The Association of SAA With Apolipoprotein B Affects Cardiovascular Risk

Cardiovascular disease (CVD) is the leading cause of death in developed nations and a major health issue in Veterans. Despite a number of different treatments, cardiovascular disease remains a major health burden, thus further treatments are needed. Individuals with obesity and/or diabetes are at particularly high risk for cardiovascular disease, and research suggests that elevated levels of serum amyloid A (SAA) may contribute to cardiovascular disease, particularly atherosclerosis. In preliminary studies in both mouse and human the investigators have identified that SAA appears to shift between lipid particles. SAA is mainly found on high density lipoprotein (HDL) particles; however, the investigators have found that in both mice and humans with obesity and/or diabetes SAA is found on low density lipoprotein (LDL) and very low density lipoprotein (VLDL) particles, and the investigators hypothesize that the presence of SAA on LDL or VLDL makes these particles more likely to cause cardiovascular disease. To determine what leads SAA to shift between lipid particles, SAA knockout mice will be injected with HDL containing SAA then blood collected at several time points over 24 hours, and the lipid particles will be isolated to measure SAA. In some experiments the investigators will compare different isoforms of SAA, different types of HDL particles, or induce expression of enzymes likely involved in shifting SAA between particles. To determine if the presence of SAA makes lipid particles bind vascular matrix more strongly, the investigators will collect carotid arteries and compare the extent of lipid particles bound to the vascular matrix in the vessel wall when the particles have or do not have SAA present. If this research confirms this hypothesis then the presence of SAA on LDL or VLDL may 1) be a new marker indicating humans at highest risk for cardiovascular disease and 2) be a new target of therapy to prevent cardiovascular disease.

Clinical burden of CVD: CVD is the leading cause of death in developed nations and the VA population is no exception. Despite decades of research, technical, and pharmacological advances, CVD remains a major public health problem. This is partly due to our impaired ability to identify subjects at greatest risk for CVD events and thus the best candidates for pharmacological risk reducing therapies, and partly due to incomplete use or efficacy of currently available therapies. Epidemiological studies have identified major risk factors for CVD including elevated LDL cholesterol, low HDL cholesterol, hypertension, smoking and diabetes. However, despite targeting individuals with these risk factors with aggressive pharmacological interventions, CVD remains a major public health problem. Furthermore, even in individuals with risk factors who are treated with pharmacological or lifestyle interventions the CVD event rates are higher than in those who never had the risk factors. Recent epidemiologic data evaluating the American Heart Association-identified cardiovascular health metrics reported that the prevalence of having CVD risk factors at ideal levels is \< 2%10; implying that \>98% of the population are candidates for risk reduction. Clearly, health systems cannot cope with pharmacological interventions for such enormous target populations. Thus, additional risk stratifying markers are needed to identify those at highest risk for events and thus at greatest likelihood of benefit. Several biomarkers, including the acute phase reactants C reactive protein (CRP) and serum amyloid A (SAA) have been studied for their role in predicting CVD events. Both CRP and SAA are chronically elevated in individuals with obesity, metabolic syndrome (MetS), diabetes, rheumatoid arthritis, lupus and other chronic inflammatory conditions associated with increased CVD rates, raising the question of whether these biomarkers merely reflect underlying risk or play a causative role in CVD. Although emerging evidence has cast doubt on the role of CRP as a causative factor the investigators and others recently demonstrated that SAA is directly atherogenic in animal models. Thus, in addition to its role as a biomarker for CVD, SAA may play a causal role in CVD.

SAA: SAA is a family of acute phase proteins synthesized primarily in the liver. In healthy individuals SAA concentrations are \< 5 mg/L but during an acute phase response SAA can increase up to 1000 mg/L for a few days, then it rapidly returns to baseline levels. However, chronic inflammatory states such as obesity, MetS, diabetes, rheumatoid arthritis etc, are associated with persistently and significantly elevated SAA concentrations of 30-100 mg/L. Acute elevations in SAA are proposed to play a major role in response to injury and inflammation, participating in cholesterol delivery to injured tissues, recruitment of inflammatory cells, and induction of tissue repair cytokines. However, the chronic elevations of SAA now prevalent in modern society likely reflect a maladaptive response and numerous studies are now examining potential roles of SAA in disease pathology. Using murine models in which acute phase SAA is over-expressed, the investigators and others demonstrated direct increases in atherosclerosis development.

