Balázs Gasz

The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.

PKA-Bad-14-3-3 and Akt-Bad-14-3-3 signaling pathways are involved in the protective effects of PACAP against ischemia/reperfusion-induced cardiomyocyte apoptosis

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system and various other tissues. PACAP has well-known anti-apoptotic effects in neuronal cell lines. Recent data suggest that PACAP exerts anti-apoptotic effects also in non-neuronal cells. The peptide is present in the cardiovascular system, and has various distinct effects. The aim of the present study was to investigate whether PACAP is protective against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Cultured cardiomyocytes were exposed to 60 min ischemia followed by 120 min reperfusion. The addition of PACAP1-38 significantly increased cell viability and decreased the ratio of apoptotic cells as measured by MTT test and flow cytometry. PACAP induced the phosphorylation of Akt and protein kinase A. In the present study we also examined the possible involvement of Akt- and protein kinase A-induced phosphorylation and thus inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. It was found that ischemia significantly decreased the levels of phosphorylated Bad, which was counteracted by PACAP. Furthermore, PACAP increased the levels of Bcl-xL and 14-3-3 protein, both of which promote cell survival, and decreased the apoptosis executor caspase-3 cleavage. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP has protective effects against ischemia/reperfusion-induced cardiomyocyte apoptosis and provides new insights into the signaling mechanisms involved in the PACAP-mediated anti-apoptotic effects.

Expression and Protective Role of Heme Oxygenase-1 in Delayed Myocardial Preconditioning

Abstract:  In the study the authors aimed to demonstrate the expression and protective effect of heme oxygenase‐1 (HO‐1) in the delayed preconditioning (PC) on cultured myocardiac cells. Neonatal rat cardiac myocytes were exposed to ischemic (ischemic medium [IM] for 20 min) and pharmacological (adenosine, epinephrine, opioid) PC. Twenty‐four hours later cells were subjected to a simulated ischemia (SI )—culturing for 3 h in IM, followed by 2‐h reperfusion in normal medium‐–and then lactate dehydrogenase (LDH), live/death ratio, and apoptosis were measured. For demonstrating the protective role of HO‐1, its enzymatic activity was competitively inhibited by administration of zinc protoporphyrin IX (ZnPPIX), and HO‐1 synthesis was blocked with HO‐1 siRNA. Cells in control group were cultured under normoxic conditions. In SI group, cells underwent only an SI without PC. HO‐1 expression in all of the groups was demonstrated with immunostaining. Our results showed a significant decrease of LDH release, apoptosis, and cell death in PC groups versus SI group, which has been risen in ZnPPIX‐ and HO‐1 siRNA‐treated groups. HO‐1 immunostaining showed an appreciable HO‐1 expression in PC groups, which was abolished with HO‐1 siRNA administration, but not in ZnPPIX group. The results therefore suggest that HO‐1 expression increases in both ischemic and pharmacological PC, and HO‐1 has cellular protective effect against cell death and apoptosis in ischemia‐reperfusion‐induced oxidative injury.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme‐binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub‐threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme‐binding properties SOUL promotes necrotic cell death by inducing mPT.

PACAP ameliorates oxidative stress in the chicken inner ear: An in vitro study

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide. Numerous studies prove that PACAP has neuroprotective effects in diverse neuronal systems in vitro and in vivo. The involvement of PACAP in visual and olfactory sensory processing has also been documented, but little is known about its effects in the auditory system. The presence of PACAP and its receptor, the specific PAC1 receptor, has been shown in the cochlea and in brain structures involved in auditory pathways. The aim of the present study was to investigate whether PACAP is protective in cochlear oxidative stress-induced cell death, which is known to play a role in several ototoxic insults. Chicken cochlear cells were exposed to 1mM H(2)O(2), which resulted in a marked reduction of cell viability and a parallel increase of apoptotic and necrotic cells assessed by MTT test, annexin V/propidium iodide flow cytometry and JC-1 apoptosis assay. Co-incubation with 100nM PACAP increased cell viability and reduced the percentage of apoptotic cells. Furthermore, oxidative stress increased the activation of caspase-3, while simultaneous PACAP treatment reduced it. In summary, our present results demonstrate that PACAP effectively protects cochlear cells against oxidative stress-induced apoptotic cell death.

