Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

C1 esterase inhibitor concentrate in 1085 Hereditary Angioedema attacks--final results of the I.M.P.A.C.T.2 study.

BACKGROUND: The placebo-controlled study International Multicentre Prospective Angioedema C1-INH Trial 1 (I.M.P.A.C.T.1) demonstrated that 20 U/kg C1 esterase inhibitor (C1-INH) concentrate (Berinert®; CSL Behring, Marburg, Germany) is effective in treating acute abdominal and facial Hereditary Angioedema (HAE) attacks. METHODS: I.M.P.A.C.T.2 was an open-label extension study of I.M.P.A.C.T.1 to evaluate the safety and efficacy of long-term treatment with 20 U/kg C1-INH for successive HAE attacks at any body location. Efficacy outcomes included patient-reported time to onset of symptom relief (primary) and time to complete resolution of all symptoms (secondary), analysed on a per-patient and per-attack basis. Safety assessments included adverse events, vital signs, viral safety and anti-C1-INH antibodies. RESULTS: During a median study duration of 24 months, 1085 attacks were treated in 57 patients (10-53 years of age). In the per-patient analysis, the median time to onset of symptom relief was 0.46 h and was similar for all types of attacks (0.39-0.48 h); the median time to complete resolution of symptoms was 15.5 h (shortest for laryngeal attacks: 5.8 h; 12.8-26.6 h for abdominal, peripheral and facial attacks). Demographic factors, type of HAE, intensity of attacks, time to treatment, use of androgens and presence of anti-C1-INH antibodies had no clinically relevant effect on the efficacy outcomes. There were no treatment-related safety concerns. No inhibitory anti-C1-INH antibodies were detected in any patient. CONCLUSIONS: A single dose of 20 U/kg C1-INH concentrate is safe and provides reliable efficacy in the long-term treatment of successive HAE attacks at any body location.

Efficacy and safety of a new immunoglobulin G product, Gammaplex(®), in primary immunodeficiency diseases.

This open-label multi-centre study evaluated a new intravenous immunoglobulin, Gammaplex®, in the treatment of 50 patients with primary immunodeficiency and significant hypogammglobulinaemia. Patients treated previously with other intravenous immunoglobulins received Gammaplex® on their same infusion schedule for 1 year; 22 were on a 21-day and 28 on a 28-day regimen (300-800 mg/kg/infusion). There were no serious, acute bacterial infections, whereas six subjects (12·0%) had at least one such infection in the 6 months before enrollment. Forty subjects (80·0%) had at least one non-serious infection; the median number of infective episodes per subject per year was 3·07. Antibiotics were taken by 38 subjects therapeutically and prophylactically by 16 at some time. Fewer than half (46·0%) missed any time off work or school because of infection or other illness. Trough immunoglobulin (Ig)G levels were above 6·00 g/l in all subjects at all assessments after 15 weeks with two exceptions. Overall, 21·2% of infusions were associated with an adverse event up to 72 h after infusion. The frequency of adverse events increased with infusion rate. Headache was the most common product-related adverse event (7·5% of 703 infusions). In conclusion, Gammaplex® is effective in primary immunodeficiency and is well tolerated.

Insights into serotonin signaling mechanisms associated with canine degenerative mitral valve disease.

Little is known about the molecular abnormalities associated with canine degenerative mitral valve disease (DMVD). The pathology of DMVD involves the differentiation and activation of the normally quiescent mitral valvular interstitial cell (VIC) into a more active myofibroblast phenotype, which mediates many of the histological and molecular changes in affected the valve tissue. In both humans and experimental animal models, increased serotonin (5-hydroxytryptamine, 5HT) signaling can induce VIC differentiation and myxomatous valve damage. In canine DMVD, numerous lines of evidence suggest that 5HT and related molecules such as transforming growth factor-beta play a critical role in the pathogenesis of this disease. A variety of investigative techniques, including gene expression, immunohistochemistry, protein blotting, and cell culture, shed light on the potential role of 5HT in the differentiation of VIC, elaboration of myxomatous extracellular matrix components, and activation of mitogen-activated protein kinase pathways. These studies help support a hypothesis that 5HT and its related pathways serve as an important stimulus in canine DMVD. This review describes the pathological characteristics of canine DMVD, the organization and role of the 5HT pathway in valve tissue, involvement of 5HT in human and experimental models of valve disease, avenues of evidence that suggest a role for 5HT in naturally occurring DMVD, and finally, a overarching hypothesis describing a potential role for 5HT in canine DMVD.

Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate.

Bioprostheses fabricated from porcine aortic valves are widely used to replace diseased heart valves. Calcification is the principal cause of the clinical failure of these devices. In the present study, inhibition of the calcification of bioprosthetic heart valve cusps implanted subcutaneously in rats was achieved through the adjacent implantation of controlled-release matrices containing the anticalcification agent ethanehydroxydiphosphonate dispersed in a copolymer of ethylene-vinyl acetate. Prevention of calcification was virtually complete, without the adverse effects of retarded bone and somatic growth that accompany systemic administration of ethanehydroxydiphosphonate.

Serum serotonin concentrations in dogs with degenerative mitral valve disease.

BACKGROUND: Increased serotonin (5HT) signaling has been implicated in valvular disease of humans and animals, including canine degenerative mitral valve disease (DMVD). High circulating 5HT concentration is a potential source of increased signaling, and serum 5HT concentrations have not been previously reported in dogs with DMVD. HYPOTHESIS: Dogs with DMVD and small breed dogs predisposed to DMVD have higher serum 5HT concentrations than large breed controls. ANIMALS: Fifty dogs affected with DMVD, 34 dogs predisposed to DMVD but without cardiac murmur or echocardiographic evidence of DMVD, and 36 healthy large breed control dogs. METHODS: Prospective analysis. Serum 5HT concentration was measured by an ELISA test. RESULTS: Median serum 5HT concentration was significantly higher in dogs with DMVD and in dogs predisposed to DMVD as compared with controls (DMVD, 765.5 ng/mL [interquartile range, 561.3-944.4]; predisposed, 774.9 ng/mL [528.3-1,026]; control, 509.8 ng/mL [320.8-708.8]; P= .0001). Subgroup analysis of predisposed dogs indicated significantly higher serum 5HT concentrations in Cavalier King Charles Spaniel (CKCS) dogs than in other breeds (CKCS, 855.0 ng/mL [635.8-1,088]; non-CKCS, 554.2 ng/mL [380.6-648.4]; P= .0023). Age, platelet count, and platelet morphology were not correlated with 5HT concentration in any group. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with DMVD had significantly higher serum 5HT concentrations when compared with large breed control dogs. Healthy CKCS dogs had significantly higher serum 5HT concentrations than other healthy dogs predisposed to DMVD. Additional investigation into a possible role of 5HT in the pathogenesis of DMVD is warranted.

Vitamin K-dependent calcium binding proteins in aortic valve calcification.

The pathogenesis of valvar calcification, which complicates the course of cardiac valve disease and also affects tissue valve prostheses, is incompletely understood. The present work explores the possible role of the vitamin K-dependent, calcium-binding amino acid, gamma-carboxyglutamic acid (Gla) in valve mineralization. Gla is normally found in the vitamin K-dependent clotting factor proteins, and is also present in unique calcium binding proteins in bone, kidney, and lung. Unique Gla-containing proteins have also been isolated from pathologic calcifications including calcium containing renal stones and calcified atherosclerotic plaque. Calcified valves including specimens with calcific aortic stenosis, calcified porcine xenograft valves, and a calcified aortic homograft valve were analyzed for Gla content, complete amino acid analysis, and tissue calcium and phosphorus levels. Normal porcine valves contained protein-bound Gla (2.0-10.6 Gla/10(4) amino acids): no Gla was present in normal valve leaflets. Furthermore, Gla levels paralleled tissue calcium content in the calcified valves. In addition, complete amino acid analysis indicated a decline in valvar collagen content plus increased acidic proteins in conjunction with valvar calcification and the presence of Gla-containing proteins. These results suggest that calcific valvar disease may result in part from vitamin K-dependent processes.

