Apelin-induced cardioprotection against ischaemia/reperfusion injury: roles of epidermal growth factor and Src.

AIM: Apelin, the ligand of the G-protein-coupled receptor (GPCR) APJ, exerts a post-conditioning-like protection against ischaemia/reperfusion injury through activation of PI3K-Akt-NO signalling. The pathway connecting APJ to PI3K is still unknown. As other GPCR ligands act through transactivation of epidermal growth factor receptor (EGFR) via a matrix metalloproteinase (MMP) or Src kinase, we investigated whether EGFR transactivation is involved in the following three features of apelin-induced cardioprotection: limitation of infarct size, suppression of contracture and improvement of post-ischaemic contractile recovery. METHOD: Isolated rat hearts underwent 30 min of global ischaemia and 2 h of reperfusion. Apelin (0.5 µm) was infused during the first 20 min of reperfusion. EGFR, MMP or Src was inhibited to study the pathway connecting APJ to PI3K. Key components of RISK pathway, namely PI3K, guanylyl cyclase or mitochondrial K+ -ATP channels, were also inhibited. Apelin-induced EGFR and phosphatase and tensing homolog (PTEN) phosphorylation were assessed. Left ventricular pressure and infarct size were measured. RESULTS: Apelin-induced reductions in infarct size and myocardial contracture were prevented by the inhibition of EGFR, Src, MMP or RISK pathway. The involvement of EGFR was confirmed by its phosphorylation. However, neither direct EGFR nor MMP inhibition affected apelin-induced improvement of early post-ischaemic contractile recovery, which was suppressed by Src and RISK inhibitors only. Apelin also increased PTEN phosphorylation, which was removed by Src inhibition. CONCLUSION: While EGFR and MMP limit infarct size and contracture, Src or RISK pathway inhibition suppresses the three features of cardioprotection. Src does not only transactivate EGFR, but also inhibits PTEN by phosphorylation thus playing a crucial role in apelin-induced cardioprotection.

Nanoprecipitated catestatin released from pharmacologically active microcarriers (PAMs) exerts pro-survival effects on MSC.

Catestatin (CST), a fragment of Chromogranin-A, exerts angiogenic, arteriogenic, vasculogenic and cardioprotective effects. CST is a very promising agent for revascularization purposes, in "NOOPTION" patients. However, peptides have a very short half-life after administration and must be conveniently protected. Fibronectin-coated pharmacologically active microcarriers (FN-PAM), are biodegradable and biocompatible polymeric microspheres that can convey mesenchymal stem cell (MSCs) and therapeutic proteins delivered in a prolonged manner. In this study, we first evaluated whether a small peptide such as CST could be nanoprecipitated and incorporated within FN-PAMs. Subsequently, whether CST may be released in a prolonged manner by functionalized FN-PAMs (FN-PAM-CST). Finally, we assessed the effect of CST released by FN-PAM-CST on the survival of MSCs under stress conditions of hypoxia-reoxygenation. An experimental design, modifying three key parameters (ionic strength, mixing and centrifugation time) of protein nanoprecipitation, was used to define the optimum condition for CST. An optimal nanoprecipitation yield of 76% was obtained allowing encapsulation of solid CST within FN-PAM-CST, which released CST in a prolonged manner. In vitro, MSCs adhered to FN-PAMs, and the controlled release of CST from FN-PAM-CST greatly limited hypoxic MSC-death and enhanced MSC-survival in post-hypoxic environment. These results suggest that FN-PAM-CST are promising tools for cell-therapy.

Redox signalling and cardioprotection: translatability and mechanism.

