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.

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes.

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H(2)O(2) (group 2), I/R (group 3), I/R and EA (group 4), H(2)O(2) coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H(2)O(2) treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H(2)O(2). GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury.

Effects of PACAP and preconditioning against ischemia/reperfusion-induced cardiomyocyte apoptosis in vitro.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are widely expressed in the nervous system and various other tissues. PACAP exerts strong anti-apoptotic effects in neuronal cell lines and, according to recent data, also in non-neuronal cells. The peptide is present in the cardiovascular system and has various distinct effects. We have demonstrated earlier that PACAP has protective effects against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Preconditioning with brief intermittent periods of ischemia is known to provide protection against ischemic injury. The aim of the present study was to investigate whether PACAP could enhance the protective effect of preconditioning against in vitro ischemic injury. Cultured cardiomyocytes were exposed to brief preconditioning ischemia followed by 2 h ischemia and 4 h reperfusion. Both PACAP treatment and preconditioning alone significantly increased cell viability and decreased the ratio of cell death. Pretreatment with PACAP was found to further reduce the level of cleaved caspase-8 but it did not lead to additional survival rate when compared to cells treated with PACAP or preconditioning alone. These results show that although both PACAP and preconditioning have a protective effect against ischemia/reperfusion-induced cardiomyocyte apoptosis, their effects are not additive.

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.

Protective effects of pituitary adenylate cyclase activating polypeptide in endothelial cells against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed neuropeptide that has various different functions in the nervous system and in non-neural tissues. Little is known about the effects of PACAP in endothelial cells. The aim of the present study was to investigate the effects of PACAP on endothelial cell survival and apoptotic signaling pathways under oxidative stress. Mouse hemangioendothelioma (EOMA) cells were exposed to 0.5mM H(2)O(2) which resulted in a marked reduction of cell viability and a parallel increase of apoptotic cells assessed by MTT test and flow cytometry. Co-incubation with 20nM PACAP1-38 increased cell viability and reduced the percentage of apoptotic cells. Flow cytometry analysis showed that oxidative stress reduced the phosphorylation of the anti-apoptotic ERK and increased the phosphorylation of the pro-apoptotic JNK and p38 MAP kinases. PACAP1-38 treatment ameliorated these changes: levels of phospho-ERK were elevated and those of phospho-JNK and p38 were decreased. All these effects were abolished by simultaneous treatment with the PACAP antagonist PACAP6-38. In summary, our results show that PACAP effectively protects endothelial cells against the apoptosis-inducing effects of oxidative stress.

Effect of cardiopulmonary bypass on cytokine network and myocardial cytokine production.

BACKGROUND: In addition to the well-investigated proinflammatory cytokine expression, there is an ever increasing interest in the field of anti-inflammatory response to cardiopulmonary bypass (CPB). Evidence suggests that myocardium serves as an important source of cytokines during reperfusion and application of CPB. The effect of coronary artery bypass graft (CABG) without CPB on myocardial cytokine production has not as yet been investigated. HYPOTHESIS: Cardiopulmonary bypass can cause long-term disturbance in pro- and anti-inflammatory cytokine balance, which may impede a patient's recovery following surgery. Therefore, the effect of CPB on the balance of the pro-/anti-inflammatory cytokines network and myocardial cytokine outflow was assessed throughout a longer period after surgery. METHODS: Twenty patients were scheduled for CABG with CPB and 10 had off-pump surgery. Blood samples were taken before, during, and over the first week following surgery. Coronary sinus blood samples were collected during surgery. The ratio of pro- and anti-inflammatory cytokines was calculated and the cytokine concentration of peripheral and coronary sinus blood were compared in both groups. RESULTS: Pro-/anti-inflammatory cytokine ratio decreased early after CPB followed by a delayed and marked increase. A more balanced ratio was present following off-pump surgery. Coronary sinus levels of certain cytokines exceeded the concentration of systemic blood in the course of CPB but not during off-pump operation. CONCLUSION: Patients show pro-inflammatory predominant cytokine balance at a later stage after CPB in contrast to those without CPB. The heart produces a remarkable amount of cytokines only in the course of surgery with CPB.

Threshold level of NF-kB activation in small bowel ischemic preconditioning procedure.

