Yasui, H., and Matsuda, T. (2003). Canine endothelial progenitor cell-lined hybrid vascular graft with nonthrombogenic potential. J Thorac Cardiovasc Surg 126, 455-464.
Hirata, K., Ishida, T., Matsushita, H., Tsao, P.S., and Quertermous, T. (2000). Regulated expression of endothelial cell-derived lipase. Biochem Biophys Res Commun 272, 90-93.
Hogg, D.S., McMurray, G., and Kozlowski, R.Z. (2002). Endothelial cells freshly isolated from small pulmonary arteries of the rat possess multiple distinct k+ current profiles. Lung 180, 203-214.
Ishida, T., Choi, S., Kundu, R.K., Hirata, K., Rubin, E.M., Cooper, A.D., and Quertermous, T. (2003). Endothelial lipase is a major determinant of HDL level. J Clin Invest 111, 347-355.
Jin, W., Sun, G.S., Marchadier, D., Octtaviani, E., Glick, J.M., and Rader, D.J. (2003). Endothelial cells secrete triglyceride lipase and phospholipase activities in response to cytokines as a result of endothelial lipase. Circ Res 92, 644-650.
Kojima, Y., Ishida, T., Sun, L., Yasuda, T., Toh, R., Rikitake, Y., Fukuda, A., Kume, N., Koshiyama, H., Taniguchi, A., et al. (2010). Pitavastatin decreases the expression of endothelial lipase both in vitro and in vivo. Cardiovasc Res.
Massaro, M., Zampolli, A., Scoditti, E., Carluccio, M.A., Storelli, C., Distante, A., and De Caterina, R. (2009). Statins inhibit cyclooxygenase-2 and matrix metalloproteinase-9 in human endothelial cells: anti-angiogenic actions possibly contributing to plaque stability. Cardiovasc Res.
Maugeais, C., Tietge, U.J., Broedl, U.C., Marchadier, D., Cain, W., McCoy, M.G., Lund-Katz, S., Glick, J.M., and Rader, D.J. (2003). Dose-dependent acceleration of high-density lipoprotein catabolism by endothelial lipase. Circulation 108, 2121-2126.
McEver, R.P., Moore, K.L., and Cummings, R.D. (1995). Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem 270, 11025-11028.
Nachman, R.L., and Jaffe, E.A. (2004). Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest 114, 1037-1040.
Paradis, M.E., Badellino, K.O., Rader, D.J., Deshaies, Y., Couture, P., Archer, W.R., Bergeron, N., and Lamarche, B. (2006). Endothelial lipase is associated with inflammation in humans. J Lipid Res 47, 2808-2813.
Qiu, G., Ho, A.C., Yu, W., and Hill, J.S. (2007). Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion. J Lipid Res 48, 385-394.
Ribatti, D., Nico, B., Vacca, A., Roncali, L., and Dammacco, F. (2002). Endothelial cell heterogeneity and organ specificity. J Hematother Stem Cell Res 11, 81-90.
Rikitake, Y., and Liao, J.K. (2005). Rho GTPases, statins, and nitric oxide. Circ Res 97, 1232-1235.
Schnittler, H.J. (1998). Structural and functional aspects of intercellular junctions in vascular endothelium. Basic Res Cardiol 93 Suppl 3, 30-39.
She, M., McGuigan, A.P., and Sefton, M.V. (2007). Tissue factor and thrombomodulin expression on endothelial cell-seeded collagen modules for tissue engineering. J Biomed Mater Res A 80, 497-504.
Strong, J.P. (1992). Atherosclerotic lesions. Natural history, risk factors, and topography. Arch Pathol Lab Med 116, 1268-1275.
Strong, J.P., Malcom, G.T., and Oalmann, M.C. (1995). Environmental and genetic risk factors in early human atherogenesis: lessons from the PDAY study. Pathobiological Determinants of Atherosclerosis in Youth. Pathol Int 45, 403-408.
Vallance, P. (1992). Endothelial regulation of vascular tone. Postgrad Med J 68, 697-701.
Vallance, P., and Chan, N. (2001). Endothelial function and nitric oxide: clinical relevance. Heart 85, 342-350.
Villar, I.C., Francis, S., Webb, A., Hobbs, A.J., and Ahluwalia, A. (2006). Novel aspects of endothelium-dependent regulation of vascular tone. Kidney Int 70, 840-853.