The morbidity and mortality from coronary artery disease (CAD) remain significant worldwide. The treatment for acute myocardial infarction has improved over the past decades, including early reperfusion of culprit coronary arteries. Although it is mandatory to reperfuse the ischaemic territory as soon as possible, paradoxically this leads to additional myocardial injury, namely ischaemia/reperfusion (I/R) injury, in which redox stress plays a pivotal role and for which no effective therapy is currently available. In this review, we report evidence that the redox environment plays a pivotal role not only in I/R injury but also in cardioprotection. In fact, cardioprotective strategies, such as pre- and post-conditioning, result in a robust reduction in infarct size in animals and the role of redox signalling is of paramount importance in these conditioning strategies. Nitrosative signalling and cysteine redox modifications, such as S-nitrosation/S-nitrosylation, are also emerging as very important mechanisms in conditioning cardioprotection. The reasons for the switch from protective oxidative/nitrosative signalling to deleterious oxidative/nitrosative/nitrative stress are not fully understood. The complex regulation of this switch is, at least in part, responsible for the diminished or lack of cardioprotection induced by conditioning protocols observed in ageing animals and with co-morbidities as well as in humans. Therefore, it is important to understand at a mechanistic level the reasons for these differences before proposing a safe and useful transition of ischaemic or pharmacological conditioning. Indeed, more mechanistic novel therapeutic strategies are required to protect the heart from I/R injury and to improve clinical outcomes in patients with CAD.

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential.

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides.

Chromogranin A (CgA) is produced by cells of the sympathoadrenal system and by human ventricular myocardium. In the clinical setting CgA has been mainly used as a marker of neuroendocrine tumors, but in the last decade a plenty of data have been published on the role of CgA and its derived peptides, particularly catestatin and vasostatin, in the regulation of cardiovascular function and diseases, including heart failure and hypertension. CgA-derived peptides, namely catestatin and vasostatin, may exert negative inotropic and lusitropic effects on mammalian hearts. As such CgA and its derived peptides may be regarded as mediators of a complex feedback system able to modulate the exaggerated release of catecholamines. This system may be also interpreted as an attempt for compensatory cardioprotective response against myocardial injury in the pre and postischemic scenarios. In fact, while vasostatin can trigger cardioprotective effects akin ischemic preconditioning (protection is triggered before ischemia), catestatin is a potent cardioprotective agent in the early post-ischemic phase, acting like a postconditioning agent (protection is triggered at the onset of reperfusion). Admittedly, the exact mechanism of cardioprotection of this system is far from being fully understood. Interestingly, both vasostatin and catestatin have shown to be able to activate multiple cardioprotective pathways. In particular, these two CgA-derived peptides may induce nitric oxide dependent pathway, which may play a pivotal role in cardioprotection against ischemia/reperfusion injury. Here, we review the literature about the cardiac effects of catestatin and vasostatin, the mechanisms of myocardial injury and protection and the role of CgA derived peptides in cardioprotection.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Intensification of GVHD prophylaxis with low-dose ATG-F before allogeneic PBSC transplantation from HLA-identical siblings in adult patients with hematological malignancies: results from a retrospective analysis.

Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III-IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications.

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4(+) T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.

Targeted inhibition of mTORC1 and mTORC2 by active-site mTOR inhibitors has cytotoxic effects in T-cell acute lymphoblastic leukemia.

The mammalian Target Of Rapamycin (mTOR) serine/threonine kinase belongs to two multi-protein complexes, referred to as mTORC1 and mTORC2. mTOR-generated signals have critical roles in leukemic cell biology by controlling mRNA translation of genes that promote proliferation and survival. However, allosteric inhibition of mTORC1 by rapamycin has only modest effects in T-cell acute lymphoblastic leukemia (T-ALL). Recently, ATP-competitive inhibitors specific for the mTOR kinase active site have been developed. In this study, we have explored the therapeutic potential of active-site mTOR inhibitors against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. The inhibitors affected T-ALL cell viability by inducing cell-cycle arrest in G(0)/G(1) phase, apoptosis and autophagy. Western blot analysis demonstrated a Ser 473 Akt dephosphorylation (indicative of mTORC2 inhibition) and a dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors. The inhibitors strongly synergized with both vincristine and the Bcl-2 inhibitor, ABT-263. Remarkably, the drugs targeted a putative leukemia-initiating cell sub-population (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, the inhibitors displayed remarkable anti-leukemic activity, which emphasizes their future development as clinical candidates for therapy in T-ALL.

