Stage-dependent activation of cell cycle and apoptosis mechanisms in the right ventricle by pressure overload.

The molecular basis of the intrinsic vulnerability of the compliant right ventricle to chronic pressure overload is poorly understood. Extensive apoptosis, possibly coupled with aberrant cell cycle reentry, in response to unrestrained biomechanical stress may account for this phenotypic flaw. To address this issue we have studied changes in expression of the cell cycle and apoptosis regulators in the right ventricle following induction of pulmonary hypertension in the rat by injection of monocrotaline. Hypertrophy, apoptosis and cell cycle events, as well as expression of their regulator genes were documented during a period of 31 days. The hypertrophy index reached 127% at day 31. At the early stage both apoptosis and cell proliferation pathways were coincidentally activated. The level of cyclin A and E transcripts steadily increased, the labeling index was 4.8% at day 31, and expression of the caspase-3 gene peaked at day 14. Until day 21 execution of apoptosis was prevented, probably by a high level of Bcl-2. At this time point Bcl-2 collapsed, cyclin D1 was upregulated, the differentiation gatekeeper p27Kip1 was downregulated, pro-caspase-3 was activated and extensive apoptosis developed. These results indicate that the right ventricle is especially vulnerable to apoptotic pressure-dependent stimuli, and that the cell cycle and apoptosis pathways were co-activated in this experimental model.

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis.

7-Ketocholesterol is a component of oxidized LDL, which plays a central role in atherosclerosis. It is a potent inducer of cell death towards a wide number of cells involved in atherosclerosis. In this study, it is reported that 7-ketocholesterol treatment induces an increase of cytosolic-free Ca(2+) in THP-1 monocytic cells. This increase is correlated with the induction of cytotoxicity as suggested from experiments using the Ca(2+) channel blockers verapamil and nifedipine. This 7-ketocholesterol-induced apoptosis appears to be associated with the dephosphorylation of serine 75 and serine 99 of the proapoptotic protein Bcl-2 antagonist of cell death (BAD). We demonstrated that this dephosphorylation results mainly from the activation of calcium-dependent phosphatase calcineurin by the oxysterol-induced increase in Ca(2+). Moreover, this Ca(2+) increase appears related to the incorporation of 7-ketocholesterol into lipid raft domains of the plasma membrane, followed by the translocation of transient receptor potential calcium channel 1, a component of the store operated Ca(2+) entry channel, to rafts.

Postischemic myocardial recovery and oxidative stress status of vitamin C deficient rat hearts.

OBJECTIVE: To investigate the role of vitamin C tissue content as a protective agent during myocardial ischemia-reperfusion injury, we have evaluated the postischemic functional recovery and free radical release of osteogenic disorder Shionogi (ODS) inherently scorbutic rat hearts and compared them to healthy Wistar rat hearts. METHODS: Isolated perfused hearts of ODS or Wistar rats underwent 30 min of a global total normothermic ischemia followed by 30 min of reperfusion. The lipid-soluble spin trap alpha-phenyl N-tert-butylnitrone (3 mM) was perfused upstream of the coronary bed. Functional parameters were recorded and samples of coronary effluents were analysed using electron spin resonance spectroscopy to characterise and quantify the amount of radical species released. RESULTS: From the onset of reperfusion, a large and long-lasting release of alkyl/alkoxyl radicals was detected, with a peak value of 29.0+/-3.2 nM obtained after 13 min, which was associated with a persistent contractile dysfunction. However, ODS rat hearts showed a higher myocardial recovery with lower left ventricular end diastolic pressure (44.34+/-1.74 vs. 55.03+/-1.57 mmHg for Wistar), higher recovery of rate pressure product (12.3+/-1.4 vs. 1.9+/-1.7x10(3) mmHg beats/min for Wistar) and shorter duration of contractile abnormalities during reperfusion (3.7+/-1.0 vs. 20.8+/-5.3 min for Wistar). Moreover, free radical release was identical in ODS rat hearts as compared to control Wistar rats. Ascorbic acid tissue content was significantly altered in ODS rats (31.9+/-3.3 vs. 591.0+/-54.9 mmol/g of tissue for Wistar) but superoxide dismutases, glutathion peroxidases and inducible heat shock protein 70 genes were up-regulated. CONCLUSIONS: This study shows that ascorbic-acid-deficient ODS rat hearts are more resistant to an ischemic insult than control Wistar rats, probably through the development of alternative protective defences, like the induction of heat shock proteins. These paradoxical results raise the question of the relative importance of each endogenous antioxidant in the cardiac resistance to ischemia-reperfusion injury.