Local delivery of dexamethasone for prevention of neointimal proliferation in a rat model of balloon angioplasty.

A periadventitial polymer system is an alternative local drug delivery technique to obtain and maintain high tissue levels of the drug at the site of vascular injury. To determine if local periadventitial delivery of dexamethasone decreases neointimal proliferation after balloon vascular injury, in three groups of Sprague-Dawley rats, 5% dexamethasone, 0.5% dexamethasone, and placebo silicone polymers were implanted around the left common carotid artery after balloon injury. In a fourth group, placebo polymers were implanted without balloon injury. Dexamethasone serum and tissue levels after polymer implantation were significantly higher in the 5% dexamethasone group compared with the 0.5% dexamethasone group. There was no neointima formation in any of the arterial segments covered with placebo polymers for 3 wk, but without balloon injury. In the arterial segments covered by the 5 and 0.5% dexamethasone polymers, there was a 76 and 75% reduction in intima/media ratios, respectively, compared with the placebo group (5% dexamethasone, 0.26 +/- 0.04; 0.5% dexamethasone, 0.27 +/- 0.03; placebo, 1.09 +/- 0.16, respectively; P < 0.0001). These results suggest that: (a) silicone polymers wrapped around the common carotid arteries for 3 wk did not, without balloon injury, stimulate neointimal proliferation in the rat model; (b) the activity of the drug-eluting polymer for suppressing intimal proliferation was chiefly, but not exclusively, site specific; and (c) transadventitial local delivery of dexamethasone at two different doses markedly inhibits neointimal proliferation after balloon vascular injury.

Gene delivery from a DNA controlled-release stent in porcine coronary arteries.

Expandable intra-arterial stents are widely used for treating coronary disease. We hypothesized that local gene delivery could be achieved with the controlled release of DNA from a polymer coating on an expandable stent. Our paper reports the first successful transfection in vivo using a DNA controlled-release stent. Green fluorescent protein (GFP) plasmid DNA within emulsion-coated stents was efficiently expressed in cell cultures (7.9% +/- 0.7% vs. 0.6% +/- 0.2% control, p < 0.001) of rat aortic smooth muscle cells. In a series of pig stent-angioplasty studies, GFP expression was observed in all coronary arteries (normal, nondiseased) in the DNA-treated group, but not in control arteries. GFP plasmid DNA in the arterial wall was confirmed by PCR, and GFP presence in the pig coronaries was confirmed by immunohistochemistry. Thus, DNA-eluting stents are capable of arterial transfection, and could be useful as delivery systems for candidate vectors for gene therapy of cardiovascular diseases.

Deletion of Rapgef6, a candidate schizophrenia susceptibility gene, disrupts amygdala function in mice.

In human genetic studies of schizophrenia, we uncovered copy-number variants in RAPGEF6 and RAPGEF2 genes. To discern the effects of RAPGEF6 deletion in humans, we investigated the behavior and neural functions of a mouse lacking Rapgef6. Rapgef6 deletion resulted in impaired amygdala function measured as reduced fear conditioning and anxiolysis. Hippocampal-dependent spatial memory and prefrontal cortex-dependent working memory tasks were intact. Neural activation measured by cFOS phosphorylation demonstrated a reduction in hippocampal and amygdala activation after fear conditioning, while neural morphology assessment uncovered reduced spine density and primary dendrite number in pyramidal neurons of the CA3 hippocampal region of knockout mice. Electrophysiological analysis showed enhanced long-term potentiation at cortico-amygdala synapses. Rapgef6 deletion mice were most impaired in hippocampal and amygdalar function, brain regions implicated in schizophrenia pathophysiology. The results provide a deeper understanding of the role of the amygdala in schizophrenia and suggest that RAPGEF6 may be a novel therapeutic target in schizophrenia.

gamma-Carboxyglutamate excretion and warfarin therapy.