A recently developed bifacial platelet-rich fibrin matrix.

Platelet-rich plasma (PRP) is used clinically in liquid or gel form to promote tissue repair. Because of the poor mechanical properties, conventional PRP is often difficult to handle when used in clinical settings and requires secure implantation in a specific site, otherwise when released growth factors could be washed out during an operation. In this study, we analyzed the end product of a recently developed commercially available system (FIBRINET), which is a dense pliable, platelet-rich fibrin matrix (PRFM). We characterized the mechanical properties of PRFM and tested whether PRFM releases growth factors and whether released factors induce the proliferation of mesenchymal stem cells (MSC). Mechanical properties as well as platelet distribution were evaluated in PRFM. PRFM demonstrated robust mechanical properties, with a tear elastic modulus of 937.3 +/- 314.6 kPa, stress at a break of 1476.0 +/- 526.3 kPa, and an elongation at break of 146.3 +/- 33.8 %. PRFM maintained its mechanical properties throughout the testing process. Microscopic observations showed that the platelets were localized on one side of the matrix. Elevated levels of PDGF-AA, PDGF-AB, EGF, VEGF, bFGF and TGF-beta1 were measured in the day 1-conditioned media (CM) of PRFM and growth factor levels decreased thereafter. BMP2 and BMP7 were not detectable. MSC culture media supplemented with 20% PRFM-CM stimulated MSC cell proliferation; at 24 and 48 hours the induction of the proliferation was significantly greater than the induction obtained with media supplemented with 20% foetal bovine serum. The present study shows that the production of a dense, physically robust PRFM made through high-speed centrifugation of intact platelets and fibrin in the absence of exogenous thrombin yields a potential tool for accelerating tissue repair.

Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms.

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair.

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

The platelet activating factor triggers preconditioning-like cardioprotective effect via mitochondrial K-ATP channels and redox-sensible signaling.

Endogenous platelet activating factor (PAF) is involved in heart ischemic preconditioning. PAF can also afford pharmacological preconditioning. We studied whether mitochondrial-ATP-sensitive K(+) (mK(ATP)) channels and reactive oxygen species (ROS) are involved in PAF-induced cardioprotection. In Group 1 control hearts, Langendorff-perfused rat hearts underwent 30 min ischemia and 2 hours of reperfusion. Group 2 hearts, before ischemia, were perfused for 19 min with PAF (2x10(-11) M); Groups 3 and 4 hearts were co-infused with PAF and N-acetyl-L-cysteine or 5-hydroxydecanoate to scavenge ROS or to block mK(ATP) channels, respectively. Left ventricular pressure and infarct size were determined. PAF-pretreatment reduced infarct size (33 +/- 4% vs 64 +/- 4.6 % of the area at risk of control hearts) and improved pressure recovery. Infarct-sparing effect of PAF was abolished by N-acetyl-L-cysteine and 5-hydroxydecanoate. Thus, the cardioprotective effect exerted by PAF-pretreatment involves activation of mK(ATP) channels and redox signaling in pre-ischemic phase.

The paradigm of postconditioning to protect the heart.

