Atherosclerosis, an inflammatory disease of the blood vessels, is the leading cause of death in the Western world. 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 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 transfected with the fat-1 gene and perform this conversion. This study will focus on the microencapsulation of transfected cells to be given orally to atherosclerotic mice.
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. 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 transfected 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. Researchers transfected Escherichia coli strain DH5 with a gene for urease derived from Klebsiella aerogenes. They microencapsulated these cells and gave them orally to rats in 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 transfected cells for the prevention of atherosclerosis in ApoE-null mice.
Hypothesis and Specific Aims
Hypothesis
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. In addition, treated mice will have smaller plaque areas than untreated mice.
Specific Aims
The objectives of this study are to:
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
Cell cultures and infection with recombinant adenovirus
The recombinant adenovirus will be obtained from Z. Kang (Department of Medicine, Massachusetts General Hospital, Boston MA). The mouse cardiac myocytes will be obtained using the Worthington Neonatal Cardiomyocyte Isolation System (WBC, 2010). The mouse cardiac myocytes will be cultured and transfected 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 transfection
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 cooling, the transfected cardiac myocytes will be added to the solution resulting in a viscous alginate-bacterial suspension. The 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 is 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. This cooling causes the droplets to gel. 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. 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 high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels will be measured.
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 heart and the aorta will be collected for future analysis (She et al., 2009). Tissue preparation will follow the methods of Ni et al. (2001) and She et al. (2009). Serial cross sections of the aortic root (approximately 10 μm thick) will be prepared (She et al., 2009). Atherosclerotic lesions will be examined at five different locations 80-120 μm apart (Ni et al., 2001; She et al., 2009). The sections will then be stained with hematoxylin and eosin for morphological analysis (She et al., 2009).
Anticipated Results, Potential Pitfalls, and Alternative Approaches
Anticipated results
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.
Potential pitfalls
One potential pitfall is that the microencapsulated fat-1 cells will not have the intended effects on the ApoE- null mice or that the effect will not be great enough to be medically beneficial. Another potential pitfall is that the mice experience a toxic reaction to the microencapsulated cells.
Alternative approaches
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.
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).
