Eric M. Billings

Heterogeneity of human macrophages in culture and in atherosclerotic plaques.

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.

Optimization of quantum mechanical molecular mechanical partitioning schemes: Gaussian delocalization of molecular mechanical charges and the double link atom method

Two new techniques for modeling chemical processes in condensed phases with combined quantum mechanical and molecular mechanical (QM/MM) potentials are introduced and tested on small, model compounds. The first technique, the double link atom (DLA) method, is an extension of the traditional, single link atom (SLA) method to avoid some of the problems with the latter method. These problems are primarily electrostatic, as the SLA method can produce an unphysical overall charge or dipole. The second technique, the delocalized Gaussian MM charge (DGMM) method, is an empirical way to include the delocalized character of the electron density of atoms in the MM region. This can be important for the electrostatic interaction of the QM region with nearby atoms in the MM region, and it can simplify the rules governing which classical interactions are included in the energies and forces. Even for very short distances, the DGMM method does not require the neglect of the MM host in the QM calculation. The DGMM method ...

3D structural model of the G-protein-coupled cannabinoid CB2 receptor

The potential for therapeutic specificity in regulating diseases and for reduced side effects has made cannabinoid (CB) receptors one of the most important G‐protein‐coupled receptor (GPCR) targets for drug discovery. The cannabinoid (CB) receptor subtype CB2 is of particular interest due to its involvement in signal transduction in the immune system and its increased characterization by mutational and other studies. However, our understanding of their mode of action has been limited by the absence of an experimental receptor structure. In this study, we have developed a 3D model of the CB2 receptor based on the recent crystal structure of a related GPCR, bovine rhodopsin. The model was developed using multiple sequence alignment of homologous receptor sub‐types in humans and mammals, and compared with other GPCRs. Alignments were analyzed with mutation scores, pairwise hydrophobicity profiles and Kyte‐Doolittle plots. The 3D model of the transmembrane segment was generated by mapping the CB2 sequence onto the homologous residues of the rhodopsin structure. The extra‐ and intracellular loop regions of the CB2 were generated by searching for homologous Cα backbone sequences in published structures in the Brookhaven Protein Databank (PDB). Residue side chains were positioned through a combination of rotamer library searches, simulated annealing and minimization. Intermediate models of the 7TM helix bundles were analyzed in terms of helix tilt angles, hydrogen‐bond networks, conserved residues and motifs, possible disulfide bonds. The amphipathic cytoplasmic helix domain was also correlated with biological and site‐directed mutagenesis data. Finally, the model receptor‐binding cavity was characterized using solvent‐accessible surface approach. Proteins 2003.

Stimulation of Signal Transducer and Activator of Transcription-1 (STAT1)-dependent Gene Transcription by Lipopolysaccharide and Interferon-γ Is Regulated by Mammalian Target of Rapamycin

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells. LY294002, a dual mTOR and PI3K inhibitor, blocked human inducible nitric oxide synthase (hiNOS) promoter activation and mRNA induction by cytokines and LPS in a PI3K-independent fashion. On gene expression analysis, LY294002 selectively blocked the induction of a subset of 14 LPS/interferon-γ (IFN-γ)-induced genes, previously characterized as signal transducer and activator of transcription-1 (STAT1)-dependent. LY294002, but not wortmannin, inhibited LPS/IFN-γ-dependent STAT1 phosphorylation at Ser-727 and STAT1 activity. Consistent with dual inhibition of mTOR and PI3K by LY294002, dominant-negative mTOR, anti-mTOR small interfering RNA, or rapamycin each inhibited phosphorylation of STAT1 only in the presence of wortmannin. LPS/IFN-γ led to the formation of a macromolecular complex containing mTOR, STAT1, as well as protein kinase Cδ, a known STAT1α kinase. Thus, LPS and IFN-γ activate the PI3K and mTOR pathways, which converge to regulate STAT1-dependent transcription of pro-apoptotic and pro-inflammatory genes in a rapamycin-insensitive manner.

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis.

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg × LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of 125I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7α1, CYP27α1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.

Differential gene expression in hematopoietic progenitors from paroxysmal nocturnal hemoglobinuria patients reveals an apoptosis/immune response in ‘normal’ phenotype cells

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder characterized clinically by intravascular hemolysis, venous thrombosis, and bone marrow failure. Despite elucidation of the biochemical and molecular defects in PNH, the pathophysiology of clonal expansion of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient cells remains unexplained. In pursuit of evidence of differences between GPI-AP-normal and -deficient CD34 cells, we determined gene expression profiles of isolated marrow CD34 cells of each phenotype from PNH patients and healthy donors, using DNA microarrays. Pooled and individual patient samples revealed consistent gene expression patterns relative to normal controls. GPI-AP-normal cells from PNH patients showed upregulation of genes involved in apoptosis and the immune response. Conversely, genes associated with antiapoptotic function and hematopoietic cell proliferation and differentiation were downregulated in these cells. In contrast, the PNH clone of GPI-AP-deficient cells appeared more similar to CD34 cells of healthy individuals. Gene chip data were confirmed by other methods. Similar gene expression patterns were present in PNH that was predominantly hemolytic as in PNH associated with aplastic anemia. Our results implicate an environmental influence on hematopoietic cell proliferation, in which the PNH clone evades immune attack and destruction, while normal cells suffer a stress response followed by programmed cell death.

