Robert D. Gerard

Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.

We employed homologous recombination in embryonic stem cells to produce mice lacking functional LDL receptor genes. Homozygous male and female mice lacking LDL receptors (LDLR-/- mice) were viable and fertile. Total plasma cholesterol levels were twofold higher than those of wild-type litter-mates, owing to a seven- to ninefold increase in intermediate density lipoproteins (IDL) and LDL without a significant change in HDL. Plasma triglyceride levels were normal. The half-lives for intravenously administered 125I-VLDL and 125I-LDL were prolonged by 30-fold and 2.5-fold, respectively, but the clearance of 125I-HDL was normal in the LDLR-/- mice. Unlike wild-type mice, LDLR-/- mice responded to moderate amounts of dietary cholesterol (0.2% cholesterol/10% coconut oil) with a major increase in the cholesterol content of IDL and LDL particles. The elevated IDL/LDL level of LDLR-/- mice was reduced to normal 4 d after the intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor driven by the cytomegalovirus promoter. The virus restored expression of LDL receptor protein in the liver and increased the clearance of 125I-VLDL. We conclude that the LDL receptor is responsible in part for the low levels of VLDL, IDL, and LDL in wild-type mice and that adenovirus-encoded LDL receptors can acutely reverse the hypercholesterolemic effects of LDL receptor deficiency.

A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure

Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. We describe >12 miRNAs that are up- or down-regulated in cardiac tissue from mice in response to transverse aortic constriction or expression of activated calcineurin, stimuli that induce pathological cardiac remodeling. Many of these miRNAs were similarly regulated in failing human hearts. Forced overexpression of stress-inducible miRNAs was sufficient to induce hypertrophy in cultured cardiomyocytes. Similarly, cardiac overexpression of miR-195, which was up-regulated during cardiac hypertrophy, resulted in pathological cardiac growth and heart failure in transgenic mice. These findings reveal an important role for specific miRNAs in the control of hypertrophic growth and chamber remodeling of the heart in response to pathological signaling and point to miRNAs as potential therapeutic targets in heart disease.

Chapter 8 Use of Recombinant Adenovirus for Metabolic Engineering of Mammalian Cells

Publisher Summary This chapter describes the utility of Adenovirus for transfer of genes involved in metabolic regulation into mammalian cells, with particular emphasis to primary cell types with low replicative activity, such as hepatocytes and cells of the islets of Langerhans. The chapter provides methods and procedures for constructing and propagating new recombinant virions. Recombinant adenoviruses have been useful for delivering genes to whole animals. Viruses have been administered as single injections via accessible blood vessels such as the external jugular vein. Adenovirus in its current form is unlikely to represent the ultimate transfer vector for human gene therapy, given problems such as the lack of integration of the viral genome and the potential for immunological response to injected virus. It is therefore likely that new research initiatives will focus on attempting to engineer “second generation” virions that combine functional features of different viruses—that is, the site-specific integration function of adeno-associated virus (AAV) with the growth and infectivity characteristics of adenovirus.

ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion.

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette (ABC) transporters that limit intestinal absorption and promote biliary excretion of neutral sterols. Mutations in either ABCG5 or ABCG8 result in an identical clinical phenotype, suggesting that these two half-transporters function as heterodimers. Expression of both G5 and G8 is required for either protein to be transported to the plasma membrane of cultured cells. In this paper we used immunofluorescence microscopy to confirm, in vivo, that G5 is localized to the apical membranes of mouse enterocytes and hepatocytes. Other ABC half-transporters function as homodimers or as heterodimers with other subfamily members. To determine whether G5 or G8 complex with other ABCG half-transporters, we co-expressed G1, G2, and G4 with either G5 or G8 in cultured cells. G1, G2, and G4 co-immunoprecipitated with G5, and G4 co-immunoprecipitated with G8, but the putative dimers were retained in the endoplasmic reticulum (ER). Adenovirus-mediated expression of either G5 or G8 in the liver of G5G8 null mice resulted in ER retention of the expressed proteins and no increase in biliary cholesterol. In contrast, co-expression of G5 and G8 resulted in transit of the proteins out of the ER and a 10-fold increase in biliary cholesterol concentration. Finally, adenoviral expression of G2 in the presence or absence of G5 or G8 failed to promote sterol excretion into bile. These experiments indicate that G5 and G8 function as obligate heterodimers to promote sterol excretion into bile.

Regulation of Hypoxia-Inducible Factor 2α Signaling by the Stress-Responsive Deacetylase Sirtuin 1

Coordinating Response to Stress Sirtuin 1 (Sirt1) (a protein deacetylase implicated in aging), senses the metabolic state of the cell and modulates the activity of substrate proteins that in turn regulate cellular transcriptional responses. In response to hypoxia, cells activate the transcription factor hypoxia-inducible factor 2 alpha (HIF-2α), which promotes adaptive responses. Dioum et al. (p. 1289; see the Perspective by Guarente) discovered a link between these two important cellular stress response systems—HIF-2α is a substrate of Sirt1. Direct interaction between Sirt1 and HIF-2α results in deacetylation of HIF-2α and enhances its transcriptional activity. In mice lacking Sirt1, the ability of HIF-2α to promote synthesis of the growth factor erythropoietin is diminished. Thus, the regulation of HIF-2α helps to coordinate responses of cells to various stresses. A deacetylase implicated in aging directly regulates a transcription factor that controls stress-responsive genes. To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2α) during hypoxia. The effect of Sirt1 on HIF-2α required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2α that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2α target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.

