Iloprost treatment summer-suspension: effects on skin thermal properties and cytokine profile in systemic sclerosis patients.

AIM: Aim of the study was to assess whether Iloprost treatment summer suspension modifies systemic cytokines levels, cutaneous thermal properties and functional response to a cold-induced stress in patients affected by systemic sclerosis (SSc). METHODS: Twenty-eight patients fulfilling the American College of Rheumatology (ACR) criteria for SSc were included in the study. Patients recorded number, duration and pain-severity of Raynaud phenomenon (RP). Pain-severity was determined by a visual analog scale. Cytokines expression and production in peripheral blood mononuclear cells and serum were evaluated by RT-PCR and ELISA assay. Basal finger temperature (Tb), distal-dorsal difference temperature (DTdd) and thermal recovery time (tr) from cold stress were measured by means of functional infrared imaging (fIR). Measurements were performed in late spring, during routine Iloprost therapy (1-3 days infusion of 0.5-2 ng/kg every month), and in late summer after a therapy-withdrawal period. RESULTS: Deterioration of SSc patients' skin thermal properties was observed in the period of therapy withdrawal (Tb reduction and tr enhancement; no DTdd differences) despite the improvement in symptoms of RP. A reduction in IL-12/23p40 gene expression was recorded after therapy withdrawal and a direct correlation between IL-12/23p40 and IL-23p19 gene expression was observed, stronger after therapy suspension. CONCLUSION: Our data suggest that Iloprost treatment summer suspension may induce the loss of the therapy beneficial effect on microcirculation despite the objective reduction of RP, thus favouring a continuous use of Iloprost in absence of severe side effects. Iloprost showed to modulate only IL-23 expression corroborating the idea that this cytokine is crucial for SSc development and progression.

An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1ß-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [(125)I]-labeled NRG-1ß to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.

Kinetic study on the effects of extremely low frequency electromagnetic field on catalase, cytochrome P450 and inducible nitric oxide synthase in human HaCaT and THP-1 cell lines.

Extremely low frequency electromagnetic fields (ELF-EMF) have been found to produce a variety of biological effects. These effects of ELF-EMF depend upon frequency, amplitude, and length of exposure, and are also related to intrinsic susceptibility and responsiveness of different cell types. Although the mechanism of this interaction is still obscure, ELF-EMF can influence cell proliferation, differentiation, cell cycle, apoptosis, DNA replication and protein expression. The aim of this study was to estimate various kinetic constants of catalase, cytochrome P450 and inducible nitric oxide synthase in response to ELF-EMF exposure in human HaCaT and THP-1 cell lines. In order to evaluate the effect of ELF-EMF on the modulation of cellular responses to an inflammatory stimulus, both cell lines were treated with lipopolysaccharide. To the best of our knowledge there is no available report on such type of kinetic study of selected enzymes in response to ELF-EMF in these cell lines. Therefore, the current study may reveal novel mechanism of ELFEMF biological interaction with the enzymological and hormonal systems of living organisms. These new insights may be important for ELF-EMF application particularly for wound healing, tissue regeneration, Parkinson's and Alzheimer's diseases.

Extremely low frequency electromagnetic fields modulate expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and cyclooxygenase-2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing.

BACKGROUND: Extremely low frequency (ELF) electromagnetic fields (EMF) are known to produce a variety of biological effects. Clinical studies are ongoing using EMF in healing of bone fractures and skin wounds. However, little is known about the mechanisms of action of ELF-EMF. Several studies have demonstrated that expression and regulation of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) are vital for wound healing; however, no reports have demonstrated a direct action of ELF-EMF in the modulation of these inflammatory molecules in human keratinocytes. OBJECTIVES: The present study analysed the effect of ELF-EMF on the human keratinocyte cell line HaCaT in order to assess the mechanisms of action of ELF-EMF and to provide further support for their therapeutic use in wound healing. METHODS: Exposed HaCaT cells were compared with unexposed control cells. At different exposure times, expression of inducible NOS (iNOS), endothelial NOS (eNOS) and COX-2 was evaluated by Western blot analysis. Modulation of iNOS and eNOS was monitored by evaluation of NOS activities, production of nitric oxide (NO) and O(2)(-) and expression of activator protein 1 (AP-1). In addition, catalase activity and prostaglandin (PG) E(2) production were determined. Effects of ELF-EMF on cell growth and viability were monitored. RESULTS: The exposure of HaCaT cells to ELF-EMF increased iNOS and eNOS expression levels. These ELF-EMF-dependent increased expression levels were paralled by increased NOS activities, and increased NO production. In addition, higher levels of AP-1 expression as well as a higher cell proliferation rate were associated with ELF-EMF exposure. In contrast, ELF-EMF decreased COX-2 expression, PGE(2) production, catalase activity and O(2)(-) production. CONCLUSIONS: Mediators of inflammation, such as reactive nitrogen and PGE(2), and keratinocyte proliferation are critical for the tissue regenerative processes. The ability of ELF-EMF to upmodulate NOS activities, thus nitrogen intermediates, as well as cell proliferation, and to downregulate COX-2 expression and the downstream intermediate PGE(2), highlights the potential therapeutic role of ELF-EMF in wound healing processes.

