Jesús Mateo

Regulation of hypoxia-inducible factor-1α by nitric oxide through mitochondria-dependent and -independent pathways

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.

Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 μM L-arginine at 70 μM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0±0.8 and 24.8±0.8 μM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

Noninvasive Assessment of Hypoxia in Rabbit Advanced Atherosclerosis Using 18F-fluoromisonidazole Positron Emission Tomographic Imaging

Background—Hypoxia is an important microenvironmental factor influencing atherosclerosis progression by inducing foam-cell formation, metabolic adaptation of infiltrated macrophages, and plaque neovascularization. Therefore, imaging plaque hypoxia could serve as a marker of lesions at risk. Methods and Results—Advanced aortic atherosclerosis was induced in 18 rabbits by atherogenic diet and double balloon endothelial denudation. Animals underwent 18F-fluoromisonidazole positron emission tomographic and 18F-fluorodeoxyglucose positron emission tomographic imaging after 6 to 8 months (atherosclerosis induction) and 12 to 16 months (progression) of diet initiation. Four rabbits fed standard chow served as controls. Radiotracer uptake of the abdominal aorta was measured using standardized uptake values. After imaging, plaque hypoxia (pimonidazole), macrophages (RAM-11), neovessels (CD31), and hypoxia-inducible factor-1&agr; were assessed by immunohistochemistry.18F-fluoromisonidazole uptake increased with time on diet (standardized uptake value mean, 0.10±0.01 in nonatherosclerotic animals versus 0.20±0.03 [P=0.002] at induction and 0.25±0.03 [P<0.001] at progression). Ex vivo positron emission tomographic imaging corroborated the 18F-fluoromisonidazole uptake by the aorta of atherosclerotic rabbits. 18F-fluorodeoxyglucose uptake also augmented in atherosclerotic animals, with an standardized uptake value mean of 0.43±0.02 at induction versus 0.35±0.02 in nonatherosclerotic animals (P=0.031) and no further increase at progression. By immunohistochemistry, hypoxia was mainly located in the macrophage-rich areas within the atheromatous core, whereas the macrophages close to the lumen were hypoxia-negative. Intraplaque neovessels were found predominantly in macrophage-rich hypoxic regions (pimonidazole+/hypoxia-inducible factor-1&agr;+/RAM-11+). Conclusions—Plaque hypoxia increases with disease progression and is present in macrophage-rich areas associated with neovascularization. 18F-fluoromisonidazole positron emission tomographic imaging emerges as a new tool for the detection of atherosclerotic lesions.

Preclinical evaluation of MR attenuation correction versus CT attenuation correction on a sequential whole-body MR/PET scanner.

ObjectivesThe application of attenuation correction for combined magnetic resonance/positron emission tomography (MR/PET) systems is still a major challenge for accurate quantitative PET. Computed tomographic attenuation correction (CTAC) is the current clinical standard for PET/computed tomographic (CT) scans. Magnetic resonance, unlike CT, has no direct information about photon attenuation but, rather, proton densities. On combined MR/PET scanners, MR-based attenuation correction (MRAC) consists of assigning empirical attenuation coefficients to MR signal intensities. The objective of the current study was to evaluate the MRAC implemented on the combined MR/PET scanner versus the CTAC with the same PET data in an animal model. Materials and MethodsAcquisition was performed using a clinically approved sequential MR/PET scanner (Philips Ingenuity TF). Computed tomographic and MR/PET images of 20 New Zealand White rabbits were retrospectively analyzed. The animals were positioned on a customized animal bed to avoid movement between the CT and MR/PET scanners. Positron emission tomographic images from both methods (MRAC and CTAC) were generated. Voxel-by-voxel and region-of-interest (ROI) analyses were performed to determine differences in standardized uptake values (SUV). Regions of interest were drawn on the coregistered CT images for the aorta, liver, kidney, spine, and soft tissue (muscle) and superimposed on the PET images. ResultsThe voxel-by-voxel comparison of PET showed excellent correlation between MRAC and CTAC SUV values (R = 0.99; P < 0.0001). The mean of the difference of SUVs between all respective MRAC and CTAC voxels was −0.94% (absolute difference [AD] ± SD, −0.06 ± 0.30), confirming slight underestimation of MRAC. The ROI-based comparison similarly showed that MRAC SUV values were underestimated compared with CTAC SUV values. The mean difference between MRAC and CTAC for all ROIs was 10.8% (AD, −0.08 ± 0.06; R = 0.99; P < 0.0001) and −9.7% (AD, −0.15 ± 0.12; R = 0.99; P < 0.0001) for the SUV mean (SUVmean) and the SUV maximum (SUVmax), respectively. The highest differences were found in the spine (SUVmean −26.1% [−0.11]) and areas close to large bones such as the back muscles (SUVmean, −16.8% [−0.04]). ConclusionsIn this study, we have compared MRAC and CTAC methods for PET attenuation correction in an animal model. We have confirmed that the MRAC method implemented on a sequential MR/PET scanner underestimates PET values by less than 10% in most regions, except the areas containing or close to large bone structures such as the spine or the back muscles. Bone segmentation is therefore suggested to be included in the MR attenuation map to minimize the quantification error of MRAC methods compared with the clinical standard CTAC. Further clinical studies need to be carried out to validate the clinical use of MRAC.

