As a research technician (2000 - 2004) in the lab of Dr. Paul Toselli, I learned and developed a surgical mouse model of atherosclerosis, used by our cardiovascular program project core investigators. This model created inflammatory lesions in the femoral arteries of transgenic and knock-out mice that could be studied for changes in the lesion architecture and gene expression. This work resulted in three publications as well as a fourth that I am currently preparing for submission as first author.
Since joining the lab of Dr. Barbara Schreiber (2004 - present), my work has focused on examining the role of Serum Amyloid A (SAA) in atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterized by the development of vascular lesions containing macrophages, smooth muscle cells, extracellular matrix and lipid. Early signs of the disease are present in young humans within the first 10 years of life. Circulating low-density lipoprotein (LDL) becomes trapped in the arterial walls, where it is partially oxidized. This form of LDL attracts monocytes which enter the walls of large arteries, differentiate into macrophages and engulf the lipid-rich lipoproteins. Lipid-laden macrophages, referred to as “foam cells” accumulate within the artery forming “fatty streak lesions”. These areas can become sites of more advanced atherosclerotic plaque formation later in life. The events leading to plaque formation include smooth muscle cell proliferation and synthesis of matrix components which can be followed by the formation of a necrotic “lipid core” and a “fibrous cap”. Lesions become clinically significant when they increase in size and restrict blood flow or more likely, become unstable (1).
An increase in circulating acute phase inflammatory proteins has been shown to be an indicator of heart disease. Serum amyloid A (SAA) is an acute phase reactant that is one such indicator (2-5). The SAA protein family consists of 12-14 kDa constitutive (SAA4) and acute phase (SAA1, SAA2 and SAA3) isoforms. Expression of the acute phase isoforms is induced during inflammation. Previous workers in my lab showed that treatment of neonatal aortic smooth muscle cells with low doses of the inflammatory cytokine, IL-1alpha, induces expression of SAA (6). The function of SAA remains uncertain however, pro-atherogenic as well as anti-atherogenic roles have been suggested (7). SAA is capable of binding HDL due to amphipathic regions, possibly disrupting reverse cholesterol transport (8). There is also evidence that SAA can exist in a lipid free form in tissues (9). It has been shown that atherosclerotic plaques contain SAA that has either been synthesized within the lesion and/or deposited from the bloodstream. Lipid-free SAA can also attract white blood cells, binding the FPRL1 G-protein coupled receptor (10). Finally, it has been shown that a high fat diet stimulates SAA production in the liver (11).
Although the presence of SAA correlates with increased risk of atherosclerosis, it is important to consider the possibility that its function could be protective. It has been shown that recombinant acute phase SAA or a peptide corresponding to the cholesterol binding region of acute phase SAA down-regulates lipid biosynthesis (12). Unpublished data from our lab suggests that the SAA-induces movement of endogenous cholesterol to the endoplasmic reticulum. It is known that cholesterol arrival at the endoplasmic reticulum inhibits cleavage of a transcription factor known as sterol response element binding protein (SREBP), which inhibits its movement to the nucleus, thereby decreasing SREBP-regulated gene transcription. SREBP is known to regulate transcription of genes involved in lipid synthesis and therefore it is hypothesized that SAA induces movement of cholesterol to the endoplasmic reticulum, which causes the decrease in lipid synthesis and accumulation. This suggests a protective role for SAA in the progression of atherosclerosis.
Gene microarray data collected from our lab show that the mRNA levels for many genes are affected by treatment of rat aortic smooth muscle cells with SAA. One such gene, of interest for its role in lipid metabolism, is secretory phospholipase A2 group IIa (sPLA2), whose expression increased upon treatment with SAA. It was also shown that the observed increase in mRNA results in elevated levels of sPLA2 protein by Western blot. As a member of the phospholipase family (PL) of enzymes, sPLA2 is important in phospholipid metabolism. This enzyme cleaves fatty acids at the sn2 position of phospholipids generating a free fatty acid and a lysophospholipid. Like SAA, this enzyme is considered an inflammatory indicator and has been implicated as both pro- and anti-atherogenic. In atherosclerotic lesions, sPLA2 has been shown to hydrolyze arterially embedded LDL and induce an inflammatory response (1,13). Cholesterol homeostasis may also depend somewhat on sPLA2. By hydrolyzing surface phospholipids on HDL, sPLA2 can enable movement of cholesterol to the surface where it is delivered to the liver and excreted (14-16).