Extremely low frequency electromagnetic field enhances human keratinocyte cell growth and decreases proinflammatory chemokine production.

BACKGROUND: Proliferation and differentiation of keratinocytes are central processes in tissue regeneration after injury. Chemokines, produced by a wide range of cell types including keratinocytes, play a regulatory role in inflammatory skin diseases. Several studies have shown that an electromagnetic field (EMF) can influence both inflammatory processes and repair mechanisms including wound healing on different tissue models. OBJECTIVES: To elucidate the effect of extremely low frequency EMF (ELF-EMF) on keratinocyte proliferation and production of chemokines [RANTES, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and interleukin (IL)-8] in order to evaluate a potential therapeutic use of magnetic fields. METHODS: The human keratinocyte cell line HaCaT was exposed at 1 mT, 50 Hz for different lengths of time and compared with unexposed control cells. Cell growth and viability were evaluated at different exposure times by cell count and trypan blue exclusion. Chemokine production and expression were analysed by enzyme-linked immunosorbent assay (ELISA) and by real-time polymerase chain reaction. Total NF-kappaB p65 was quantified by ELISA. RESULTS: Significantly increased growth rates were observed after 48 h of EMF exposure as compared with control cells, while no difference in cell viabilities were detected. Gene expression and release of RANTES, MCP-1, MIP-1 alpha and IL-8 were significantly reduced after 72 h of exposure. NF-kappaB levels became almost undetectable after only 1 h of EMF exposure, and were inversely correlated with cell density. CONCLUSIONS: Our results show that ELF-EMF modulates chemokine production and keratinocyte growth through inhibition of the NF-kappaB signalling pathway and thus may inhibit inflammatory processes. ELF-EMF could represent an additional therapeutic approach in the treatment of skin injury.

Eosinophil recruiting chemokines are down-regulated in peripheral blood mononuclear cells of allergic patients treated with deflazacort or desloratadine.

Chemokines are cytokines with chemotactic properties on leukocyte subsets whose modulation plays a key role in allergic inflammatory processes. To better understand the possible anti-inflammatory effects of histamine-1 receptor antagonists in allergic asthma, we studied the mRNA expression of a set of chemokines known to be involved in the eosinophils-basophils activation as well as recruitment and T-cell signaling events, before and after corticosteroid or antihistamine treatment in PBMCs from allergic-asthmatic patients ex vivo. Twelve patients were enrolled, all of whom were allergic to Parietaria judaica and suffering for mild persistent asthma: six were treated with desloratadine (10 mg/day), and six with deflazacort (12 mg/day). Before and after the treatment, PBMC samples were collected from each patient and analyzed for the expression of encoding mRNAs for several chemokines, I-309 (CCL1), MCP-1 (CCL2), MIP1-alpha (CCL3), MIP1-beta (CCL4), RANTES (CCL5), IL-8 (CXCL8), IP-10 (CXCL10), Lymphotactin (XCL1). Clinical and functional improvements were seen after 3 weeks of therapy; this was associated with a reduced expression in the mRNA levels for the chemokines RANTES, MIP1-alpha and MIP1-beta with either the corticosteroid or the antihistamine, compared to the pre-treatment levels. Chemokine downregulation was statistically significant in both groups of patients. These findings suggest that certain antihistamines may act as down-modulators of allergic inflammation, possibly through a negative regulation of the chemokines involved in activation and attraction of eosinophils. Our results suggest that clinical trials with long follow-ups may be useful in evaluating histamine-1 receptor antagonists as add-on therapy to steroids in the treatment of asthma.

Expression of glycoprotein 90K in human malignant pleural mesothelioma: correlation with patient survival.