Effect of acetylsalicylic acid on nuclear factor-kappaB activation and on late preconditioning against infarction in the myocardium.

Nuclear factor-κB (NF-κB) plays an essential role in the intracellular signal transduction of the second window of protection (SWOP). Acetylsalicylic acid (ASA) blocks NF-κB-dependent gene activation in leukocytes and endothelial cells through preventing phosphorylation and subsequent degradation of the inhibitor IκB-α. This study investigated the effect of ASA on the late phase of ischemic preconditioning (PC) against myocardial infarction and on the activation of NF-κB in the preconditioned myocardium. Conscious rabbits were subjected to 4 cycles of 5 minutes of coronary occlusion and 5 minutes of reperfusion together with 3 different doses of ASA (5 mg/kg; 25 mg/kg; 130 mg/kg). After 30 minutes of reperfusion we determined the activation of NF-κB with an electrophoretic mobility shift assay (EMSA). Twenty-four hours later, after 30 minutes of test ischemia, we performed infarct size analysis using triphenyltetrazolium-chloride (TTC) staining. Neither 5 mg/kg (antithrombotic dose) nor 25 mg/kg (analgesic/antipyretic dose) of ASA interfered with the NF-κB activation and the protective effect of late preconditioning against myocardial infarction. In contrast, NF-kB activation and late PC effect were completely abrogated by 130 mg/kg of ASA. Our results suggest that nonselective doses of NSAIDs should be used with caution in patients with atherosclerotic cardiovascular disease because they may deprive the heart of its innate defensive response.

PACAP Inhibits Oxidative Stress-Induced Activation of MAP Kinase-Dependent Apoptotic Pathway in Cultured Cardiomyocytes

Abstract:  The present article investigated the effect of pituitary adenylate cyclase‐activating polypeptide (PACAP) on oxidative stress‐induced apoptosis in neonatal rat cardiomyocytes. Our results show that PACAP decreased the ratio of apoptotic cells following H2O2 treatment. PACAP also diminished the activity of apoptosis signal‐regulating kinase. These effects of PACAP were counteracted by the PACAP antagonist PACAP6‐38. In summary, our results show that PACAP is able to attenuate oxidative stress‐induced cardiomyocyte apoptosis and suggest that its cardioprotective effect is mediated through inhibition of the MAP kinase‐dependent apoptotic pathway.

Cardioprotective action of urocortin in early pre- and postconditioning

Abstract:  Pre‐ and postconditioning are powerful endogenous adaptive phenomenon of the organism whereby different stimuli enhance the tolerance against various types of stress. Urocortin (Ucn), member of the corticotropin‐releasing factor (CRF) family has potent effects on the cardiovascular system. The aim of this article was to investigate the action of Ucn on cultured cardiomyocytes in the process of pre‐ and postconditioning. Isolated neonatal rat ventricular myocytes were preconditioned with adenosine, simulated ischemia, and Ucn (10‐min treatment followed by 10‐min reperfusion/recovery). For detecting the effect of alternative types of preconditioning, necrosis enzyme (lactate dehydrogenase [LDH]) release, vital staining (trypan blue), and ratio of apoptosis/necrosis were examined after cardiac cells were exposed to 3‐h sustained ischemia and 2‐h reperfusion. Same parameters were measured in the postconditioned groups (30‐ or 60‐min ischemia followed by postconditioning with 10‐min ischemic stimulus or Ucn and 2‐h reperfusion). Cells exposed to 3‐h ischemia followed by 2‐h reperfusion were shown as control. Our results show that LDH release a number of trypan blue‐stained dead cells and the ratio of apoptotized and necrotized cells was decreased in all preconditioned groups compared with control group. In postconditioned groups LDH content of culture medium, trypan blue‐positive cardiomyocytes, and the rate of apoptotic/necrotic cells was reduced contrasted with non‐postconditioned group. We can conclude that preconditioning with Ucn induced such a powerful cell protective effect as adenosine and ischemia. Furthermore, postconditioning with Ucn after 60‐min ischemia was more cardioprotective than ischemic postconditioning.

