First-in-Human Study in Healthy Subjects with FR104, a Pegylated Monoclonal Antibody Fragment Antagonist of CD28.

FR104 is a monovalent pegylated Fab' Ab, antagonist of CD28, under development for treatment of transplant rejection and autoimmune diseases. In contrast to CD80/86 antagonists (CTLA4-Ig), FR104 selectively blunts CD28 costimulation while sparing CTLA-4 and PD-L1 coinhibitory signals. In the present work, FR104 has been evaluated in a first-in-human study to evaluate the safety, pharmacokinetics, pharmacodynamics, and potency of i.v. administrations in healthy subjects. Sixty-four subjects were randomly assigned to four single ascending dose groups, two double dose groups and four single ascending dose groups challenged with keyhole limpet hemocyanin. Subjects were followed up over a maximum of 113 d. Overall, the pharmacokinetics of FR104 after a single and double infusions was approximately linear at doses ≥0.200 mg/kg. CD28 receptor occupancy by FR104 was saturated at the first sampling time point (0.5 h) at doses above 0.02 mg/kg and returned to 50% in a dose-dependent manner, by day 15 (0.020 mg/kg) to 85 (1.500 mg/kg). FR104 was well tolerated, with no evidence of cytokine-release syndrome and no impact on blood lymphocyte subsets. Inhibition of anti-keyhole limpet hemocyanin Ab response was dose-dependent in FR104 recipients and was already apparent at a dose of 0.02 mg/kg. Abs to FR104 were detected in 22/46 (48%) of FR104 recipients and only 1/46 (2.2%) was detected during drug exposure. In conclusion, selective blockade of CD28 with FR104 was safe and well tolerated at the doses tested. The observed immunosuppressive activity indicated that FR104 has potential to show clinical activity in the treatment of immune-mediated diseases.

Systemic and vascular oxidation limits the efficacy of oral tetrahydrobiopterin treatment in patients with coronary artery disease.

BACKGROUND: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease. METHODS AND RESULTS: Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue. CONCLUSIONS: Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states.

Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model.

BACKGROUND: The interest in non-antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic-resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection. MATERIALS AND METHODS: Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti-adhesive effect on H. pylori was determined first in a cell model using the mucus-producing gastric epithelial cell line NCI-N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day -2 to day 4 post-infection. Bacterial loads were determined by using quantitative real-time PCR and the standard plate count method. RESULTS: The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 µg/mL. In the anti-adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti-adhesive effects and even caused an increase in bacterial load in the stomach. CONCLUSIONS: The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.

Role of oxidative stress and apoptosis in the cellular response of murine macrophages upon Leishmania infection.

Leishmania parasites are able to survive in the macrophage, one of the most hostile environments of the vertebrate host. The present study investigated how Leishmania infection influences these host cell defence mechanisms. Macrophages were infected with antimony-susceptible and -resistant Leishmania strains. Free radical production in Leishmania-infected macrophages was measured by electron paramagnetic resonance. Apoptosis was detected with fluorescence microscopy using Annexin-V FITC labelling and with Western blotting to detect caspase-3 cleavage. Independent of their drug susceptibility profile or species background, all studied Leishmania strains induced a similar increase in free radical production in macrophages. O2 ●- production was significantly elevated during phagocytosis of the stationary phase promastigotes. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage. Leishmania donovani infection led to phosphatidylserine externalization only in RAW 264.7 cells. After an initial burst of O2 ●- during phagocytosis of promastigotes, amastigotes protect themselves by decreasing the O2 ●- production to the basal level. An increased NO production was observed 6 h after infection. Finally, induction of cell death is probably not essential in the survival of the parasite within the macrophage.

Intrinsic susceptibility of Giardia duodenalis assemblage subtypes A(I), A(II), B and E(III) for nitric oxide under axenic culture conditions.

The antigiardial effects of nitric oxide (NO·) have been reported in vitro, but only for assemblage A(I) lab strains. This study investigated the intrinsic NO· susceptibility of different assemblage subtypes. The susceptibility (IC50) for NO· released by MAHMA NONOate was studied for three lab (WB, G1 and GS/M-83-H7) and six field isolates of assemblage subtypes A(I), A(II), B and E(III). Tests were performed in phosphate-buffered saline supplemented with L-cysteine HCl, trypticase peptone, powder bovine bile and 20% inactivated foetal calf serum (for assemblages A and E) or human serum (for assemblage B), adjusted to pH 7.3, to support adequate trophozoite survival. Flow cytometry with fluorescein diacetate and propidium iodide as viability indicators was used to determine trophozoite viability. This study indicated that the NO· susceptibilities of assemblage A lab and field strains (subtypes A(I) and A(II)) were fully comparable, indicating that the NO· susceptibility of the lab strains remained representative for their genotype. The trophozoites of assemblages B and E(III) showed comparable NO· susceptibilities that were markedly higher than the susceptibilities of assemblage subtypes A(I) and A(II). This study suggests a role for the assemblage subtype in defining NO· susceptibilities. The underlying mechanisms still need to be elucidated, but assemblage-linked differences in the expression of the genes coding for flavohemoglobin or A-type flavoprotein may certainly deserve further attention.

