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Atherosclerosis, an inflammatory disease of the blood vessels, is the leading cause of death in the Western world. This condition results in the formation of plaques, which can lead to myocardial infarctions and strokes. Omega-3 fatty acids have been shown to promote good cardiovascular health and prevent atherosclerosis. They have also been shown to prevent obesity, a risk factor for cardiovascular disease. The typical American diet, though, is not sufficient in omega-3 fatty acids (anti-inflammatory) and is high in omega-6 fatty acids, which are pro-inflammatory. Mammals lack the ability to synthesize omega-3 fatty acids, but some lower organisms have the ability to convert omega-6 to omega-3 fatty acids via the fat-1 gene. Previous research has shown that mammalian cells can be infected with the fat-1 gene and perform this conversion. This study will focus on the microencapsulation of infected cells to be given orally to atherosclerotic mice.
First, a recombinant adenovirus containing the fat-1 gene will need to be constructed. This recombinant adenovirus will then be used to infect mouse cardiac myocytes, which will express the fat-1 gene. These infected cells will then be microencapsulated for oral delivery into ApoE-null mice. The ApoE-null mice will be put on a Western diet (high in cholesterol and fat), which will induce atherosclerosis. One group of mice will receive the microencapsulated fat-1 cells, while another group will be used as a control. Throughout the study, mice will be weighed and their blood tested for cholesterol and triglycerides. At the end of this 16-week study, the mice will be sacrificed and their aortas will be examined to determine the extent of atherosclerosis.
Background and Significance
Atherosclerosis is the leading cause of death in Western society. Atherosclerosis is an inflammatory disease, which results in the formation of plaques in the blood vessels. Myocardial infarctions and strokes can occur due to plaque rupture and thrombosis (Glass and Witztum, 2001). Risk factors for developing atherosclerosis include hypertension, hypercholesterolemia (Ross, 1999), obesity (Hubert et al., 1983), and genetics (Lahoz et al., 2001). The gene for Apolipoprotein E (ApoE), in particular, has been widely studied and linked to cardiovascular disease (Lahoz et al., 2001).
Omega-3 fatty acids can help reduce the incidences of cardiovascular events such as arrhythmias, inflammation, hypertension, and atherothrombosis (Masson et al., 2007; Marik and Varon, 2009). In addition, omega-3 fatty acids can help lower heart rate and blood pressure, which helps lower the risk of cardiovascular disease. Omega-3 fatty acids can also reduce triglyceride levels in blood serum, the formation of blood clots, and irregular heartbeats (Hooper et al., 2006). The omega-3 fatty acids eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) are precursors to certain eicosanoids such as prostaglandins, thromboxanes, and leukotrienes. These eicosanoids are responsible for some of the cardiovascular benefits of omega-3 fatty acid consumption. They are anti-inflammatory, antithrombotic, and antiarrhythmic compounds (Covington, 2004).
In addition, omega-3 fatty acids can help prevent/treat cardiovascular disease by preventing and alleviating obesity. Omega-3 fatty acids can help prevent obesity by effecting lipid metabolism. They can reduce lipid uptake by suppressing lipoprotein lipase, an enzyme that hydrolyzes triglycerides. Omega-3 fatty acids, specifically EPA, promote oxidation of mitochondrial fatty acids. In addition, omega-3 fatty acids can decrease lipid synthesis by inhibiting fatty acid synthase, which is involved in the synthesis of fatty acids (Li et al., 2008). Previous research has shown that the omega-3 fatty acid EPA has the potential to lower lipid concentrations in blood serum (Mitsuyoshi et al., 1991). Animal studies have shown that supplementing a high-fat obesity-inducing diet with omega-3 fatty acids can reduce body fat accumulation. In other animal studies, scientists have shown that supplementing an obese mouse's diet with omega-3 fatty acids caused a loss in body weight (Buckley and Howe, 2009). The typical American diet, though, does not contain a sufficient amount of omega-3 fatty acids, but instead is high in omega-6 fatty acids (Simopoulos, 2002). The ratio of omega-6 to omega-3 fatty acids in the typical American diet is 14:1 (Marik and Varon, 2009). One way to address this problem is to convert these omega-6 fatty acids into omega-3 fatty acids.