Ischemic preconditioning (IPC), which is obtained by exposure to brief periods of vascular occlusion, improves organ tolerance to prolonged ischemia. The aim of this study was to evaluate the threshold level of NF-kB activation in small intestine during an IPC procedure. Various intestinal IPC were performed on 20 Wistar rats in seven groups: group I (GI, nonpreconditioned); group II (GII, 1-minute ischemia and 1-minute reperfusion); group III (GIII, two cycles of 1-minute ischemia and 1-minute reperfusion); group IV (GIV, 2-minutes ischemia and 2-minutes reperfusion); group V (GV, two cycles of 2-minute ischemia and 2-minute reperfusion); group VI (GVI, 5-minute ischemia and 10-minute reperfusion); group VII (GVII, two cycles of 5-minute ischemia and 10-minute reperfusion). Bowel biopsies were collected after laparotomy (control) as well as at 30, 60, and 120 minutes following IPC. We determined the cytoplasmic and nuclear NF-kB by a chemiluminescence-based ELISA method. Our results showed low, constant NF-kB levels in GI. In the preconditioned groups (GII-GVII), NF-kB was significantly elevated at 30 minutes following IPC (P < .05 vs control). After 1 hour, NF-kB activity decreased to the control level. However, 2 hours after IPC both forms of NF-kB were elevated significantly again, which was independent of the number of IPC cycles (P < .05 vs control). Our experiments revealed that one cycle of 1-minute ischemia and 1-minute reperfusion is a critical threshold level for NF-kB activation during small bowel IPC. Longer and more IPC cycles did not result in further elevation of NF-kB activation.

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. 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.

Expression of CD97 and adhesion molecules on circulating leukocytes in patients undergoing coronary artery bypass surgery.

OBJECTIVE: Leukocyte activation is thought to be responsible for the adverse effects and postoperative complications following cardiopulmonary bypass (CPB). A novel cell surface molecule, CD97, is a sensitive marker of leukocyte and primary lymphocyte activation. The present study aimed to determine the activation of different leukocyte subsets by comparing the expression of CD97 and adhesion molecules (CD11, CD18) in patients receiving coronary surgery with or without CPB. METHODS: 30 patients were enrolled and scheduled for coronary bypass surgery under CPB (20 patients, group A) and with off-pump (OP) operation (10 patients, group B). Blood samples were taken before and during surgery, and over the following first week. RESULTS: Here, we report an early decrease in CD97 expression of granulocytes (PMN) and monocytes (MC) followed by an intensive increase reaching the maximum on postoperative days 2 and 3 in patients operated with CPB. The rate of active CD97-positive lymphocytes showed a marked, gradual increase until postoperative day 3 and remained elevated up to day 7 after CPB. OP surgery resulted in moderate alteration in the presence of CD97 on PMN, MC and lymphocytes. The expression of adhesion molecules was similar to CD97 in all leukocyte subsets. CONCLUSION: The findings about CD97 expression suggest considerable leukocyte activation following coronary bypass with CPB compared to OP surgery. The collected data show that the lymphocytes are highly activated and involved in leukocyte sequestration after CPB. Moreover, the importance of CD97 in CPB-related inflammatory response can be stated.

Bradykinin and angiotensin-converting enzyme inhibition in cardioprotection.

OBJECTIVES: To show that angiotensin-converting enzyme (ACE) inhibition potentiates subthreshold ischemic preconditioning (IPC) via the elevation of bradykinin activity, leading to a fully delayed cardioprotective response. METHODS: On day 1 of the experiment, pigs were subjected to sham (group 1, controls) or IPC protocols. In groups 2 and 3, 4x5 min and 2x2 min of IPC, respectively, were elicited by occluding the left anterior descending coronary artery with percutaneous transluminal coronary angioplasty inflatable balloon catheter. Group 4 was subjected to the ACE inhibitor perindoprilate only. In group 5, the pigs were pretreated with perindoprilate (0.06 mg/kg) and then subjected to 2x2 min IPC. In group 6, intracoronary HOE 140 (a selective bradykinin B(2) receptor antagonist) was added before the perindoprilateaugmented subthreshold (2x2 min) PC stimulus. On the second day, all animals underwent 40 min left anterior descending coronary artery ligation and 3 h reperfusion, followed by infarct size analysis using triphenyl tetrazolium chloride staining. RESULTS: THE RATES OF INFARCT SIZE AND RISK ZONE WERE THE FOLLOWING IN THE EXPERIMENTAL GROUPS: group 1, 42.8%; group 2,19.5% (P<0.05); group 3, ischemia/reperfusion (I/R) 33.4%; group 4, I/R 18.4% (P<0.05); group 5, I/R 31.2%; and group 6, I/R 36.3%. A significant increase of nuclear factor kappa B activation in groups 2 and 4 was seen. CONCLUSIONS: Results confirm that ACE inhibitors do not give total pharmacological IPC, but they enhance the induction effect of small ischemic insults, which raises the ischemic tolerance of myocardium. It was determined that enhanced bradykinin activity leads to downstream nuclear factor kappa B activation in this model.