In vitro and in vivo prophylactic and curative activity of the triterpene saponin PX-6518 against cutaneous Leishmania species.

OBJECTIVES: The oleanane triterpene saponin PX-6518, with known potent in vitro and in vivo activity against Leishmania donovani, was investigated for its spectrum against the cutaneous species Leishmania mexicana, Leishmania panamensis and Leishmania major. METHODS: In vitro activity was based on the reduction of amastigotes in primary peritoneal mouse macrophages. BALB/c mice were injected with 2 × 10(6) amastigotes in the base of the tail (L. panamensis and L. major) or the foot (L. mexicana) and subcutaneously treated with PX-6518 [1-10 mg/kg body weight (BW)] or Pentostam(®) (250 mg/kg BW Sb(V) eq). Evolution of skin lesions was monitored in a prophylactic dose-finding study, and early curative [6 weeks post-infection (pi)] and late curative (>8-10 weeks pi) studies. RESULTS: While moderate susceptibility to PX-6518 was obtained in vitro (IC(50): 1-4 µg/mL), excellent in vivo activity was demonstrated. In the prophylactic study (six administrations on alternate days, starting at 1 day pi), PX-6518 was 100% effective at 1 mg/kg BW against L. mexicana and L. panamensis, whereas L. major lesions could be prevented at 2 mg/kg BW. In the early curative (1 mg/kg BW once a week for 4 weeks) and late curative (1 mg/kg BW twice a week for 4 weeks) studies, PX-6518 completely healed L. mexicana and L. panamensis lesions, whereas L. major lesions were reduced by ∼ 50%. CONCLUSIONS: This study demonstrates that PX-6518 possesses potent and broad-spectrum prophylactic and curative efficacy against cutaneous leishmaniasis in the BALB/c mouse model. L. major was the least susceptible species tested and parasitological cure could not be obtained.

Transcription profiles of aortic smooth muscle cells from atherosclerosis-prone and -resistant regions in young apolipoprotein E-deficient mice before plaque development.

BACKGROUND/AIMS: Site-specific atherosclerosis is generally attributed to differential gene expression in endothelial cells. We investigated whether the transcriptome of smooth muscle cells is different between atherosclerosis-prone and atherosclerosis-resistant regions in apolipoprotein E-deficient (apoE-/-) mice before plaque development, and in C57Bl/6 mice. METHODS: De-endothelialized aortas (both strains: 3 males, 3 females, age 4 months) were divided into atherosclerosis-prone (AA: ascending aorta, aortic arch and proximal 2 mm of thoracic aorta) and -resistant (CTA: central thoracic aorta, i.e. 6 mm distal from the proximal 2 mm) regions. The transcriptome of these two regions was compared using whole-genome mouse microarrays. RESULTS: Microarray analysis revealed differential expression (>2-fold difference) of 70 and 244 genes in C57Bl/6 and apoE-/- mice. This was confirmed for 6 genes using the real-time quantitative polymerase chain reaction. Up- or downregulation in the AA was observed for 33 and 37 genes in C57Bl/6, and for 186 and 58 genes in apoE-/- mice, respectively. The 201 genes that showed exclusively differential expression in apoE-/- mice were related to atherosclerotic processes, such as cell adhesion, proliferation, differentiation, motility, cell death, lipid metabolism and immune responses. CONCLUSION: Our findings indicate that smooth muscle cells display an altered transcriptome at atherosclerosis-prone locations before actual lesion development.

An alternative, sensitive method to detect Helicobacter pylori DNA in feces.

BACKGROUND: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. MATERIALS AND METHODS: The newly proposed method uses selective hybridization of target DNA with biotin-labeled probes, followed by DNA isolation with streptavidin-coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. RESULTS: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10-fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. CONCLUSIONS: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.

MTHFR 677 C>T Polymorphism reveals functional importance for 5-methyltetrahydrofolate, not homocysteine, in regulation of vascular redox state and endothelial function in human atherosclerosis.

