Srinivasa T. Reddy

Inflammatory/Antiinflammatory Properties of High-Density Lipoprotein Distinguish Patients From Control Subjects Better Than High-Density Lipoprotein Cholesterol Levels and Are Favorably Affected by Simvastatin Treatment

Background—The inflammatory/antiinflammatory properties of HDL were compared with HDL cholesterol in 2 groups of patients and in age- and sex-matched control subjects. Methods and Results—Group 1 consisted of 26 patients not yet taking a statin who presented with coronary heart disease (CHD) or CHD equivalents by National Cholesterol Education Program Adult Treatment Panel III criteria studied before and 6 weeks after 40 mg/d of simvastatin. Group 2 consisted of 20 patients with documented CHD and HDL cholesterol ≥84 mg/dL. The inflammatory/antiinflammatory properties of HDL were determined by the ability of the subject’s HDL to alter LDL-induced monocyte chemotactic activity (MCA) in a human artery wall coculture. Induction of MCA by a control LDL was determined in the absence or presence of the subject’s HDL. Values in the absence of HDL were normalized to 1.0. Values >1.0 after the addition of HDL indicated proinflammatory HDL; values <1.0 indicated antiinflammatory HDL. Group 1 values before simvastatin were LDL cholesterol, 118±24 mg/dL; HDL cholesterol, 57±13 mg/dL; triglycerides, 125±64 mg/dL; and high-sensitivity C-reactive protein (hs-CRP), 1.7±1.9 mg/L; and MCA values were 1.38±0.91, compared with 0.38±0.14 for control subjects (P =1.5×10−5). After simvastatin, values were LDL cholesterol, 73±24 mg/dL; HDL cholesterol, 61±14 mg/dL; triglycerides, 99±52 mg/dL; and hs-CRP, 1.3±1.3 mg/L; and MCA values were 1.08±0.71. In group 2, values were LDL cholesterol, 108±34 mg/dL; HDL cholesterol, 95±14 mg/dL; triglycerides, 89±44 mg/dL; and hs-CRP, 0.8±0.7 mg/L; and MCA values were 1.28±0.29, compared with 0.35±0.11 for control subjects (P =1.7×10−14). Similar results were obtained with the cell-free assay. Conclusions—The inflammatory/antiinflammatory properties of HDL distinguished patients from control subjects better than HDL cholesterol and were improved with simvastatin.

Differential effects of prostaglandin derived from ω-6 and ω-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion

Omega-6 (ω-6) polyunsaturated fatty acids (PUFA), abundant in the Western diet, are precursors for a number of key mediators of inflammation including the 2-series of prostaglandins (PG). PGE2, a cyclooxygenase (COX) metabolite of arachidonic acid, a ω-6 PUFA, is a potent mediator of inflammation and cell proliferation. Dietary supplements rich in ω-3 PUFA reduce the concentrations of 2-series PG and increase the synthesis of 3-series PG (e.g., PGE3), which are believed to be less inflammatory. However, studies on cellular consequences of increases in 3-series PG in comparison to 2-series PG have not been reported. In this study, we compared the effects of PGE2 and PGE3 on (i) cell proliferation in NIH 3T3 fibroblasts, (ii) expression and transcriptional regulation of the COX-2 gene in NIH 3T3 fibroblasts, and (iii) the production of an inflammatory cytokine, IL-6, in RAW 264.7 macrophages. PGE3, unlike PGE2, is not mitogenic to NIH 3T3 fibroblasts. PGE2 and PGE3 both induce COX-2 mRNA via similar signaling mechanisms; however, compared with PGE2, PGE3 is significantly less efficient in inducing COX-2 gene expression. Furthermore, although both PGE2 and PGE3 induce IL-6 synthesis in RAW 264.7 macrophages, PGE3 is substantially less efficient compared with PGE2. We further show that increasing the ω-3 content of membrane phospholipid results in a decrease in mitogen-induced PGE2 synthesis. Taken together, our data suggest that successful replacement of ω-6 PUFA with ω-3 PUFA in cell membranes can result in a decreased cellular response to mitogenic and inflammatory stimuli.

HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms

The lipoprotein HDL has two important roles: first, it promotes reverse cholesterol transport, and second, it modulates inflammation. Epidemiological studies show that HDL-cholesterol levels are inversely correlated with the risk of cardiovascular events. However, many patients who experience a clinical event have normal, or even high, levels of HDL cholesterol. Measuring HDL-cholesterol levels provides information about the size of the HDL pool, but does not predict HDL composition or function. The main component of HDL, apolipoprotein A-I (apo A-I), is largely responsible for reverse cholesterol transport through the macrophage ATP-binding cassette transporter ABCA1. Apo A-I can be damaged by oxidative mechanisms, which render the protein less able to promote cholesterol efflux. HDL also contains a number of other proteins that are affected by the oxidative environment of the acute-phase response. Modification of the protein components of HDL can convert it from an anti-inflammatory to a proinflammatory particle. Small peptides that mimic some of the properties of apo A-I have been shown in preclinical models to improve HDL function and reduce atherosclerosis without altering HDL-cholesterol levels. Robust assays to evaluate the function of HDL are needed to supplement the measurement of HDL-cholesterol levels in the clinic.

Oral D-4F Causes Formation of Pre-β High-Density Lipoprotein and Improves High-Density Lipoprotein–Mediated Cholesterol Efflux and Reverse Cholesterol Transport From Macrophages in Apolipoprotein E–Null Mice

Background— These studies were designed to determine the mechanism of action of an oral apolipoprotein (apo) A-I mimetic peptide, D-4F, which previously was shown to dramatically reduce atherosclerosis in mice. Methods and Results— Twenty minutes after 500 μg of D-4F was given orally to apoE-null mice, small cholesterol-containing particles (CCPs) of 7 to 8 nm with pre-β mobility and enriched in apoA-I and paraoxonase activity were found in plasma. Before D-4F, both mature HDL and the fast protein liquid chromatography fractions containing the CCPs were proinflammatory. Twenty minutes after oral D-4F, HDL and CCPs became antiinflammatory, and there was an increase in HDL-mediated cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol transport from intraperitoneally injected cholesterol-loaded macrophages in vivo. In addition, oral D-4F significantly reduced lipoprotein lipid hydroperoxides (LOOH), except for pre-β HDL fractions, in which LOOH increased. Conclusions— The mechanism of action of oral D-4F in apoE-null mice involves rapid formation of CCPs, with pre-β mobility enriched in apoA-I and paraoxonase activity. As a result, lipoprotein LOOH are reduced, HDL becomes antiinflammatory, and HDL-mediated cholesterol efflux and reverse cholesterol transport from macrophages are stimulated.

Human Paraoxonase-3 Is an HDL-Associated Enzyme With Biological Activity Similar to Paraoxonase-1 Protein but Is Not Regulated by Oxidized Lipids

Abstract— Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL) and participates in the prevention of low density lipoprotein (LDL) oxidation. Two other paraoxonase (PON) family members, namely, PON2 and PON3, have been identified. In this study, we report the cloning and characterization of the human PON3 gene from HepG2 cells. Tissue Northern analysis identifies an ≈1.3-kb transcript for PON3 primarily in the liver. PON3-specific peptide antibodies detect an ≈40-kDa protein associated with HDL and absent from LDL. Pretreatment of cultured human aortic endothelial cells with supernatants from HeLa Tet On cell lines overexpressing PON3 prevents the formation of mildly oxidized LDL and inactivates preformed mildly oxidized LDL. In contrast to PON1, PON3 is not active against the synthetic substrates paraoxon and phenylacetate. Furthermore, PON3 expression is not regulated in HepG2 cells by oxidized phospholipids and is not regulated in the livers of mice fed a high-fat atherogenic diet.

Identification of biomarkers for ovarian cancer using strong anion-exchange ProteinChips: Potential use in diagnosis and prognosis

One hundred eighty-four serum samples from patients with ovarian cancer (n = 109), patients with benign tumors (n = 19), and healthy donors (n = 56) were analyzed on strong anion-exchange surfaces using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry technology. Univariate and multivariate statistical analyses applied to protein-profiling data obtained from 140 training serum samples identified three biomarker protein panels. The first panel of five candidate protein biomarkers, termed the screening biomarker panel, effectively diagnosed benign and malignant ovarian neoplasia [95.7% sensitivity, 82.6% specificity, 89.2% accuracy, and receiver operating characteristic (ROC) area under the curve of 0.94]. The other two panels, consisting of five and four candidate protein biomarkers each, effectively distinguished between benign and malignant ovarian neoplasia and were therefore referred to as validation biomarker panel I (81.5% sensitivity, 94.9% specificity, 88.2% accuracy, and ROC = 0.94) and validation biomarker panel II (72.8% sensitivity, 94.9% specificity, 83.9% accuracy, and ROC = 0.90). The three ovarian cancer biomarker protein panels correctly diagnosed 41 of the 44 blinded test samples: 21 of 22 malignant ovarian neoplasias [10 of 11 early-stage ovarian cancer (I/II) and 11 of 11 advanced-stage ovarian cancer (III/IV)], 6 of 6 low malignant potential, 5 of the 6 benign tumors, and 9 of 10 normal patient samples. In conclusion, we have discovered three ovarian cancer biomarker protein panels that, when used together, effectively distinguished serum samples from healthy controls and patients with either benign or malignant ovarian neoplasia.

