Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.

Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition.

CC-chemokines are important mediators in the pathogenesis of atherosclerosis. Atherosclerosis progression is reduced by high-level, short-term inhibition of CC-chemokine activity, for example by adenoviral gene transfer. However, atherosclerosis is a chronic condition where short-term effects, while demonstrating proof-of-principle, are unlikely to provide maximum therapeutic benefit. Accordingly, we generated a recombinant lentivirus, lenti35K, encoding the broad-spectrum CC chemokine inhibitor, 35K, derived from the vaccinia virus. To investigate the effects of prolonged broad-spectrum chemokine inhibition on atherosclerosis, lenti35K, or lentiGFP or PBS were delivered to 6-week-old ApoE knockout (ApoE-KO) mice by hydrodynamic injection. Sustained lentiviral transduction and transgene expression were demonstrated by 35K mRNA and viral DNA in liver tissue, and recombinant 35K protein circulating in the plasma, 3 months after gene transfer. Plasma from lenti35K animals had reduced chemokine activity compared with plasma from lentiGFP or PBS-treated animals. Histologic analysis of aortic sinus sections revealed that atherosclerotic plaque area in lenti35K mice was significantly reduced compared with both lentiGFP and PBS controls. Furthermore, plaque macrophage content was substantially reduced in lenti35K mice. Lentiviral 35K gene transfer is a promising experimental strategy to reduce atherosclerosis progression, and demonstrates the potential of long-term CC-chemokine inhibition as a potential therapeutic target in atherosclerosis.

Programmed gene rearrangements altering gene expression.

Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens). Two major mechanisms for expression control are DNA inversion and DNA transposition. In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression. In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion. Gene rearrangements altering gene expression have mainly been found in some unicellular organisms. They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus. The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way.

Adenovirus-mediated gene transfer of a secreted form of human macrophage scavenger receptor inhibits modified low-density lipoprotein degradation and foam-cell formation in macrophages.

BACKGROUND: Macrophage scavenger receptors (MSRs) play an important role in the pathogenesis of atherosclerosis. Therefore, local modulation of MSR activity could have a beneficial effect on atherogenesis. METHODS AND RESULTS: We cloned a secreted "decoy" MSR (sMSR) that contains an extracellular portion of the human MSR type AI and constructed an adenoviral vector that directs high-level expression of sMSR in macrophages under the control of the human CD68 promoter. Expression of the sMSR protein inhibited the degradation of (125)I-labeled acetylated LDL and oxidized LDL by murine macrophages up to 90%. sMSRs also reduced acetylated LDL degradation in MSR knockout mouse peritoneal macrophages by 60% to 80%, which suggests that the decoy construct can compete for the uptake mediated via other related scavenger receptors. In addition, sMSRs inhibited foam-cell formation in murine macrophages in the presence of cytochalasin D. The mechanism of inhibition is through ligand binding to the sMSRs, which prevents the ligand binding to MSRs on cell membranes. CONCLUSIONS: The demonstration that recombinant adenovirus-mediated gene transfer of decoy sMSRs can block foam-cell formation suggests a possible new strategy for gene therapy of atherosclerosis and for the treatment of lipid accumulation after arterial manipulations.

Trypanosoma brucei variant-specific glycoprotein gene chromatin is sensitive to single-strand-specific endonuclease digestion.

Active variant surface glycoprotein (VSG) gene chromatin is preferentially digested by the restriction enzyme HinfI in nuclei of bloodstream variants of Trypanosoma brucei. HinfI sensitivity of VSG gene chromatin is not observed in nuclei of relapse variants in which the VSG gene has been inactivated in situ. Active VSG gene chromatin is preferentially degraded by the single-strand-specific endonucleases S1 and Bal31. This sensitivity is not the result of pre-existing single-strand breaks or a detectably altered nucleosomal organization. Trypanosome nuclei in which the run-on transcription of VSG genes has been specifically shut down have been used to show that Hinfl and Bal31 sensitivity is not dependent upon continued transcription of the VSG gene. The presence of single-stranded DNA regions within VSG gene chromatin is consistent with a model in which VSG genes are activated by increased torsional stress.

Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene.

We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.

5' structural motifs and Xenopus beta globin gene activation.

We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.

Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

Globin gene switching: a paradigm or what?

The delineation of the beta-globin locus control region has led to a new understanding of the developmental regulation of the beta-globin gene cluster. It now seems that globin gene switching is effected through the sequential and mutually exclusive interaction of the locus control region with the embryonic, fetal and adult stage specific globin genes.

Glutaredoxin 2a overexpression in macrophages promotes mitochondrial dysfunction but has little or no effect on atherogenesis in LDL-receptor null mice.