Ischaemic preconditioning limits the damage induced by subsequent ischaemia/reperfusion (I/R). However, preconditioning is of little practical use as the onset of an infarction is usually unpredictable. Recently, it has been shown that the heart can be protected against the extension of I/R injury if brief (10-30 sec.) coronary occlusions are performed just at the beginning of the reperfusion. This procedure has been called postconditioning (PostC). It can also be elicited at a distant organ, termed remote PostC, by intermittent pacing (dyssynchrony-induced PostC) and by pharmacological interventions, that is pharmacological PostC. In particular, brief applications of intermittent bradykinin or diazoxide at the beginning of reperfusion reproduce PostC protection. PostC reduces the reperfusion-induced injury, blunts oxidant-mediated damages and attenuates the local inflammatory response to reperfusion. PostC induces a reduction of infarct size, apoptosis, endothelial dysfunction and activation, neutrophil adherence and arrhythmias. Whether it reduces stunning is not clear yet. Similar to preconditioning, PostC triggers signalling pathways and activates effectors implicated in other cardioprotective manoeuvres. Adenosine and bradykinin are involved in PostC triggering. PostC triggers survival kinases (RISK), including Akappat and extracellular signal-regulated kinase (ERK). Nitric oxide, via nitric oxide synthase and non-enzymatic production, cyclic guanosine monophosphate (cGMP) and protein kinases G (PKG) participate in PostC. PostC-induced protection also involves an early redox-sensitive mechanism, and mitochondrial adenosine-5' -triphosphate (ATP)-sensitive K(+) and PKC activation. Protective pathways activated by PostC appear to converge on mitochondrial permeability transition pores, which are inhibited by acidosis and glycogen synthase kinase-3beta (GSK-3beta). In conclusion, the first minutes of reperfusion represent a window of opportunity for triggering the aforementioned mediators which will in concert lead to protection against reperfusion injury. Pharmacological PostC and possibly remote PostC may have a promising future in clinical scenario.

Transfusion recipient identification.

Recent reports from different haemovigilance systems indicate that errors in the whole-blood transfusion chain - from initial recipient identification to final blood administration - occur with a frequency of approximately 1 in 1000 events. Although mistakes occur also within the blood transfusion service, about two-thirds of errors are associated with incorrect blood recipient identification at the patient's bedside. To prevent the potentially fatal consequences of such mistakes, specific tools have been developed, including patient identification bracelets with barcodes and/or radio frequency identification devices, mechanical or electronic locks preventing access to bags assigned to other patients, and palm computers suitable for transferring blood request and administration data from the patient's bedside to the blood transfusion service information system in real time. The effectiveness of these systems in preventing mistransfusion has been demonstrated in a number of studies.

Mitochondrial ATP-sensitive channel opener does not induce vascular preconditioning, but potentiates the effect of a preconditioning ischemia on coronary reactive hyperemia in the anesthetized goat.

Preconditioning ischemia (PI) increases the speed of the initial vasodilatation (vascular preconditioning) of a subsequent coronary reactive hyperemia (CRH) and reduces total hyperemic flow (THF). We investigated whether changes in CRH similar to those induced by PI are obtained with diazoxide, a mitochondrial ATP-sensitive K+ channel opener, and whether diazoxide influences the effects of a subsequent PI on CRH. In anesthetized goats, flow was recorded from the left circumflex coronary artery (LCCA). CRH and PI were obtained with 15-s and 5-min LCCA occlusions, respectively. CRH was studied before and after PI, before and after diazoxide (2.5 mg/kg i.v.) as well as before and after PI was induced after diazoxide pre-treatment. After PI, the time to peak (ttp) of CRH and THF decreased by 51+/-13% and 23+/-8%, respectively. Diazoxide did not change CRH. After diazoxide and PI, when basal flow had returned to the control level, the ttp of CRH was reduced as after PI alone (-45+/-12%), whereas THF was reduced to a greater extent (-41+/-9% versus -23+/-8%; P<0.01). In conclusion, PI alters CRH by decreasing THF and reducing the ttp of CRH. Whilst diazoxide does not reproduce the effects of PI on CRH, pre-treatment with diazoxide potentiates the effects of PI on THF.

Involvement of nitric oxide in ischemic preconditioning.