The urinary excretion of gamma-carboxyglutamic acid (Gla), the amino acid involved in the vitamin K-dependent calcium binding of prothrombin and clotting factors VII, IX, and X, was studied in warfarin-anticoagulated patients. An isotope dilution procedure was developed for the measurement of free urinary Gla with the use of prior anion-exchange chromatography to separate and concentrate the free Gla from whole urine and subsequent automated amino acid analysis. Eight subjects on stable warfarin anticoagulant therapy and 11 comparable control subjects with normal coagulation were examined. Urinary Gla excretion was reduced in patients on warfarin anticoagulant therapy (p = 0.001) and the urinary Gla level correlated (r = -0.73, p = 0.001) with plasma prothrombin time. It is concluded that decreased urinary Gla in warfarin-treated patients is related to coagulation status and may be a clinically useful parameter.

Treatment of status epilepticus with lorazepam.

Twenty-one episodes of status epilepticus (SE) were each treated with 1 to 9 mg (mean, 4 mg) of intravenous lorazepam. All patients with generalized tonic-clonic ( GTC ) SE responded within 15 minutes. Nine (82%) of the 11 patients with episodes of partial SE with altered responsiveness responded poorly. Respiratory depression occurred in five instances (two requiring intubation) and was associated with transient loss of brain-stem reflexes, hypotension, and decorticate posturing in three cases. Generalized tonic-clonic SE was transformed into partial SE with altered responsiveness in three patients. In an additional four patients, marked lethargy developed. Lorazepam appears effective in controlling GTC SE but only occasionally effective in partial SE with altered responsiveness.

Azathioprine toxicity in neuromuscular disease.

Azathioprine toxicity was examined in 64 consecutively treated patients with various neuromuscular diseases. Reversible leukopenia was seen in 14 patients (22%). Hepatotoxicity developed in six patients (9%), and a systemic reaction characterized by fever, abdominal pain, nausea, vomiting, and anorexia occurred in eight patients (12%). Toxic effects limited the dose of azathioprine in 27 patients (42%) and led to discontinuation of therapy in 13 (20%). Macrocytosis developed in 20% of patients, but did not require an adjustment in the dose. Two patients received allopurinol and azathioprine; both developed reversible leukopenia and macrocytosis. Patients with hematologic and hepatic toxicity, but not those with systemic toxicity, successfully tolerated retreatment with azathioprine. Toxicity was delayed as long as 56 weeks after starting azathioprine in some patients.

The identification of the vitamin K-dependent bone protein osteocalcin as one of the gamma-carboxyglutamic acid containing proteins present in calcified atherosclerotic plaque and mineralized heart valves.

Calcium binding proteins containing gamma-carboxyglutamic acid (Gla) have previously been demonstrated to occur in calcified atherosclerotic plaque and calcified cardiac valves. Experiments were carried out to determine if one of the Gla-containing proteins in human cardiovascular calcifications is the vitamin K-dependent bone protein, osteocalcin. A radio-immunoassay for human osteocalcin was employed, and EDTA extractions of calcified atheromata, and aortic valves as well as relevant noncalcified material were analyzed. Tissue calcium levels were also determined, as were Gla levels as a measure of total vitamin K-dependent protein content. Osteocalcin was present at low levels in all calcified cardiovascular tissues (4.5-175.7 ng osteocalcin/mg protein) with trace levels or nondetectable amounts present in noncalcified tissue. Osteocalcin accounted for a small proportion of the total protein-bound Gla (0.01-0.05%). The relationship of osteocalcin to the other Gla-containing proteins of atherosclerotic plaque including atherocalcin, the principal extractable Gla-containing protein of calcified plaque, is discussed.