AIMS: Reactive oxygen species (ROS)-mediated formation of mixed disulfides between critical cysteine residues in proteins and glutathione, a process referred to as protein S-glutathionylation, can lead to loss of enzymatic activity and protein degradation. Since mitochondria are a major source of ROS and a number of their proteins are susceptible to protein-S-glutathionylation, we examined if overexpression of mitochondrial thioltranferase glutaredoxin 2a (Grx2a) in macrophages of dyslipidemic atherosclerosis-prone mice would prevent mitochondrial dysfunction and protect against atherosclerotic lesion formation. METHODS AND RESULTS: We generated transgenic Grx2aMac(LDLR-/-) mice, which overexpress Grx2a as an EGFP fusion protein under the control of the macrophage-specific CD68 promoter. Transgenic mice and wild type siblings were fed a high fat diet for 14 weeks at which time we assessed mitochondrial bioenergetic function in peritoneal macrophages and atherosclerotic lesion formation. Flow cytometry and Western blot analysis demonstrated transgene expression in blood monocytes and peritoneal macrophages isolated from Grx2aMac(LDLR-/-) mice, and fluorescence confocal microscopy studies confirmed that Grx2a expression was restricted to the mitochondria of monocytic cells. Live-cell bioenergetic measurements revealed impaired mitochondrial ATP turnover in macrophages isolated from Grx2aMac(LDLR-/-) mice compared to macrophages isolated from non-transgenic mice. However, despite impaired mitochondrial function in macrophages of Grx2aMac(LDLR-/-) mice, we observed no significant difference in the severity of atherosclerosis between wildtype and Grx2aMac(LDLR-/-) mice. CONCLUSION: Our findings suggest that increasing Grx2a activity in macrophage mitochondria disrupts mitochondrial respiration and ATP production, but without affecting the proatherogenic potential of macrophages. Our data suggest that macrophages are resistant against moderate mitochondrial dysfunction and rely on alternative pathways for ATP synthesis to support the energetic requirements.

Chemokines and myeloid cell recruitment.

Chemokine-chemokine receptor interactions mediate constitutive leukocyte trafficking and leukocyte recruitment to sites of infection and inflammation. We suggest that a multiplicity of leukocyte chemoattractants exists to increase the selectivity of leukocyte recruitment in a range of physiological and pathological settings.

Rabbit atherosclerotic lesions express scavenger receptor AIII mRNA, a naturally occurring splice variant that encodes a non-functional, dominant negative form of the macrophage scavenger receptor.

Macrophage class A scavenger receptor types I and II (SR-AI and II) mediate the uptake of oxidized LDL in atherosclerotic lesions. The recently described type III receptor (SR-AIII), which lacks amino acids encoded by exon 10 of the SR-A gene, is unable to mediate the uptake of ligands and acts as a dominant negative regulator in the trimeric SR-A molecule. To find out whether SR-AIII might play a role in the regulation of SR-A activity in the arterial wall, we studied its expression in normal and atherosclerotic aortic intima-medias of Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed New Zealand white (NZW) rabbits. SR-A mRNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a SR-AIII-specific primer pair and with a primer pair suitable for both SR-AI and III. Very low SR-AI expression and no SR-AIII expression was found in the lesion-free aortic intima-medias of WHHL rabbits and control NZW rabbits. WHHL rabbit fatty streaks contained abundant SR-AI expression and low-level SR-AIII expression. In contrast, the numerous fatty streaks and fatty plaques appearing in the aortas of cholesterol-fed (14 weeks) NZW rabbits, and the fatty plaques of WHHL rabbits contained clearly detectable SR-AIII expression in addition to the abundant SR-AI expression. In addition, SR-AIII mRNA was detected in NZW and WHHL rabbit livers. The results suggest that in advanced atherosclerotic lesions, cells may protect themselves from the excessive uptake of oxidized lipoproteins by generating SR-A molecules which cannot bind modified LDL.

Atherosclerosis: role of chemokines and macrophages.

Atherosclerosis is a pathological process that takes place in the major arteries and is the underlying cause of heart attacks, stroke and peripheral artery disease. The earliest detectable lesions, called fatty streaks, contain macrophage foam cells that are derived from recruited monocytes. More-advanced atherosclerotic lesions, called fibro-fatty plaques, are the result of continued monocyte recruitment and smooth muscle cell migration and proliferation. Variable numbers of CD4+ T cells are found in atherosclerotic lesions, and cytokines secreted by T helper 1 (Th1)- or Th2-type cells can have a profound influence on macrophage gene expression within atherosclerotic plaques. This review briefly addresses the key features of macrophage biology and discusses the factors that influence the growth and development of atherosclerotic lesions (atherogenesis). It then considers the potential role of chemokines in mediating monocyte recruitment and macrophage differentiation within atherosclerotic lesions.