Lora J. Kasselman

The gut microbiome and elevated cardiovascular risk in obesity and autoimmunity

Cardiovascular disease associated with obesity and autoimmunity is the leading cause of death in these populations and significant residual risk remains despite current treatment approaches. Obesity, type 1 diabetes mellitus (T1DM), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) are linked to chronic inflammation, and subjects with these disorders have characteristic shifts in their gut microbiome composition. Recent data suggest that alterations in gut microbial and metabolic composition may be responsible, in part, for induction of chronic inflammation, thus promoting cardiovascular disease. Common microbiome changes observed in obesity, T1DM, RA, and SLE include a decrease in the ratio of bacteria, such as Gram-positive Firmicutes to Gram-negative Bacteroidetes, as well as an overabundance or depletion of certain species, including Prevotella copri. The consequent effects of these shifts include alterations in the metabolic composition of the gut, hyper-activation of toll-like receptor 4 (TLR-4), upregulation of inflammatory pathways, e.g. c-Jun N-terminal kinase and nuclear factor-kappa B (NFκB), increased intestinal permeability, increased C-reactive protein, and increased levels of trimethylamine N-oxide (TMAO). Differential microbiome compositions may also explain sex differences observed in autoimmunity, where a male gut microbiome promotes anti-inflammatory processes as compared to a female pro-inflammatory gut microbiome. Intervention at the level of the microbiota appears to attenuate symptoms in these inflammatory syndromes with probiotic treatment, such as Lactobacilli, playing a uniquely beneficial role in restoring intestinal health, decreasing inflammation, and reducing cardiovascular disease. This review will discuss obesity, T1DM, RA, and SLE in the context of how each unique microbiome profile contributes to elevated cardiovascular risk.

Amyloid toxicity in Alzheimer’s disease

Abstract A major feature of Alzheimer’s disease (AD) pathology is the plaque composed of aggregated amyloid-β (Aβ) peptide. Although these plaques may have harmful properties, there is much evidence to implicate soluble oligomeric Aβ as the primary noxious form. Aβ oligomers can be generated both extracellularly and intracellularly. Aβ is toxic to neurons in a myriad of ways. It can cause pore formation resulting in the leakage of ions, disruption of cellular calcium balance, and loss of membrane potential. It can promote apoptosis, cause synaptic loss, and disrupt the cytoskeleton. Current treatments for AD are limited and palliative. Much research and effort is being devoted to reducing Aβ production as an approach to slowing or preventing the development of AD. Aβ formation results from the amyloidogenic cleavage of human amyloid precursor protein (APP). Reconfiguring this process to disfavor amyloid generation might be possible through the reduction of APP or inhibition of enzymes that convert the precursor protein to amyloid.

CKD, arterial calcification, atherosclerosis and bone health: Inter-relationships and controversies

Mineral bone disease (MBD) is a common complication of chronic kidney disease (CKD) characterized by disruption of normal mineral homeostasis within the body. One or more of the following may occur: hypocalcemia, hyperphosphatemia, secondary hyperparathyroidism (SHPT), decreased vitamin D and vascular calcification (VC). The greater the decrease in renal function, the worse the progression of CKD-MBD. These abnormalities may lead to bone loss, osteoporosis and fractures. CKD-MBD is a major contributor to the high morbidity and mortality among patients with CKD. Another well-known complication of CKD is cardiovascular disease (CVD) caused by increased atherosclerosis and VC. CVD is the leading cause of morbidity and mortality in CKD patients. VC is linked to reduced arterial compliance that may lead to widened pulse pressure and impaired cardiovascular function. VC is a strong predicator of cardiovascular mortality among patients with CKD. Elevated phosphorus levels and increased calcium-phosphorus product promote VC. Controlling mineral disturbances such as hyperphosphatemia and SHPT is still considered among the current strategies for treatment of VC in CKD through restriction of calcium based phosphate binders in hyperphosphatemic patients across all severities of CKD along with dietary phosphate restriction and use of calciminetics. Additionally, Vitamin D insufficiency is common in CKD and dialysis patients. The causes are multifactorial and a serious consequence is SHPT. Vitamin D compounds remain the first-line therapy for prevention and treatment of SHPT in CKD. Vitamin D may also have atheroprotective effects on the arterial wall, but clinical studies do not show clear evidence of reduced cardiovascular mortality with vitamin D administration. This review discusses the issues surrounding CKD-MBD, cardiovascular disease and approaches to treatment.

