Atherosclerosis An Inflammatory Disease Of The Blood Vessels Biology Essay
Atherosclerosis, an inflammatory disease of the blood vass, is the taking cause of decease in the Western universe. This status consequences in the formation of plaques, which can take to myocardial infarctions and shots. Omega-3 fatty acids have been shown to advance good cardiovascular wellness and prevent coronary artery disease. They have besides been shown to forestall fleshiness, a hazard factor for cardiovascular disease. The typical American diet, though, is non sufficient in omega-3 fatty acids ( anti-inflammatory ) and is high in omega-6 fatty acids, which are proinflammatory.
Mammals lack the ability to synthesise omega-3 fatty acids, but some lower beings have the ability to change over omega-6 to omega-3 fatty acids via the fat-1 cistron. Previous research has shown that mammalian cells can be infected with the fat-1 cistron and execute this transition. This survey will concentrate on the microencapsulation of septic cells to be given orally to atherosclerotic mice.First, a recombinant adenovirus incorporating the fat-1 cistron will necessitate to be constructed. This recombinant adenovirus will so be used to infect mouse cardiac myocytes, which will show the fat-1 cistron. These septic cells will so be microencapsulated for unwritten bringing into ApoE-null mice. The ApoE-null mice will be put on a Western diet ( high in cholesterin and fat ) , which will bring on coronary artery disease. One group of mice will have the microencapsulated fat-1 cells, while another group will be used as a control.
Throughout the survey, mice will be weighed and their blood tested for cholesterin and triglycerides. At the terminal of this 16-week survey, the mice will be sacrificed and their aortas will be examined to find the extent of coronary artery disease.
Background and Significance
Atherosclerosis is the taking cause of decease in Western society. Atherosclerosis is an inflammatory disease, which consequences in the formation of plaques in the blood vass.
Myocardial infarctions and shots can happen due to plaque rupture and thrombosis ( Glass and Witztum, 2001 ) . Hazard factors for developing coronary artery disease include high blood pressure, hypercholesteremia ( Ross, 1999 ) , fleshiness ( Hubert et al. , 1983 ) , and genetic sciences ( Lahoz et al. , 2001 ) . The cistron for Apolipoprotein E ( ApoE ) , in peculiar, has been widely studied and linked to cardiovascular disease ( Lahoz et al. , 2001 ) .
Omega-3 fatty acids can assist cut down the incidences of cardiovascular events such as arrhythmias, redness, high blood pressure, and atherothrombosis ( Masson et al. , 2007 ; Marik and Varon, 2009 ) . In add-on, omega-3 fatty acids can assist lower bosom rate and blood force per unit area, which helps lower the hazard of cardiovascular disease. Omega-3 fatty acids can besides cut down triglyceride degrees in blood serum, the formation of blood coagulums, and irregular pulses ( 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 ingestion. They are anti-inflammatory, antithrombotic, and antiarrhythmic compounds ( Covington, 2004 ) .In add-on, omega-3 fatty acids can assist prevent/treat cardiovascular disease by forestalling and relieving fleshiness. Omega-3 fatty acids can assist forestall fleshiness by set uping lipid metamorphosis. They can cut down lipid consumption by stamp downing lipoprotein lipase, an enzyme that hydrolyzes triglycerides. Omega-3 fatty acids, specifically EPA, promote oxidization of mitochondrial fatty acids.
In add-on, omega-3 fatty acids can diminish lipid synthesis by suppressing fatty acerb 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 potency to take down lipid concentrations in blood serum ( Mitsuyoshi et al. , 1991 ) . Animal surveies have shown that supplementing a high-fat obesity-inducing diet with omega-3 fatty acids can cut down organic structure fat accretion. In other carnal surveies, scientists have shown that supplementing an corpulent mouse ‘s diet with omega-3 fatty acids caused a loss in organic structure weight ( Buckley and Howe, 2009 ) . The typical American diet, though, does non incorporate a sufficient sum of omega-3 fatty acids, but alternatively 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 manner to turn to this job is to change over these omega-6 fatso acids into omega-3 fatty acids.Mammals lack the ability to change over omega-6 fatty acids to omega-3 fatty acids, but some lower organisms possess this ability. The tinea Caenorhabditis elegans contains a cistron called fat-1. This cistron encodes a desaturase enzyme, which allows the tinea to change over 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 incorporating the fat-1 cistron. In this in vitro survey, scientists were able to demo that these cells could change over omega-6 to omega-3 fatty acids ( Kang et al. , 2001 ) .Previous research has shown that genetically engineered cells can be microencapsulated for unwritten bringing. In one survey, research workers infected Escherichia coli strain DH5 with a cistron for urease derived from Klebsiella aerogenes. They microencapsulated these cells and gave them orally to rats enduring from nephritic failure.