The expression of the tumour-associated glycoprotein 90K in patients with malignant pleural mesothelioma (MM) has not been described. This study used enzyme-linked immunoassay (ELISA) to measure 90K in pleural effusions (PEs) and sera from patients with MM (n=28), lung cancer (LC) (n=14) and benign pleural disease (BPD) (n=15). Immunohistochemistry was used to investigate 90K expression in MM and LC tissue sections. The expression of 90K was further evaluated in vitro by ELISA and western blot analysis of conditioned media and cellular extracts of MM, LC and normal human mesothelial (NHM) cell cultures. Finally, the relationships between 90K expression in MM and patient age and survival were studied. The mean 90K level was significantly higher (p<0.05) in PEs of MM patients (11.0+/-6.6 microg/ml) than in LC (6.1+/-3.2 microg/ml) or BPD (6.2+/-5.0 microg/ml) patients. Immunohistochemistry showed a positive reaction for 90K in MM biopsy sections and positive staining limited to inflammatory infiltrates in LC sections. The level of 90K was significantly higher in cell culture media of MM than of LC or NHM (p<0.001). Bands representing proteins with molecular weight of approximately 90 kDa were detected by western blot in MM cellular extracts. An inverse correlation between PE 90K levels and MM patient age (r=-0.45; p=0.017) and a positive correlation between serum 90K levels and MM patient survival (r=0.62; p=0.006) were detected by linear regression analysis. Kaplan-Meier univariate analysis showed increased survival probability for MM patients with serum 90K level >7.3 microg/ml (log rank, p<0.05). This is the first report in MM of the expression of 90K and of its potential diagnostic and prognostic application.

Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications.

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.

AIT-082 is neuroprotective against kainate-induced neuronal injury in rats.

4-[[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid (AIT-082) is an hypoxanthine derivative that stimulates in vitro neurite outgrowth and the production of adenosine and neurotrophins from astrocytes. These effects may predict an in vivo neuroprotective activity of the drug. Thus, we evaluated whether AIT-082 protected against a long-term excitotoxicity of hippocampal neurons following status epilepticus induced in rats by i.p. injection of kainate (12 mg/kg). The epileptogenic effect of kainate was evaluated by monitoring behavioral signs and by electroencephalographic (EEG) recording (80% of the animals showed status epilepticus with a latency of 96.8 +/- 7.4 min starting from the injection). In surviving rats (40% of the injected animals) the neurotoxic effect was evaluated by measuring glutamic acid decarboxylase (GAD) activity, as an index of loss of hippocampal GABAergic neurons, by evaluating the body weight after 7 days and by histological examination of hippocampi. The GAD activity was reduced by 44 +/- 8%, and neuronal loss (about 70%) was found in the CA3c, the CA1 area, and in the dentate gyrus. A single dose of diazepam (20 mg/kg; i.p., 20 min before the kainate injection) almost completely inhibited both seizures and neurotoxicity, ensuring survival of animals. AIT-082 (60 mg/kg/day; i.p., for 7 days, starting from 20 min before the kainate injection) did not modify the seizures caused by kainate but, like diazepam, it decreased kainate-induced mortality, the reduction of GAD activity, and the loss of hippocampal neurons. These data confirm that AIT-082 is of potential interest for the experimental therapy of neurodegenerative disorders.

Basic fibroblast growth factor in mesothelioma pleural effusions: correlation with patient survival and angiogenesis.

The expression of angiogenic factors may represent useful markers for diagnosis and prediction of disease outcome. Basic fibroblast growth factor (b-FGF) is a potent angiogenic factor which promotes in vitro growth of endothelial cells and in vivo vessel formation. We investigated the expression of b-FGF in patients affected with malignant and non-malignant pleural diseases and presenting clinically with non-specific signs and symptoms. We also studied the relationships between the expression of b-FGF in patients with malignant pleural mesothelioma (MM) and tumour aggressiveness, assessed as tumour vessel density (TVD), or patient survival. Basic-FGF was measured by immunoassay in the serum and pleural effusions (PE) of 37 patients. Of these, MM was diagnosed in 15/37 patients while the remaining patients had either peripheral lung adenocarcinoma (PLA) or benign inflammatory pleural disease (BPD). The mean b-FGF level measured 8.5+/-6.1 pg/ml in the PE of the malignant group (MM + PLA) and 23.9+/-19.8 in the PE of the non-malignant group (BPD) (p=0.001). The mean b-FGF level was significantly lower in the PE of MM patients (6.9+/-5.2 pg/ml) compared to BPD patients (p=0.004). Linear regression analysis showed a significant inverse correlation (r=-0.59; p=0.041) between b-FGF levels found in MM PE and patient survival. A noteworthy relationship between high serum b-FGF levels and reduced survival was also observed (r=-0.57; p=0.052). Interestingly, both serum (r=0.48; p=0.114) and PE (r=0.26; p=0.413) b-FGF levels in MM patients correlated poorly with TVD. Our data indicate that b-FGF is significantly more expressed in non-malignant compared to malignant PE, this difference being particularly evident between MM and BPD. Our results also suggest that high b-FGF levels correlate with poor MM patient survival through mechanisms which may be independent of b-FGF angiogenic potential.

Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma.

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946+/-14 pg/ml vs. 180+/-17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase-related VEGF receptors Flt-1 and KDR. Recombinant human VEGF phosphorylated both Flt-1 and KDR and increased proliferation of all four MM cell lines in a dose-dependent fashion. Neutralizing antibodies against either VEGF, Flt-1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt-1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non-malignant pleural disease (1885.7+/-894.9 pg/ml vs. 266.9+/-180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth.

Prognostic significance of presence and reduplication of basal lamina in malignant pleural mesothelioma.

The prognosis of patients with malignant pleural mesothelioma (MM) is dependent more on tumor extension and differentiation than on therapeutic effects. Reduplication of the basal lamina (RBL) is an ultrastructural feature of some benign and malignant tumors that has been inversely correlated with aggressiveness and was recently described in MM. To investigate whether RBL is important for predicting the survival of patients with MM, transmission electron microscopy was used to identify the presence of basal lamina or RBL in biopsy specimens obtained by thoracoscopy from 35 patients. Cox's regression analysis was used to study the relation of these ultrastructural features to survival. Better outcomes were found for patients whose tumors expressed either basal lamina (HR 0.48; 95% CI, 0.09-2.47) or RBL (HR 0.38; 95% CI 0.12-1.22) compared with the reference category, where basal lamina or RBL was not found. The expression of basal lamina and RBL is an important novel prognostic factors in MM. HUM PATHOL 31:1341-1345.

Simian virus-40 sequences are a negative prognostic cofactor in patients with malignant pleural mesothelioma.

Several biochemical and clinical factors have been shown to correlate with survival in human malignant pleural mesothelioma (MM). Nevertheless, average survival of 4 to 10 months from diagnosis is sometimes not sufficient for full expression of these factors. Several studies have reported SV40 sequences in MM, suggesting a possible pathogenic role. We investigated whether the presence of these sequences had any effect on MM patient survival. For this study, we used polymerase chain reaction and Southern blot analysis to search for and identify SV40 DNA in biopsy samples from 83 MM patients. These cases were divided according to histology: 62/83 (74. 7%) had epithelioid morphology (EMM) and 21/83 (25.3%) had either biphasic or sarcomatous morphology (B/SMM). SV40 positivity was significantly associated with B/SMM growth pattern (chi-squared test = 5.03, P = 0.025). Kaplan-Meier univariate analysis confirmed the independent effect of histology on MM survival (log-rank test = 13.9, P < 0.001) and showed a trend for increased survival in SV40-negative patients (log-rank test = 2.83, P = 0.09). Most importantly, Cox's regression model showed that SV40-positive status affected the predictive value of histology on patient survival. In particular, when SV40 expression was added to the B/SMM histotype, Cox's regression model showed a significant increase in hazard ratio (HR) with respect to SV40-negative B/SMM (HR = 4.25, 95% CI = 2.00-9. 00, likelihood ratio test = 14.31, P < 0.001). We conclude that SV40 expression is significantly associated with B/SMM histology and represents an important prognostic cofactor when associated with the tumor subtype in MM patients.

Adenovirus-mediated wild-type p53 overexpression reverts tumourigenicity of human mesothelioma cells.

Pleural malignant mesothelioma (MM) shows poor survival, regardless of tumour stage at diagnosis. MM is unresponsive to present treatment regimens and new protocols are desperately needed. The localised nature, the potential accessibility, and the relative lack of distant metastases make MM a particularly attractive candidate for somatic gene therapy. A common target for cancer gene therapy is the tumour suppressor protein p53. p53 does not seem to be mutated or deleted in MM, but it can be inactivated by binding to other proteins, like mdm2 and SV40 large T antigen. We tested the effects of a replication-deficient adenoviral vector carrying wild-type p53 cDNA in human MM cells. Our results show that >95% of MM cells were efficiently infected with 25 multiplicity of infection (MOI) of vector. Wild-type p53 was effectively expressed resulting in >80% inhibition of proliferation in MM cells. AdCMV.p53 infection induced apoptosis while controls did not show any evident morphological alterations. Ex vivo p53 gene transfer experiments inhibited tumourigenesis in nude mice. In vivo, direct intratumour injection of AdCMV.p53 arrested tumour growth and prolonged survival of treated mice. These results indicate that p53-gene therapy should be strongly exploited for clinical trials in MM patients.

