Nicolas Bousette

Inducible Activation of TLR4 Confers Resistance to Hyperoxia-Induced Pulmonary Apoptosis

TLRs are essential mediators of host defense against infection via recognition of unique microbial structures. Recent observations indicate that TLR4, the principal receptor for bacterial LPS, may also be activated by noninfectious stimuli including host-derived molecules and environmental oxidant stress. In mice, susceptibility to ozone-induced lung permeability has been linked to the wild-type allele of TLR4, whereas deficiency of TLR4 predisposes to lethal lung injury in hyperoxia. To precisely characterize the role of lung epithelial TLR4 expression in the host response to oxidant stress, we have created an inducible transgenic mouse model that targets the human TLR4 signaling domain to the airways. Exposure of induced transgenic mice to hyperoxia revealed a significant reduction in pulmonary apoptosis compared with controls. This phenotype was associated with sustained up-regulation of antiapoptotic molecules such as heme oxygenase-1 and Bcl-2, yet only transient activation of the transcription factor NF-κB. Specific in vivo knockdown of pulmonary heme oxygenase-1 or Bcl-2 expression by intranasal administration of short interfering RNA blocked the effect of TLR4 signaling on hyperoxia-induced lung apoptosis. These results define a novel role for lung epithelial TLR4 as a modulator of cellular apoptosis in response to oxidant stress.

Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by α-crystallin-B

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.

Effect of endothelin receptor antagonist on lung allograft apoptosis and NOSII expression

BACKGROUND It is postulated that apoptosis contributes to ischemia-reperfusion graft dysfunction after lung transplantation. The purpose of this study was to determine whether the improvement in lung function that we previously observed with the use of an endothelin-1 (ET-1) receptor antagonist after ischemia-reperfusion injury is associated with a reduction in inducible nitric oxide synthase (NOSII) expression and programmed cell death. METHODS Left lung canine allotransplantation was performed. Harvested lung blocks were preserved with modified Eurocollins solution and stored at 4 degrees C for 18 to 20 hours. Lung allografts were tested for the expression of NOSII by immunohistochemistry, and extent of apoptosis by terminal dUTP nick end-labeling (TUNEL). Animals blindly received either an intravenous infusion of saline (control) or the ET-1 receptor antagonist (SB209670) (15 microg/kg/min). Infusion began 30 minutes pretransplantation and continued to 6 hours posttransplantation. RESULTS Immunohistochemical analysis demonstrated significantly stronger NOSII immunostaining in the allografts of the saline control group (36.5%+/-3.6%) compared with native right lungs (6.9%+/-1.3%, p < 0.001) or the ET-receptor antagonist treatment group (9.6%+/-1.4%, p < 0.001). The TUNEL staining revealed a significantly stronger labeling in the allografts of the saline treatment control group (40.7%+/-6.2%) compared with native right lungs (5.0%+/-0.6%, p < 0.005) or the ET receptor antagonist treatment group (14.1%+/-2.8%, p < 0.01). CONCLUSIONS We conclude that treatment of lung allografts with the ET-1 receptor antagonist SB209670 reduces the area of NOSII expression and the extent of apoptosis, factors known to contribute to the process of prolonged ischemia-reperfusion injury.

Urotensin II Receptor Knockout Mice on an ApoE Knockout Background Fed a High-Fat Diet Exhibit an Enhanced Hyperlipidemic and Atherosclerotic Phenotype

Rationale: Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases. Objective: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis. Methods and Results: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice. Conclusions: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor–mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.