These cells were able to successfully take carbamide from the blood of the rats ( Chang and Prakash, 1998 ) . Orally delivered microencapsulated cells have besides been studied as an unwritten immunisation method ( Katz et al. , 2003 ) . Microencapsulation can be used for unwritten bringing of fat-1 septic cells for the bar of coronary artery disease in ApoE-null mice.
Hypothesis and Specific Aims
HypothesisMicroencapsulated genetically engineered fat-1 cells can be delivered orally to ApoE-null mice to forestall coronary artery disease.
Mice treated with the genetically engineered cells will hold lower organic structure weight, cholesterin degrees, and triglycerides than untreated mice. Specifically for cholesterin degrees, treated mice will hold low LDL ( low-density lipoprotein ) degrees and high HDL ( high-density lipoprotein ) degrees when compared to the untreated mice. In add-on, treated mice will hold smaller plaque countries than untreated mice.
Specific AimsThe aims of this survey are to:Concept a recombinant adenovirus incorporating the fat-1 cistron,Genetically engineer mouse cardiac myocytes to show the fat-1 cistron,Microencapsulated these genetically engineered cells for unwritten bringing into ApoE-null mice,Show that these microencapsulated cells can forestall atherogenesis in ApoE-null mice.
Research Design and Methods
Construction of the recombinant adenovirusThe recombinant adenovirus will be constructed following the methods of He et Al. ( 1998 ) and Kang et Al. ( 2001 ) . A plasmid ( pCE8 ) incorporating the complementary DNA for the fat-1 cistron will be purchased from Washington State University. In order to let go of the complementary DNA from the plasmid, a dual digestion will be performed utilizing the limitation enzymes EcoRI and KpnI ( Kang et al.
, 2001 ) . Gel cataphoresis will be performed to insulate the complementary DNA after digestion ( the complementary DNA should be about 1391 bp in length ) ( NCBI, 2009 ) . The fat-1 complementary DNA will so be inserted into a shuttle vector ( Kang et al. , 2001 ) . The shuttle vector ( pAdTrack-CMV ) will be purchased from Addgene ( Addgene, 2009 ) . The limitation enzyme PmeI will so be used to linearise the end point plasmid. The plasmid will so be contransformed into E. coli BJ5183 cells with an adenoviral anchor plasmid utilizing electroporation ( He et al.
, 1998 ) . The E. coli BJ5183 cells and the adenoviral anchor plasmid ( pAdEasy-1 ) will be obtained from ATCC ( ATCC, 2009 ) . Cells exhibiting Kantrex opposition will be selected ( He et al. , 1998 ) .Digestion with the limitation enzymes PacI, SpeI, and BamHI will be used to corroborate recombination. The selected plasmids will so be linearized utilizing the limitation enzyme PacI. The linearized plasmid will so 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 yearss ( He et al. , 1998 ) .
Cell civilizations and infection with recombinant adenovirusThe mouse cardiac myocytes will be obtained utilizing the Worthington Neonatal Cardiomyocyte Isolation System ( WBC, 2010 ) . The mouse cardiac myocytes will be cultured and infected utilizing the methods described by Zhou et Al. ( 2000 ) and Zhu et Al. ( 2000 ) . The stray cardiac myocytes will be suspended in a minimum indispensable medium ( MEM, M1018, Sigma-Aldrich ) , which will incorporate 1.2 mM Ca ions, 2.
5 % preselected foetal bovine serum ( PFBS ) , and 1 % penicillin-streptomycin ( PS ) . The myocytes will so be allowed to pelletize for about 10 min. The supernatant will so be removed and the myocytes will be washed two more times with this medium ( Zhou et al.