Mammals lack the ability to convert omega-6 fatty acids to omega-3 fatty acids, but some lower organisms possess this ability. The roundworm Caenorhabditis elegans contains a gene called fat-1. This gene encodes a desaturase enzyme, which allows the roundworm to convert omega-6 to omega-3 fatty acids (Kang et al., 2004). Previous research has shown that rat cardiac myocytes can be successfully infected with a recombinant adenovirus containing the fat-1 gene. In this in vitro study, scientists were able to show that these cells could convert omega-6 to omega-3 fatty acids (Kang et al., 2001).
Previous research has shown that genetically engineered cells can be microencapsulated for oral delivery. In one study, researchers infected Escherichia coli strain DH5 with a gene for urease derived from Klebsiella aerogenes. They microencapsulated these cells and gave them orally to rats suffering from renal failure. These cells were able to successfully remove urea from the blood of the rats (Chang and Prakash, 1998). Orally delivered microencapsulated cells have also been studied as an oral immunization method (Katz et al., 2003). Microencapsulation can be used for oral delivery of fat-1 infected cells for the prevention of atherosclerosis in ApoE-null mice.
Hypothesis and Specific Aims
Microencapsulated genetically engineered fat-1 cells can be delivered orally to ApoE-null mice to prevent atherosclerosis. Mice treated with the genetically engineered cells will have lower body weight, cholesterol levels, and triglycerides than untreated mice. Specifically for cholesterol levels, treated mice will have low LDL (low-density lipoprotein) levels and high HDL (high-density lipoprotein) levels when compared to the untreated mice. In addition, treated mice will have smaller plaque areas than untreated mice.
The objectives of this study are to:
Construct a recombinant adenovirus containing the fat-1 gene,
Genetically engineer mouse cardiac myocytes to express the fat-1 gene,
Microencapsulated these genetically engineered cells for oral delivery into ApoE-null mice,
Show that these microencapsulated cells can prevent atherogenesis in ApoE-null mice.
Research Design and Methods
Construction of the recombinant adenovirus
The recombinant adenovirus will be constructed following the methods of He et al. (1998) and Kang et al. (2001). A plasmid (pCE8) containing the cDNA for the fat-1 gene will be purchased from Washington State University. In order to release the cDNA from the plasmid, a double digestion will be performed using the restriction enzymes EcoRI and KpnI (Kang et al., 2001). Gel electrophoresis will be performed to isolate the cDNA after digestion (the cDNA should be approximately 1391 bp in length) (NCBI, 2009). The fat-1 cDNA will then be inserted into a shuttle vector (Kang et al., 2001). The shuttle vector (pAdTrack-CMV) will be purchased from Addgene (Addgene, 2009). The restriction enzyme PmeI will then be used to linearize the resultant plasmid. The plasmid will then be contransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid using electroporation (He et al., 1998). The E. coli BJ5183 cells and the adenoviral backbone plasmid (pAdEasy-1) will be obtained from ATCC (ATCC, 2009). Cells exhibiting kanamycin resistance will be selected (He et al., 1998).
Digestion with the restriction enzymes PacI, SpeI, and BamHI will be used to confirm recombination. The selected plasmids will then be linearized using the restriction enzyme PacI. The linearized plasmid will then be transfected into an adenovirus packaging cell line (He et al, 1998). The adenovirus packaging cell line 293 will be used and will be obtained from Microbix Biosystems MBI, 2008). The recombinant adenoviruses will be used for infection after 7-10 days (He et al., 1998).
Cell cultures and infection with recombinant adenovirus
The mouse cardiac myocytes will be obtained using the Worthington Neonatal Cardiomyocyte Isolation System (WBC, 2010). The mouse cardiac myocytes will be cultured and infected using the methods described by Zhou et al. (2000) and Zhu et al. (2000). The isolated cardiac myocytes will be suspended in a minimal essential medium (MEM, M1018, Sigma-Aldrich), which will contain 1.2 mM calcium ions, 2.5% preselected fetal bovine serum (PFBS), and 1% penicillin-streptomycin (PS). The myocytes will then be allowed to pelletize for approximately 10 min. The supernatant will then be removed and the myocytes will be washed two more times with this medium (Zhou et al., 2000). Next, the myocytes will be plated on culture dishes precoated with 10 μg/mL mouse laminin (Zhu et al., 2000). Each plate will have 0.5-1 x 104 cells/cm2 and contain MEM with 2.5% PFBS and 1% PS. The medium will be changed to a PFBS-free MEM after one hour of incubation at 37°C in a 5% CO2 incubator. The medium will need to be changed every 48 hours (Zhou et al., 2000).