The gene for sPLA2 has been identified and sequenced in mice, rats and humans (17-19). CCAAT/enhancer binding protein (C/EBP) binding sites have been identified and shown to play a key role in regulation of the gene’s transcription (20, 21). C/EBPs are a family of bZip transcription factors controlling the proliferation and differentiation of many different cell types. They have also been implicated in control of genes associated with the acute phase inflammatory response activated by IL-6. Interestingly, data generated from the gene array in our lab suggest that SAA also activates expression of IL-6. With regards to sPLA2 gene transcription, C/EBP beta can displace single stranded binding proteins that inhibit transcription of the gene (22). Other transcription factor binding sites identified within the proximal promoter include nuclear factor kappa B (NF- kB) and peroxisome proliferator activated receptor (PPAR) (20, 21).
As discussed above, another critical element of smooth muscle cell function that impacts on the progression of atherosclerosis is the ability of this cell type to synthesize extracellular matrix components including elastin. Elastin is a key matrix component of arteries, giving them flexibility to adapt to changes in blood pressure and smooth muscle cell contraction. There is evidence that elastin turnover, potentially due to the generation of bioactive peptides, leads to plaque weakening in atherosclerotic vessels. It was demonstrated that the levels of tropoelastin were higher and cross-linked elastin accumulation was lower in human atheroma vs. non-diseased vessels (23) . A recent study found that symptomatic human carotid plaques had more elastin than non-symptomatic plaques and that the elastin associated with the plaques was primarily of “intermediate size” (24) . Interestingly, SAA down-regulates elastin expression as shown by gene microarray and Northern blot analysis. It has been shown that C/EBP beta down-regulates tropoelastin gene transcription in rat pulmonary fibroblasts (25) and therefore, it may play a role in SAA-induced down-regulation of elastin expression. Interestingly, putative SREBP elements are present within the human and rat elastin promoters as shown using Transfac 7.0, and it is of interest to determine if they are active elements and responsive to SAA treatment.
SAA’s ability to alter cholesterol trafficking and gene regulation in vascular smooth muscle cells makes it an intriguing candidate for a potential role in the progression of atherosclerosis. My objective is to consider its role on smooth muscle cell function as it may impact on disease progression. I intend to consider the molecular mechanisms whereby SAA affects expression of sPLA2 and tropoelastin using an in vitro model of aortic smooth muscle cell culture. (top)
1. Lusis AJ. Atherosclerosis. Nature. 2000;407:233-241.
2. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000;342:836-843.
3. Ridker PM, Rifai N, Pfeffer M, Sacks F, Lepage S, Braunwald E. Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction. Circulation. 2000;101:2149-2153.
4. Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation. 2000;101:1767-1772.
5. Sacks FM, Ridker PM. Lipid lowering and beyond: results from the CARE study on lipoproteins and inflammation. Cholesterol and Recurrent Events. Herz. 1999;24:51-56.
6. Kumon Y, Sipe JD, Brinckerhoff CE, Schreiber BM. Regulation of extrahepatic apolipoprotein serum amyloid A (ApoSAA) gene expression by interleukin-1 alpha alone: synthesis and secretion of ApoSAA by cultured aortic smooth muscle cells. Scand J Immunol. 1997;46:284-291.
7. Schreiber BM. Serum amyloid A; in search of function. Amyloid. 2002;9:276-278.
8. Cabana VG, Lukens JR, Rice KS, Hawkins TJ, Getz GS. HDL content and composition in acute phase response in three species: triglyceride enrichment of HDL a factor in its decrease. J Lipid Res. 1996;37:2662-2674.
9. Stone MJ. Amyloidosis: a final common pathway for protein deposition in tissues. Blood. 1990;75:531-545.
10. Su SB, Gong W, Gao JL, Shen W, Murphy PM, Oppenheim JJ, Wang JM. A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells. J Exp Med. 1999;189:395-402.
11. Liao F, Andalibi A, deBeer FC, Fogelman AM, Lusis AJ. Genetic control of inflammatory gene induction and NF-kappa B-like transcription factor activation in response to an atherogenic diet in mice. J Clin Invest. 1993;91:2572-2579.
12. Schreiber BM, Veverbrants M, Fine RE, Blusztajn JK, Salmona M, Patel A, Sipe JD. Apolipoprotein serum amyloid A down-regulates smooth-muscle cell lipid biosynthesis. Biochem J. 1999;344 Pt 1:7-13.
13. Pruzanski W, Stefanski E, Kopilov J, Kuksis A. Mitogenic effect of lipoproteins on human vascular smooth muscle cells: the impact of hydrolysis by gr II A phospholipase A(2). Lab Invest. 2001;81:757-765.