Role of nitric oxide synthases in the infarct size-reducing effect conferred by heat stress in isolated rat hearts.

Nitric oxide (NO) donors are known to induce both delayed cardioprotection and myocardial heat stress protein (HSP) expression. Moreover, heat stress (HS), which also protects myocardium against ischaemic damages, is associated with a NO release. Therefore, we have investigated the implication of NO in HS-induced resistance to myocardial infarction, in the isolated rat heart model. Rats were divided in six groups (n=10 in each group), subjected or not to heat stress (42 degrees C internal temperature, 15 min) and treated or not with nitro-L-arginine-methylester (L-NAME) a non-selective inhibitor of NO synthase isoforms, or L-N(6)-(1-imino-ethyl)lysine (L-NIL), a selective inhibitor of the inducible NO synthase. Twenty-four hours after heat stress, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Infarct-to-risk ratio was significantly reduced in HS (18.7+/-1.6%) compared to Sham (33.0+/-1.7%) hearts. This effect was abolished in L-NAME-treated (41.7+/-3.1% in HS+L-NAME vs 35.2+/-3.0% in Sham+L-NAME ) and L-NIL-treated (36.1+/-3.4% in HS+L-NIL vs 42.1+/-4.6% in Sham+L-NIL) groups. Immunohistochemical analysis of myocardial HSP 27 and 72 showed an HS-induced increase of these proteins, which was not modified by L-NAME pretreatment. We conclude that NO synthases, and in particular the inducible isoform, appear to play a role in the heat stress-induced cardioprotection, independently of HSP 27 and 72 levels. Further investigations are required to elucidate the precise role of HSPs in this adaptive response.

The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival.

The presence of tyrosinase mRNA in the peripheral blood cells of melanoma patients has been recently studied as a possible marker of haematogenous dissemination. However, considerable variations in the rates of detection have been noted. We determined the presence of tyrosinase mRNA-positive circulating cells using reverse transcriptase-polymerase chain reaction (RT-PCR) in 35 patients with stage I melanoma, two patients with stage II melanoma and two patients with stage III melanoma. Among the patients with stage 1, 13 were tested before and after surgery (< 1 h). Twenty healthy subjects served as negative controls. Out of the melanoma patients, the tyrosinase gene was expressed in three of the 52 samples tested. Tyrosinase mRNA was present in the circulating cells of only one patient with stage I melanoma after intra-congenital naevi resection. However, two other stage I patients developed rapidly lethal metastasis within the following 6 months, despite the lack of detectable tyrosinase mRNA. None of stage II patients were positive for the tyrosinase transcripts, while both patients with stage III melanoma showed enzyme expression. Our results confirm those of previous studies, showing that a small proportion of stage I melanoma patients have tyrosinase-positive circulating cells. Moreover, the lack of tyrosinase mRNA detection in the blood does not necessarily exclude metastatic progression. Therefore, this study indicates that the detection of tyrosinase mRNA-positive circulating cells by RT-PCR is not a predictive biomarker of a metastasis risk in patients with stage I melanoma.

High circulating levels of 7beta- and 7alpha-hydroxycholesterol and presence of apoptotic and oxidative markers in arterial lesions of normocholesterolemic atherosclerotic patients undergoing endarterectomy.

In previous investigations, we found that 7beta-hydroxycholesterol had potent pro-apoptotic, and pro-oxidative properties. So, we asked whether the circulating level of this oxysterol was enhanced in atherosclerotic patients undergoing endarterectomy of the superficial femoral artery. To this end, 7beta-hydroxycholesterol serum concentrations were determined and compared with common lipid parameters in atherosclerotic patients, and in healthy subjects. 7alpha-hydroxycholesterol was simultaneously measured to evaluate the reliability of the method used for oxysterol analysis. On normal and atherosclerotic arterial fragments from patients, markers of oxidation (4-hydroxynonenal (4-HNE) adducts), and apoptosis (activated caspase-3; condensed/fragmented nuclei) were studied. Interestingly, high serum concentrations of 7beta- and 7alpha-hydroxycholesterol were found in normocholesterolemic atherosclerotic patients. However, in statin-treated patients, the circulating levels of 7beta- and 7alpha-hydroxycholesterol tend towards normal values. Therefore, 7beta- as well as 7alpha-hydroxycholesterol could be more appropriate markers of lipid metabolism disorders than cholesterol or LDL in normocholesterolemic patients with atherosclerosis of the lower limbs, and statins could normalize their serum concentrations. At the arterial level, apoptotic cells were mainly identified in low grade lesions and no statin effects were found on oxidation and apoptosis.

