Sleep EEG changes after middle cerebral artery infarcts in mice: different effects of striatal and cortical lesions.

STUDY OBJECTIVES: Hemispheric stroke in humans is associated with sleep-wake disturbances and sleep electroencephalogram (EEG) changes. The correlation between these changes and stroke extent remains unclear. In the absence of experimental data, we assessed sleep EEG changes after focal cerebral ischemia of different extensions in mice. DESIGN: Following electrode implantation and baseline sleep-wake EEG recordings, mice were submitted to sham surgery (control group), 30 minutes of intraluminal middle cerebral artery (MCA) occlusion (striatal stroke), or distal MCA electrocoagulation (cortical stroke). One and 12 days after stroke, sleep-wake EEG recordings were repeated. The EEG recorded from the healthy hemisphere was analyzed visually and automatically (fast Fourier analysis) according to established criteria. MEASUREMENTS AND RESULTS: Striatal stroke induced an increase in non-rapid eye movement (NREM) sleep and a reduction of rapid eye movement sleep. These changes were detectable both during the light and the dark phase at day 1 and persisted until day 12 after stroke. Cortical stroke induced a less-marked increase in NREM sleep, which was present only at day 1 and during the dark phase. In cortical stroke, the increase in NREM sleep was associated in the wake EEG power spectra, with an increase in the theta and a reduction in the beta activity. CONCLUSION: Cortical and striatal stroke lead to different sleep-wake EEG changes in mice, which probably reflect variable effects on sleep-promoting and wakefulness-maintaining neuronal networks.

Atherosclerotic mice exhibit systemic inflammation in periadventitial and visceral adipose tissue, liver, and pancreatic islets.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease of major conduit arteries. Similarly, obesity and type 2 diabetes mellitus are associated with accumulation of macrophages in visceral white adipose tissue and pancreatic islets. Our goal was to characterize systemic inflammation in atherosclerosis with hypercholesterolemia, but without obesity. METHODS AND RESULTS: We compared 22-week-old apolipoprotein E knockout (ApoE(-/-)) with wild-type mice kept for 14 weeks on a high cholesterol (1.25%) diet (CD, n=8) and 8-week-old ApoE(-/-) with wild-type mice kept on a normal diet (ND, n=8). Hypercholesterolemic, atherosclerotic ApoE(-/-) mice on CD exhibited increased macrophages and T-cells in plaques and periadventitial adipose tissue that revealed elevated expression of MIP-1alpha, IL-1beta, IL-1 receptor, and IL-6. Mesenteric adipose tissue and pancreatic islets in ApoE(-/-) mice showed increased macrophages. Expression of IL-1beta was enhanced in mesenteric adipose tissue of ApoE(-/-) mice on CD. Furthermore, these mice exhibited steatohepatitis with macrophage and T-cell infiltrations as well as increased MIP-1alpha and IL-1 receptor expression. Blood glucose, insulin and total body weight did not differ between the groups. CONCLUSIONS: In hypercholesterolemic lean ApoE(-/-) mice, inflammation extends beyond atherosclerotic plaques to the periadventitial and visceral adipose tissue, liver, and pancreatic islets without affecting glucose homeostasis.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Development of glycopeptide-intermediate resistance by Staphylococcus aureus leads to attenuated infectivity in a rat model of endocarditis.

Glycopeptide-intermediate resistant Staphylococcus aureus (GISA) are characterized by multiple changes in the cell wall and an altered expression of global virulence regulators. We investigated whether GISA are affected in their infectivity in a rat model of experimental endocarditis. The glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 and its laboratory-derived GISA M1V16 were examined for their ability to (i) adhere to fibrinogen and fibronectin in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonize aortic vegetations in rats, and (iv) compete for valve colonization by co-inoculation. Both GISA M1V16 and M1V2 adhered similarly to fibrinogen and fibronectin in vitro. In rats, GISA M1V16 was cleared faster from the blood (P < 0.05) and required 100-times more bacteria than parent M1V2 (10(6) versus 10(4)CFU) to infect 90% of vegetations. GISA M1V16 also had 100 to 1000-times lower bacterial densities in vegetations. Moreover, after co-inoculation with GISA M1V16 and M1V2Rif, a rifampin-resistant variant of M1V2 to discriminate them in organ cultures, GISA M1V16 was out-competed by the glycopeptide-susceptible counterpart. Thus, in rats with experimental endocarditis, GISA showed an attenuated virulence, likely due to a faster clearance from the blood and a reduced fitness in cardiac vegetations. The GISA phenotype appeared globally detrimental to infectivity.