Myocyte attachment to the plates should be achieved approximately after one hour of incubation. Adenovirus-mediated gene transfer will then be performed. First, the medium and unattached myocytes will be removed from the plates. A half volume of the PFBS-free MEM will then be added to each plate (Zhou et al., 2000) with an appropriate titer of the recombinant adenovirus (Zhu et al., 2000). After one to two more hours of incubation, another half volume of the PFBS-free MEM will be added to each plate (Zhou et al., 2000).
Test for proper infection
After adenoviral infection with the fat-1 gene, the infected cardiac myocytes will be grown on normal medium supplemented with 10 µM 18:2n-6 and 20:4n-6 (Kang et al., 2001). Non-infected cardiac myocytes will also be cultured under the same conditions to serve as a control. The cells will be incubated for 48 h before being analyzed for fatty acid content (Kang et al., 2001).
Fatty acid content of the cells will be analyzed using gas chromatography. Samples of infected and non-infected cells will be freeze-dried prior to analysis. These freeze-dried samples will be analyzed using the method of Chi et al. (2007). A small sample of the freeze-dried cells (approximately 20 mg) will be mixed with 4 mL of a solution of methanol, concentrated sulfuric acid, and chloroform (1.7:0.3:2.0 v/v). A known amount of heptadecanoic acid (C17:0) will be added to each sample to act as an internal standard. Tubes containing the cell and solution mixture will be heated in a water bath at 90°C for 40 min. The tubes will then be removed from the water bath and allowed to cool to room temperature. Next, 1 mL of distilled water will be added to each tube and each tube will be vortexed for approximately 1 min. The mixture will then be allowed to settle, separating into two phases. The bottom phase, which contains the fatty acid methyl esters, will be pipetted into a microcentrifuge tube. Anhydrous Na2SO4 will be added to each microcentrifuge tube to ensure that water has been removed from the sample. The samples will be centrifuged at 10,000 rpm for 8 min to settle the Na2SO4. Next, the liquid will be transferred from the microcentrifuge tubes to a vial for analysis using gas chromatography (Chi et al., 2007).
Microencapsulation of genetically engineered cells
Microencapsulation of genetically engineered cells will be performed following the method of Prakash and Chang (1995). A solution containing 0.9% (w/v) sodium alginate and 0.1 mol/L calcium chloride will be autoclaved at 121°C for 15 min. After the solution cools to room temperature, the infected cardiac myocytes will be added to the solution resulting in a viscous alginate-myocyte suspension. This suspension will be passed through a 23-gauge needle using a syringe pump while compressed air is pushed through a 16-gauge needle. The compressed air will be used to shear the droplets as they come out of the 23-gauge needle (Prakash and Chang, 1995).
The droplets will then be cooled in a cold 1.4% calcium chloride solution for 15 min with gentle stirring to prevent the droplets from sticking. This cooling causes the droplets to gel into beads. Next, a 0.05% polylysine buffer in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer saline will be used to coat the gelled beads for 10 min. The beads will then be washed with HEPES and a 0.1% alginate solution will be used to coat the beads for 4 min. The gel inside the capsules will then be liquefied by washing the capsules in a 3% citrate bath (Prakash and Chang, 1995).
Mouse strain and diet
Apo-E null male mice will be obtained from Taconic (TF, 2010). Twenty mice will be obtained with 10 receiving the microencapsulated engineered cells and 10 receiving no additional supplementation. These sample sizes were determined using JMP with an alpha level of 0.05 and a power of 0.8. The mice will be fed a western diet to induce atherogenesis consisting of 10% fat and 1.25% cholesterol (She et al., 2009).