SV40, JC and BK expression in tissue, urine and blood samples from patients with malignant and nonmalignant pleural disease.

BACKGROUND: Polyomaviruses are expressed in both human tumors and immunodepressed patients. Malignant and nonmalignant pleural effusions create an environment that could favor the expression of opportunistic viral infections. We studied if SV40, JC, and BK viral DNA can be amplified from biopsies obtained from different pleural diseases. MATERIALS AND METHODS: DNA was extracted from mesotheliomas (MM), nonspecific inflammatory and tubercular pleural biopsies, blood and urinary sediments from patients with MM, and pleural effusion cytological specimens. SV40, JC and BK viral early regions were amplified by PCR and analyzed by Southern Blot hybridization with specific probes. RESULTS: SV40 was positive in 9/23 MM, 5/18 tubercular and 1/7 nonspecific inflammatory biopsies, and 5/12 pleural effusion cytological specimens. JC was positive in 2/23 MM and in 7/15 urinary sediments. All blood samples were negative and BK was also negative in all samples. CONCLUSIONS: Tissue specific factors, characteristic of MM and TB, may contribute to expression of SV40 in these diseases.

Modulation of integrin expression on mesotheliomas: the role of different histotypes in invasiveness.

The in vitro and in vivo integrin expression in human pleural malignant mesothelioma (MM) of three different histotypes was studied. Cell lines from MM of epithelioid (E1), fibrous (F1), byphasic histotype (B1) and normal mesothelial cells (NM) were analysed for the surface expression of alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 subunits and alphavbeta5 integrins. We found that alpha6, beta4 subunits and alphavbeta5, weakly detectable on NM cells, were expressed on MM cells. The beta3 subunit, well expressed on NM cells, was absent on MM cells. Differential expression among histotypes was observed, the MM-E1 was the least and the MM-B1 the most positive. Specimens for each MM histotype, were analysed by immunohistochemistry. The alpha6 and alphav subunits were more evident on the epithelioid histotype. Intense staining for beta3 and beta4 subunits, was found in all MM, particularly in invading cells, while the alpha5, and alphavbeta5 integrins were variously expressed. The different histotypes can affect the in vitro integrin expression and may indicate a preferential involvement of some subunits in vivo during MM tumor progression.

[Simvastatin and ischemia-reperfusion damage: its effects on apoptotic myocyte death and on the endothelial expression of nitric-oxide synthetase in an experimental model of the isolated rat heart]. / Simvastatina e danno da ischemia-riperfusione: effetti sulla morte apoptotica dei miociti e sull'espressione dell'ossido nitrico-sintetasi endoteliale nel modello sperimentale del cuore isolato di ratto.

Recent studies have suggested that simvastatin may exert endothelial-protective and anti-ischemic effects via nitric oxide (NO) mechanisms. The aim of this study was to evaluate, in isolated working rat hearts, the effect of acute simvastatin administration on endothelial and inducible NO-synthase (eNOS and iNOS) mRNA and on myocytic apoptosis after ischemia-reperfusion. We used isolated working rat hearts submitted to 15 min global, no-flow, normothermic ischemia and 180 min reperfusion. To detect myocytic apoptosis we used DNA agarose gel electrophoresis and Tunel technique; eNOS and iNOS expression were evaluated by multiplex reverse transcriptase-polymerase chain reaction; glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as standard. The eNOS and iNOS mRNAs were expressed as G3PDH/eNOS and G3PDH/iNOS densitometric ratio (BioRad Gel Doc 1000). Hearts were divided into four groups: A) hearts excised and used as histological controls; B) untreated hearts submitted to ischemia and reperfusion; C) actinomicin D-treated (1.5 mg/kg) hearts, perfused with 25 microM simvastatin, subjected to ischemia and reperfusion; D) hearts treated with simvastatin 25 microM and submitted to ischemia and reperfusion. In Group B we evidenced a significant myocytic apoptotic damage, reduced in groups C and D. In Group B an increase in G3PDH/eNOS ratio vs Group A was detected; in Group D a reduction in G3PDH/eNOS ratio vs Group B occurred; no significant changes were observed between groups C and D. As for G3PDH/iNOS ratio, it was significantly increased in Group D with respect to groups A and B. Our data suggest that simvastatin in acute may modulate NO-synthase mRNA expression (induction of eNOS mRNA by means of post-transcriptional mechanisms and inhibition of iNOS postischemic overexpression) and reduce myocytic apoptosis.