BACKGROUND: The role of circulating homocysteine as an atherosclerosis risk factor has recently been questioned. However, 5-methyl-tetrahydrofolate (5-MTHF), the circulating metabolite of folic acid participating in homocysteine metabolism, has direct effects on vascular function. We sought to distinguish the effects of plasma versus vascular tissue 5-MTHF and homocysteine on vascular redox and endothelial nitric oxide bioavailability in human vessels. METHODS AND RESULTS: We used the methyl tetrahydrofolate reductase (MTHFR) gene polymorphism 677C>T as a model of chronic exposure of the vascular wall to varying 5-MTHF levels in 218 patients undergoing coronary artery bypass graft surgery. Vascular superoxide, vascular 5-MTHF, and total homocysteine were determined in saphenous veins and internal mammary arteries obtained during surgery. Nitric oxide bioavailability was evaluated by organ bath studies on saphenous vein rings. MTHFR genotype was a determinant of vascular 5-MTHF (not vascular homocysteine). Both MTHFR genotype and vascular 5-MTHF were associated with vascular nitric oxide bioavailability and superoxide generated by uncoupled endothelial nitric oxide synthase. In contrast, vascular homocysteine was associated only with NADPH-stimulated superoxide. CONCLUSIONS: Genetic polymorphism 677 C>T on MTHFR affects vascular 5-MTHF (but not homocysteine) and can be used as a model to distinguish the chronic effects of vascular 5-MTHF from homocysteine on vascular wall. Vascular 5-MTHF, rather than plasma or vascular homocysteine, is a key regulator of endothelial nitric oxide synthase coupling and nitric oxide bioavailability in human vessels, suggesting that plasma homocysteine is an indirect marker of 5-MTHF rather than a primary regulator of endothelial function.

Preoperative sCD40L levels predict risk of atrial fibrillation after off-pump coronary artery bypass graft surgery.

BACKGROUND: The risk of atrial fibrillation (AF) after coronary bypass surgery has been related to redox state, inflammation, and ischemia. Platelet activation is common to all of these pathways. We investigated the relation between AF and preoperative soluble CD40 ligand (sCD40L), a proinflammatory marker released by activated platelets. Furthermore, we studied the role of inflammation, endothelial function, and redox state in this relation. METHODS AND RESULTS: sCD40L levels were measured in 144 patients in sinus rhythm the day before off-pump coronary artery surgery. Systemic inflammation was assessed from levels of C-reactive protein and soluble intercellular adhesion molecule-1, and endothelial function was assessed from the brachial artery flow-mediated dilatation response. Graft samples were collected during surgery to assess vascular redox state. AF occurred in 33% of patients after surgery, with 3% still in AF after 6 weeks. Preoperative sCD40L levels were significantly higher in those who developed in-hospital AF (odds ratio for a 1-SD increase in log[sCD40L]=1.97; 95% CI, 1.21 to 3.22; P=0.007; after adjustment for age, sex, Euroscore, and total duration of operation). sCD40L and vascular superoxide levels were higher in patients still in AF at 6 weeks, and endothelial function was lower, although the small number of events precluded statistical analysis in this group. Systemic endothelial function, redox state, and preoperative markers of systemic inflammation were not associated with in-hospital postoperative AF. CONCLUSIONS: Preoperative platelet activation, as assessed by sCD40L levels, is a novel predictor of postoperative AF, independent of systemic endothelial function, vascular redox state, and systemic inflammation.

Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis.

BACKGROUND: Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear. AIMS: We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advanced atherosclerosis. METHODS AND RESULTS: Paired samples of saphenous veins (SVs) and internal mammary arteries (IMAs) were collected from 201 patients undergoing coronary bypass surgery, and serum ADMA was measured pre-operatively. The vasomotor responses of SV segments to acetylcholine (ACh) and bradykinin (Bk) were evaluated ex vivo. Vascular O2- was measured in paired SV and IMA by lucigenin-enhanced chemiluminescence. The l-NAME-inhibitable as well as the NADPH-stimulated vascular O2- generation was also determined by chemiluminescence. High serum ADMA levels were associated with decreased vasorelaxation of SV to ACh (P < 0.05) and Bk (P < 0.05). Similarly, high serum ADMA was associated with higher total O2- production in both SVs and IMAs (P < 0.05) and greater L-NAME-inhibitable vascular O2- (P < 0.05). However, serum ADMA was not associated with NADPH-stimulated vascular O2-. In multivariable linear regression, serum ADMA was independently associated with vascular O2- in both SVs [beta (SE): 0.987 (0.412), P = 0.019] and IMAs [beta (SE): 1.905 (0.541), P = 0.001]. Asymmetrical dimethylarginine was also independently associated with maximum vasorelaxation in response to both ACh [beta (SE): 14.252 (3.976), P = 0.001] and Bk [beta (SE): 9.564 (3.762), P = 0.013]. CONCLUSION: This is the first study that demonstrates an association between ADMA and important measures of vascular function, such as vascular O2- production and NO bioavailability directly in human vessels. Although serum ADMA has no effect on NADPH-stimulated superoxide in intact vessels, it is associated with greater eNOS uncoupling in the human vascular endothelium of patients with coronary artery disease.