Carboxyglutamic acid (Gla) containing proteins of human calcified atherosclerotic plaque solubilized by EDTA. Molecular weight distribution and relationship to osteocalcin.

Proteins containing the calcium binding amino acid, gamma-carboxyglutamic acid (Gla), are abundant in calcified human atherosclerotic plaque, but are detectable only at trace levels in the normal arterial wall and non-mineralized atherosclerotic lesions. These proteins have been incompletely characterized, and their role in the pathophysiology of atherosclerosis is not known. The present study sought to determine the overall molecular weight distribution of the calcified plaque Gla-protein fraction solubilized by EDTA demineralization and the possible relationship of these proteins to the bone Gla-protein, osteocalcin. Calcified atheromata were demineralized with EDTA (0.5 M, pH 6.9) for 7 days and the dialyzed EDTA extract subjected to procedures which specifically labeled the protein-bound Gla-residues with tritium. The EDTA solubilized Gla-protein fraction (19.5% of the total Gla) was separated by gel filtration high performance liquid chromatography which demonstrated a single broad radiolabeled Gla-protein peak with an approximate molecular weight of 6000 daltons. In addition the EDTA solubilized atherosclerotic Gla-proteins could be distinguished from the bone Gla-protein, osteocalcin (molecular weight = 5700 daltons), by reverse phase HPLC and specific radioimmunoassays for osteocalcin. It is concluded that the Gla-proteins of human calcified atherosclerotic plaque solubilized with EDTA demineralization consist of a heterogeneous 6000 dalton fraction, which is apparently unrelated to the bone Gla-protein, osteocalcin.

Novel delivery of antiarrhythmic agents.

Conventional antiarrhythmia therapy by oral or intravenous routes of administration is often ineffective and results in drug-associated complications and toxicity. In addition, poor bioavailability and a high first-pass effect limit therapeutic applications of several investigational antiarrhythmic compounds, which are otherwise more potent and less toxic than available agents. The regional nature of the several cardiac diseases, such as ischaemia, restenosis or heart valve calcification, may require a high concentration of drug at the location of the disease, which by conventional routes may not be attainable due to systemic toxicity of the drug. Localised cardiac delivery of antiarrhythmic agents, based on drug-polymer implants, may have several advantages, including enhanced drug effects and reduced systemic drug toxicity. Computer-assisted automated feedback systems may further enhance the usefulness of this therapy in the clinical setting. Before clinical application of this method of drug delivery further study will be required, but it is hypothesised that pharmacokinetic variability for drugs delivered in this manner will be reduced and therefore efficacy and toxicity will be more predictable.

Mechanism of calcification of porcine bioprosthetic aortic valve cusps: role of T-lymphocytes.

Calcification of glutaraldehyde-preserved porcine aortic valve bioprosthetic cusps limits their success as cardiac valve substitutes. Subcutaneous implants of porcine bioprostheses in rats and rabbits have provided a convenient experimental tool to study this calcification process. Previous clinical research has suggested that the host's immune response to the porcine xenograft tissue may contribute to the calcific degeneration. To investigate the possible contribution of the immune response, porcine bioprosthetic cusps implanted subcutaneously in congenitally athymic (nude) BALBc mice and normal controls were analyzed biochemically and histologically after retrieval at 21 days. Calcification was comparable in implants retrieved from athymic (calcium 95.5 +/- 24.5 micrograms/mg) and normal mice (calcium 102.3 +/- 4.66 micrograms/mg). Explants from nude mice demonstrated fewer adherent cells than those from normal animals, but the morphologic characteristics of the calcification were the same in both groups, with dystrophic mineralization of the spongiosa predominating. Thus, normal T-lymphocyte function is not necessary for porcine bioprosthetic calcification, and immunologic processes do not contribute to this process.

Onset and progression of calcification in porcine aortic bioprosthetic valves implanted as orthotopic mitral valve replacements in juvenile sheep.