Apolipoprotein A-I Mimetic Peptides

Despite identical amino acid composition, differences in class A amphipathic helical peptides caused by differences in the order of amino acids on the hydrophobic face results in substantial differences in antiinflammatory properties. One of these peptides is an apolipoprotein A-I (apoA-I) mimetic, D-4F. When given orally to mice and monkeys, D-4F caused the formation of pre-beta high-density lipoprotein (HDL), improved HDL-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. In apolipoprotein E (apoE)-null mice, D-4F increased reverse cholesterol transport from macrophages. Oral D-4F reduced atherosclerosis in apoE-null and low-density lipoprotein (LDL) receptor-null mice. In vitro when added to human plasma at nanomolar concentrations, D-4F caused the formation of pre-beta HDL, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. Physical-chemical properties and the ability of various class A amphipathic helical peptides to activate lecithin cholesterol acyltransferase (LCAT) in vitro did not predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. We conclude that the antiinflammatory properties of different class A amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides, which determines the ability of the peptides to sequester inflammatory lipids. These differences appear to be too subtle to predict efficacy based on physical-chemical properties alone. However, understanding these physical-chemical properties provides an explanation for the mechanism of action of the active peptides.

Mechanisms of Disease: proatherogenic HDL—an evolving field

It is well known that, in large populations, HDL-cholesterol levels are inversely related to the risk of atherosclerotic clinical events; however, in an individual, the predictive value of an HDL-cholesterol level is far from perfect. As a result, other HDL-associated factors have been investigated, including the quality and function of HDL in contradistinction to the level of HDL-cholesterol. Regarding their quality, HDL particles are highly heterogeneous and contain varying levels of antioxidants or pro-oxidants, which results in variation in HDL function. It has been postulated that HDL functions to promote reverse cholesterol transport. Recent studies support this role for HDL but also indicate that HDL is a modulator of systemic inflammation. In the absence of inflammation, HDL has a complement of antioxidant enzymes that work to maintain an anti-inflammatory state. In the presence of systemic inflammation, these antioxidant enzymes can be inactivated and HDL can accumulate oxidized lipids and proteins that make it proinflammatory. Under these conditions the main protein of HDL, apolipoprotein A-I, can be modified by reactive oxygen species. This modification impairs the ability of HDL to promote cholesterol efflux by the ATP-binding cassette transporter A-1 pathway. Animal studies and small-scale human studies suggest that measures of the quality and novel functions of HDL might provide an improved means of identifying subjects at increased risk for atherosclerotic events, compared with the current practice of only measuring HDL-cholesterol levels. The quality and function of HDL are also attractive targets for emerging therapies.

The early detection of ovarian cancer: from traditional methods to proteomics. Can we really do better than serum CA-125?

Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. More than 80% of patients present with advanced disease, with 5 year survival rates between 15% and 45%. In contrast, the survival rate for stage I disease, with malignancy confined to the ovary, is approximately 95%. Given the discrepancy in survival outcomes between early- and late-stage disease, strategies that would allow for the detection of ovarian cancer in its early stages would hold promise to significantly improve the mortality rate from ovarian cancer. Unfortunately, current screening methods for the detection of early stage ovarian cancer are inadequate. However, several recent proteomics-based biomarker discovery projects show promise for the development of highly sensitive and specific markers for gynecological malignancies, including ovarian cancer. In this review, we hope to provide an overview of the early detection ovarian cancer from traditional methods to recent promises in the proteomics pipeline.

Prostaglandin Synthase-1 and Prostaglandin Synthase-2 Are Coupled to Distinct Phospholipases for the Generation of Prostaglandin D2 in Activated Mast Cells

Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.