In vivo testing of a novel adjustable glaucoma drainage device.

PURPOSE: We report on the in vivo testing of a novel noninvasively adjustable glaucoma drainage device (AGDD), which features an adjustable outflow resistance, and assess the safety and efficiency of this implant. METHODS: Under general anesthesia, the AGDD was implanted on seven white New Zealand rabbits for a duration of 4 months under a scleral flap in a way analogous to the Ex-PRESS device and set in an operationally closed position. The IOP was measured on a regular basis on the operated and control eyes using a rebound tonometer. Once a month the AGDD was adjusted noninvasively from its fully closed to its fully open position and the resulting pressure drop was measured. The contralateral eye was not operated and served as control. After euthanization, the eyes were collected for histology evaluation. RESULTS: The mean preoperative IOP was 11.1 ± 2.4 mm Hg. The IOP was significantly lower for the operated eye (6.8 ± 2 mm Hg) compared to the nonoperated eye (13.1 ± 1.6 mm Hg) during the first 8 days after surgery. When opening the AGDD from its fully closed to fully open position, the IOP dropped significantly from 11.2 ± 2.9 to 4.8 ± 0.9 mm Hg (P < 0.05). CONCLUSIONS: Implanting the AGDD is a safe and uncomplicated surgical procedure. The fluidic resistance was noninvasively adjustable during the postoperative period with the AGDD between its fully closed and fully open positions.

Dietary fish oil is antihypertrophic but does not enhance postischemic myocardial function in female mice.

Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac-specific overexpression of angiotensinogen that develop normotensive cardiac hypertrophy and littermate wild-type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 wk. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30 min), and reperfusion arrhythmia incidence were assessed. FO diet suppressed cardiac growth by 5% and 10% in WT and TG, respectively (P < 0.001). The extent of mechanical recovery [rate-pressure product (RPP) = beats/min x mmHg] of FO-fed WT and TG hearts was similar (50 +/- 7% vs. 45 +/- 12%, 30 min reperfusion), and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention were assessed. The extent of mechanical recovery of FO-fed OVX WT and TG (RPP, 50 +/- 4% vs. 64 +/- 8%) was not enhanced compared with CTR-fed mice (RPP, 60 +/- 11% vs. 80 +/- 8%, P = 0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary-intact mice or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity postischemia in a murine ex vivo heart model.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

Cardiac dysfunction and impaired compensatory response to pressure overload in mice deficient in stem cell antigen-1.

Stem cell antigen-1 (Sca-1) has been used to identify cardiac stem cells in the mouse heart. To investigate the function of Sca-1 in aging and during the cardiac adaptation to stress, we used Sca-1-deficient mice. These mice developed dilated cardiomyopathy [end-diastolic left ventricular diameter at 18 wk of age: wild-type (WT) mice, 4.2 mm ± 0.3; Sca-1-knockout (Sca-1-KO) mice, 4.6 mm ± 0.1; ejection fraction: WT mice, 51.1 ± 2.7%; Sca-1-KO mice, 42.9 ± 2.7%]. Furthermore, the hearts of mice lacking Sca-1 demonstrated exacerbated susceptibility to pressure overload [ejection fraction after transaortic constriction (TAC): WT mice, 43.5 ± 3.2%; Sca-1-KO mice, 30.8% ± 4.0] and increased apoptosis, as shown by the 2.5-fold increase in TUNEL(+) cells in Sca-1-deficient hearts under stress. Sca-1 deficiency affected primarily the nonmyocyte cell fraction. Indeed, the number of Nkx2.5(+) nonmyocyte cells, which represent a population of cardiac precursor cells (CPCs), was 2-fold smaller in Sca-1 deficient neonatal hearts. In vitro, the ability of CPCs to differentiate into cardiomyocytes was not affected by Sca-1 deletion. In contrast, these cells demonstrated unrestricted differentiation into cardiomyocytes. Interestingly, proliferation of cardiac nonmyocyte cells in response to stress, as judged by BrdU incorporation, was higher in mice lacking Sca-1 (percentages of BrdU(+) cells in the heart after TAC: WT mice, 4.4 ± 2.1%; Sca-1-KO mice, 19.3 ± 4.2%). These data demonstrate the crucial role of Sca-1 in the maintenance of cardiac integrity and suggest that Sca-1 restrains spontaneous differentiation in the precursor population. The absence of Sca-1 results in uncontrolled precursor recruitment, exhaustion of the precursor pool, and cardiac dysfunction.

Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.