Influence of deep hypothermia on the tolerance of the isolated cardiomyocyte to ischemia-reperfusion.

The influence of deep hypothermia (4 degrees C) during a substrate-free, hypoxia-reoxygenation treatment was investigated on cardiomyocytes (CM) prepared from newborn rat heart in culture in an in vitro, substrate-free model of ischemia-reperfusion. The transmembranous potentials were recorded with standard microelectrodes. The contractions were monitored photometrically. The RNA messenger (mRNA) and protein expression for protein (HSP70) were analysed by RT-PCR (reverse transcriptase-polymerase chain reaction) and Western blotting, respectively. Simulated ischemia (SI) caused a gradual decrease and then a cessation of the spontaneous electromechanical activity. During the reoxygenation, the CM recovered normal function, provided that SI did not exceed 2.5 h. When SI duration was increased up to 4 h, reoxygenation failed to restore the spontaneous electromechanical activity. Conversely, the exposure of the CM to SI together with deep hypothermia decreased the functional alterations observed, and provided a complete electromechanical recovery after 2.5 h as well as after 4 h of SI. Deep hypothermia alone failed to induce HSP70 mRNA and protein production. On the contrary, HSP70 mRNA production increased after 2.5 and 4 h of deep hypothermia followed by 1 h of rewarming, proportionally to the duration of the cooling period. This augmentation in mRNA was associated with a rise in HSP70 protein content. In summary, it appeared that deep hypothermia exerts a strong cytoprotective action during SI only, whereas cooling CM before SI has no beneficial effect on subsequent SI. Moreover, these results suggested the persistence of a signaling system and/or transduction in deeply cooled, functionally depressed cells. Finally, CM in culture appeared to be a model of interest for studying heart graft protection against ischemia-reperfusion and contributed to clarifying the molecular and cellular mechanisms of deep hypothermia on myocardium.

Levels of nitric oxide in the heart after experimental myocardial ischemia.

The effect of myocardial ischemia on nitric oxide (NO) production is controversial in part because of indirect NO quantification. In the present study, direct quantification of NO was investigated in an in vivo rat model of myocardial ischemia (MI). A NO spin-trapping technique using electron spin resonance (ESR) spectroscopy was used to study NO production in the ischemic and in the nonischemic area of the rat heart 2, 8, or 24 h after left main coronary artery ligation. The method was based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine-dithiocarbamate (MGD) and Fe(II) to form a stable NO-FeMGD complex that gives rise to a characteristic triplet ESR spectrum. This metal-chelator complex was administered half an hour before sacrifice of the rats. A large and time-dependent increase of the ESR signal corresponding to the NO-FeMGD complex was observed 8 h (11.6 +/- 0.9 arbitrary units [AU]) and 24 h (29.7 +/- 2.9 AU) in the ischemic area after MI. On the contrary, no ESR triplet was observed in the nonischemic region of the heart and in sham-operated rats. NO blood derivative levels (nitrosylhemoglobin and plasma nitrites and nitrates) were unchanged compared with sham-operated rats. Previous administration of aminoguanidine, a NO synthase inhibitor, in animals subjected to a 24-h ischemia resulted in a complete abolition in the NO-FeMGD spectrum in the ischemic area. These findings directly demonstrated an increase of the NO-FeMGD levels during in vivo myocardial ischemia that appeared to be specifically localized in the ischemic area.

Upregulation of the netrin receptor (DCC) gene during activation of b lymphocytes and modulation by interleukins.

The DCC (deleted in colon cancer) gene has a brain restricted high expression pattern. It encodes a transmembrane protein of the immunoglobulin superfamily identified as the netrin-1 receptor. It might be a member of the so called "brain-lymphoid" molecules, which control key cell surface events. To test this hypothesis we have assessed the DCC mRNA level in human normal and malignant myeloid and lymphoid cells. A high mRNA content has been observed only in mature B cells at the secreting or presecreting stage. Expression of DCC was also assessed in the anti-CD40 model of immunopoiesis. Activation of purified tonsillar B cells by anti-CD 40 antibody strongly increased the DCC mRNA level and this effect was dramatically enhanced by the association of IL-2 + IL-10, which is a potent and selective in vitro inducer of the B cell memory phenotype. In contrast no effect has been detected after activation of T cells by anti-CD3. These data suggest that the DCC encoded netrin receptor is involved in B cell immunopoiesis.