, 2000 ) . Following, the myocytes will be plated on civilization dishes precoated with 10 ?g/mL mouse laminin ( Zhu et al. , 2000 ) . Each home base will hold 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 hr of incubation at 37 & A ; deg ; C in a 5 % CO2 brooder. The medium will necessitate to be changed every 48 hours ( Zhou et al.
, 2000 ) .Myocyte fond regard to the home bases should be achieved about after one hr of incubation. Adenovirus-mediated cistron transportation will so be performed. First, the medium and unattached myocytes will be removed from the home bases.
A half volume of the PFBS-free MEM will so be added to each home base ( Zhou et al. , 2000 ) with an appropriate titre 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 home base ( Zhou et al. , 2000 ) .Trial for proper infectionAfter adenoviral infection with the fat-1 cistron, the septic 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 besides be cultured under the same conditions to function as a control. The cells will be incubated for 48 H before being analyzed for fatty acid content ( Kang et al. , 2001 ) .Fatty acerb content of the cells will be analyzed utilizing gas chromatography.
Samples of septic and non-infected cells will be lyophilized anterior to analysis. These lyophilized samples will be analyzed utilizing the method of Chi et Al. ( 2007 ) . A little sample of the lyophilized cells ( about 20 milligram ) will be assorted with 4 milliliters of a solution of methyl alcohol, concentrated sulphuric acid, and trichloromethane ( 1.7:0.
3:2.0 v/v ) . A known sum of heptadecanoic acid ( C17:0 ) will be added to each sample to move as an internal criterion. Tubes incorporating the cell and solution mixture will be heated in a H2O bath at 90 & A ; deg ; C for 40 min. The tubings will so be removed from the H2O bath and allowed to chill to room temperature. Next, 1 milliliter of distilled H2O will be added to each tubing and each tubing will be vortexed for about 1 min. The mixture will so be allowed to settle, dividing into two stages.
The bottom stage, which contains the fatty acid methyl esters, will be pipetted into a microcentrifuge tubing. Anhydrous Na2SO4 will be added to each microcentrifuge tubing to guarantee that H2O has been removed from the sample. The samples will be centrifuged at 10,000 revolutions per minute for 8 min to settle the Na2SO4. Next, the liquid will be transferred from the microcentrifuge tubes to a phial for analysis utilizing gas chromatography ( Chi et al. , 2007 ) .Microencapsulation of genetically engineered cellsMicroencapsulation of genetically engineered cells will be performed following the method of Prakash and Chang ( 1995 ) . A solution incorporating 0.
9 % ( w/v ) Na alginate and 0.1 mol/L Ca chloride will be autoclaved at 121 & A ; deg ; C for 15 min. After the solution cools to room temperature, the septic cardiac myocytes will be added to the solution ensuing in a syrupy alginate-myocyte suspension. This suspension will be passed through a 23-gauge acerate leaf utilizing a syringe pump while compressed air is pushed through a 16-gauge acerate leaf. The tight air will be used to shear the droplets as they come out of the 23-gauge acerate leaf ( Prakash and Chang, 1995 ) .The droplets will so be cooled in a cold 1.4 % Ca chloride solution for 15 min with soft stirring to forestall the droplets from lodging.
This chilling 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 surface the gelled beads for 10 min. The beads will so be washed with HEPES and a 0.
1 % alginate solution will be used to surface the beads for 4 min. The gel inside the capsules will so be liquefied by rinsing the capsules in a 3 % citrate bath ( Prakash and Chang, 1995 ) .Mouse strain and dietApo-E void male mice will be obtained from Taconic ( TF, 2010 ) . Twenty mice will be obtained with 10 having the microencapsulated engineered cells and 10 having no extra supplementation. These sample sizes were determined utilizing JMP with an alpha degree of 0.05 and a power of 0.8.
The mice will be fed a western diet to bring on atherogenesis dwelling of 10 % fat and 1.25 % cholesterin ( She et al. , 2009 ) .Mouse weight, cholesterin, and triglyceride degreesThe mice will be weighed daily over the class of the survey to find the effects of the microencapsulated cells on fleshiness in the ApoE-null mice. Blood samples will besides be taken every two hebdomads to find the cholesterin and triglyceride degrees of the mice.