One single bout of low-intensity isometric handgrip exercise reduces blood pressure in healthy pre- and hypertensive individuals.

BACKGROUND: The aim of this study was to investigate the acute effect of one single session of isometric handgrip exercise (IHG) on blood pressure (BP) during daily life activities in healthy adults. METHODS: Fifteen healthy adults with pre- or stage 1 hypertension (10 men; mean age 48±7.1 years) completed two experimental sessions in random order: one control (rest for 15 minutes) and one low intensity IHG session (4×2 minutes sustained contractions at 30% of maximal volutional contraction interspersed with 1-minute rest intervals). Blood pressure was recorded before each intervention and following the intervention BP was recorded for one hour in the office and thereafter for 6 hours during their daily activities by means of an ambulatory BP device. Physical activity (number of steps and total energy expenditure) was assessed by means of a SenseWear mini device. RESULTS: Systolic BP was higher the first minute after the isometric handgrip exercise but quickly returned to baseline levels after 15 minutes in the office. Over the 7-hour period, systolic blood pressure was significantly lower (-5.4±7.3 vs. +0.23±6.9; P<0.05) after IHG compared to the control session. Although not significant, a tendency was observed towards a lower diastolic blood pressure after IHG compared to control (P=0.09). Physical activity behavior was similar on both days (P>0.05). CONCLUSIONS: This study demonstrates that one single bout of IHG can result in an overall reduction in BP during daily activities. More research is needed to confirm these findings and to explain possible mechanisms responsible for these observed changes.

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice.

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta.

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.

Gene delivery strategies targeting stable atheromatous plaque.

Conventional therapeutic options to treat chronic angina pectoris are pharmacological interventions, coronary bypass surgery (CABG) and percutaneous coronary intervention (PCI). In animal models, it was shown that gene delivery strategies harbour an exciting potential to support and maybe even replace conventional anti-angina treatments, but the translation of the basic science to clinical practise appears problematic. Gene therapy targeting key elements of neointima formation (e.g. cell cycle regulators, metalloproteinases, inflammation and oxidative stress) reduces vein graft and stent failure in experimental models. Additionally, systemic gene delivery of genes targeting NO production, oxidative stress, inflammation and foam cell formation has been shown to prevent atherosclerosis in different animal models. During CABG the vein graft can be transfected ex vivo and during PCI, a stent carrying transfection vectors can be deployed. Both strategies result in the induction of local transgene expression at the site of interest. This limits unwarranted transgene expression and the toxicity seen with systemic gene delivery. However, with the development of new transfection vectors, able to induce local transgene expression without detrimental side effects, systemic anti-inflammatory and anti-oxidative, gene delivery could be a powerful tool in secondary prevention.

HMGCoA reductase inhibition reverses myocardial fibrosis and diastolic dysfunction through AMP-activated protein kinase activation in a mouse model of metabolic syndrome.

AIMS: The metabolic syndrome (MS) leads to myocardial fibrosis (MF) and diastolic dysfunction. Statins have proven beneficial effects in MS, but their impact on cardiac remodelling is uncertain. We examined the effects and mechanisms of chronic statin treatment on cardiac remodelling, e.g. fibrosis and diastolic properties. METHODS AND RESULTS: We used a mouse model deficient in leptin and the LDL-receptor (DKO) that reproduces this MS phenotype. DKO mice (12 weeks) were treated with rosuvastatin (R) for 6 months vs. placebo. Morphometric and echocardiographic measurements showed that R reduced cardiac mass and increased left-ventricular end-diastolic diameter despite unchanged cardiomyocyte dimensions. Similarly, R had no effect on the hypertrophic response to neurohormones in isolated cardiomyocytes. Conversely, R reversed the age-dependent development of MF as well as mRNA expression of TGF-ß1 and several pro-fibrotic markers (procollagen type I, its carboxy-terminal proteinase, Lysyl oxidase). R similarly inhibited the pro-fibrotic effects of TGF-ß1 on procollagen type I, alpha Smooth Muscle Actin expression and migratory properties of cardiac fibroblasts in vitro. In parallel, R increased the activation of AMP-activated protein kinase (AMPK), a known inhibitor of fibrosis, in vivo and in vitro, and the anti-fibrotic effects of R were abrogated in fibroblasts transfected with AMPKα1/α2 siRNA. The reversal of MF by R in DKO mice was accompanied with improved diastolic properties assessed by P-V loop analysis (slope of EDPVR, dP/dt min and cardiac output). CONCLUSION: In this model of MS, statin treatment reverses myocardial remodelling and improves ventricular relaxation through AMPK-mediated anti-fibrotic effects.

