Up-regulation of heme oxygenase-1 after infarct initiation reduces mortality, infarct size and left ventricular remodeling: experimental evidence and proof of concept.

BACKGROUND: Up-regulation of HO-1 by genetic manipulation or pharmacological pre-treatment has been reported to provide benefits in several animal models of myocardial infarction (MI). However, its efficacy following MI initiation (as in clinical reality) remains to be tested. Therefore, this study investigated whether HO-1 over-expression, by cobalt protoporphyrin (CoPP) administered after LAD ligation, is still able to improve functional and structural changes in left ventricle (LV) in a rat model of 4-week MI. METHODS: A total of 144 adult male Wistar rats were subjected to either left anterior coronary artery ligation or sham-operation. The effect of CoPP treatment (5 mg/kg i.p. at the end of the surgical session and, then, once a week for 4 weeks) was evaluated on the basis of survival, electro- and echocardiography, plasma levels of B-type natriuretic peptide (BNP), endothelin-1 and prostaglandin E2, coronary microvascular reactivity, MI size, LV wall thickness and vascularity. Besides, the expression of HO-1 and connexin-43 in different LV territories was assessed by western blot analysis and immunohistochemistry, respectively. RESULTS: CoPP induced an increased expression of HO-1 protein with >16 h delay. CoPP treatment significantly reduced mortality, MI size, BNP concentration, ECG alterations, LV dysfunction, microvascular constriction, capillary rarefaction and restored connexin-43 expression as compared to untreated MI. These functional and structural changes were paralleled by increased HO-1 expression in all LV territories. HO activity inhibition by tin-mesoporphyrin abolished the differences between CoPP-treated and untreated MI animals. CONCLUSIONS: This is the first report demonstrating the putative role of pharmacological induction of HO-1 following coronary occlusion to benefit infarcted and remote territories, leading to better cardiac function in a 4-week MI outcome.

Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression.

BACKGROUND: Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression. RESULTS: In an ischemia/reperfusion (I/R) rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod) and thioredoxin reductase (trxr1) upon short (4 h) and long (72 h) reperfusion times in the right ventricle (RV), and in the ischemic/reperfused (IRR) and the remote region (RR) of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR). In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb), Glyceraldehyde-3-P-dehydrogenase (gapdh), Ribosomal protein L13A (rpl13a), Tyrosine 3-monooxygenase (ywhaz), Beta-glucuronidase (gusb), Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt), TATA binding box protein (tbp), Hydroxymethylbilane synthase (hmbs), Polyadenylate-binding protein 1 (papbn1). According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs) without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary. CONCLUSIONS: This study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in each region.

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle.

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.

In vivo microspectroscopy monitoring of chromium effects on the photosynthetic and photoreceptive apparatus of Eudorina unicocca and Chlorella kessleri.

In microorganisms and plants, chromium (Cr) is not essential for any metabolic process, and can ultimately prove highly deleterious. Due to its widespread industrial use, chromium has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of specific algal populations able to tolerate high levels of Cr compounds. The varying Cr-resistance mechanisms displayed by microorganisms include biosorption, diminished accumulation, precipitation, reduction of Cr(6+) to Cr(3+), and chromate efflux. In this paper we describe the effects of Cr(6+) (the most toxic species) on the photosynthetic and photoreceptive apparatus of two fresh water microalgae, Eudorina unicocca and Chlorella kessleri. We measured the effect of this heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr(6+) effects are very different in the two algae. In E. unicocca the metal induced a complete pheophinitization of the chlorophylls and a modification of the carotenoids present in the eyespot after only 120 h of exposition at a concentration equal or greater than 40 microM, which is the limit for total Cr discharge established by US EPA regulations. In C. kessleri, chromium concentrations a hundred times higher than this limit had no effect on the photosynthetic machinery. The different tolerance level of the two algae is suggested to be due to the different properties of the mucilaginous envelope and cell wall covering, respectively, the colonies of Eudorina and the single cells of Chlorella, which binds chromium cations to a different extent.

Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor.

This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90A). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor.

Modulation of erythrocyte sensitivity to oxidative stress by transient hyperhomocysteinemia in healthy subjects and in patients with coronary artery disease.