COX-2-dependent and independent effects of COX-2 inhibitors and NSAIDs on proatherogenic changes in human monocytes/macrophages

It is the second decade of controversy regarding the cardiovascular effects of cyclo-oxygenase-2 (COX-2) inhibitors. At this time, celecoxib is the only available COX-2-specific inhibitor for treatment of pain and inflammation. Therefore, the present study was designed primarily to determine the impact of celecoxib on cholesterol handling (uptake via scavenger receptors and efflux from the cells) and foam cell formation in human THP-1 macrophages, followed by comparison to rofecoxib and other non-steroidal anti-inflammatory drugs (NSAIDs). THP-1 human macrophages and peripheral blood mononuclear cells were incubated with: celecoxib, rofecoxib, naproxen (at 5, 10, 25 µM) and acetaminophen (0.5 mM, 1 mM)±oxidized low-density lipoprotein (oxLDL, 25 µg/mL). Scavenger receptors: CD36, LOX-1, SR-A1, and CXCL16 and cholesterol efflux proteins: ATP-binding cassette transporter (ABC) A1 and G1, and 27-hydroxylase were detected. The adhesion of monocytes to cultured endothelial cells with/ without COX-2 inhibitors/NSAIDs was also analyzed. The presence of celecoxib and rofecoxib (at high concentrations) significantly decreased expression of 27-hydroxylase and ABCA1, interfering with normal cholesterol outflow from macrophages. Acetaminophen and the non-specific COX inhibitor naproxen had no significant effect on these proteins. Only celecoxib had a profound effect on the class B scavenger receptor CD36 and the class E receptor LOX1. We demonstrate that in contrast to celecoxib, rofecoxib and naproxen increased adhesive properties of monocytes to endothelial cells. This work might contribute to our understanding of multiple mechanisms underlying elevated cardiovascular risk upon the use of COX-2 inhibitors and uncover new possibilities to enhance the safety profile of existing COX-2 inhibitors.

BMI-DEPENDENT EFFECTS OF ADIPOSE TISSUE EXOSOMES ON HUMAN MACROPHAGE CHOLESTEROL TRANSPORT GENE EXPRESSION

Obesity, a major risk for atherosclerotic cardiovascular disease, has quadrupled in adolescents in the last 30 years. How adipose tissue influences the pathological process of atherosclerosis is not well understood. This study compares cholesterol efflux gene expression in human macrophages exposed

MP1: CORRECTING ATHEROGENIC EFFECTS OF LUPUS PLASMA ON MACROPHAGES WITH RESVERATROL AND MYCOPHENOLATE

Purpose of Study Premature atherosclerosis with coronary artery disease is a major cause of morbidity in Systemic Lupus Erythematosus (SLE). SLE patient plasma induces a pro-atherogenic profile of cholesterol transport genes in macrophages. A common immunosuppressive treatment for SLE, mycophenolate (MMF) reduces scavenger receptors thus reducing lipid influx. We have demonstrated atheroprotective properties of the polyphenol resveratrol on cholesterol efflux. This study determines whether MMF and resveratrol work synergistically to regulate cholesterol transport in macrophages exposed to pro-atherogenic SLE plasma. Methods Used THP-1 human macrophages (106/ml) were incubated in 10% SLE plasma with: media (control); MMF (1 µg/ml); resveratrol (50 µM); and MMF+resveratrol. After 24 h incubation, total RNA and protein were isolated. Message level of scavenger receptors CD36, LOX1, and SRA1; and efflux proteins 27-hydroxylase, ATP binding cassette transporter (ABC)A1, and ABCG1 were evaluated by QRT-PCR and confirmed by immunoblot. Cholesterol efflux was measured by Amplex Red Cholesterol Assay kit run±cholesterol esterase. Summary of Results In 10% SLE plasma, MMF suppressed efflux genes ABCA1 and ABCG1 (58.38±3.5% and 72.98±3.3%) vs. SLE plasma alone (p<0.0001) while MMF+resveratrol corrected this suppression. In SLE plasma, MMF+resveratrol decreased ScrA1 and LOX-1 by 15±2.5% and 47±1.0%, respectively vs. resveratrol alone (p<0.0001). SLE plasma promoted cholesterol accumulation in THP-1 macrophages and prevented efflux into medium. It increased the ratio of cholesterol esters to free cholesterol (ChE/FC). Resveratrol decreased intracellular cholesterol and restored ChE/FC ratios to that of cells in healthy control plasma. Conclusions MMF and resveratrol exhibit complimentary effects on macrophages exposed to SLE plasma. Both agents combined restore cholesterol influx and efflux gene expression to that of cells treated with control plasma. Resveratrol additionally reverses cholesterol accumulation caused by SLE plasma. Further evaluation of resveratrol+MMF in atherosclerosis in SLE may lead to improved treatment.