SAA and apolipoprotein B (apoB) containing lipoproteins: SAA is a lipid binding apolipoprotein and lipid-free SAA has not been found in vivo. The dogma is that SAA is exclusively an HDL associated lipoprotein; however, the investigators and others have reported SAA on apoB-containing lipoproteins in both mice and humans. Several studies have reported on a complex termed SAA-LDL associated with components of MetS, remnant like particle cholesterol, smoking status, lifestyle interventions, and statin treatment. These studies suggest that SAA-LDL is a risk factor for CVD. In new preliminary studies the investigators demonstrate that SAA has a differential lipoprotein association in diabetes, and in post-prandial lipoprotein metabolism, and the investigators demonstrate that the presence of SAA on apoB-lipoproteins augments their proteoglycan binding, a key step in atherosclerosis development. Thus, emerging evidence suggests that the presence of SAA on apoB-lipoproteins may be a novel CVD risk factor, play a causal role in atherosclerosis, and thus be a therapeutic target.

Post-prandial apoB-lipoprotein metabolism: The various lipoproteins are defined based on size and density criteria, as well as by their protein constituents. However, even within each lipoprotein class there is considerable heterogeneity, as the particles undergo continuous remodeling. Briefly, lipids consumed in the diet associate with apoB-48 to form chylomicrons, which are transported in intestinal lymphatics before entering the bloodstream. Various enzymes act on newly formed chylomicrons shifting lipids and proteins between chylomicrons and HDLs before the chylomicron remnants are taken up by the liver. The liver re-packages the lipids into VLDL particles containing apoB-100. The hydrolysis of VLDL results in smaller apoB-100 particles called VLDL remnants or intermediate density lipoproteins (IDLs). Collectively, these particles are termed triglyceride rich lipoproteins (TGRLs).

Ongoing remodeling of TGRLs by various lipases leads to the formation of LDL. LDL can be taken up by peripheral tissues, including the vasculature, or by the liver. The sub-endothelial retention of apoB-containing particles initiates atherosclerosis.

Post-prandial lipoproteins and CVD: Elevated levels of LDL cholesterol and low levels of HDL cholesterol are documented risk factors for CVD and contribute causally to atherogenesis. However, individuals with obesity, MetS and diabetes do not typically have elevated LDL cholesterol; their dyslipidemia is characterized by elevated triglycerides and low HDL cholesterol. The role of triglycerides as a CVD risk factor remains controversial; however, post-prandial triglycerides may be a more significant risk factor than fasting triglycerides. As humans spend most of their lives in the post-prandial state, there is ongoing interest in the role of post-prandial lipoprotein metabolism in CVD risk. However, most studies have relied on fasting lipoprotein samples; triglycerides are the lipoprotein component most affected by food consumption. The mechanisms accounting for the excess prevalence of CVD in MetS and diabetic subjects beyond that predicted by the traditional CVD risk factors remain unclear; however, insulin resistant states are characterized by increased intestinal apoB48 production, increased TGRL production and delayed lipoprotein clearance, which may contribute to CVD prevalence. Retention of apoB- containing lipoproteins in the vascular wall by the ionic interaction between apoB and proteoglycans, leads to the initiation of atherosclerosis. Lipolysis of VLDL more than doubles its ability to cross the endothelium and deposit lipids in the subendothelial space. TGRLs have proportionately more triglyceride than cholesterol: however, their size means that they can deposit 5-20 times more cholesterol per particle in the subendothelial space compared to an LDL particle. Increased TGRL production and delayed particle clearance increases the likelihood of particle retention and cholesterol deposition in the subendothelial space. The investigators have novel preliminary data demonstrating that the presence of SAA on apoB-containing lipoproteins increases their proteoglycan binding. The investigators propose that the increased presence of SAA on apoB-containing post-prandial lipoproteins in insulin resistant states increases the atherogenicity of these particles and could be a mechanism accounting for the increased CVD prevalence in insulin resistant states such as MetS and diabetes.