Rationale and study design of the CardioGene Study: genomics of in-stent restenosis

BACKGROUND AND AIMS in-stent restenosis is a major limitation of stent therapy for atherosclerosis coronary artery disease. The CardioGene Study is an ongoing study of restenosis in bare mental stents (BMS) for the treatment of coronary artery disease. The overall goal is to understand the genetic determinants of the responses to vascular injury that result in the development of restenosis in some patients but not in others. Gene expression profiling at transcriptional and translational levels provides global assessment of gene activity after vascular injury and mechanistic insight. Furthermore, the delineation of genetic biomarkers would be of value in the clinical setting of risk-stratify patients prior to stent therapy. Prospective risk stratification would allow for the rational selection of specialized treatments against the development of in-stent restenosis (ISR), such as drug-eluting stents. SETTING Patients are enrolled at two sites in the US with high-volume cardiac catheterization facilities: the William Beaumont Hospital in Royal Oak, MI, USA, and the Mayo Clinic in Rochester, MN, USA. STUDY DESIGN Two complementary study designs are used to understand the molecular mechanisms of restenosis and the genetic biomarkers predictive of restenosis. First, 350 patients are enrolled prospectively at the time of stent implantation. Blood is sampled prior to stent placement and afterwards at 2 weeks and 6 months. The clinical outcome of restenosis is determined 6 and 12 months after stent placement. The primary outcome is clinical restenosis at 6 months. The major secondary outcome is clinical restenosis at 12 months. Second, a corollary case-control analysis will be carried out with the enrollment of an additional 250 cases with a history of recurrent restenosis after treatment with BMS. Controls for this analysis are derived from the prospective cohort. PATIENTS AND METHODS Consecutive patients presenting to the cardiac catheterization laboratory are screened, informed about the study and enrolled after signing the consent form. Enrollment has been completed for the prospective cohort, and enrollment of the additional group is ongoing. A standardized questionnaire is used to collect clinical data primarily through direct patient interview to assess medical history, medication use, functional status, family history, environmental factors, and social history. Further data are abstracted from the medical charts and catheterization reports. A total of 276 clinical variables are collected per individual at baseline, and 49 variables are collected at each of the 6- and 12-month follow-up visits. A Clinical Events Committee adjudicates clinical outcomes. Blood samples are processed at each clinical enrollment site using standardized operating procedures. From each blood sample, several aliquots are prepared and stored of peripheral blood mononuclear cells, granulocytes, platelets, serum, and plasma. Additionally, a portion of each patients leukocytes is cryopreserved for future cell-line creation. Samples are frozen and shipped to the National Heart, Lung and Blood Institute (NHLBI). Additional materials generated in the analysis of the samples at the NHLBI are frozen and stored, including isolated genomic DNA, total RNA, reverse transcribed cDNA libraries and labeled RNA hybridization mixtures used in microarray analysis. Per individual in the prospective cohort, high-quality transcript profiles of peripheral blood mononuclear cells at each time of blood sampling are obtained using Affymetrix U133A microarrays (Affymetrix, Santa Clara, CA, USA). Per chip, this yields 495,930 features per individual per time of sampling. This represents expression levels for 22,283 genes per patients oer time of blood sampling, including 14,500 well-characterized human genes. Proteomics of plasma is performed with multidimensional liquid chromatography and tandem mass spectrometry. Protein expression is examined similarly to mRNA expression as a measure of gene expression. Genotyping is performed in two manners. First, those genes showing differential expression at the levels of mRNA and protein are investigated using a candidate gene approach. Specific variants in known gene regulatory regions, such as promoters, are sought initially, as those variants may explain differences in expression level. Second, a genome-wide scan is used to identify genetic loci that are associated with ISR. Those regions identified are further examined for genes that show differential expression in the mRNA microarray profiling or proteomics investigations. These genes are finely investigated for candidate SNPs and other gene variants. Complementary genomic and proteomic approaches are expected to be robust. Integration of data sets is accomplished using a variety of informatics tools, organization of gene expression into functional pathways, and investigation of physical maps of up- and downregulated sets of genes. CONCLUSIONS The CardioGene Study is designed to understand ISR. Global gene and protein expression profiling define molecular phenotypes of patients. Well-defined clinical phenotypes will be paired with genomic data to define analyses aimed to achieve several goals. These include determining blood gene and protein expression in patients with ISR, investigating the genetic basis of ISR, developing predictive gene and protein biomarkers, and the identification of new targets for treatment.

Chemokine-Enhanced Chemotaxis of Lymphangioleiomyomatosis Cells with Mutations in the Tumor Suppressor TSC2 Gene

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

Mesenchymal–epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations (“two-hit” cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal–epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.

Detection of Venous Thromboembolism by Proteomic Serum Biomarkers

Background Available blood assays for venous thromboembolism (VTE) suffer from diminished specificity. Compared with single marker tests, such as D-dimer, a multi-marker strategy may improve diagnostic ability. We used direct mass spectrometry (MS) analysis of serum from patients with VTE to determine whether protein expression profiles would predict diagnosis. Methods and Results We developed a direct MS and computational approach to the proteomic analysis of serum. Using this new method, we analyzed serum from inpatients undergoing radiographic evaluation for VTE. In a balanced cohort of 76 patients, a neural network-based prediction model was built using a training subset of the cohort to first identify proteomic patterns of VTE. The proteomic patterns were then validated in a separate group of patients within the cohort. The model yielded a sensitivity of 68% and specificity of 89%, which exceeded the specificity of D-dimer assay tested by latex agglutination, ELISA, and immunoturbimetric methods (sensitivity/specificity of 63.2%/60.5%, 97.4%/21.1%, 97.4%/15.8%, respectively). We validated differences in protein expression between patients with and without VTE using more traditional gel-based analysis of the same serum samples. Conclusion Protein expression analysis of serum using direct MS demonstrates potential diagnostic utility for VTE. This pilot study is the first such direct MS study to be applied to a cardiovascular disease. Differences in protein expression were identified and subsequently validated in a separate group of patients. The findings in this initial cohort can be evaluated in other independent cohorts, including patients with inflammatory conditions and chronic (but not acute) VTE, for the diagnosis of VTE.