Inhibitory Role of Plasminogen Activator Inhibitor-1 in Arterial Wound Healing and Neointima Formation A Gene Targeting and Gene Transfer Study in Mice

BACKGROUND Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation. METHODS AND RESULTS Neointima formation was evaluated in PAI-1-deficient (PAI-1(-/-)) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001+/-0.001 mm2 in PAI-1(+/+) and 0.016+/-0.008 mm2 in PAI-1(-/-) mice within 1 week after electric injury (P<.02) and 0.055+/-0.008 mm2 in PAI-1(+/+) and 0.126+/-0.006 mm2 in PAI-1(-/-) mice within 3 weeks after mechanical injury (P<.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1(-/-) smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1(-/-) mice were intravenously injected with replication-defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61+/-8 microg/mL with 2x10(9) plaque-forming units (pfu) virus. Luminal stenosis was 35+/-13% in control AdRR5-treated (2x10(9) pfu) and suppressed to 5+/-5% in AdCMVPAI-1-treated (6x10(8) pfu) PAI-1(-/-) mice (P<.002). CONCLUSIONS By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.

Crystal structure of the receptor-binding domain of adenovirus type 5 fiberprotein at 1.7 Å resolution

BACKGROUND Adenoviral infection begins with the binding of virion to the surface of host cells. Specific attachment is achieved through interactions between host-cell receptors and the adenovirus fiber protein and is mediated by the globular carboxy-terminal domain of the adenovirus fiber protein, termed the carboxy-terminal knob domain. RESULTS The crystal structure of the carboxy-terminal knob domain of the adenovirus type 5 (Ad5) fiber protein has been determined at 1.7 A resolution. Each knob monomer forms an eight-stranded antiparallel beta-sandwich structure. In the crystal lattice, the knob monomers form closely interacting trimers which possess a deep surface depression centered around the three-fold molecular symmetry axis and three symmetry-related valleys. CONCLUSIONS The amino acid residues lining the wall of the central surface depression and the three symmetry-related floors of the valleys are strictly conserved in the knob domains of Ad5 and adenovirus type 2 (Ad2) fiber proteins, which share the same cellular receptor. The beta-sandwich structure of the knob monomer demonstrates a unique folding topology which is different from that of other known antiparallel beta-sandwich structures. The large buried surface area and numerous polar interactions in the trimer indicate that this form of the knob protein is predominant in solution, suggesting a possible assembly pathway for the native fiber protein.

The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent

Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002)

Human Endothelial Nitric Oxide Synthase Gene Transfer Inhibits Vascular Smooth Muscle Cell Proliferation and Neointima Formation After Balloon Injury in Rats

BACKGROUND Loss of endothelial NO production after arterial injury may contribute to restenosis, characterized by neointima formation and elastic recoil. Adenovirus-mediated transfer of the gene encoding NO synthase (NOS) in balloon-injured arteries may restore NO production and inhibit neointima formation. METHODS AND RESULTS After balloon injury, rat carotid arteries were transduced with 3x10(10) pfu/mL recombinant adenovirus carrying the human endothelial constitutive NOS cDNA (AdCMVceNOS, n=8) or no cDNA (AdRR5, n=8). ceNOS expression was confirmed by immunoblot analysis of vascular extracts and was localized by immunostaining in 30% of medial smooth muscle cells (SMCs) and in the adventitia of AdCMVceNOS-transduced arteries. Vascular cGMP levels were reduced from 3.9 pmol/g wet wt in uninjured arteries to 0.7 pmol cGMP/g after AdRR5 but were restored after ceNOS gene transfer (3.8 pmol cGMP/g wet wt, P<.05 versus AdRR5). Intima-to-media ratio 2 weeks after injury was significantly reduced (0.19+/-0.02 in AdCMVceNOS-infected versus 0.69+/-0.07 in AdRR5-infected arteries, P<.05). In vitro, BrdU incorporation of AdCMVceNOS-infected SMCs was reduced by 28% compared with AdRR5-infected SMCs. Transduced cells from injured carotid arteries subjected to FACS sorting showed a significantly lower BrdU labeling index in ceNOS-infected rats (29+/-6% versus 43+/-5% and 45+/-4% in control, injured, and AdRR5-infected rats, respectively, P<.05). CONCLUSIONS AdCMVceNOS gene transfer to balloon-injured rat carotid arteries restores vascular NO production and reduces neointima formation, at least in part because of an antiproliferative effect on medial SMCs. Adenovirus-mediated ceNOS gene transfer might reduce arterial restenosis after balloon angioplasty.

Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486

microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.