Calpain and caspase-3 inhibitors reduce infarct size and post-ischemic apoptosis in rat heart without modifying contractile recovery.

Overactivation of proteases play a key role in the development of ischemia reperfusion (IR) myocardial injury. Calpains are calcium-dependent cysteine proteases and have been implicated in post-ischemic cell death. Moreover, activation of caspases, another family of proteases, represents an important step in the apoptotic process. We investigated the effect of leupeptin and calpain inhibitor-1 (CAI-1), two calpain inhibitors and of a caspase-3 inhibitor, Ac-DEVD-CHO, on functional recovery, myocardial infarct size and apoptosis in isolated rat hearts (Langendorff technique) subjected to 30 min of global ischemia and 120 min of reperfusion. Each inhibitor was added to the perfusion medium 10 min before ischemia and during the first 30 min of reperfusion. IR was associated with mechanical dysfunction and myocardial infarction. Apoptosis induced by this sequence was demonstrated by DNA ladder and TUNEL staining. Whereas leupeptin, CAI-1 or Ac-DEVD-CHO did not modify post-ischemic function, they significantly reduced infarct size and cardiomyocyte positive TUNEL staining. Our findings suggest that calpain and caspase-3 inhibitors may protect heart from the development of cell death induced by IR; this effect could be due, at least in part, to the reduction of apoptosis. However, in our experimental conditions, these inhibitors did not afford improvement of post-ischemic myocardial function.

Pattern of superoxide dismutase enzymatic activity and RNA changes in rat heart ventricles after myocardial infarction.

A multiplex reverse transcription polymerase chain reaction assay was designed to measure manganese superoxide dismutase (MnSOD) and CuZnSOD mRNAs in the left and right ventricles of rat hearts after myocardial infarction induced by occlusion of the left coronary artery. These data were compared with changes in enzymatic activities. In the left ventricle, Mn-SOD RNA increased significantly at 6 hours, peaked at 12 hours (490 +/- 38 arbitrary units), and progressively decreased (127 +/- 21 arbitrary units at 48 hours). In contrast, there was a steady accumulation of transcripts in the right ventricle up to 48 hours. In both ventricles, the changes in the MnSOD mRNA and protein content were not associated with proportional variations in enzymatic activity. There was no characteristic alteration of the CuZnSOD system in either ventricle over the 48-hour period. These results demonstrate that infarction selectively activates the MnSOD gene in the viable myocardium of both ventricles. They suggest that MnSOD may be involved in the adaptive response of myocytes to the overloading stress.

Changes in HSP70 and P53 expression are related to the pattern of electromechanical alterations in rat cardiomyocytes during simulated ischemia.

The objective was to relate the response of the HSP70 and P53 genes to the cessation and the recovery of cardiac muscle cell functions when submitted to ischemia-reperfusion. We have measured the electromechanical activity, the released enzymes and HSP70 RNA and protein levels in cultured neonatal rat cardiomyocytes (CM) in a substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. In parallel the expression of the two genes P53 (the key apoptosis regulator gene) and P21/Waf1 (the P53 target gene) has been evaluated. The functional recovery during post-'ischemic' reoxygenation was associated with an overexpression of HSP70 and P53 lasting until the functional parameters reverted back to the normal, prehypoxic values. In contrast, extending the substrate-free hypoxic treatment worsens the dysfunction of the cardiac muscle cell and, in these conditions, reoxygenation failed to restore cell functions and to activate HSP70. Finally, in the conditions of reversible 'ischemic' cell injury, an early and transitory activation of P53 was associated with the functional recovering process of the CM submitted to simulated ischemia. These observations are suggestive of a contributive role of both HSP70 and P53 to a cytoprotective program activated by reoxygenation in post-'ischemic' CM.

Antioxidative properties of pyruvate and protection of the ischemic rat heart during cardioplegia.

