Norman C. W. Wong

Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors

ABSTRACT Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1β, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 × 1011 particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-α), RANTES, IP-10, MIP-1β, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-α and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 × 1011 particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b+ cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.

RVX-208: A Small Molecule That Increases Apolipoprotein A-I and High-Density Lipoprotein Cholesterol In Vitro and In Vivo

OBJECTIVES The aim of this study was to determine whether a novel small molecule RVX-208 affects apolipoprotein (apo)A-I and high-density lipoprotein cholesterol (HDL-C) levels in vitro and in vivo. BACKGROUND Increased apoA-I and HDL-C levels are potential therapeutic targets for reducing atherosclerotic disease. METHODS HepG2 cells were treated with 0 to 60 mumol/l RVX-208 followed by assays for apoA-I and HDL-C production. For in vivo studies, African green monkeys (AGMs) received 15 to 60 mg/kg/day RVX-208, and the serum was analyzed for lipoprotein levels, HDL-subparticle distribution, cholesterol efflux, and activity of lipid-modifying enzymes. A phase I clinical trial was conducted in healthy volunteers (given 1 to 20 mg/kg/day of RVX-208) to assess safety, tolerability, and pharmacokinetics. RESULTS The RVX-208 induced apoA-I messenger ribonucleic acid and protein synthesis in HepG2 cells, leading to increased levels of pre-beta-migrating and alpha-lipoprotein particles containing apoA-I (LpA-I) in spent media. Similarly, in AGMs, RVX-208 treatment for 63 days increased serum apoA-I and HDL-C levels (60% and 97%, respectively). In addition, the levels of pre-beta(1)-LpA-I and alpha1-LpA-I HDL-subparticles were increased as well as adenosine triphosphate binding cassette AI, adenosine triphosphate binding cassette G1, and scavenger receptor class B type I-dependent cholesterol efflux. These changes were not mediated by cholesteryl-ester-transfer protein. Treatment of humans for 1 week with oral RVX-208 increased apoA-I, pre-beta-HDL, and HDL functionality. CONCLUSIONS RVX-208 increases apoA-I and HDL-C in vitro and in vivo. In AGMs, RVX-208 raises serum pre-beta(1)-LpA-I and alpha-LpA-I levels and enhances cholesterol efflux. Data in humans point to beneficial features of RVX-208 that might be useful for treating atherosclerosis.

Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-κB

ABSTRACT The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVβgal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5′-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVβgal was dependent on the −161- to −96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVβgal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-κB at position −113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-κB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVβgal and an adenoviral vector expressing IκBα resulted in suppression of adCMVβgal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-κB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-κB.

Effects of Glucose and Insulin on Rat Apolipoprotein A-I Gene Expression

We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mm) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (−474 to −7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning −425 to −376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (−411 to −404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

RVX-208, an Inducer of ApoA-I in Humans, Is a BET Bromodomain Antagonist

Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.

Adenovirus Vector-Induced Inflammation: Capsid-Dependent Induction of the C-C Chemokine RANTES Requires NF-κB

Adenovirus vectors for gene therapy activate responses in the host that result in acute inflammation of transduced tissues. Our previous studies in vivo demonstrate that chemokines, including the C-C chemokine RANTES (regulated on activation, normal T cell expressed and secreted), contribute to the acute inflammation induced by adenovirus vectors. Various first-generation adenovirus vectors, including adCMV beta gal, were equally capable of inducing the expression of RANTES 3 hr after transduction in epithelial HeLa and REC cells. Deletional analysis of the human RANTES promoter revealed that induction by adCMV beta gal required the elements spanning base pairs -90 to -25 of the gene. Electrophoretic mobility shift assays demonstrated that nuclear extracts from adCMV beta gal-transduced HeLa cells bound to an NF-kappa B site at position -54. Overexpression of I-kappa B alpha suppressed adCMV beta gal induction of RANTES, confirming that this process was dependent on the nuclear translocation of NF-kappa B. The coxsackievirus-adenovirus receptor (CAR)-independent, serotype 3 adenovirus was equally capable of inducing the expression of RANTES in HeLa cells. This observation suggested that binding to CAR was not specifically required in adenovirus vector-induced RANTES expression. The use of RGD peptides to block adCMV beta gal interactions with alpha(v)-integrins reduced RANTES expression but also transduction efficiency. In CAR-deficient P815 cells, binding of adCMV beta gal to alpha(v)-integrins without efficient cell transduction did not result in increased RANTES expression. Expression of human CAR in P815 cells increased the binding and transduction efficiency of adCMV beta gal and resulted in RANTES expression in these cells. These results suggest that the induction of RANTES by adenovirus vectors is dependent on efficient interaction with its cell surface receptors and vector internalization. Understanding the biology of the host response to adenovirus vectors will impact the design of future generations of these agents aimed at reducing their immunogenicity and improving their safety.