Targeting vascular redox biology through antioxidant gene delivery: a historical view and current perspectives.

Oxidative stress, resulting from a deregulated equilibrium between superoxide and nitric oxide (NO) production, contributes to the progression of different vascular diseases such as atherosclerosis, hypertension, ischemia/reperfusion injury and restenosis. Despite disappointing results of various oral antioxidant treatment trials, promising findings have been reported using gene delivery of enzymes to improve NO bioavailability and decrease oxidative stress in animal models for vascular diseases. NO production can be increased by overexpression of endothelial NO synthase (eNOS) in the vascular wall. However, the complex regulation of NOS needs to be carefully considered in the context of gene therapy along with the availability of its cofactor tetrahydrobiopterin and eNOS uncoupling. Furthermore, preclinical studies demonstrated that gene delivery of antioxidative vascular wall-specific enzymes, such as heme oxygenase-1, superoxide dismutase, catalase and glutathione peroxidase, has the potential to attenuate oxidative stress and inhibit atherosclerosis. Another option is to transfect vascular disease patients with secreted antioxidants such as high density lipoprotein-associated enzymes or soluble scavenger receptors. The advantage of the latter is that gene delivery of these enzymes and receptors does not need to be endothelium specific. Nonetheless, techniques to deliver genes specifically to the vascular wall are under development and hold interesting perspectives for the treatment of vascular diseases in the future. The patents relevant to gene delivery are also discussed in this review article.

Gene therapy targeting inflammation in atherosclerosis.

The extensive cross-talk between the immune system and vasculature leading to the infiltration of immune cells into the vascular wall is a major step in atherogenesis. In this process, reactive oxygen species play a crucial role, by inducing the oxidation of LDL and the formation of foam cells, and by activating a number of redox-sensitive transcriptional factors such as nuclear factor kappa B (NFkappa B) or activating protein 1 (AP1), that regulate the expression of multiple pro/anti inflammatory genes involved in atherogenesis. Delivery of genes encoding antioxidant defense enzymes (e.g. superoxide dismutase, catalase, glutathione peroxidase or heme oxygenase- 1) or endothelial nitric oxide synthase (eNOS), suppress atherogenesis in animal models. Similarly, delivery of genes encoding regulators of redox sensitive transcriptional factors (e.g. NF-kappa B, AP-1, Nrf2 etc) or reactive oxygen species scavengers have been successfully used in experimental studies. Despite the promising results from basic science, the clinical applicability of these strategies has proven to be particularly challenging. Issues regarding the vectors used to deliver the genes (and the development of immune responses or other side effects) and the inability of sufficient and sustained local expression of these genes at the target-tissue are some of the main reasons preventing optimism regarding the use of these strategies at a clinical level. Therefore, although premature to discuss about effective "gene therapy" in atherosclerosis at a clinical level, gene delivery techniques opened new horizons in cardiovascular research, and the development of new vectors may allow their extensive use in clinical trials in the future.

Leishmania-macrophage interactions: insights into the redox biology.

Leishmaniasis is a neglected tropical disease that affects about 350 million individuals worldwide. The protozoan parasite has a relatively simple life cycle with two principal stages: the flagellated mobile promastigote living in the gut of the sandfly vector and the intracellular amastigote within phagolysosomal vesicles of the vertebrate host macrophage. This review presents a state-of-the-art overview of the redox biology at the parasite-macrophage interface. Although Leishmania species are susceptible in vitro to exogenous superoxide radical, hydrogen peroxide, nitric oxide, and peroxynitrite, they manage to survive the endogenous oxidative burst during phagocytosis and the subsequent elevated nitric oxide production in the macrophage. The parasite adopts various defense mechanisms to cope with oxidative stress: the lipophosphoglycan membrane decreases superoxide radical production by inhibiting NADPH oxidase assembly and the parasite also protects itself by expressing antioxidant enzymes and proteins. Some of these enzymes could be considered potential drug targets because they are not expressed in mammals. In respect to antileishmanial therapy, the effects of current drugs on parasite-macrophage redox biology and its involvement in the development of drug resistance and treatment failure are presented.