Blood samples will be taken from the retro-orbital plexis of the mice ( Kaplan et al. , 2001 ) . Both HDL and LDL degrees will be measured. Hypothesis proving, with an alpha degree of 0.05, will be performed to find if the consequences are statistically important. The void hypothesis will be that the mean weight, cholesterin degree, and triglyceride degree of the treated mice are equal to those of the untreated mice. The alternate hypothesis for mean weight, LDL degree, and triglyceride degree is that the norms for each of these parametric quantities of treated mice will be less than the norms of untreated mice.
The alternate hypothesis for mean HDL degree is that the mean HDL degree of treated mice will be greater than the norm for untreated mice.Quantification of coronary artery diseaseThe mice will be sacrificed after 16 hebdomads to find the extent of coronary artery disease. Following, the mouse will be perfused with ice-cold PBS and 4 % paraformaldehyde. The Black Marias 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 bosom to the iliac bifurcation ( Paigen et al. , 1987 ) .
The aortas will be opened longitudinally and pinned level on Styrofoam ( Paigen et al. , 1987 ; She et al. , 2009 ) .
The aorta will so be fixed and stained with Oil ruddy O ( She et al. , 2009 ) . Staining will be performed utilizing a buffered solution of formol and Oil ruddy 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 country stained by Oil ruddy O will be determined utilizing Image Pro Plus package ( She et al. , 2009 ) . Hypothesis proving, with an alpha degree of 0.05, will be used to prove for statistical significance. The void hypothesis will be that the lesion country in treated and untreated mice will be equal.
The alternate hypothesis will be that the lesion country in the treated mice is less than the lesion country in untreated mice.
Anticipated Results, Potential Pitfalls, and Alternative Approaches
Anticipated consequencesA recombinant adenovirus incorporating the fat-1 cistron will be successfully constructed. This recombinant adenovirus can so be used to successfully infect mouse cardiac myocytes to show the fat-1 cistron.
In add-on, these genetically engineered cells will be successfully microencapsulated for unwritten bringing.Mice treated with the microencapsulated fat-1 cells will hold lower organic structure weight, cholesterin degrees, and triglycerides than untreated mice. The LDL degrees in treated mice will be low and the HDL degrees will be high when compared to the untreated mice. The treated mice will besides hold smaller plaque countries than untreated mice.
All consequences in this survey are anticipated to be statistically important.Potential booby trapsOne possible booby trap is that the microencapsulated fat-1 cells will non be effectual at change overing the omega-6 to omega-3 fatty acids in the ApoE-null mice or that the consequence will non be great adequate to be medically good. If the microencapsulated fat-1 cells are uneffective when delivered orally, other methods of cell bringing will be considered such as nidation of the microencapsulated cells.Another possible booby trap is that the mice experience a toxic reaction to the microencapsulated cells. In this instance, alteration of the microencapsulated cells can be taken in order to do them less toxic. These alterations can include altering the stuffs used for microencapsulation or altering the infected cell line.
Alternate attacksOne alternate attack is to engraft the microencapsulated cells alternatively of presenting them orally. Implantation may be more effectual, but is an invasive method of intervention. In add-on, nidation of the microencapsulated cells can ensue in immune response, which can take to the decease of the microencapsulated cell ( Chang and Prakash, 1998 ) .Another attack to forestalling atherogenesis with omega-3 fatty acids would be diet alteration and supplementation.
The chief nutrient beginning of omega-3 fatty acids, though, is fatty fish, which leads to other wellness concerns. Many fatty fish contain toxic compounds such as methylmercury, dioxins, and polychlorinated biphenyls. Consumption of these toxins can do neurological harm and leads to increased hazards of malignant neoplastic disease ( Hooper et al. , 2006 ) .Diet alteration may go a more appealing option in the hereafter with the building of transgenic animate beings. Presently the fat-1 cistron derived from C.
elegans has been use to bring forth transgenic mice ( Kang et al. , 2004 ) and hogs ( Lai et al. , 2006 ) . The transgenic hogs were found to hold high degrees 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 look of the fat-1 cistron in transgenic animate beings, more dietetic options for omega-3 fatty acids will go available.