Mouse weight, cholesterol, and triglyceride levels
The mice will be weighed daily over the course of the study to determine the effects of the microencapsulated cells on obesity in the ApoE-null mice. Blood samples will also be taken every two weeks to determine the cholesterol and triglyceride levels of the mice. Blood samples will be taken from the retro-orbital plexis of the mice (Kaplan et al., 2001). Both HDL and LDL levels will be measured. Hypothesis testing, with an alpha level of 0.05, will be performed to determine if the results are statistically significant. The null hypothesis will be that the average weight, cholesterol level, and triglyceride level of the treated mice are equal to those of the untreated mice. The alternative hypothesis for average weight, LDL level, and triglyceride level is that the averages for each of these parameters of treated mice will be less than the averages of untreated mice. The alternative hypothesis for average HDL level is that the average HDL level of treated mice will be greater than the average for untreated mice.
Quantification of atherosclerosis
The mice will be sacrificed after 16 weeks to determine the extent of atherosclerosis. Next, the mouse will be perfused with ice-cold PBS and 4% paraformaldehyde. The hearts and the aortas will be collected for future analysis (She et al., 2009). Evaluation of atherosclerotic lesions in the whole aorta will follow the methods of She et al. (2009) and Paigen et al. (1987). The aortas will be excised from the junction with the heart to the iliac bifurcation (Paigen et al., 1987). The aortas will be opened longitudinally and pinned flat on styrofoam (Paigen et al., 1987; She et al., 2009). The aorta will then be fixed and stained with Oil red O (She et al., 2009). Staining will be performed using a buffered solution of formalin and Oil red O. The styrofoam will be floated in the solution with the aorta face down (Paigen et al., 1987). After staining, the aorta will be transferred to a slide. The area stained by Oil red O will be determined using Image Pro Plus software (She et al., 2009). Hypothesis testing, with an alpha level of 0.05, will be used to test for statistical significance. The null hypothesis will be that the lesion area in treated and untreated mice will be equal. The alternative hypothesis will be that the lesion area in the treated mice is less than the lesion area in untreated mice.
Anticipated Results, Potential Pitfalls, and Alternative Approaches
A recombinant adenovirus containing the fat-1 gene will be successfully constructed. This recombinant adenovirus can then be used to successfully infect mouse cardiac myocytes to express the fat-1 gene. In addition, these genetically engineered cells will be successfully microencapsulated for oral delivery.
Mice treated with the microencapsulated fat-1 cells will have lower body weight, cholesterol levels, and triglycerides than untreated mice. The LDL levels in treated mice will be low and the HDL levels will be high when compared to the untreated mice. The treated mice will also have smaller plaque areas than untreated mice. All results in this study are anticipated to be statistically significant.
One potential pitfall is that the microencapsulated fat-1 cells will not be effective at converting the omega-6 to omega-3 fatty acids in the ApoE-null mice or that the effect will not be great enough to be medically beneficial. If the microencapsulated fat-1 cells are ineffective when delivered orally, other methods of cell delivery will be considered such as implantation of the microencapsulated cells.
Another potential pitfall is that the mice experience a toxic reaction to the microencapsulated cells. In this case, modification of the microencapsulated cells can be taken in order to make them less toxic. These modifications can include changing the materials used for microencapsulation or changing the infected cell line.
One alternative approach is to implant the microencapsulated cells instead of delivering them orally. Implantation may be more effective, but is an invasive method of treatment. In addition, implantation of the microencapsulated cells can result in immune response, which can lead to the death of the microencapsulated cell (Chang and Prakash, 1998).
Another approach to preventing atherogenesis with omega-3 fatty acids would be diet change and supplementation. The main food source of omega-3 fatty acids, though, is fatty fish, which leads to other health concerns. Many fatty fish contain toxic compounds such as methylmercury, dioxins, and polychlorinated biphenyls. Consumption of these toxins can cause neurological damage and leads to increased risks of cancer (Hooper et al., 2006).
Diet modification may become a more appealing alternative in the future with the construction of transgenic animals. Currently the fat-1 gene derived from C. elegans has been use to generate transgenic mice (Kang et al., 2004) and pigs (Lai et al., 2006). The transgenic pigs were found to have high levels of omega-3 fatty acids in their meat when compared to their wild-type littermates (Lai et al., 2006). As more research is conducted in the expression of the fat-1 gene in transgenic animals, more dietary options for omega-3 fatty acids will become available.