Declan McLaughlin

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-β3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase-induced cardiomyopathy: involvement of mitofusin-2

The anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.

An investigation into the potential use of nanoparticles as adjuvants for the production of polyclonal antibodies to low molecular weight compounds

Two nanoparticle based adjuvants were assessed for their ability to produce polyclonal antibodies in rabbits to low molecular weight target analytes, i.e. veterinary drugs banned from use in food producing animals. The nanoparticles, Montanide IMS 251 and amphiphilic poly (γ-glutamic acid) were compared against a mineral oil adjuvant, Montanide ISA 50, which had previously been shown to be successful in producing antibodies to haptens whilst being safe to use with respect to the welfare of the host animals. The adjuvants were assessed for their tendency to cause adverse effects to the host animals and by the quality of the antibodies generated in terms of assay sensitivity. None of the three adjuvants employed in the trial generated any measurable adverse effects in the host animals. While the mineral oil adjuvant produced higher titres of antibodies the nanoparticle adjuvants were found to produce antibodies of statistically comparable sensitivity. Based on IC(50) values, six antisera displayed potential to detect the required level of the target compounds; five of these were produced by rabbits immunised with the two different nanoparticle adjuvants. As antibody sensitivity is the main performance criteria of an analytical immunoassay, it can be concluded that the nanoparticle adjuvants under evaluation are fit for the purpose described in this study.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

MODULATION OF OXIDATIVE STRESS INDUCED APOPTOSIS AND AUTOPHAGY IN CARDIOMYOCYTES BY INTERMEDIN

The heart is subjected to oxidative stress in conditions of increased reactive oxygen species (ROS) production, such as doxorubicin (DOX) chemotherapy. A major cause of associated ventricular dysfunction is cardiomyocyte loss through apoptosis, while autophagic processes can also be detrimental. Intermedin (IMD) has emerged as a major counter-regulatory peptide with cytoprotective properties. Here we examined its potential to be upregulated and attenuate indices of cardiomyocyte death by a mechanism involving NADPH oxidase-derived superoxide. In isolated adult C57BL/6J wild type (WT) mouse ventricular cardiomyocytes, DOX (5×10−7 M) increased preproIMD mRNA expression 3.5-fold, which was significantly decreased in NOX2-deficient cells. Similarly, IMD mRNA was upregulated by the direct pro-oxidant, H2O2 (10−7 M) in both WT and HL-1 cardiomyocytes, as was NOX2; DOX increased IMD protein (immuno-cytochemistry). Superoxide production stimulated by DOX or H2O2 (by ∼35%, lucigenin-enhanced chemiluminescence), was abolished by IMD (10−10 M–10−8 M) in both WT and HL-1 cardiomyocytes. Apoptosis in HL-1 cells, shown by DOX-induced increases in caspase 3/7 activity (3.5-fold), was decreased significantly by IMD (10−9 M), which also increased cell viability. Increased autophagosome formation after serum starvation or DOX treatment (by ∼50%, Cyto-ID fusion with LC3 protein), was decreased to control values by IMD (10−9 M). Confocal imaging showed numerous cytoplasmic punctate structures with DOX, whereas addition of IMD showed a diffuse LC-3 staining pattern similar to control. These findings indicate that a NOX2-mediated increase in IMD in cardiomyocytes could have a potential autocrine effect, acting at nM concentration to reduce levels of superoxide and so limit cell death by apoptotic and autophagic mechanisms.

42 Identification of an inherent mechanism of NADPH oxidase signalling in doxorubicin cardiotoxicity

Anthracyclines used in cancer therapeutics, including doxorubicin (DOX), cause both short- and long-term cardiotoxicity associated with increased myocardial oxidative stress. Chronic DOX treatment increases superoxide anion production in vivo, which is derived largely from Nox2 NADPH oxidase and contributes to key processes underlying cardiac dysfunction. The aim of this study was to identify possible mechanisms involved in the signal relay from increased Nox2 activity to development of disease phenotype. In this context, we performed a whole-genome gene expression array (Illumina MouseWG-6 v2.0) of ventricular tissue from DOX-treated wild type vs Nox2KO mice. Pathway analysis (GeneGo Metacore) revealed the apoptotic process most significant to the data set (p=0.00005). Applying the 63 relevant genes (FDR cut-off, adjusted p<0.05) in a network analysis showed significant mRNA increases in peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and the mitochondrial membrane protein, mitofusin-2, known to be upregulated by PGC-1α. Since mitofusin-2 independent of its pro-fusion effect can form a functional unit with the Bcl-2 family member, Bax, the potential involvement of mitofusin-2 in DOX-mediated apoptosis was investigated. In HL-1 cardiomyocytes, superoxide production was significantly increased by 13±3% by DOX (10–7M; 3 h) and H2O2 generated increases of 66%±22% and 44%±17% (10–7M; 3 h, 20 h) along with marked upregulation of Nox2 mRNA at 3 h (2.9-fold). DOX increased caspase 3/7 activity 3.0-fold at 20 h. In a similar time frame, DOX increased mitofusin-2 mRNA at 3 h and protein at 24 h. NOX2-mediated upregulation of mitofusin-2 and its pleiotropic effect on apoptosis may play a part in DOX-associated cardiomyocyte dysfunction.