BACKGROUND: The pathobiological mechanisms by which Hcy can promote atherothrombosis are not completely understood. Many observations suggest that oxidative consequences of hyperhomocysteinemia have a distinct role in the development of occlusive vascular disease. The aim of this work was to investigate whether sensitivity of erythrocytes to chemically induced oxidative stress in both healthy subjects and patients with clinically ascertained atherosclerosis was modified during the transient increase in homocysteine driven by methionine load. METHODS: Erythrocyte sensitivity to oxidative stress during transient hyperhomocysteinemia was assessed by cumene hydroperoxide-induced alpha-tocopherol consumption before and after methionine load in 31 healthy subjects and 23 patients with coronary artery disease. RESULTS: Decreased sensitivity to oxidative challenge ("Type-1" response) after methionine load was more frequent in healthy subjects (35% vs 13% in patients), while increased sensitivity ("Type-2" response) was more frequent in patients (22% vs 6% in healthy subjects). No variation in sensitivity to oxidative challenge throughout the loading test ("Non-variant" response) was detected in either group (58% in healthy subjects and 65% in patients). The distribution of these responses was significantly different between healthy subjects and patients and independent of basal and post-load increase in homocysteine. Plasma lipoperoxides, erythrocyte alpha-tocopherol and glutathione content before methionine load were significantly different between patients and healthy subjects; however only the redox potential of the GSSG/GSH couple was significantly different in the different groups of response. CONCLUSIONS: The higher frequency of "Type-2" response in patients with respect to healthy subjects suggests that methionine load reveals individual factors that may contribute to the pathogenesis of atherosclerosis.

Absorption microspectroscopy, theory and applications in the case of the photosynthetic compartment.

We performed microspectroscopic evaluation of the pigment composition of the photosynthetic compartments of both algae and higher plants. The feasibility of microspectroscopy for discriminating among species and/or phylogenetic groups was tested on laboratory cultures. Gaussian bands decompositions, and a fitting algorithm, together with fourth-derivative transformation of absorbance spectra, provided a reliable discrimination among chlorophylls, phycobiliproteins and carotenoids. Comparative analysis of absorption spectra highlighted the evolutionary grouping of the algae into three main lineages in accordance with the most recent endosymbiotic theories.

The effect of Ginkgo biloba in isolated ischemic/reperfused rat heart: a link between vitamin E preservation and prostaglandin biosynthesis.

The effect of Ginkgo biloba extract (EGb 761) was studied in rat hearts submitted to ischemia/reperfusion. Isolated hearts perfused in Langendorff mode were subjected to 60 minutes of global ischemia and 15 minutes of reperfusion. EGb 761 was administered by chronic or acute treatment: intra-peritoneal injections of 5 mg/Kg extract for 5 days, or 100 mg /L extract addition to the perfusion buffer, respectively. In hearts not treated with EGb 761, ischemia induced a 20% decrease in the concentration of membrane alpha-tocopherol. This effect was not worsened by reperfusion. alpha-tocopherol consumption was accompanied by about 650% increase in 6-ketoPGF1alpha release within 3 minutes of reperfusion. Moreover, ischemia induced activation of transcription factor NF-kappaB, as compared with the untreated group. In both chronic and acute treatment with EGb 761, heart concentration of alpha-tocopherol was completely spared during ischemia as much as after reperfusion, and a significant decrease of 6-ketoPGF1alpha release was observed at 3 minutes of reperfusion. Nuclear translocation of NF-kappaB was lowered during ischemia. EGb 761 might act as direct free radical scavenger or as tocopheryl radical recycler; in both cases sparing membrane vitamin E should affect phospholipase A2 activity. Finally, EGb 761, by lowering ROS produced during ischemia, challenges nuclear translocation of NF-kappaB.

In vitro modulation of intracellular oxidative stress of endothelial cells by diagnostic cardiac ultrasound.

Diagnostic cardiac ultrasound are commonly assumed to pose no hazard to the patient-but this is not synonymous with being biologically inert. The production of intracellular reactive oxygen species (ROS) on endothelial cells is a key modulator of atheroprotective (at low level) and atherogenic (at high levels) actions. The aim of the study was to evaluate in vitro the effects on intracellular ROS of endothelial cells after ultrasound exposure of variable duration with commercially available cardiac imaging systems. Endothelial cells fluorescence was evaluated in vitro after sham (transducer off) exposure to ultrasound and after 5', 15' and 30' of ultrasound irradiation with second harmonic 1.3/2.6 MHz cardiac ultrasound scan (mechanical index 1.5). Intracellular ROS were 83 at baseline, and rose to 86, 112 and 122 fluorescence intensity at 1 h incubation after 5', 15' and 30' of ultrasound exposure respectively (P<0.01 for 30' versus baseline and 5' comparison). There were microscopic signs of endothelial damage only following 30' stage. Ultrasound exposure induced significant DNA laddering and LDH leakage after 15' of ultrasound exposure. Effects on endothelial cells could be reproduced by adding exposed extracellular medium to unexposed cells, and could be prevented removing exposed medium from cell culture or pretreating the medium with catalase. Cardiac ultrasound of current clinical diagnostic use increases intracellular oxidative stress on endothelial cells in vitro. This increase is accompanied by morphological evidence of endothelial damage only after longer exposure times, persists 1 h after withdrawal of ultrasound, and can be modulated over a wide range according to the duration of ultrasound exposure. Free radical production in the extracellular medium is the likely mediator of ultrasound effect.