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In the present study we have identified that the expression of endothelial lipase in the endothelial hybrid cell line EAhy were significantly reduced by the statins (HMG-CoA reductase inhibitors), Simvastatin and Pravastatin. The positive control (THP-1) used in this study was based on previous work investigating the effects of Atorvastatin on endothelial lipase in THP-1 macrophages, therefore giving a result that can be hypothesised.
Using the cell extracts from human THP-1 macrophage as a positive control, it has been demonstrated that THP-1 cells synthesise endothelial lipase, as THP-1 cells showed a dark, dense band at the molecular weight point of 57kDa.(Figure 4a 5) Bartels et al investigated endothelial lipase mRNA in human atherosclerotic regions in THP-1 macrophages, where the expression of EL mRNA was increased, as quantified by the process of RT-PCR.(Bartels et al., 2007).In THP-1 macrophages, whilst investigating the consequence of EL on low density lipoprotein (LDL) binding and cell association, EL overexpression markedly increased intracellular lipid accumulation, portraying an accelerating role in foam cell formation. (Qiu and Hill, 2007b)
The result obtained from the western blot x-ray film showing the band which showed the expression of endothelial lipase on EAhy cells when treated with TNFa (Figure 4a 3)was thinner than the negative control (DMEM alone) and postitive control (THP-1 cells) bands. It has been reported that proinflammatory cytokines such as tumor necrosis factor-Î± and interleukin (IL)-1Î², have been shown to upregulate EL expression in endothelial cells and smooth muscle cells (Hirata et al., 2000), (Jin et al., 2003)The explanation for the weaker band seen when Eahy cells were treated by TNFa could be due to the fact that the human endothelial-like immortalised cell line EA.hy is derived from the fusion of HUVEC with the lung carcinoma cell line A549 (BARANSKA et al., 2005) and is not a primary endothelial cell. Lidington et al (1989) compared the endothelial cell lines HMEC-1, ECV304 and EaHy926 with human umbilical vein endothelial cells (HUVEC) and significant differences were found in the ability to respond to cytokines; the greatest difference being induction of VCAM-1 and E-selectin in response to TNF-alpha which were detectable in HUVEC but not detectable in the EAhy cells. Hirat et al (2000) showed that EDL mRNA levels were increased in HUVEC and human coronary artery endothelial cells (HCAEC) when stimulated by TNF-a and IL-1b. Fluid shear stress and cyclic stretch were found to increase EDL mRNA levels in endothelial cells and Northern blots were evaluated for EDL mRNA levels by hybridization showing a 6.5-fold overall increasing EDL levels, compared to the constitutively active control gene cyclophilin. These studies indicate that EL maybe closely associated with inflammation and involved in the development of atherosclerosis.
The endothelial lipase in Eahy cells, upregulated by TNFa was decreased by the statins (HMG-CoA reductase inhibitors), pravastatin and simvastatin, showing simvastatin to have the strongest effect in reducing endothelial lipase in EAhy cells (Figure 4a 1&2). There was not a massive reduction of endothelial lipase when the Eahy cells were treated with statins, however a slight reduction was observed in comparison to the positive control. The pleiotropic effects of statins have been studied in numerous cell models; however studies displaying the effects of statins on endothelial lipase are limited, with the first work on this topic to be done by the scientists Qiu and Hill (2007). In this study THP-1 macrophages were treated with a series of known activators (Rho and LXRa) and inhibitors (Rho) of the NF-KB pathway in the absence and presence of atorvastatin. It was demonstrated that NF-kB inhibition by SN50 was associated with a 30% reduction of EL expression with atorvastatin in human THP-1 macrophages, indicating that NF-kB plays an important role in EL expression with atorvastatin treatment (Qiu and Hill, 2007a) A recent study by Kojima et al (2010) showed similar results, however the effect of Pitavastatin on endothelial lipase were shown to be decreased in vitro and vivo. Pitavastatin was shown to suppress cytokine treated endothelial lipase expression in endothelial cells. Inhibition of RhoA activity by a Rho kinase inhibitor decreased endothelial lipase levels in endothelial cells and in mouse tissues, suggesting that Pitavastatin can reduce the expression of endothelial lipase by inhibiting the activity of RhoA in vitro and vivo(Kojima et al., 2010) This study revealed that Pitvastatin suppressed the cytokine treated endothelial lipase expression in endothelial cells supporting my findings ( Figure 4 1&2). Simvastatin was seen to have a much stronger affect in reducing endothelial lipase than pravastatin (Figure 4 1& 2) as total cholesterol was normalized in 77.8% of the patients (28 of 36) after simvastatin treatment and in 68.9% of the patients (23 of 36) after pravastatin treatment in patients with hypercholesterolemia, indicating that simvastatin is a stronger statin than Pravastatin. (Sasaki et al., 1997) These studies indicate that statins can reduce the expression of endothelial lipase that may be closely associated with inflammation and involved in the development of atherosclerosis.
It is clear from the experiment that endothelial lipase can be detected as a dark band were seen at the point of 57kDa (molecular weight of endothelial lipase), however non- specific bands were also displayed on the x-ray film (Figure 4a) which can be explained by saying that the non- specific bands may have been protein that is a different member of the same family. Non- specific bands can be avoided by reducing the concentration of the primary antibody, reduce the amount of protein loaded into gel (25µl) and increasing the number of washes (x5).
Limitations of experimental protocol
The first immunoblot assay of samples of EAhy cells with treatments of cytokine and statin produced an x-ray film with bands, indicating the band for endothelial lipase. However, unfortunately the second and third immunoblot assay showed a dark high background x-ray film showing no production of bands.
Certain aspects of the protocol were altered whilst the study was being carried out, in order for accurate results to be obtained. There was a decreased amount of protein loaded into the gel from 30 µl to 25µl to ensure that too much protein was not loaded into the gel. Also, the number of washes was increased from 4 to 5 and the samples were blocked overnight to decrease blocking efficiency. The exposure time of the x-ray film on the nitrocellulose membrane was decreased from 5 mins to 2mins, to ensure thst the x-ray film was not exposed for too long displaying a dark, high background. The remainder of the protocol stayed the same, except for the few new changes. Despite the new changes in the protocol no results were obtained in the next two immunoblot assays.
This study demonstrated that pravastatin and simvastatin reduced cytokine treated endothelial lipase expression in EAhy cells. Further studies using primary endothelial cells, e.g. HUVEC and other cytokines (IL-6) and statins need to be conducted to see if the same results are produced. Using fluid shear stress and cyclic stretch would give increased levels of endothelial lipase mRNA endothelial cells, so different techniques can be used to quantify EL mRNA such as Northern blots or RT-PCR to give a more accurate result in the amount of endothelial lipase expressed. Using RhoA inhibitors and NFkÎ² inhibitors which are known to inhibit endothelial lipase would allow us to characterise the mechanism by which statins reduce endothelial lipase in endothelial cells. The results from this study may help us to understand how to design better drugs against heart diseases such as atherosclerosis in the future, as such observations offers a wider understanding into the development of new therapeutic drugs used to reduce the occurrence cardiovascular diseases.