New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans.

Diagnosis of pulmonary arterial hypertension (PAH) is difficult due to the lack of specific clinical symptoms and biomarkers, especially at early stages. We compared plasma metabolic fingerprints of PAH patients (n = 20) with matched healthy volunteers (n = 20) using, for the first time, untargeted multiplatform metabolomics approach consisting of high-performance liquid and gas chromatography coupled with mass spectrometry. Multivariate statistical analyses were performed to select metabolites that contribute most to groups’ classification (21 from liquid in both ionization modes and 9 from gas chromatography-mass spectrometry). We found metabolites related to energy imbalance, such as glycolysis-derived metabolites, as well as metabolites involved in fatty acid, lipid and amino acid metabolism. We observed statistically significant changes in threitol and aminomalonic acid in PAH patients, which could provide new biochemical insights into the pathogenesis of the disease. The results were externally validated on independent case and control cohorts, confirming up to 16 metabolites as statistically significant in the validation study. Multiplatform metabolomics, followed by multivariate chemometric data analysis has a huge potential for explaining pathogenesis of PAH and for searching potential and new more specific and less invasive markers of the disease.

Cardiovascular imaging: what have we learned from animal models?

Cardiovascular imaging has become an indispensable tool for patient diagnosis and follow up. Probably the wide clinical applications of imaging are due to the possibility of a detailed and high quality description and quantification of cardiovascular system structure and function. Also phenomena that involve complex physiological mechanisms and biochemical pathways, such as inflammation and ischemia, can be visualized in a non-destructive way. The widespread use and evolution of imaging would not have been possible without animal studies. Animal models have allowed for instance, (i) the technical development of different imaging tools, (ii) to test hypothesis generated from human studies and finally, (iii) to evaluate the translational relevance assessment of in vitro and ex-vivo results. In this review, we will critically describe the contribution of animal models to the use of biomedical imaging in cardiovascular medicine. We will discuss the characteristics of the most frequent models used in/for imaging studies. We will cover the major findings of animal studies focused in the cardiovascular use of the repeatedly used imaging techniques in clinical practice and experimental studies. We will also describe the physiological findings and/or learning processes for imaging applications coming from models of the most common cardiovascular diseases. In these diseases, imaging research using animals has allowed the study of aspects such as: ventricular size, shape, global function, and wall thickening, local myocardial function, myocardial perfusion, metabolism and energetic assessment, infarct quantification, vascular lesion characterization, myocardial fiber structure, and myocardial calcium uptake. Finally we will discuss the limitations and future of imaging research with animal models.

Noninvasive Assessment of Hypoxia in Rabbit Advanced Atherosclerosis Using 18F-fluoromisonidazole PET Imaging