HDL metabolism: Like VLDL and LDL, HDL comprises a range of particles; however, HDL does not contain apoB, instead containing apoA-I. HDL is often separated into two major classes by size and density: the large HDL2 and the smaller HDL3. As discussed above, HDL undergoes continuous lipid interchange with various apoB-containing lipoprotein particles. A change in lipoprotein structure or composition by various enzymes is termed remodeling. HDL is typically thought to be an atheroprotective lipoprotein due to its ability to transport cholesterol away from the periphery back to the liver. In addition, HDL has a number of other beneficial properties including anti-inflammatory and anti-oxidative functions. In insulin resistant states HDL levels tend to be low, and some studies suggest its beneficial properties are reduced. Remodeling of lipoproteins affects their functionality and half-life; for example, the remodeling of HDL by CETP (which transfers triglycerides from TGRL to HDL and cholesterol ester from HDL to TGRL) predisposes HDL to enhanced catabolism and is thought to contribute to the lower levels of HDL seen in insulin resistant states. Although the paradigm is that SAA is a HDL associated lipoprotein, in preliminary studies the investigators have found SAA on apoB particles in insulin resistant persons in the post-prandial period. However, it is not clear how SAA associates with either HDL or apoB-lipoprotein particles.

SAA lipoprotein association: In the setting of an acute phase response SAA levels can increase up to 1000-fold; however, even at these highly elevated levels SAA remains exclusively found on HDL particles. Thus, there is no evidence of a "maximum capacity" of HDL for SAA. How SAA associates with either HDL or apoB-lipoprotein particles is not fully understood. SAA is thought to be produced by the liver in a lipid-free form and bind lipoproteins extracellularly, or in plasma SAA has been shown to induce HDL biogenesis via ATP binding cassette 1 (ABCA1), which may be a major mechanism by which SAA associates with HDL. Murine studies using knockout mice demonstrated that in the absence of HDL, SAA was found on apoB-particles. However, the investigators and others have reported SAA on apoB particles despite the presence of HDL. In new preliminary studies the investigators found that the remodeling of HDL led to the liberation of both lipid-poor apoA-I and lipid-poor SAA, and that lipid-poor SAA associates with apoB particles. Thus, the remodeling of HDL, particularly in the post-prandial period, may lead to SAA shifting from HDL to apoB particles; alternately, SAA could associate with apoB particles during their hepatic secretion. Both HDL remodeling and hepatic apoB- particle secretion are increased in insulin resistant conditions.

Role of lipoprotein-proteoglycan interactions in atherogenesis: There are several hypotheses as to what triggers the initiation of atherosclerosis, with the "Response to Retention" hypothesis well supported by biomedical evidence. As outlined in this theory, early fatty streak lesions are initiated by deposition of atherogenic lipoproteins (LDLs and TGRLs) in the subendothelial matrix by their retention by extra cellular matrix proteoglycans. Studies show that lipoproteins migrate in and out of the subendothelial space, but once bound to proteoglycans these lipoproteins are retained in this region, become more susceptible to oxidation and other modifications, and are taken up by macrophages leading to the formation of foam cells. TGRLs may be even more atherogenic than LDLs as they don't need modification to be taken up by macrophages, and deliver 5-20 times more cholesterol than LDL on per particle basis. The investigators have demonstrated the presence of SAA on apoB-containing lipoprotein particles in mice, and recently confirmed this in humans. In preliminary studies the investigators demonstrate that the presence of SAA on apoB-lipoproteins enhances their proteoglycan binding. The investigators propose that the presence of SAA on apoB-containing lipoproteins enhances their retention increasing atherogenesis.

Eligibility Criteria:
Inclusion Criteria:

Up to 80 U.S. veterans age 50-75 will be recruited in the following three groups:

* Obese (BMI 27-45 kg/m2), metabolically healthy, (25-30 subjects)
* Obese (BMI 27-45 kg/m2), metabolic syndrome, (25-30 subjects)
* Obese (BMI 27-45 kg/m2), diabetic, (25-30 subjects)

Exclusion Criteria:

The use of:

* Statins (we will not exclude subjects on lipid lowering medications if they are willing to discontinue them for 1-2 weeks prior to participation)
* Fibrates
* Niacin
* Anti-inflammatory drugs including Thiazolidinediones, non-steroidal anti-inflammatories (NSAID), aspirin, steroids
* Estrogen replacement

Conditions such as:

* Acute illness
* Chronic inflammatory illness (such as psoriasis, rheumatoid arthritis, lupus, etc.)
* Infections
* Impaired renal function (eGFR \< 60 ml/min)
* Hypo- or hyperthyroidism (subjects biochemically euthyroid on levothyroxine therapy are permitted)
* Gastrointestinal dysfunction

Lifestyles including:

* Use of tobacco products
* Consumption of \> 3 drinks /day