Formation of oxygen free radicals during heart transplantation seems to be related to the alterations occurring during ischemia and reperfusion and could explain the short preservation time of donor hearts. The aim of our study was (a) to analyze the protective effects of pyruvate during cold cardioplegia and ischemia/reperfusion sequence, and (b) to investigate in vitro the radical scavenging properties of this compound. After 30 min of perfusion, isolated working rat hearts were arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused with Krebs-Henseleit buffer for 45 min. Pyruvate (2 mM) was added to Krebs-Henseleit, cardioplegic, and storage solutions, and functional parameters were recorded throughout the experiments. In a second part, control hearts and hearts treated with pyruvate were cannulated via the aorta and perfused for 30 min by the Langendorff method, arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused for 45 min by the Langendorff method. Malonedialdehyde and alpha-tocopherol levels were determined on heart homogenate. In situ detection of apoptotic cells also was performed on tissue samples (left ventricle) at the end of the ischemia/reperfusion sequence. To demonstrate in vitro the antioxidant effects of pyruvate, we monitored (a) its hydroxyl radical scavenging properties by using electron paramagnetic resonance (EPR) spectroscopy, and (b) the decrease of fluorescence of allophycocyanin, in the presence of a Fenton system (H2O2/Cu2+). Ischemia for 4 h, followed by myocardial reperfusion, resulted in substantially reduced mechanical function. Hearts subjected to this ischemia and pretreated with pyruvate showed a significant improvement in the function recovery. After the ischemia/reperfusion protocol, no significant decrease of malonedialdehyde levels was shown on hearts treated with pyruvate. However, alpha-tocopherol levels were higher in the pyruvate group compared with the control group. At the end of the reperfusion period, levels of apoptotic cells were significantly lower in hearts treated with pyruvate compared with control hearts. EPR studies showed that pyruvate was an efficient hydroxyl scavenger, with a median inhibitory concentration (IC50) of 8 mM. The allophycocyanin assay also showed a dose-dependent effect of pyruvate against hydroxyl radicals. In conclusion, these findings showed that pyruvate could prevent reperfusion injuries in the isolated heart, probably by its antioxidative properties. The application of pyruvate may contribute to the preservation of hearts for organ transplantation.

False-positive results caused by cotton swabs in commercial Aspergillus antigen latex agglutination test.

While testing for Aspergillus spp. using a commercially available latex agglutination test on a brain biopsy wrapped in a cotton swab, false-positive results were obtained. Subsequent application of the test on lavage samples obtained using cotton and synthetic swabs resulted positive with the cotton swabs only. This suggests that epitopes cross-reactive with Aspergillus galactomannan may be present in cotton.

Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas.

Microsatellite instability has been proposed as an alternative pathway of colorectal carcinogenesis. The aim of this study was to evaluate the interest of immunohistochemistry as a new tool for highlighting mismatch repair deficiency and to compare the results with a PCR-based microsatellite assay. A total of 100 sporadic proximal colon adenocarcinomas were analysed. The expression of hMLH1, hMSH2 and hMSH6 proteins evaluated by immunohistochemistry was altered in 39% of the cancers, whereas microsatellite instability assessed by PCR was detected in 43%. There was discordance between the two methods in eight cases. After further analyses performed on other tumoural areas for these eight cases, total concordance between the two techniques was observed (Kappa=100%). Our results demonstrate that immunohistochemistry may be as efficient as microsatellite amplification in the detection of unstable phenotype provided that at least two samples of each carcinoma are screened, because of intratumoural heterogeneity.

Downregulation and nuclear relocation of MLP during the progression of right ventricular hypertrophy induced by chronic pressure overload.

The cardiac LIM domain protein MLP plays a crucial role in the architecture and mechanical function of cardiac myocytes. Mice lacking the MLP gene develop cardiac hypertrophy, dilated cardiopathy and heart failure. We investigated whether downregulation of MLP is induced by pressure overload and contributes to the physiopathology of cardiac hypertrophy and failure. We studied this mechanism in rat right ventricles submitted to pulmonary arterial hypertension, because it is known that this ventricle is very vulnerable to the deleterious effects of pressure overload. During the progression of cardiac hypertrophy to failure over a 31 days period there was a dramatic decrease by 50% of the MLP transcripts level. Consistently, immunohistochemistry detected very weak protein signals in the cytoplasms of cardiomyocytes at the failing stage, but myocytes nuclei were heavily labeled. The nuclear relocation was confirmed by the immunodetection of MLP on the nuclear and cytosolic fractions. This nuclear localization is the hallmark of a retro-differentiated phenotype, since it has been observed only in differentiating myoblasts. These changes were associated with ultrastructural disorganization of the myofibrils similar to that observed in MLP -/- mice. Therefore, MLP dowregulation occurring during gene reprogramming may critically contribute to mechanical failure of the myocardium.