Wierzbicki, A.S., and Mikhailidis, D.P. (2002). Beyond LDL-C--the importance of raising HDL-C. Curr Med Res Opin 18, 36-44.
Wu, K.K., and Thiagarajan, P. (1996). Role of endothelium in thrombosis and hemostasis. Annu Rev Med 47, 315-331.
Treatment of statins in 6-well plate
Confluent cells from HUVEC-c ell concentration of 7.2 x 105 cells/ml were re-suspended in 2ml of M199 medium, and were transferred to 6-well plate and were labelled as followed:
Well 1: cells alone negative control: no statin or cytokines
Well 2: cytokine (TNF-a)
Well 3: TNF-a and pravastatin
Well 4: TNF-a and fluvastatin
Well 5: TNF-a and simvastatin
Well 6: Empty- cells alone
Stock solution of statins:
* In 1ml to 40µM in 1ml to 20µM
* 1000/40 = 25 dilution factor Or 1000/20 = 50 dilution factor
* In 2ml: 2000/25 = 80µl in 2ml: 2000/50 = 40µl
80µl (was added in experiment 1 and 2), 40µl (was added in experiment 3), of fluvastatin, pravastatin and simvastatin were added to the cells as shown above. The final concentration of the statins was 40µM and 20µM.
Laemmli buffer recipe:
300ml of hot (65oC) Laemmli buffer (pH 6.8) was prepared as followed:
* 50mM Tris-HCl
* 0.8mM sodium pyrophosphate
* 5mM EDTA
* 2% SDS
* 10% glycerol
* 0.1% bromophenol blue
* 50mM DTT (dithiothreitol)
SDS-PAGE gel description in more detail:
Resolution of proteins by SDS-PAGE
A BioRad mini transfer kit was used for the wet transfer of proteins to nitrocellulose membrane. Nitrocellulose membrane was pre-wet in blotting buffer for 15min each time. A transfer sandwich was prepared containing sponge, blotter paper, resolving gel and the pre-wet nitrocellulose membrane. This sandwich was inserted into a transfer tank filled with blotting buffer. The whole tank was engulfed with ice to prevent overheating and connected to the power supply at 100V for 90min.
Detection of protein
Once transfer was completed, the nitrocellulose membrane was placed in 20ml of blocking buffer of 10% of BSA in TBS and NATT buffer containing detergent (Tween 20) at concentration of 0.02%-0.05%, dependent on the protein being detected, it was left at room temperature for 2h, this allowed blocking of all non-specific proteins binding sites on the blot. The membrane was then incubated with the primary antibody (rabbit anti-human EL polyclonal antibody) diluted in 1:1000 in 0.2% of BSA in NATT buffer. This was left overnight at room temperature. The following day the membrane was washed by performing 4-5 room temperature washes (15 min each) with TBS and Tween to remove the antibody. The membrane was then incubated with the secondary antibody (anti-rabbit IgG HRP conjugated) diluted 1:1000 in 0.2% BSA in NATT buffer for 1h at room temperature. The antibody was removed and the blot subjected to 4-5 washes with NATT buffer.
SDS-PAGE gel recipe:
Resolving gel (10%):
Buffer 1 6ml
Buffer 1 (resolving gel) pH= 8.4
1.5M Tris Base
0.4% w/v SDS
Staking gel (10%):
Buffer 2 3.75ml
Buffer 2 (stacking gel) pH= 6.8
0.5M Tris Base
0.4% w/v SDS
SDS (for final conc of 0.1%)
NATT Buffer or TBS-T (Tris Base Salline-T) pH = 7.4
Luminal stock 1ml
Tris base (1M, pH=8.5) 10ml
Continue to 100ml with water
H2O2 (30%) 64µl
Tris base (1M, pH= 8.5) 10ml
Continue to 100ml with water
To check if that stock of ECL1 and 2 work properly:
Mix 2ml of ECL 1and 2ml of ECL 2 , then add 1-2µl of secondary antibody to the mixture. The mixture color should become yellowish.
250mM of Luminol (MW =177.2) in DMSO (0.866g in 20ml) stored at -20oC
p-Coumaric acid stock:
90mM p-Coumaric (MW=164.2) in DMSO (0.295g in 20ml) stored at -20oC
* Developing solution -50ml developer and 150ml of water
* fixer solution -50ml fixer and 150ml water