11: THE ADENOSINE A2A RECEPTOR AGONIST UK-432,097 STIMULATES ANTI-ATHEROGENIC REVERSE CHOLESTEROL TRANSPORT PROTEINS IN THP-1 HUMAN MACROPHAGES

Purpose of Study Methotrexate (MTX) is an anti-rheumatic drug with atheroprotective properties mediated through adenosine release and activation of the adenosine A2A receptor (A2AR). A2AR ligation increases reverse cholesterol transport via upregulation of cholesterol efflux proteins ATP-binding cassette transporter (ABC)A1 and ABCG1, liver X receptor (LXR) and cholesterol 27-hydroxylase. MTX is non-specific and associated with adverse effects on liver and kidney. Therefore, this study examines the anti-atherogenic efficacy of a specific A2AR agonist, UK-432,097, a drug with an established safety profile in humans. Methods Used THP-1 human macrophages were incubated in the following conditions: (1) RPMI media (untreated control); (2) dimethyl sulfoxide vehicle control; (3) UK-432,097 (100 nM); (4) ZM-241385 (1 µM) (A2AR antagonist)+UK-432,097 (100 nM). Gene expression analysis was performed using QRT-PCR for cholesterol efflux genes, normalized to the housekeeping gene GAPDH. Western blotting was performed using specific antibodies. All data were analyzed by one-way ANOVA with P values <0.05 considered significant. Summary of Results Following 6 h exposure to UK-432,097, mRNA and protein levels of ABCA1 increased by 88.75±5.4% and by 56.34±12.4% above control (P<0.01), respectively. ABCG1 expression increased by 58.42±6.32% and 65.45±5.24% vs. control (P<0.01), respectively. Following 18 h incubation in UK-432,097, 27-hydroxylase mRNA and protein increased by 46.45±3.4% and 50.27±8.9% (P<0.01), respectively. Message and protein level of LXRα were upregulated to 155.80±4.9% and 157.98±12.9% (n=3, P<0.01), respectively. A2AR blockade with ZM-241385 negated the effect of UK-432,097. UK-432,097 decreased oxidized LDL uptake by 28.9% in THP-1 macrophages (P<0.01). Conclusions This study demonstrates that UK-432,097 increases anti-atherogenic reverse cholesterol transport proteins with concomitant reduction in oxidized lipid accumulation in THP-1 macrophages. Since MTX is already being used in clinical trials to reduce cardiovascular risk, our results encourage further studies of specific A2AR agonists as cardioprotective treatment in high risk individuals.

