Jurgen Seppen

Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression

Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.

Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

BACKGROUND/AIMSnLentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes.nnnMETHODSnA hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors.nnnRESULTSnThe HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3+/-1.7-fold and 2.3-6.0-fold in various other cell types. A role of HBV x protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5+/-1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54+/-13% and increased fluorescence 2.8+/-0.6-fold. The PPT increased transduction to 47+/-11% and increased fluorescence 2.3+/-0.4-fold. The vector with PPT and HBV elements transduced 68+/-10% of hepatocytes and increased fluorescence synergistically, 17+/-6 fold.nnnCONCLUSIONSnThis study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.

T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells

AIMnTreatment of disorders of the sinus node or the atrioventricular node requires insights into the molecular mechanisms of development and homoeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogramme terminally differentiated working cardiomyocytes into pacemaker cells.nnnMETHODS AND RESULTSnTBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice using tamoxifen. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na(+) channel (Na(V)1.5; I(Na)), and inwardly rectifying K(+) ion channels (K(ir) genes; I(K1)). Concordantly, we observed conduction slowing in these hearts and reductions in I(Na) and I(K1) in cardiomyocytes isolated from these hearts. The reduction in I(K1) resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current I(f) was observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes.nnnCONCLUSIONSnTBX3 reprogrammes terminally differentiated working cardiomyocytes and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce I(K1) density to enable diastolic depolarization are promising TBX3 characteristics that may facilitate biological pacemaker formation strategies.

Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer

Bilirubin is glucuronidated by bilirubin UDP-glucuronyltransferase (UGT1A1) before biliary excretion. Because bilirubin is toxic, patients with Crigler–Najjar type I (CN), who have no UGT1A1 activity, suffer severe brain damage early in childhood. The Gunn rat is the model for CN type 1. Gunn rat fetuses were injected with 107 transducing units of UGT1A1 lentiviral vector at the end of the third trimester on embryonic day 19. Serum bilirubin of injected Gunn rats was lowered by 45% compared to untreated controls. This decrease was highly significant (P < 106) and was sustained for more than a year. In treated Gunn rats, bilirubin glucuronides were present in bile and UGT1A1 protein was detected in tissue. Liver, intestine, stomach, pancreas, and other organs were transduced and mostly contained 1% or less vector copies per genome. Tissue distribution was variable among experimental animals but high transduction levels were seen in pancreas and intestine in most animals. Immunohistochemistry of these organs revealed transduction of pancreatic acinar cells and intestinal epithelium. Injection of a lentiviral UGT1A1 vector into third-trimester Gunn rat fetuses corrects the metabolic deficiency and mediates a reduction of serum bilirubin levels that would be therapeutic in humans.

A mutation which disrupts the hydrophobic core of the signal peptide of bilirubin UDP-glucuronosyltransferase, an endoplasmic reticulum membrane protein, causes Crigler-Najjar type IIs

Crigler‐Najjar (CN) disease is caused by a deficiency of the hepatic enzyme, bilirubin UDP‐glucuronosyltransferase (B‐UGT). We have found two CN type II patients, who were homozygous for a leucine to arginine transition at position 15 of B‐UGT1. This mutation is expected to disrupt the hydrophobic core of the signal peptide of B‐UGT1. Wild type and mutant B‐UGT cDNAs were transfected in COS cells. Mutant and wild type mRNA were formed in equal amounts. The mutant protein was expressed with 0.5% efficiency, as compared to wild type. Mutant and wild type mRNAs were translated in vitro. Wild type transferase is processed by microsomes, no processing of the mutant protein was observed.

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMSnProgressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts.nnnMETHODSnTo investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts.nnnRESULTSnOverexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice.nnnCONCLUSIONSnATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Apical Gene Transfer into Quiescent Human and Canine Polarized Intestinal Epithelial Cells by Lentivirus Vectors

ABSTRACT Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.