The purpose of this study was to characterize the onset and progression of mineralization in porcine bioprosthetic valves implanted in sheep and to test the hypothesis that such valves simulate calcification that is observed clinically and in other experimental models. Hancock I porcine aortic bioprosthetic valves (Medtronic Heart Valve Division, Irvine, Calif.) were implanted as orthotopic mitral valve replacements in juvenile sheep, retrieved after 1 to 124 days, and analyzed as follows: gross inspection, radiography, light, transmission, and surface scanning electron microscopy, and calcium analysis by absorption spectroscopy. Mineralization increased with increasing time after implantation in both valve cusps and adjacent aortic wall. Mean cuspal calcification was 80 micrograms/mg in valves removed after 3 to 4 months. Nevertheless, considerable variability among valves was apparent in the level of calcification noted at specific time intervals. Virtually all aspects of the morphologic characteristics were identical to those previously noted for clinical explants and experimental specimens, both subcutaneous and circulatory. In particular, ultrastructural examination revealed that the earliest calcific deposits were associated with devitalized cuspal connective tissue cells and their fragments. Collagen calcification was sparse. Both surface scanning and transmission electron microscopy indicated a lack of endothelial or blood-derived cells on the valves at all sampling times. We conclude that porcine bioprosthetic valves implanted as mitral valves in sheep provide a useful calcification model, simulating morphologic and pathobiologic events that occur clinically and in noncirculatory models. However, sufficient specimen replicates must be done to overcome variability in calcification among valves and sampling sites.

Antimineralization treatments for bioprosthetic heart valves. Assessment of efficacy and safety.

Since calcification limits the durability of contemporary bioprosthetic heart valves, antimineralization treatments are being widely investigated. Potential antimineralization treatments must have sustained prevention of mineralization without adverse effects. The preclinical investigation of efficacy and safety of antimineralization treatments comprises four essential steps: (1) subcutaneous implantation in small animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals.

Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy. Experimental studies of optimal dosages and therapeutic durations.

Ethanehydroxydiphosphonate therapy was studied for prevention of calcification of bioprosthetic heart valve cusps (from glutaraldehyde-preserved porcine aortic valves) implanted subcutaneously in 3-week-old male rats. Animals received daily subcutaneous injections of the drug (1, 5, 10, 15, or 25 mg/kg/24 hr) for 21 days with maximal inhibition of bioprosthetic heart valve calcification at a dosage of 15 mg/kg/24 hr (calcium level of diphosphonate-treated bioprostheses 3.5 +/- 0.5 micrograms/ml; calcium level of control bioprostheses, 161.2 +/- 5.0 micrograms/mg), but with irreversibly diminished bone and somatic growth. A dosage optimum was observed at 10 mg/kg/24 hr with significant inhibition of bioprosthetic heart valve calcification (at 21 days, the calcium level was 16.4 +/- 3.6 micrograms/mg) and an absence of adverse effects on epiphyseal development and overall growth. Bioprosthetic heart valves retrieved from animal receiving ethanehydroxydiphosphonate (15 mg/kg/24 hr) for only the first week after implantation had significantly more calcification after 21 days than did bioprostheses from animals treated for 2 or 3 weeks. Bioprostheses explanted after 110 days from animals receiving the drug (15 mg/kg/24 hr) for the first 3 weeks had calcification equivalent to that of untreated control rats. Diphosphonate (15 mg/kg/24 hr) was most efficacious when initiated within 48 hours of bioprosthesis implantation, but was totally ineffective if administered after 1 week. It is concluded that ethanehydroxydiphosphonate optimally prevents bioprosthesis calcification without significant adverse effects on epiphyseal development and overall somatic growth at a dosage of 10 mg/kg/24 hr in rat subdermal implants, but it must be administered by continuous daily injections beginning within 48 hours of the implantation; this approach should be pursued in further long-term circulatory experimental studies because of its possible clinical relevance.