14. Pruzanski W, de Beer FC, de Beer MC, Stefanski E, Vadas P. Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2. Biochem J. 1995;309 ( Pt 2):461-464.
15. Bamberger M, Lund-Katz S, Phillips MC, Rothblat GH. Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture. Biochemistry. 1985;24:3693-3701.
16. Collet X, Perret BP, Simard G, Vieu C, Douste-Blazy L. Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins. Biochim Biophys Acta. 1990;1043:301-310.
17. Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, Cromlish WA. A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains. J Biol Chem. 1995;270:22378-22385.
18. Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow EP, Tizard R, Pepinsky RB. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem. 1989;264:5768-5775.
19. Ohara O, Ishizaki J, Nakano T, Arita H, Teraoka H. A simple and sensitive method for determining transcription initiation site: identification of two transcription initiation sites in rat group II phospholipase A2 gene. Nucleic Acids Res. 1990;18:6997-7002.
20. Andreani M, Olivier JL, Berenbaum F, Raymondjean M, Bereziat G. Transcriptional regulation of inflammatory secreted phospholipases A(2). Biochim Biophys Acta. 2000;1488:149-158.
21. Antonio V, Brouillet A, Janvier B, Monne C, Bereziat G, Andreani M, Raymondjean M. Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors. Biochem J. 2002;368:415-424.
22. Fan Q, Paradon M, Salvat C, Bereziat G, Olivier JL. C/EBP factor suppression of inhibition of type II secreted phospholipase A2 promoter in HepG2 cells: possible role of single-strand binding proteins. Mol Cell Biol. 1997;17:4238-4248.
23. Krettek A, Sukhova GK, Libby P. Elastogenesis in human arterial disease: a role for macrophages in disordered elastin synthesis. Arterioscler Thromb Vasc Biol. 2003;23:582-587.
24. Goncalves I, Moses J, Dias N, Pedro LM, Fernandes e Fernandes J, Nilsson J, Ares MP. Changes related to age and cerebrovascular symptoms in the extracellular matrix of human carotid plaques. Stroke. 2003;34:616-622.
25. Kuang PP, Goldstein RH. Regulation of elastin gene transcription by interleukin-1 beta-induced C/EBP beta isoforms. Am J Physiol Cell Physiol. 2003;285:C1349-1355. (top)
Growing up in Brunswick, Maine, I became interested in science during my junior and senior years of high school. I pursued my interests at the University of New Hampshire where I received a Bachelor of Science degree in Microbiology. After completing my degree program, I was sure that I wanted to pursue a graduate degree, but I wasn’t sure of what area I should focus on. I applied for a job at Boston University School of Medicine as a technician and was hired in February of 2000 to work in the lab of Dr. Paul Toselli as a technician working for the Cardiovascular Program Project core. My work in Dr. Toselli’s lab involved learning and carrying out a difficult femoral artery injury on mice to be analyzed as a model of atherosclerosis. As I worked, I began to take the biochemistry core courses and became interested in obtaining my degree in that area. I first enrolled in the master’s program in 2003 and eventually transferred into the Ph.D. program in 2004 as a member of Dr. Barbara Schreiber’s lab. Since entering Dr. Schreiber’s lab, I have studied the effects of Serum Amyloid A (SAA) treatment on neonatal rat vascular smooth muscle cells.
In my more than seven years as a member of the biochemistry department, I have been active within the department as a member of the Biochemistry Student Organization (BSO) and a student member of the Student Affairs Committee. I have been active within the Division of Graduate Medical Sciences (GMS) as well. Along with students from other departments, I co-founded the Graduate Medical Sciences Student Organization (GMSSO) in 2004 to address student issues among the entire GMS student body. I served as vice president of this organization from 2005 to 2006 while we worked with the division office to create and participate in student events. We have also raised awareness of issues with the student health care coverage and hosted a career fair in the spring of 2007. In addition to these activities I have participated in student recruitment events including those in the department of biochemistry as well as MA candidate recruitment for the GMS division.
While conducting my doctoral research I was also able to gain valuable teaching experience as an instructor for Boston University's CityLab Academy. CityLab Academy is a nine month academic and laboratory skills training program for high school graduates looking to enter a career in biotechnology. As an instructor for their Cell Culture Techniques course, I was responsible for every aspect of course planning, management and laboratory management. It was a challenging experience, but fun. It was a learning experience for me as well as the students and it gave me a new respect for teachers.
During my time at Boston University, I have also been able to start a family. I rely on the support of my wife Kelly and have been strengthened by the joy of raising our two daughters Hailey and Margaret. I am thankful for their patience during these challenging years of graduate school. (top)