In ischemic preconditioning, nitric oxide (NO) limits the extension of a subsequent infarct and protects against ischemia/reperfusion-induced endothelial dysfunction, arrhythmias and myocardial stunning. The protective activity concerns both the first and the second window of protection. The antiarrhythmic effect is attributed to microvessel dilation and to the production of cyclic guanosine monophosphate in the myocardium. The limitation of the infarct size is likely to depend on the opening of the mitochondrial adenosine triphosphate-sensitive potassium channels, to which NO participates via the activation of a protein kinase C (PKC). The endothelial protection involves an NO-mediated reduction in neutrophil adherence to the coronary endothelium and platelet aggregation and is accompanied by an enhanced response to vasodilator stimuli. During preconditioning ischemia, NO is released from the coronary endothelium as a result of bradykinin-induced activation of B2 endothelial receptors. In addition to the early protection, endothelium-derived NO is also responsible for a signaling cascade which leads to the activation of myocardial inducible NO synthase, which in turn is responsible for the release of NO involved in the delayed protection. The signaling cascade includes the activation of PKC-epsilon, tyrosine kinase and some mitogen-activated protein kinases. It has been suggested that the activation of PKC-epsilon is mediated by peroxynitrite produced by the combination of NO and the superoxide anion, the latter being generated during reperfusion which follows preconditioning ischemia.

Ischemic preconditioning: from the first to the second window of protection.

In many species one or more brief coronary occlusions limit the injuries which a subsequent ischemia-reperfusion can produce in the myocardium. A similar protection has been observed in the majority of organ systems. A first period or window of protection can lasts up to 3 hours and is followed by a second window of protection (SWOP) which begins about 24 hours after the brief coronary occlusions and lasts about 72 hours. Increase of the release of endogenous agents such as adenosine and nitric oxide (NO) may be responsible for both windows through the activation of a protein-kinase C (PKC) which in turn activates ATP sensitive potassium (K+(ATP)) channels. Nitric oxide is also reported to act directly on K+(ATP) channels. Recently, it has been suggested that the channels involved in the protection are mitochondrial rather than sarcolemmal. In SWOP the origin of NO is attributed to the activity of an inducible NO-synthase. Free oxygen radicals released during preconditioning are likely to take part in the delayed protection through the production of peroxynitrite which activates PKC and through the increase of the activity of antioxidant enzymes such as Mn superoxide-dismutase. The production of heat shock proteins is considered a marker rather than a mechanism of SWOP.

Role of calcium-sensitive K(+) channels and nitric oxide in in vivo coronary vasodilation from enhanced perfusion pulsatility.

BACKGROUND: In vitro studies support K(+)(Ca) channel-induced smooth muscle hyperpolarization as underlying acetylcholine-mediated (or bradykinin-mediated) vasodilation that persists despite combined nitric oxide (NO) and PGI(2) inhibition. We tested the hypothesis that these channels are activated by enhanced pulsatile perfusion in vivo and contribute substantially to vasodilation from this stimulus. METHODS AND RESULTS: The canine left descending coronary artery was perfused with whole blood at constant mean pressure, and physiological flow pulsatility was set at 40 or 100 mm Hg by computer servo-pump. Cyclooxygenase was inhibited by indomethacin. Mean flow increased +18+/-2% (P:<0.0001) with enhanced pulsatility. This response declined approximately 50% by blocking NO synthase (L-NMMA) or K(+)(Ca) [charybdotoxin (CbTX)+apamin (AP)]. Combining both inhibitors virtually eliminated the flow rise. Inhibiting either or both pathways minimally altered basal coronary flow, whereas agonist-stimulated flow was blocked. Bradykinin-induced dilation declined more with CbTX+AP than with L-NMMA (-66% versus -46%, P:=0.03) and was fully blocked by their combination. In contrast, acetylcholine-induced dilation was more blunted by L-NMMA than by CbTX+AP (-71% versus -44%, P:<0.002) and was not fully prevented by the combination. Substituting iberiotoxin (IbTX) for CbTX greatly diminished inhibition of pulse pressure and agonist flow responses (with or without NOS inhibition). Furthermore, blockade by IbTX+AP was identical to that by AP alone, supporting a minimal role of IbTX-sensitive large-conductance K(+)(Ca) channels. CONCLUSIONS: K(+)(Ca) activation and NO comodulate in vivo pulsatility-stimulated coronary flow, supporting an important role of a hyperpolarization pathway in enhanced mechanovascular signaling. Small- and intermediate-conductance K(+)(Ca) channels are the dominant species involved in modulating both pulse pressure- and bradykinin-induced in vivo coronary dilation.