A Mutant High-Density Lipoprotein Receptor Inhibits Proliferation of Human Breast Cancer Cells

High-density lipoprotein (HDL) stimulates the growth of many types of cells, including those of breast cancer. High levels of HDL are associated with an increased risk of breast cancer development. A scavenger receptor of the B class (SR-BI)/human homolog of SR-BI, CD36, and LIMPII analogous-1 (CLA-1) facilitates the cellular uptake of cholesterol from HDL and thus augments cell growth. Furthermore, HDL is also believed to have antiapoptotic effects on various cell types, and this feature adds to its ability to promote cell growth. These collaborative roles of HDL and CLA-1 prompted us to assess the function of these components on human breast cancer cells. In this study, we created a mutant CLA-1 (mCLA) that lacked the COOH-terminal tail to determine its potential role in breast cancer cell growth. Expression of mCLA inhibited the proliferation of breast cancer cell line MCF-7. This inhibitory action of mCLA required the transcriptional factor activator protein-1 (AP-1), and the mutant receptor also affected the antiapoptotic features of HDL. The effect of HDL on AP-1 activation and [3H]thymidine incorporation was abrogated by wortmannin, a specific inhibitor of phosphoinositide 3-kinase. Furthermore, the dominant negative mutant of Akt abolished the ability of HDL to activate AP-1. These findings raise the possibility that the inhibitors of the effects of HDL may be of therapeutic value for breast cancer.

Obesity-related Changes in High-density Lipoprotein Metabolism

Obesity is associated with a 3‐or‐more‐fold increase in the risk of fatal and nonfatal myocardial infarction ( 1 , 2 , 3 , 4 , 5 , 6 ). The American Heart Association has reclassified obesity as a major, modifiable risk factor for coronary heart disease ( 7 ). The increased prevalence of premature coronary heart disease in obesity is attributed to multiple factors ( 8 , 9 , 10 ). A principal contributor to this serious morbidity is the alterations in plasma lipid and lipoprotein levels. The dyslipidemia of obesity is commonly manifested as high plasma triglyceride levels, low high‐density lipoprotein cholesterol (HDLc), and normal low‐density lipoprotein cholesterol (LDLc) with preponderance of small dense LDL particles ( 7 , 8 , 9 , 10 ). However, there is a considerable heterogeneity of plasma lipid profile in overweight and obese people. The precise cause of this heterogeneity is not entirely clear but has been partly attributed to the degree of visceral adiposity and insulin resistance. The emergence of glucose intolerance or a genetic predisposition to familial combined hyperlipidemia will further modify the plasma lipid phenotype in obese people ( 11 , 12 , 13 , 14 , 15 ).

Expression of Human Scavenger Receptor B1 on and in Human Platelets

Objective—The abundance of HDL particles correlates inversely with the incidence of coronary heart disease. The human scavenger receptor B1 (hSR-B1/CLA-1) is a receptor for HDL. Expression of hSR-B1/CLA-1 mRNA and protein in human platelets was determined using reverse transcriptase–polymerase chain reaction and Western blot, respectively. Presence of the protein on the surface of platelets was shown using flow cytometry. Methods and Results—Immunochemical staining for hSR-B1/CLA-1 showed that it was expressed in megakaryocytes, the platelet precursors of human bone marrow. These findings prompted us to ask whether hSR-B1/CLA-1 was differentially expressed on platelets obtained from patients with atherosclerotic disease compared with those in control subjects. Our findings showed that abundance of hSR-B1/CLA-1 was significantly reduced on the surface of platelets from patients with atherosclerotic disease. The reduced levels of hSR-B1/CLA-1 were associated with increased cholesterol ester content in platelets from patients with atherosclerotic disease compared with control subjects. A negative correlation existed between hSR-B1/CLA-1 expression and platelet aggregation. In summary, our studies show that the HDL receptor hSR-B1/CLA-1 is expressed in platelets and their precursor, the megakaryocyte. The levels of hSR-B1/CLA-1 expression correlate inversely with cholesterol ester content and platelet aggregation. Conclusion—These findings suggest that determining the level of hSR-B1/CLA-1 expression on the platelets may be a useful clinical marker for atherosclerotic diseases.

Epidermal Growth Factor Induction of Apolipoprotein A-I Is Mediated by the Ras-MAP Kinase Cascade and Sp1

Insulin induces apolipoprotein A-I,apoA-I gene transcription via a membrane receptor with intrinsic tyrosine kinase activity. This finding prompted us to ask whether the gene is stimulated by epidermal growth factor (EGF), EGF a peptide hormone that binds to another member of the receptor superfamily with tyrosine kinase activity. Our data showed that like insulin, EGF increased abundance of apoA-I protein and transcription of the gene in human hepatoma, Hep G2 cells. The effects of both hormones appeared direct because their induction of apoA-I gene transcription was not affected by the protein synthesis inhibitor, cycloheximide. Although both insulin and EGF stimulate apoA-I expression, each hormone binds to a distinct membrane receptor thus suggesting differential intracellular signaling. Therefore, we used a panel of inhibitors to define the pathway(s) that mediate the actions of these hormones. Whereas, the actions of EGF required only the Ras-mitogen-activated protein, MAP kinase, those of insulin were mediated by equal participation of both the Ras-MAP kinase and protein kinase C, PKC cascades. Despite differences in signaling pathways triggered by each hormone receptor, the activation ofapoA-I transcription required the participation of a single transcription factor, Sp1. Furthermore, EGF induction of transcription was attenuated by mutating the MAP kinase site at amino acid, Thr266 rendering Sp1 phosphorylation deficient. In summary, EGF stimulation of apoA-I expression is mediated solely by the Ras-MAP kinase cascade and enhanced activity of this pathway requires Sp1 with an intact phosphorylation site at Thr266. However, insulin induction of this gene is different and requires both Ras-MAP kinase and PKC pathways but their actions are also mediated by Sp1.