Robert S. Geske

Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice

To understand the normal, physiological role of the c-src proto-oncogene, a null mutation was introduced into the gene by homologous recombination in mouse embryonic stem cells. Two independent targeted clones were used to generate chimeras that transmitted the mutated allele to their offspring. Intercrossing of heterozygotes gave rise to live born homozygotes, but most of these mice died within the first few weeks of birth. Histological and hematological examination of the homozygous mutants did not reveal detectable abnormalities in the brain or platelets, where src is most highly expressed. However, these mutants were deficient in bone remodeling, indicating impaired osteoclast function, and developed osteopetrosis. These results demonstrate that src is not required for general cell viability (possibly because of functional overlap with other tyrosine kinases related to src) and uncover an essential role for src in bone formation.

Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors

BACKGROUND Efficient methods of introducing genes into myocardial cells must be developed before local somatic cell gene therapy can be implemented against myocardial disease. Although adenoviral (Ad5) vectors have been used to target rodent hearts and plasmid DNA has been directly injected into the myocardium of rats and dogs, the amounts of recombinant protein produced by these procedures have not been reported, and adenoviral vectors have not been used in large mammalian hearts. METHODS AND RESULTS Replication-deficient recombinant adenoviral vectors carrying either the luciferase or lacZ reporter genes were injected directly into the ventricular myocardium of adult domestic swine for evaluation of reporter gene expression. This procedure did not affect regional myocardial function as assessed by systolic wall thickening using ultrasonic crystals. Luciferase activity was detected 3 days after injection, increased markedly at 7 days, and then declined progressively at 14 and 21 days. Luciferase production was comparable in the right and left ventricular walls and increased with increasing amounts of virus, reaching 61 +/- 21 ng at the highest dose examined (3.6 x 10(9) plaque-forming units). The injection of 200 micrograms of plasmid DNA (pRSVL) produced levels of luciferase comparable to 1.8 x 10(8) plaque-forming units of recombinant Ad5; however, when normalized to the number of genes injected, the adenovirus was 140,000 times more efficient than plasmid DNA. Histochemical analysis of beta-galactosidase activity produced by a second Ad5 vector demonstrated that nearly all (> 95%) of the stained cells were cardiomyocytes and that the percentage of cardiomyocytes infected by the virus could be quite high in microscopic regions adjacent to the needle track (up to 75% in fields of 60 to 70 cells); however, Ad5-infected cells were rarely observed farther than 5 mm from the injection site. Furthermore, the Ad5 vector induced pronounced leukocytic infiltration that was far in excess of that seen after injection of vehicle alone. CONCLUSIONS This study demonstrates for the first time that direct intramyocardial injection of replication-deficient adenovirus can program recombinant gene expression in the cardiomyocytes of a large animal species with relevance to human physiology. The efficiency of adenovirus-mediated gene transfer is far superior to that of plasmid DNA injection, and this method appears to be capable of producing more recombinant protein. However, the cell-mediated immune response to the Ad5 vector and the limited distribution of reporter gene expression suggest that less immunogenic recombinant vectors and more homogeneous administration methods will be required before Ad5 vectors can be successfully used for phenotypic modulation.

Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis.

BACKGROUND Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty. METHODS AND RESULTS The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes. CONCLUSIONS The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.

Blindness and auditory impairment caused by loss of the sodium bicarbonate cotransporter NBC3

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.

Local Overexpression of Thrombomodulin for In Vivo Prevention of Arterial Thrombosis in a Rabbit Model

-Endothelial thrombomodulin plays a critical role in hemostasis by binding thrombin and subsequently converting protein C to its active form, a powerful anticoagulant. Thrombomodulin thus represents a central mechanism by which patency is maintained in normal vessels. However, thrombomodulin expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion. An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on in vivo thrombus formation was subsequently examined in a stasis/injury model of arterial thrombosis. The construct prevented arterial thrombosis formation in all animals, while viral and nonviral controls typically developed occluding thrombi. By histological analysis, nonviral controls exhibited intravascular thrombus occluding a mean of 70.52+/-3.72% of available lumen, while viral controls reached 86. 85+/-2.82% thrombotic occlusion; in contrast, Adv/RSV-THM reduced thrombosis to 28.61+/-3.31% of lumen in cross section. No significant intima-to-media ratio was observed in the thrombomodulin group relative to controls. Local infiltration of granulocytes and macrophages significantly decreased in the Adv/RSV-THM group relative to controls, while neutrophilic infiltration increased in viral controls relative to nonviral controls. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery, without the inflammatory damage that has limited many other adenoviral applications.

Thrombomodulin Overexpression to Limit Neointima Formation

BACKGROUND-These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. METHODS AND RESULTS-An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76+/-0.06%, whereas viral controls reached 0.77+/-0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47+/-0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. CONCLUSIONS-This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Neutrophil Sequestration and Pulmonary Dysfunction in a Canine Model of Open Heart Surgery With Cardiopulmonary Bypass Evidence for a CD18-Dependent Mechanism

BACKGROUND Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the beta 2 integrin CD18, a leukocyte adhesion molecule, mediates this response. METHODS AND RESULTS Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466 +/- 75 versus 516 +/- 43 PMN/mm2 alveolar surface area, mean +/- SEM, P < .001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H2O2 (305 +/- 45 versus 144 +/- 48 amol H2O2 per phagocyte per 20 minutes, P < .05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457 +/- 20 versus 246 +/- 49 mm Hg, P < .05), and they had a significantly increased lung wet-to-dry weight ratio (5.38 +/- 0.14 versus 4.54 +/- 0.15, P = .003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412 +/- 73 PMN/mm2, P < .001) and significantly inhibited PMN production of H2O2 (146 +/- 18 amol H2O2 per phagocyte per 20 minutes, P < .05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445 +/- 28 mm Hg, P < .05) and tended to have less lung water accumulation after bypass (4.99 +/- 0.20). CONCLUSIONS Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism.

Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland.

Lactose, the major carbohydrate of human milk, is synthesized in the Golgi from glucose and UDP-galactose. The lactating mammary gland is unique in its requirement for the transport of glucose into Golgi. Glucose transporter-1 (GLUT1) is the only isoform of the glucose transporter family expressed in mammary gland. In most cells, GLUT1 is localized to the plasma membrane and is responsible for basal glucose uptake; in no other cell type is GLUT1 a Golgi resident. To test the hypothesis that GLUT1 is targeted to Golgi during lactation, the amount and subcellular distribution of GLUTl were examined in mouse mammary gland at different developmental stages. Methods including immunohistochemistry, immunofluorescence, subcellular fractionation, density gradient centrifugation, and Western blotting yielded consistent results. In virgins, GLUTl expression was limited to plasma membrane of epithelial cells. In late pregnant mice, GLUT1 expression was increased with targeting primarily to basolateral plasma membrane but also with some intracellular signal. During lactation, GLUT1 expression was further increased, and targeting to Golgi, demonstrated by colocalization with the 110-kD coatomer-associated protein β-COP, predominated. Removal of pups 18 d after delivery resulted in retargeting of GLUT1 from Golgi to plasma membrane and a decline in total cellular GLUTl within 3 h. In mice undergoing natural weaning, GLUT1 expression declined. Changes in the amount and targeting of GLUT1 during mammary gland development are consistent with a key role for GLUT1 in supplying substrate for lactose synthesis and milk production.

Dexamethasone Enhancement of Hyperoxic Lung Inflammation in Rats Independent of Adhesion Molecule Expression

Infants and adults on oxygen often are treated with glucocorticoids in an attempt to reduce lung inflammatory injury. However, glucocorticoids hasten the development of hyperoxic lung injury in some animal models. The purpose of this study was to test the hypothesis that dexamethasone alters the lung inflammatory responses to hyperoxia exposure. We studied male Sprague-Dawley rats, placing them in >95% oxygen immediately after administration of 0, 0.1, 1, or 10 mg/kg of dexamethasone. At 0, 24, or 48 hr of exposure to hyperoxia, extravascular lung water contents were measured, and lung inflammatory responses were assessed by lung myeloperoxidase activities, lung neutrophil counts, and lung expression of E-Selectin and intercellular adhesions molecule-1 (ICAM-1). Dexamethasone, independent of exposure to hyperoxia, led to marked increases in lung neutrophil counts, without increases in lung myeloperoxidase activities or increases in the expression of the adhesion molecules. Hyperoxia exposure also enhanced lung neutrophil accumulation, and extravascular lung water increased earlier in animals exposed to hyperoxia and dexamethasone than in those exposed to hyperoxia alone. In conclusion, the increase in lung neutrophils in dexamethasone-treated rats without enhanced expression of E-Selectin or intracellular adhesions molecule-1 suggests that dexamethasone leads to lung neutrophil accumulation by its effect on neutrophils. The more rapid development of hyperoxic lung injury associated with earlier lung neutrophil accumulation suggests that dexamethasone-induced lung neutrophil sequestration primes the lung for the development of hyperoxic lung injury and supports further the conclusion that lung inflammation contributes significantly to the development of hyperoxic lung injury.

Endotoxin Induces Glutathione Reductase Activity in Lungs of Mice

ABSTRACT: Glutathione reductase catalyzes the NADPH-dependent conversion of glutathione disulfide to glutathione and helps protect the lung from injury by reactive oxygen. In animals allowed to breathe nearly 100% oxygen, the activities of other antioxidants in the lung can be induced by treatment with endotoxin, and this induction is associated with increased tolerance to hyperoxia. The purpose of this study was to see whether glutathione reductase activity in the lungs of mice increased with endotoxin treatment alone. We studied 60 FVB mice (20 males and 40 females). Half received endotoxin (500 μg/kg) intraperitoneally at time 0 and 24 h, and the controls received an equal volume of saline. At 48 h we killed the mice and removed their lungs. Treatment of mice with endotoxin increased glutathione reductase activily in the lung 55% (0.035 ± 0.005 to 0.054 ± 0.010 μmo NADPH reduced/min/mg prolein; mean ± SD; endoloxin differenl from conlrol, p < 0.001). The increase in aclivily was Ihe same for male and female mice. We measured Ihe specific prolein for glutathione reductase by Weslern analysis and mRNA for glutathione reductase using a slol-blol analysis and found lhat both increased roughly 2-fold with endoloxin treatmenl. This suggesls lhat endotoxin Ireatment resulted in either increased rale of transcription of glulathione reductase mRNA or increased mRNA slabilily. We conclude that endoloxin treatment increases glutathione reductase aclivily in the lung and thai Ihis increase in activity may play a role in subsequent protection from hyperoxia.