Controlled reperfusion decreased reperfusion induced oxidative stress and evoked inflammatory response in experimental aortic-clamping animal model

UNLABELLED Revascularization after long term aortic ischaemia in vascular surgery induces reperfusion injury accompanied with oxidative stress and inflammatory responses. The hypothesis of this study was that the aortic occlusion followed by controlled reperfusion (CR) can reduce the ischaemia-reperfusion injury, the systemic and local inflammatory response induced by oxidative stress.Animal model was used. CONTROL GROUP animals underwent a 4-hour infrarenal aortic occlusion followed by continuous reperfusion. Treated group: animals were treated with CR: after a 4-hour infrarenal aortic occlusion we made CR for 30 minutes with the crystalloid reperfusion solution (blood: crystalloid solution ratio 1:1) on pressure 60 Hgmm. Blood samples were collected different times. The developing oxidative stress was detected by the plasma levels of malondialdehyde, reduced glutathion, thiol groups and superoxide dismutase. The inflammatory response was measured by phorbol myristate acetate-induced leukocyte reactive oxygen species production and detection of change in myeloperoxidase levels. The animals were anaesthetized one week after terminating ligation and biopsy was taken from quadriceps muscle and large parenchymal organs.CR significantly reduced the postischaemic oxydative stress and inflammatory responses in early reperfusion period. Pathophysiological results: The rate of affected muscle fibers by degeneration was significantly higher in the untreated animal group. The infiltration of leukocytes in muscle and parenchymal tissues was significantly lower in the treatedgroup.CR can improve outcome after acute lower-limb ischaemia. The results confirm that CR might be also a potential therapeutic approach in vascular surgery against reperfusion injury in acute limb ischaemia. Supported by OTKA K108596.

The role of GST polymorphism in reperfusion induced oxidative stress, inflammatory responses and clinical complications after surgical and percutaneous coronary intervention

BACKGROUND Patients having coronary artery disease treated by coronary bypass or PCI procedure are exposed to tissue damage because of the phenomenon called reperfusion injury. Reperfusion injury can be characterized/monitored by oxidative stress parameters, inflammatory markers and by post-operative complication rate. OBJECTIVE Beyond the obvious factors determining its severity (affected myocardial mass, ischaemic time, collateral circulation etc.) we examined the GST enzyme groups most cardio selective member, GSTP1 and its genetic polymorphism if there is any genetically determined preventive effect on the above-mentioned parameters. MATERIALS AND METHODES We have performed randomized prospective study in the Heart Institute of Pecs with 862 patients, treated by coronary bypass or PCI procedure. Blood samples were taken a day before, one hour, one day, one week after the operation. Leucocyte count (WBC), myeloperoxidase (MPO), thiol group (SH); Superoxide dismutase (SOD), malondialdehyde (MDA), reduced Glutathione (GSH) level was checked in different periods of time as a comparison. The onset of myocardial damage and the corresponding necro enzyme level changes were registered in the perioperative period. Our patients GSTP1 allele pair combinations (A, B, or C) were determined by real time PCR method. RESULTS In patients with GSTP1 AA genotype we have found significance level reaching plasma concentration rise in SOD and MDA, and drop in GSH, SH. The CKMB concentration rise in the post-operative 24 hours was significantly higher in the GSTP1 AA group. CONCLUSIONS According to our results the AA allele combination can be considered as a risk factor. GSTP1-AA allele pair has negative effect on ischemia-reperfusion tolerance of the heart. In case of cardiovascular interventions, the study of GST enzyme polymorphisms can be an independent risk stratification factor in determining the perioperative risk in the future.