Background—Hypoxia is an important microenvironmental factor influencing atherosclerosis progression by inducing foam-cell formation, metabolic adaptation of infiltrated macrophages, and plaque neovascularization. Therefore, imaging plaque hypoxia could serve as a marker of lesions at risk. Methods and Results—Advanced aortic atherosclerosis was induced in 18 rabbits by atherogenic diet and double balloon endothelial denudation. Animals underwent 18F-fluoromisonidazole positron emission tomographic and 18F-fluorodeoxyglucose positron emission tomographic imaging after 6 to 8 months (atherosclerosis induction) and 12 to 16 months (progression) of diet initiation. Four rabbits fed standard chow served as controls. Radiotracer uptake of the abdominal aorta was measured using standardized uptake values. After imaging, plaque hypoxia (pimonidazole), macrophages (RAM-11), neovessels (CD31), and hypoxia-inducible factor-1&agr; were assessed by immunohistochemistry.18F-fluoromisonidazole uptake increased with time on diet (standardized uptake value mean, 0.10±0.01 in nonatherosclerotic animals versus 0.20±0.03 [P=0.002] at induction and 0.25±0.03 [P<0.001] at progression). Ex vivo positron emission tomographic imaging corroborated the 18F-fluoromisonidazole uptake by the aorta of atherosclerotic rabbits. 18F-fluorodeoxyglucose uptake also augmented in atherosclerotic animals, with an standardized uptake value mean of 0.43±0.02 at induction versus 0.35±0.02 in nonatherosclerotic animals (P=0.031) and no further increase at progression. By immunohistochemistry, hypoxia was mainly located in the macrophage-rich areas within the atheromatous core, whereas the macrophages close to the lumen were hypoxia-negative. Intraplaque neovessels were found predominantly in macrophage-rich hypoxic regions (pimonidazole+/hypoxia-inducible factor-1&agr;+/RAM-11+). Conclusions—Plaque hypoxia increases with disease progression and is present in macrophage-rich areas associated with neovascularization. 18F-fluoromisonidazole positron emission tomographic imaging emerges as a new tool for the detection of atherosclerotic lesions.

Protein corona and phospholipase activity drive selective accumulation of nanomicelles in atherosclerotic plaques

ApoB-100 and Phosphatidylcholine-specific phospholipase C (PC-PLC) are important contributors to atherosclerosis development. ApoB-100 is the main structural protein of LDL, being directly associated with atherosclerosis plaque generation. PC-PLC is highly expressed in atherosclerosis lesions and contributes to their progression. We show how phosphatidylcholine-coated nanomicelles can be used for specific characterisation of atherosclerosis plaque. Results show that ApoB-100 in the protein corona of the nanomicelle targets the particles to atherosclerotic areas in apolipoprotein E-/- mice. Furthermore, PC-PLC selectively removes the polar heads from the phospholipid coating of the nanomicelles leading to their accumulation. To fully characterise the behaviour of the nanomicelles, we developed multimodal probes using a nanoemulsion step. Hybrid imaging revealed plaque accumulation of the nanomicelles and colocalisation with PC-PLC expression and ApoB-100 in the plaque. This study shows how protein corona composition and enzyme-driven nanomaterial accumulation can be used for detection of atherosclerosis.

In Vivo 18 F-FDG-PET Imaging in Mouse Atherosclerosis

Positron emission tomography (PET) is an important technique in cardiovascular research. Vascular inflammation detected by fluorodeoxyglucose (FDG)-PET has been shown to predict cardiovascular (CV) events independent of traditional risk factors and is also highly associated with overall burden of atherosclerosis. The use of PET imaging in mouse models of atherosclerosis is challenged by the reduced size of the scanned organs. However, the last generation of dedicated PET scanners has an improved spatial resolution (<1 mm) and increased sensitivity allowing those studies to be performed. Here, we describe a procedure to perform FDG-PET experiments in atherosclerosis mouse models, the required equipment for animal handling and imaging, and the tools and procedures for image analysis and validation of the results.

Magnetic Resonance Imaging of the Atherosclerotic Mouse Aorta

Plaque development has been extensively studied using magnetic resonance imaging (MRI) in animal models of rapidly progressing atherosclerosis, such as apolipoprotein E-knockout (apoE-KO) mice. Preclinical MRI plays a significant role in the study of experimental atherosclerosis. Currently, MRI is capable of detecting luminal narrowing, plaque size, and morphology with high accuracy and reproducibility, providing reliable measurements of plaque burden. Therefore, MRI offers a noninvasive approach to serially monitor the progression of the disease. Compared with other imaging modalities, MRI appears to have the greatest potential for plaque characterization, through the use of multiple contrast weightings (e.g., T1, T2, and proton density). Here, we illustrate a standard procedure to image the aorta of atherosclerotic mice using noninvasive MRI.