1: COMPARATIVE EFFECT OF SELECTIVE AND NON-SELECTIVE COX-2 INHIBITORS ON LIPID ACCUMULATION IN HUMAN MACROPHAGES

Purpose of Study It is the second decade of controversy regarding the cardiovascular (CV) effects of cycloxygenase-2 (COX-2) inhibitors. COX-2 inhibitors possess anti-inflammatory and analgesic effects comparable with conventional non-steroidal anti-inflammatory drugs, but produce fewer gastrointestinal adverse effects. Here we demonstrate that only selective COX-2 inhibitors cause disruption of the delicate balance between cholesterol efflux and influx that leads to lipid overload and macrophage foam cell formation (FCF). Methods Used THP-1 human macrophages were incubated with: celecoxib (10 µM, 25 µM); rofecoxib (10 µM, 25 µM); naproxen (10 µM, 25 µM); acetaminophen (0.5 mM, 1 mM)±oxidized low density lipoprotein (oxLDL, 25 µg/ml, 48 h) or 5 µg/ml (Dil)-oxLDL. FCF (% oil red O stained cells) and oxLDL accumulation were determined (fluorescent intensity). Scavenger receptors: CD36, LOX-1, SR-A1 and CXCL16 and cholesterol efflux proteins: ATP-binding cassette transporter (ABC) A1 and ABCG1 were detected in macrophages by QRT-PCR and immunocytochemistry. Summary of Results Celecoxib decreased ABCA1 and ABCG1 message in a concentration dependent manner: 68.2±13.36% for ABCA1 and 65.7±13.36% for ABCG1 (control set at 100%, n=6, P<0.01). Neither naproxen nor acetaminophen significantly affected expression of cholesterol efflux proteins. Both specific and nonspecific COX-2 inhibitors had a significant impact on expression of scavenger receptors CD36, LOX-1 and SR-A1–nearly double control (n=6, P<0.05). However, only specific COX-2 inhibitors significantly increased FCF in THP-1 differentiated macrophages (62.2±5.2% for celecoxib and 56.3±3.4% for rofecoxib vs. 33.5±5.1% for untreated cells, P<0.05). Conclusions Here we report that only specific COX-2 inhibitors might contribute to atherogenesis by promoting lipid overload and lipoprotein accumulation. This may explain, in part, the increased CV risk in patients taking COX-2 inhibitors for extended periods. Despite increased scavenger receptor expression, naproxen and acetaminophen do not impact lipid content, perhaps because efflux pathways remain intact.

3: INHIBITION OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS RESTORES CHOLESTEROL EFFLUX IN THP-1 HUMAN MACROPHAGES EXPOSED TO PLASMA FROM TYPE 1 DIABETES MELLITUS PATIENTS

Purpose of Study Advanced glycation end products (AGE), proteins formed by nonenzymatic glycation, are prevalent in patients with diabetes mellitus (DM) and contribute to the development of atherosclerosis. We have demonstrated that plasma from patients with type 1 DM (T1DM) decreases expression of cholesterol efflux proteins in cultured THP-1 macrophages compared to healthy control (HC) plasma. Here we explore a mechanism that restores cholesterol efflux in T1DM plasma through blockade of the receptor for AGE (RAGE). Methods Used Carboxy-methyl-lysine-modified proteins (CML-MP), the most prevalent AGE in vivo, were measured in the plasma of 20 pediatric T1DM patients and 20 sex and age-matched HC by ELISA. THP-1 macrophages (106/ml) were incubated for 18 h in RPMI media in the presence of 10% plasma from each enrolled patient in triplicate±anti-RAGE antibody. Cholesterol efflux proteins: ATP binding cassette transporter (ABC)A1 and 27-hydroxylase were quantified by real-time RT-PCR using specific primers for each gene. Summary of Results The level of CML-MP was significantly elevated in T1DM plasma (1.65±1.3 ng/ml) versus HC plasma (1.05±0.4 ng/ml) (P<0.05, n=20). ABCA1 expression was significantly lower in macrophages exposed to T1DM plasma (1.108±0.8 U) versus HC plasma (1.624±0.6) (P<0.05, n=20). Exposure of THP-1 macrophages to T1DM plasma downregulated 27-hydroxylase mRNA to 1.94±0.9 U in T1DM versus 3.4±2.9 U in HC (P<0.01, n=20). Inactivation of RAGE before exposure to T1DM plasma increased ABCA1 expression by 20%. However, we observed no effect on the level of 27-hydroxylase. Conclusions We demonstrate that elevated AGE in plasma of T1DM patients inhibits cholesterol efflux and suppresses intracellular cholesterol processing via 27-hydroxylase and ABCA1 in naïve macrophages. RAGE inactivation restores mRNA level of the ABCA1 transporter, but not the 27-hydroxylase enzyme. These findings impart new targets for prevention of cardiovascular disease in DM and suggest that factors other than AGE may impact cholesterol transport.